DISTRIBUTION OF NTTRERGIC NERVES IN THE RAT ADRENAL GLAND:

PLASTICITY IN AGING AND DISEASE

Thesis Submitted to the University of London for

the Degree of Doctor of Philosophy in

the Faculty of Science

by

Mekbeb Afework, B.Sc., M. Phil.

Department of Anatomy and Developmental Biology

University College London

June 1995

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ABSTRACT

This thesis presents the localization and characterization of the

recently discovered nitric oxide (NO)-synthesizing nerves in the

adrenal glands of rats, and their changes in various conditions which

cause alteration in adrenal nerve and/or glandular activities. The

study was conducted mainly by qualitative analysis of the NO-

synthesizing enzyme, nitric oxide synthase (NOS) immunohistochemically

and/or by the use of reduced nicotinamide adenine dinucleotide

phosphate (NADPH)-diaphorase histochemistry which also marks the site

of neuronal NOS immunoreactivity. In parts of the study, where

quantification of the levels of NOS was required, computer assisted

image analysis and biochemical assay for measuring the activity of NOS

were also used.

In the adult rat adrenal glands NOS immunoreactivity occurred in a population

of nerve cell bodies and fibres, where NADPH-diaphorase staining also colocalized. In

the adrenal cortical cells NADPH-diaphorase staining alone was observed without

NOS immunoreactivity. As found from surgical extrinsic and intrinsic adrenal

denervation studies, the NOS-immunoreactive nerve fibres in the gland have both

extrinsic and intrinsic origins. The extrinsic nitrergic nerve fibres innervate adrenal

chromaffin and ganglion cells. The intrinsic nitrergic nerve fibres which arise from the

local neurons innervate adrenal blood vessels and the zona glomerulosa of cortex.

Colocalization studies revealed that a population of NADPH-diaphorase stained NO-

synthesizing adrenal intrinsic neuronal cell bodies contained vasoactive intestinal

peptide and neuropeptide Y, but not calcitonin gene-related peptide, substance P,

tyrosine hydroxylase or calretinin.

Developmental studies showed that NOS-immunoreactive nerve fibres were

present in adrenal glands of rats at all ages examined from the 16^ ̂day of gestation up

to 2 years of age. A considerable degree of variation in the distribution of the

immunoreactive nerve fibres in both medulla and the zona glomerulosa of cortex was

observed at different ages. The NOS-containing neuronal cell bodies within the

adrenal gland were found from the 2 0 ^ ̂ day of gestation onwards, and increased in

their number to reach to that of adult levels by the 4^ ̂ day after birth. In the glands

from the aging rats their number was increased by 28.6 % above the adult levels . NOS

immunoreactivity in the chromaffin cells was observed only in the glands of the aging

rats.

Guanethidine, 6 -hydroxydopamine or capsaicin treatments did not

cause any change in the adrenal NOS immunoreactivity and NADPH-

diaphorase staining. In contrast, reserpine treatment for 7 days

caused a significant decrease in the NOS immunoreactivity in the nerve

fibres that innervate the chromaffin and ganglion cells.

Hypophysectomy did not cause any change in the NOS immunoreactivity,

although it eliminated most of the NADPH-diaphorase staining adrenal

cortical cells. Streptozotocin-induced diabetes of 8 weeks duration

caused an increase in the NOS-immunoreactive nerve fibres and induced

NOS immunoreactivity in a small number of adrenal cortical cells. It

also caused an increase in the intensity of NADPH-diaphorase staining

in the adrenal cortical cells. A significant degree of prevention of

such diabetic-induced increase in both NOS and NADPH-diaphorase was

found with ganglioside treatment. At a later stage (12 weeks of

diabetes duration) the effect of streptozotocin-induced diabetes was a

decrease in the NOS-immunoreactive nerve fibres but an increase in the

occurrence of NOS-immunoreactive adrenal cortical cells. The intensity

of NADPH-diaphorase staining in the cortical cells was still increased.

CONTENTS

ABSTRACT......................................................................................................................2

CONTENTS......................................................................................................................4

ACKNOWLEDGEMENTS...........................................................................................9

LIST OF TABLES AND FIGURES............................................................................ 10

PUBLICATIONS ARISING FROM THE WORK PRESENTED

IN THIS THESIS............................................................................................................ 12

LIST OF ABBREVIATIONS....................................................................................... 13

PREFACE........................................................................................................................15

SECTION 1: INTRODUCTION.................................................................................. 19

1:1. Historical background to adrenal gland research.............................................. 20

1:2. Morphology of adrenal gland.................................................................................21

1:3. Development and aging of adrenal medulla.........................................................24

1:4. Development and aging of adrenal cortex............................................................25

1:5. Biosynthesis and secretion of adrenal gland hormones................................... 26

1:5:1. Catecholamines........................................................................................... 26

1:5:2. Steroids........................................................................................................ 28

1:6. Innervation of adrenal gland................................................................................ 29

1:6:1. Extrinsic adrenal innervation.................................................................... 29

1:6:2. Intrinsic adrenal innervation..................................................................... 32

1:6:3. Course and distribution o f nerve fibres and

terminals within the adrenal cortex ..........................................................32

1:6:4. Course and distribution o f nerve fibres and

terminals within the adrenal medulla .......................................................32

1:7. Neuromediators in adrenal gland..........................................................................33

1:7:1. Acetylcholine................................................................................................33

1:7:2. Biogenic amines...........................................................................................34

1:7:3. Adenosine 5'-triphosphate.........................................................................35

1:7:4. Neuropeptides..............................................................................................36

1:7:4:1. Enkephalins..................................................................................36

1:7:4:2. Vasoactive intestinal peptide ......................................................37

1:7:4:3. Somatostatin................................................................................39

1:7:4:4. Neurotensin.................................................................................. 39

1:7:4:5. Calcitonin gene-relatedpeptide ............................................... 40

1:7:4:6. Neuropeptide Y ............................................................................41

1:7:4:7. Substance P ..................................................................................42

1:7:4:8. Bombesin...................................................................................... 43

1:7:4:9. Galanin........................................................................................ 44

1:8. Nitric oxide................................................................................................................45

1:8:1. Biosynthesis o f nitric oxide: nitric oxide synthase ................................... 47

1:8:2. Functional significance o f nitric oxide ......................................................49

1:8:2:1. Nitric oxide in the cardiovascular system ................................. 50

1:8:2:2. Nitric oxide in immunity and inflammation..............................51

1:8:2:3. Nitric oxide in neural transmission............................................51

1:9. Plasticity in adrenal gland innervation and expression of

neuromediators........................................................................................................ 53

1:9:1. During development and aging .................................................................. 53

1:9:2. During stress ................................................................................................55

1:9:3. After selective surgical denervations.........................................................56

1:9:3:1. Extrinsic adrenal denervation............................................ 56

1:9:3:2. Intrinsic adrenal denervation.....................................................57

1:9:3:3. Hypophysectomy..........................................................................57

1:9:4 During chronic exposure to drugs ..............................................................58

1:9:4:1. Reserpine..................................................................................... 58

1:9:4:2. 6-hydroxydopamine................................................................... 58

1:9:4:3. Guanethidine................................................................................59

1:9:4:4. Capsaicin..................................................................................... 59

1:9:5. In diabetes mellitus..................................................................................... 59

SECTION 2: MATERIALS AND METHODS...........................................................62

2:1. Animals used in this study...................................................................................... 63

2:2. Maintenance of the animals.................................................................................... 63

2:3. Tissue processing...................................................................................................... 63

2:3:1. Preparation o f adrenal sections................................................................. 63

2:3:2. Immunohistochemistry................................................................................64

2:3:3. NADPH-diaphorase histochemistry.........................................................66

2:4. Microscopy and computer assisted image analysis............................................. 67

2:5. Biochemical assay of nitric oxide synthase activity............................................ 68

2:6. Surgical procedures.................................................................................................70

2:6:1. Extrinsic adrenal denervation.................................................................... 70

2:6:2. Intrinsic adrenal denervation..................................................................... 71

2:6:3. Hypophysectomy......................................................................................... 72

2:7. Drug treatment......................................................................................................... 73

2:7:1. Reserpine.....................................................................................................73

2:7:2. Guanethidine and 6-hydroxydopamine.................................................... 73

2:7:3. Capsaicin..................................................................................................... 74

2:8. Induction of diabetes................................................................................................75

2:9. Source of Antibodies, Chemicals and Reagents.................................................. 77

SECTION 3: RESULTS..................................................................................................80

3:1. Distribution and characteristics of nitric oxide-synthesizing

nerves in the rat adrenal gland..............................................................................81

3:1:1. Colocalization o f nitric oxide synthase and

NADPH-diaphorase in rat adrenal g land .............................................. 82

3:1:2. Distribution and colocalization o f nitric oxide synthase

and NADPH-diaphorase in adrenal gland o f developing, adult

and aging Sprague-Dawley ra ts ..............................................................93

3:1:3. The intra-adrenal distribution o f intrinsic

and extrinsic nitrergic nerve fibres in the r a t ........................................110

3:1:4. Colocalization o f neuropeptides and NADPH-diaphorase

in the intra-adrenal neuronal cell bodies and

fibres o f the r a t .........................................................................................121

3:1:5. Calretinin immunoreactivity in adrenal gland o f

developing, adult and aging Sprague-Dawley ra ts ............................. 133

3:2. Changes in the expression of nitric oxide synthase and

NADPH-diaphorase in the adrenal gland of rats Iffdevelopment,-

aging and altered adrenal gland acti vity .̂....................................................... 146

3:2:1. Effect o f reserpine treatment and hypophysectomy on

the nitric oxide synthase immunoreactivity and

NADPH-diaphorase staining in the rat adrenal g land ........................ 147

3:2:2. Increase in nitric oxide synthase and NADPH-diaphorase

in the adrenal gland o f streptozotocin-diabetic Wistar

rats and its prevention by ganglioside....................................................157

SECTION 4: GENERAL DISCUSSION....................................................................181

4:1. Adrenal nitric oxide-synthesizing nerves........................................................ 182

4:2. Nitric oxide in the regulation of adrenal medullary secretion........................184

7

4:3. Nitric oxide in the regulation of adrenal cortical secretion............................188

4:4. Nitric oxide in the regulation of adrenal blood flow ....................................... 190

4:5. Plasticity in nitrergic innervation........................................................................193

REFERENCES............................................................................................................. 198

ACKNOWLEDGEMENTS

I am extremely grateful to my supervisor Professor Geoffrey Bumstock for giving me the opportunity to pursue my education under his supervision by arranging the cost of the study. I thank him for his enthusiastic and invaluable guidance of the research work and his helpful constructive comments during the preparation of the manuscript.

I thank Dr Annette Tomlinson for the helpful discussions throughout the course of the study and the constructive comments in the preparation of the manuscript. I would also like to thank Dr Abebech Belai for teaching me the technique of immunohistochemistry, and the collaborative work in the injections of the animals for the studies reported in Section 3:2:2 and the helpful discussions and encouragements throughout the course of this study.

My sincere gratitude goes to my other collaborators Dr Vera Ralevic who injected the animals for the studies reported in Section 3:1:3 and Dr Jill Lincoln who performed the biochemical assay presented in Section 3:2:2.1 thank Jane Pendjiky and Christopher Sym for their help in the photographic work. I would also like to thank Dr Peter Abrahams, Dr Christopher Dean and the late Professor John Pegington for their initiation of my study in this department and their encouragements; Dr Barbara Pittam for administrative support; Annie Evans for her encouragements and support; Tim Robson for keeping up with the material supplies; Dr John Rogers and Dr Ulrich Forstermann for providing the calretinin and some of the NOS antisera, respectively; all members of the Bumstock's research group and everybody whose contribution was helpful in the course of the study.

This study was supported in part by the grants from Overseas Research Students award, University College London Dean's Scholarship and Department of Anatomy and Developmental Biology's Research Studentship.

LIST OF FIGURES AND TABLES

Pages

Section 3:1:1.

Fig. 1 .......................................................... 8 8

Fig. 2 ............................................................................................ 89

Section 3:1:2.

Fig. 3 .......................................................................................... 101

Fig. 4 .......................................................................................... 102

Fig. 5 .......................................................................................... 103

Fig. 6 .......................................................................................... 104

Table 1 .......................................................................................105

Section 3:1:3

Fig. 7 .......................................................................................... 116

Fig. 8 .......................................................................................... 117

Fig. 9 .......................................................................................... 118

Section 3:1:4

Fig. 1 0 ........................................................................................ 127

Fig. 1 1 ........................................................................................ 128

Fig. 1 2 ........................................................................................ 129

Section 3:1:5

Fig. 1 3 ........................................................................................ 140

Fig. 1 4 ........................................................................................ 141

Fig. 1 5 ........................................................................................ 142

Section 3:2:1

Fig. 1 6 ........................................................................................ 152

Fig. 1 7 ........................................................................................ 153

Table 2 .......................................................................................154

10

Section 3:2:2

Fig. 1 8 ...........................................................................................168

Fig. 1 9 .......................................................................................... 169

Fig. 2 0 .......................................................................................... 170

Fig. 2 1 .......................................................................................... 171

Fig. 2 2 .......................................................................................... 172

Table 3 ......................................................................................... 173

Table 4 ......................................................................................... 174

Table 5 ......................................................................................... 175

11

PUBLICATIONS ARISING FROM THE WORK PRESENTED IN THIS

THESIS

Afework, M., Tomlinson, A., Belai, A. and Bumstock, G. (1992) Colocalization of nitric

oxide synthase and NADPH-diaphorase in rat adrenal gland. NeuroReport 3, 893-

896.

Afework, M., Tomlinson, A. and Bumstock, G. (1994) Distribution and colocalization

of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing,

adult and aging Sprague-Dawley rats. Cell Tissue Res. 276,133-141.

Afework, M., Ralevic, V. and Bumstock, G. (1995) The intra-adrenal distribution of

intrinsic and extrinsic nitrergic nerve fibres in the rat. Neurosci Lett 190,109-112.

Afework, M. and Bumstock, G. (1995) Colocalization of neuropeptides and NADPH-

diaphorase in the intra-adrenal neuronal cell bodies and fibres of the rat. Cell Tissue

Res in press.

Afework, M. and Bumstock, G. (1995) Calretinin immunoreactivity in adrenal glands

of developing, adult and aging Sprague-Dawley rats. Int J Dev Neurosci in press.

Afework, M. and Bumstock, G. (1995) Effect of reserpine treatment and

hypophysectomy on the nitric oxide synthase immunoreactivity and NADPH-

diaphorase staining in the rat adrenal gland. Manuscript.

Afework, M., Lincoln, J., Belai, A. and Bumstock, G. (1995) Increase in nitric oxide

synthase and NADPH-diaphorase in the adrenal gland of streptozotocin-diabetic

Wistar rats and its prevention by ganglioside. Manuscript.

12

LIST OF ABBREVIATIONS

A cells .................... adrenaline-storing and secreting cells

A ChE......................acetylcholinesterase

ACTH .....................adrenocorticotrophic hormone

A T P ........................ adenosine 5 -triphosphate

BH4 ........................ tctrahydrobiopterine

cA M P..................... cyclic adenosine monophosphate

cGM P..................... cyclic guanosine monophosphate

CGRP...................... calcitonin gene-related peptide

ChAT......................choline acetvl transferasecp m ........................ count per minuteD BH ....................... dopamine p-nyaroxyiase

DOPA....................dihydroxyphenylalanine

dpm .........................disintegration per minute

E ..............................embryonic day

ED RF..................... endothelium-derived relaxing factor

FA D .......................flavin adenine dinucleotide

FG F........................fibroblast growth factor

F ig ...........................figure

Figs........................ figures

FM N...................... flavin mononucleotide

G TP....................... guanosine triphosphate

h ............................. hour

..........................tritiated hydrogen

IDDM ................... insulin-dependent diabetes mellitus

EPS ....................... lipopolysaccharides

13

L T P .......................long term potentiation

m in ........................minute

N A ........................ noradrenaline-storing and secreting cells

NADPH................reduced nicotinamide adenine dinucleotide phosphate

L-N A M E.............N^-nitro-L-arginine methyl ester

N G F......................nerve growth factor

NIDDM ................non-insulin-dependent diabetes mellitus

L-N M M A........... N^-monomethyl-L-arginine

L -N N A ................N^-nitro-L-arginine

N O ....................... nitric oxide

N O S..................... nitric oxide synthase

N PY ..................... neuropeptide Y

6 -O H D A .............. 6 -hydroxydopamine

PB S...................... phosphate-buffered saline

PG P...................... protein gene product

PNM T.................. phenylethanolamine-N-methyltransferase

SGC cells .............small granule containing cells

S P ........................ substance P

T H ........................ tyrosine hydroxylase

V IP .......................vasoactive intestinal peptide

14

PREFACE

15

The paired adrenals are small pyramid-shaped organs located at the superior

pole of the kidneys. Each adrenal is composed of an inner part, the adrenal medulla and

an outer part, the adrenal cortex that is surrounded by a capsule. The general function

of the adrenal gland is to enable the individual to cope with stress conditions. The

catecholamine secretion by the adrenal medulla has a widespread effect in the body

system in mobilizing glucose and fatty acids for energy and preparing the heart, lungs,

and muscles for action during acute stress and injury. During chronic stress, the steroid

hormones of the cortex protect against overreaction from the body's responses to stress.

They are also important in immune defence and inflammation. In addition, particularly

during stress conditions due to prolonged deprivation of food and fluid, the adrenal

steroids stimulate gluconeogenesis to maintain the supply of glucose and increase

sodium reabsorption to maintain body fluid content.

Classically, secretion of catecholamines from the adrenal medulla was

considered to be under the sole control of the cholinergic preganglionic sympathetic

innervation (Elliott 1913; Hoshi 1926; Feldberg, Minz and Tisudzimura 1934). It was

also thought that humoral factors were exclusively responsible for the control of

adrenal cortical function (Hechter 1949; Hayenes, Savard and Dorfman 1952).

However, recent discoveries on the occurrence of a growing number of neuroactive

substances, in both adrenal medullary nerves and chromaffin cells (Kondo 1985;

Pelto-Huikko 1989), have suggested that several neuromediators are involved in

regulating the process of synthesis and secretion of various neuroactive substances by

the adrenal chromaffin cells. In the adrenal cortex, morphological studies provided

evidence for the presence of nerve fibres and endings containing various mediators (see

Charlton 1990). Functional studies have shown the involvement of these nerves in the

regulation of adrenal cortical activities (see Vinson, Hinson and Toth 1994). Secretion

from the adrenal gland is also found to be influenced by adrenal blood flow (Vinson

and Hinson 1992).

The major neuroactive substances found in the adrenal neuronal components

16

and/or chromaffin cells include acetylcholine, biogenic amines, adenosine triphosphate

(ATP) and neuropeptides. In addition, a substance with possible functional role(s),

whose synthesis in the adrenal gland was recently recognized is nitric oxide (NO). It is

also a recent discovery that NO which is a noxious unstable gas has been found to be

secreted by mammalian cells with widespread functions in the cardiovascular system,

immune system, inflammation and neurotransmission. Furthermore, changes in its

levels of production has been associated with various pathophysiological

complications.

NO is synthesized by a family of enzymes called nitric oxide synthases which

catalyze the conversion of L-arginine and molecular oxygen to NO and L-citrulline

(see Forstermann, Schmidt, Pollock, Sheng, Mitchell, Warner, Nakane and Murad

1991). Immunohistochemically, nitric oxide synthase (NOS) has been localized in a

wide range of cells that synthesize NO, and has been colocalized with the

histochemical staining for reduced nicotinamide adenine dinuceleotide phosphate

(NADPH)-diaphorase. In the adrenal gland, the presence of NOS was originally

reported from a biochemical study which demonstrated the activity of the enzyme in

rat and bovine adrenal glands (Palacios, Knowles, Palmer and Moncada 1989).

Recently, immunohistochemical and enzyme histochemical studies have shown its

occurrence in rat adrenal medullary nerves (Bredt, Hwang and Snyder 1990; Dawson,

Bredt, Fotuchi, Hwang and Snyder 1991). These studies, however, were brief and apart

from the demonstration of the existence of NOS in the gland, they did not characterize

the nature and extent of its distribution. The aim of the study presented in this thesis

was therefore to fully investigate and characterize cellular elements containing the

neuronal NOS isoform in the adrenal glands of rats in health and during various

conditions causing altered adrenal nerve and/or glandular activities.

In this thesis, a literature survey on the historical background and current

knowledge of the adrenal gland and NO is given in the Introduction (Section 1). The

17

Materials and Methods section (Section 2) describes the general background of the

techniques and the protocols employed in the present study. These include

immunohistochemistry, NADPH-diaphorase histochemistry and an enzyme

biochemical method for assay of NOS activity. The various experimental approaches

employed in the present study to cause alteration in the adrenal innervation and/or

glandular secretions are also described in this section.

The results are presented in two main parts. The first part deals with studies on

the demonstration and characterization of the NO-synthesizing nerves in the adrenal

glands of rats. These include: a detailed account of the distribution of NOS

immunoreactivity and its colocalization with NADPH-diaphorase staining in the adult

rat adrenal gland (Section 3:1:1); an account of the changes in the distribution of NOS

that occur during development and aging (Section 3:1:2); intra-adrenal distribution

and characterization of the extrinsic and intrinsic NOS-containing nerve fibres as

determined by surgical denervations of extrinsic and intrinsic adrenal nerves, and by

chemical sympathectomy and sensory denervation (Section 3:1:3); studies on

colocalization of NOS with a number of neuropeptides and the rate limiting enzyme in

the catecholamine synthesis, tyrosine hydroxylase (TH) (Section 3:1:4) and with the

recently discovered calcium binding protein, calretinin Section 3:1:5. As this study is

the first to describe the distribution of calretinin in the adrenal gland, a detailed account

of its occurrence and changes in development and aging has also been examined in

Section 3:1:5.

In part II of the Results section, changes occurring in the adrenal NOS and

NADPH-diaphorase following alteration of the state of the adrenal innervation and/or

glandular secretions by reserpine treatment and hypophysectomy (Section 3:2:1) and

by streptozotocin-induced diabetes (Section 3:2:2) are examined.

The separate experimental work is discussed individually in each study. In

addition, a discussion of broader issues is given in the General Discussion section

(Section 4).

18

SECTION 1

INTRODUCTION

19

1:1. Historical background to adrenal gland research

In 1563 Bartholomeus Eustachius gave the first description of the adrenal glands

under the title "glandulae Renibus Incumbentes". The anatomy and functions of the

adrenals, however, remained a puzzle for nearly three centuries while they were

described with various names and supposed functions. Casper Bartholinus (1611) gave

an account of the adrenals and illustrated them in a woodcut as hollow capsule-like

organs filled with black bile. This led to the belief that the adrenal glands have ducts

which connect them to the kidneys and/or the gonads (see Rolleston 1936).

Later, Cuvier (1805) discovered the adrenal glands as solid organs, each formed

of two distinct tissues, rather than as previously thought of, a capsule with a cavity.

The anatomy of the gland was correctly identified as having a smaller central part, the

adrenal medulla, and a larger outer part, the adrenal cortex, surrounded by a fibrous

connective tissue capsule.

In 1855, Thomas Addison described disease of the adrenal glands and pointed

out their indispensability for life. This initiated a functional approach towards the study

of the adrenal glands and the following year, Brown-Sequard (1856) verified their

importance with the demonstration that adrenalectomy in various animals would result

in death.

Henlé (1865) found brown colouration of the adrenal medullary tissue upon

treatment with chromate salts and introduced the term chromaffin reaction.

Subsequently, a pressor substance was demonstrated to be contained in adrenal

medullary extract (Oliver and Schafer 1894) and was implicated for the histological

reactivity of the tissue with chromate salts (Moore 1895). The search for the

identification of this pressor substance in the medullary extract was carried out and in

1901 Takamine and Aldrich, working independently, isolated a crystalline substance

and named it adrenaline and epinephrine, respectively. It was, however, later in the mid

20

1900s that a second pressor substance found in the adrenal medulla was identified and

termed noradrenaline or norepinephrine (Holtz, Credener and Kroneberg 1947;

Bergstrorm, von Euler and Hamberg 1949; von Euler and Hamberg 1949).

The observations that removal of the interrenal body, in some fishes, was fatal

indicated that the indispensability of the adrenal glands for life is related to the cortex

but not the medulla (Biedle 1913). The essential substance found in the adrenal cortex

was then identified to be lipid extracts which maintained adrenalectomized cats

indefinitely (Hartman and Brownell 1930; Swingle and Pfiffner 1930). Later,

numerous steroids were isolated from the cortical extracts and the adrenal cortex was

discovered to have three main groups of steroids each of which consisted of multiple

corticosteroids (Pfiffiier 1942; Mason 1964; Gaunt 1975). Mineralocorticoids, mainly

secreted by cells of the zona glomerulosa are one of such steroids (Sarason 1943;

Deane and Creep 1946; Deane, Shaw and Creep 1948; Creep and Deane 1949).

Aldosterone is the primary mineralocorticoid. Glucocorticoids, secreted by cells of

zona fasciculata and zona reticularis are the second group of adrenal corticosteroids

(Sarason 1943; Deane and Creep 1946; Deane et al. 1948). In humans and most

mammals the primary glucocorticoid is cortisol, while in some rodents it is

corticosterone (Bush 1953, Sandor, Fazekas and Robinson 1976). The third group of

corticosteroids secreted from adrenal cortex are the adrenal androgens. They are

believed to be secreted from the zona reticularis of the adrenal cortex. Testosterone is

the physiologically potent androgen (Bondy 1985).

1:2. Morphology of adrenal gland

It is only in mammals that the adrenal cortex and medulla are found intimately

associated. In the other vertebrate classes, the two tissues may be completely separate

or intermingled to a varying degree (Bourne 1961; Coupland 1965). When separate, the

homologue of the adrenal medulla is simply considered as part of the chromaffin

21

tissue, while that of the adrenal cortex is called interrenal tissue.

The adrenal cortex forms the higher proportion of the organ. In rat, the cortex

accounts for about 90 % of the weight, whereas the adrenal medulla forms the

remaining 10 %. However, calculated medullary weight and volume have been shown

to vary by as much as 30 % with different methods of fixation and embedding

(Tomlinson, Durbin and Coupland 1987). The parenchyma of the adrenal cortex is

formed from steroid synthesizing and secreting epithelial cells, arranged in cords or

cylinders with varying architecture to form the three adrenal cortical zones. These are

an outer zona glomerulosa, an intermediate zona fasciculata and an inner zona

reticularis. In many mammalian species, cells of the juxtamedullary zone of the cortex

are sometimes found interdigitated with the medullary chromaffin cells and in

histological sections may appear as islands of cortical cells dispersed in the medulla

(Coupland 1965).

The adrenal medulla is mainly composed of the chromaffin cells arranged in an

anastomosing network in close relation to capillaries. It represents the largest aggregate

of the chromaffin tissue, which is embryologically derived from the neuroectoderm,

and is usually innervated by the preganglionic sympathetic nerve fibres and contains a

high concentration of catecholamines which usually results in the chromaffin reaction

(Coupland 1989). The chromaffin cells contain a large number of vesicles, which are

called chromaffin vesicles or granules. Schwann cells, vascular and connective tissue

elements, and species variable numbers of ganglion cells and islets or cords of cortical

cells are additional cellular components found in the adrenal medulla (Coupland 1965).

In most mammals, there are two types of principal adrenal medullary secretory

cells: the adrenaline-storing and secreting (A) cells and the noradrenaline-storing and

secreting (NA) cells (Eranko 1952; Hillarp and Hokfelt 1953; Coupland 1965). In

adrenals fixed with glutaraldehyde and postfixed with osmium tetroxide, the A cells

contain vesicles filled with an evenly distributed medium electron dense granules,

while the NA cells contain electron dense granules which in rodents are usually located

22

asymmetrically within the vesicle (Coupland 1965). The relative proportions of A to NA

cells vary with species and age of the animal. In most postnatal mammals A cells

predominate, although a variable but smaller amount of NA cells are also always

present (Coupland 1984).

Another group of chromaffin cells called the small granule-containing (SGC)

cells are also found in the adrenal medulla of many animal species (Unsicker 1973a;

Gorgas and Bock 1976; Coupland, Kobayashi and Tomlinson 1977; Unsicker, Kabura-

Fliih and Zwarg 1978). The SGC cells contain membrane bound granules of variably

moderate to low electron density, with few high electron dense cores. They are of the

same size as typical chromaffin cells but have smaller vesicles, a higher nucleus to

cytoplasm ratio and a great capacity to form processes. In rat, they account for not

more than 1 % of the chromaffin cell's population (Tomlinson and Coupland 1990).

The SGC cells are considered to be cells of chromaffin cell lineage, morphologically

intermediate between typical chromaffin cells and neurons (Coupland 1984).

The blood supply to the adrenal gland is by multiple small arteries which arise

from the abdominal aorta or one of its major branches between the point where it enters

abdomen and the renal arteries (Lever 1952; Coupland 1975; Coupland and Selby

1976). Several small arteries approach the adrenals where they divide and redivide to

form a capsular plexus. Many cortical arteries arise from this plexus, penetrate the

capsule and form another plexus in the subcapsular region and zona glomerulosa, from

which several capillaries branch and run through the zonae fasciculata and reticularis.

These capillaries join and rejoin progressively to form larger channels in the medulla

which eventually open into the central vein. Additionally, there are a few cortical

arteries which directly traverse the capsule and outer region of the cortex to join the

capillary plexus in the zona fasciculata, or take a looped course back towards the

surface before joining the cortical capillary plexus (Flint 1900; Lever 1952).

Although there are some investigators who favour the existence of a vascular

23

corticomedullary portal circulation (Pohrecky and Wurtman 1971; Henderon and Daniel

1978), morphological studies did not reveal any intra-adrenal portal system (Flint

1900; Bennett and Kilham 1940; Gersh and Grollman 1941, Dempster 1974; Coupland

1975; Coupland and Selby 1976; Sparrow and Coupland 1987; Murakami, Oukouchi,

Uno, Ohtsuka and Taguchi 1989b). Instead, another group of arteries, penetrate the

capsule and pass straight through the cortex without branching and reach to the

medulla. Here they form a medullary capillary plexus which ultimately joins the

peripheral radicles of the central vein. The central vein also receives other minor veins

of periadrenal origin to form the single adrenal vein, which drains into the posterior

vena cava, the left renal vein or one of its branches, or into the adrenolumbar vein

(Johnstone 1957; Coupland 1965).

1:3. Development and aging of adrenal medulla

Adrenal chromaffin cells develop from the neural crest cells (Le Douarin 1980).

This was demonstrated by transplanting the neural crest and tube from quail embryo

into the corresponding regions of chick embryo and tracing the fate of the cells with

the help of their distinct nucleolus. It was found that the migratory behaviour of the

neural crest cells depended on the pathway available while the phenotype of the cells

was determined by environmental factors. Glucocorticoids secreted from the adrenal

cortex, favour differentiation of the sympathoadrenal precursors of the neural crest

cells migrating to the adrenal gland into chromaffin cells. On the other hand, fibroblast

growth factor (FGF) and nerve growth factor (NGF) determine the neural phenotype in

the neural crest cells migrating to other regions (Le Douarin 1980; Anderson 1993).

Early in development, the migrating adrenal medullary precursor cells invade

the dorsomedial aspect of the adrenal cortical anlage and pass through three

developmental stages (Coupland 1952). The first stage is one of "primitive

sympathetic" cells with a round basophilic nucleus surrounded by a thin rim of

cytoplasm. The second stage comprises cells called "parasympathetic" or

24

phaeochromoblasts of elongated cells with an elongated nucleus. The third stage is the

chromaffin cells or phaeochromocytes, which are round to polyhedral cells displaying

the chromaffin reaction and have a rounded nucleus. Such later cell types showing the

chromaffin reaction may be found as early as 12-15 weeks in the human foetus, 20

days in foetal rabbit and 12 days in foetal rat (Coupland 1980).

During aging the adrenal medulla shows proliferative lesions (Fleischmann

1965; Haley 1976; Hollander and Snell 1976; Cheng 1980). In the rat, lesions occur as

nodular proliferations on a diffuse hyperplastic background (Tischeler, DeLellis,

Perlman, Allen, Costopoulos, Lee, Nunnemacher, Wolfe and Bloom 1985). Such

lesions are particularly observed after 1 year (Gillman, Gilbert and Spence 1953), and

their occurrence is influenced by several factors such as genetic, humoral, nourishment

and gender (Cheng 1980). In a lesioned adrenal medulla, there is occurrence of both

adrenaline and noradrenaline granules within the same chromaffin cell (Chalfe and

Perlman 1976). Moreover, the appearance of synaptic type vesicles with electron dense

contents in a number of NA cells of medulla of the aging rat suggested the

differentiation of adrenal chromaffin cells towards the neural phenotype in aging

(Coupland and Tomlinson 1989).

1:4. Development and aging of adrenal cortex

Adrenocortical primordia organize early in foetal life from the local mesoderm.

The process of growth, differentiation and cell renewal of the adrenal cortex has been a

matter of controversy. There were two principal hypotheses: the cell migration

hypothesis originally proposed by Gottschau (1883), and the zonal hypothesis

proposed by Chester Jones (1948) where each zone is thought to replenish its cells

independently. However, the observations from the regenerating capacity of the

adrenocortical cells in the enucleated adrenals (Ingle and Higgins 1938; Greep and

Deane 1949) supported the cell migration hypothesis. This was further proved with

25

studies from injection of radiolabelled thymidine which showed a continual centripetal

migration of subcapsular cells (Bertholet 1980; Zajicek, Ariel and Arber 1986). During

the migration, the cells are transformed progressively into the different cell types of the

zonae glomerulosa, fasciculata and reticularis. The migration ends in the zona

reticularis where the cells ultimately degenerate and die.

In aging rat, there is hypertrophy of the zonae fasciculata and reticularis

(Rebuffat, Belloni, Rocco, Andreis, Neri, Malendowicz, Gottardo, Mazzocchi and

Nussdorfer 1992). There is also an increase in the occurrence of nodular hyperplasia

(Attia 1985). In addition, an increase in the levels of lipid droplets contained in cells of

all the three zones and lipofÆin contained in cells of the zonae rticularis and

fasciculata is documented (Carsia and Malamed 1989; Belloni, Rebuffat,

Malendowicz, Mazzochi, Rocco and Nussdorfer 1992; Rebuffat et aL 1992).

1:5. Biosynthesis and secretion of adrenal gland hormones

1:5:1. Catecholamines

Adrenaline is synthesised from tyrosine in four enzyme catalyzed sequential

steps of dihydroxyphenylalanine (DOPA), dopamine, noradrenaline and adrenaline

(Blaschko 1939; Demis, Blaschko and Welch 1956; Masuoko, Schott, Akawie and

Clark 1956; Undenfriend and Wyngaarden 1956; Goodall and Kirshner 1957; Kirshner

1975). The conversion of tyrosine into DOPA is catalyzed by tyrosine hydroxylase

(TH). This is the rate limiting step in the pathway. DOPA is transformed into dopamine

by the enzyme DOPA decarboxylase. Both TH and DOPA decarboxylase have been

shown to C c t f j r in the cytosol, and therefore, the formations of DOPA and

dopamine take place in the cell cytoplasm. Dopamine is taken into the granules and

converted into noradrenaline by the enzyme dopamine 6̂ -hydroxylase (DBH) only

found within the chromaffin granules. In A cells noradrenaline leaks out of the

chromaffin granules and is converted into adrenaline in the cell cytoplasm by the

enzyme phenylethanolamine-N-methyltransferase (PNMT). From the cell cytoplasm,

26

adrenaline is taken up and stored in the chromaffin granules. The chromaffin vesicles

also contain nucleotides, chromogranins, lipids, glycoproteins, calcium,

mucopolysaccharides, ascorbic acid, and a number of neuroactive peptides (Winkler

1976, Winkler, Apps and Fischer-Colbrie 1986).

The secretion of catecholamines from the adrenal chromaffin cells is effected

mainly by acetylcholine released from the sympathetic preganglionic nerves.

Acetylcholine is believed to initiate opening of the calcium channels located on the

plasma membrane and brings an inward movement of calcium from extracellular fluid

to raise intracellular calcium levels which leads to the movement of chromaffin

granules towards the plasma membrane for subsequent fusion and exocytosis (Baker

and Knight 1978,1981; Knight and Baker 1982; Burgoyne 1991).

Acetylcholine receptors on the chromaffin cells are both nicotinic and

muscarinic (Feldberg et aL 1934). There is, however, species variation in the relative

distribution of this two subtypes of cholinergic receptors (Malmejac 1964; Rubin and

Miele 1968; Liang and Perlman 1979; Knight and Baker 1986). In the cat, selective

muscarinic agonists have been shown to release principally adrenaline, while nicotinic

agonists cause release of mainly noradrenaline (Douglas and Poisner 1965; Rubin and

Miele 1968). However, the absence of such preferential secretion with activation of

either of the receptor in many other species has led to the conclusion that dual control

may be peculiar to the cat (Unger and Phillips 1983). Both nicotinic and muscarinic

acetylcholine receptors contribute to activation of secretion of catecholamines

(Kirpekar, Prat and Shiavone 1982; Role and Perlman 1983; Wakade and Wakade

1983). The extent to which each receptor is involved depends on the concentration of

acetylcholine and the animal species, and it appears that it is mainly the nicotinic

receptor that is engaged in active secretion of catecholamines. The muscarinic

receptors are activated by a relatively low concentration of acetylcholine to cause an

efflux of calcium from the cell and an increase in intracellular cyclic guanosine

27

monophosphate (cGMP) levels that is inhibitory to nicotine-mediated release of

catecholamines (Schneider, Cline and Lemaire 1979; Derome, Tseng, Mercier,

Lemaire and Lemaire 1981). At relatively higher concentration of acetylcholine the

inhibition of release is overcome by activation of the nicotinic receptors. In addition to

acetylcholine, several other neuromediators (see section 1:7) and glucocorticoids have

also been shown to influence the biosynthesis and secretion of adrenal medullary

hormones.

1:5:2. Steroids

The starting material for the synthesis of adrenal steroid hormones is cholesterol

(Hechter, Solomon, Zaffaroni and Pincus 1953; Werbin and Chaikoff 1961; Krum,

Morris and Bennett 1964). Adrenal cortical cells synthesize cholesterol, or obtain it

from blood plasma, and mainly store it as cholesterol ester in the lipid droplets. When

required, the synthesis of steroid hormones occurs by a series of reactions involving

oxidation-reduction and hydroxylation, which are catalyzed respectively, by a

hydroxysteroid dehydrogenase system and a series of mixed function oxidases

(hydroxylases), utilising O2 , NADPH, adrenodoxin and cytochrome P-450 (Hyano,

Saba, Dorfman and Hechter 1956; Gower 1975; Samuels and Nelson 1975; Fraser

1992). Since the enzymes are located either in the mitochondria or the endoplasmic

reticulum, the metabolic intermediates move back and forth between the two organelles

during the reaction process. Cholesterol is released from the lipid droplets by the action

of cholesterol ester hydrolase and passes into the mitochondria, where it is converted

into a common intermediate precursor called pregnenolone. The pregnenolone passes

out of the mitochondria and enters into the endoplasmic reticulum and is converted into

androstenedione, 11-deoxycortisol and 11-deoxycorticosterone. Androstenedione is

further converted into the androgens, while 11-deoxycortisol and 11-

dexycorticosterone are transferred back into the mitochondria and yield cortisol and

corticosterone, respectively, as well as aldosterone.

28

Adrenocorticotrophic hormone (ACTH) regulates the synthesis of cortisol and

corticosterone (Hechter 1949; Haynes et al. 1952; Saffran, Grad and Bayliss 1952).

Binding of ACTH into receptors on adrenocortical cells activate adenylate cyclase

which increases intracellular levels of cyclic adenosine monophosphate (cAMP)

(Schuster 1974). The elevated levels of cAMP lead to phosphorylation of cholesterol

ester hydrolase and subsequent activation of this enzyme, to cause cleavage of the fatty

acid residues from the cholesterol ester (Jefcoate, DiBartolomeis, Williams and

McNamara, 1987; Simpson and Waterman 1988). In addition, cAMP triggered by

chronic response to ACTH regulates the expression of genes encoding steroidogenic

enzymes for a longer term maintenance of optimal levels of steroid hydroxylase

enzymes (Simpson and Waterman 1988,1992).

The renin-angiotensin system, extracellular ions and ACTH are the major

factors regulating the synthesis of aldosterone (Quinn and Williams 1988, 1992).

ACTH and prolactin are thought to control the production of adrenal androgens (Bondy

1985). In addition, the splanchnic nerves and several neuromediators have also been

demonstrated to have modulatory effects on adrenal steroidogenesis (see section 1:7).

1:6. Innervation of adrenal gland

1:6:1. Extrinsic adrenal innervation

The observation of a similar response of the gut upon stimulation of either the

greater splanchnic nerve or directly the adrenal gland led Jakobj (1892) to suggest that

the greater splanchnic nerve may innervate the gland. Dreyer (1899) provided more

direct evidence by demonstrating an increased pressor activity of the blood in the

adrenal vein of dog following stimulation of the greater splanchnic nerve. The

contribution of the lesser splanchnic nerve and branches arising from the lumbar

sympathetic chain in the innervation of the gland was later shown by Elliott (1912).

Several investigators have demonstrated that the splanchnic innervation to the gland is

29

ipsilateral (Elliott 1912; Cannon, Lewis and Britton 1926; Hollinshead 1936; Maycock

and Heslop 1939). However, a minor contralateral splanchnic innervation has also been

reported (Kahn 1911).

The observation that the secretory response of the adrenal medulla to electrical

stimulation of the splanchnic nerve could be abolished by nicotine, which also

produces sympathetic ganglionic block, indicated that the primary innervation to the

gland was preganglionic (Elliott 1913). A comparable degree of degeneration of nerve

fibres in the adrenal gland following section of either the greater splanchnic nerve or

branches from the coeliac ganglion to the gland, indicated that the adrenal nerve fibres

pass through the ganglion without synaptic interruption and further supported the

preganglionic nature of the major innervation to the gland (Hoshi 1926).

Studies from tracing of degenerating nerve fibres following nerve fibre

transection showed that the adrenal preganglionic nerves leave the spinal cord via the

anterior roots of third thoracic to second lumbar spinal nerves (Elliott 1913; Hoshi

1926; Swinyard 1937; Young 1939; MacFarland and Davenport 1941). Furthermore,

evidence from chromatolitic cell changes upon adrenal medullectomy, demonstrated

that the adrenal preganglionic nerve fibres originate from neurons located in the

intermediolateral cell column of the spinal cord (Cummings 1969). This was further

proved by studies involving injection of nerve tracing substances into the adrenal

medulla and locating the retrogradely labelled preganglionic cell bodies in the

intermediolateral cell column of thoracic and upper lumbar spinal cord segments, with

the majority of the neurons concentrated in the mid and lower thoracic regions (Ellison

and Clark 1975; Schramm, Adair, Stribling and Grey 1975; Hasse, Contestabile and

Flumerfelt 1982; Holets and Elde 1982; Bacon and Smith 1988; Kesse, Parker and

Coupland 1988; Strack, Sawyer, Marubio and Loewy 1988; Parker, Mohamed and

Coupland 1990b). The majority of the studies demonstrated an ipsilateral location of

the preganglionic neurons in the spinal cord. However, a bilateral location with a small

number of neurons found contralaterally has also been reported (Ross, Smolen and

30

Cherry 1980; Holets and Elde 1982).

Studies with nerve fibre tracer injection into the gland, have also indicated that a

small number of neurons located in the prcvcrtebral sympathetic chain in rat, guinea

pig and marmoset as well as the parasympathetic dorsal motor nuclei of the vagus in

rat and guinea pig have their terminals in the gland (Kesse et al. 1988; Coupland,

Parker, Kesse and Mohamed 1989; Parker et at. 1990b).

Earlier findings of a small number of degenerating nerve endings following

removal of the thoracic dorsal root ganglia suggested the presence of sensory nerve

fibres in the gland (Pines and Narowtschatowa 1931; Kiss 1951). More direct

morphological evidence was, however, obtained recently with the help of injection of

nerve fibre tracer substance into the gland and locating the retrogradely labelled

neurons in the ipsilateral dorsal root ganglia and the bilateral sensory vagal ganglia in

rat, guinea pig and marmoset (Afework 1988; Mohamed, Parker and Coupland 1988;

Coupland et al. 1989; Parker, Afework and Coupland 1990a; Parker, Kesse, Mohamed

and Afework 1993). The spinal sensory nerve fibres originate from the upper thoracic

to mid sacral dorsal root ganglia, and the majority of the neurons are located in the

lower thoracic ganglia.

Higher centres located in the brain stem and hypothalamus are implicated in the

regulation of adrenal medullary secretion. Electrical stimulation of various parts of the

hypothalamus and the midbrain causes catecholamine secretion in cat (Folkow and von

Euler 1954; Robinson, Culberson and Carmichael 1983; Stoddard-Apter, Siegel and

Levin 1983; Goadsby 1985) and rat (Gauthier and Reader 1982; Matsui 1984).

Stimulation of medulla oblongata has also been shown to result in catecholamine

secretion in rat (Matsui 1979). Moreover, transneuronal cell body labelling with

pseudorabies virus has provided morphological evidence for five centres within the

hypothalamus and brain stem that have connections with adrenal medullary nerve

fibres (Strack, Sawyer, Platt and Loewy 1989; Wesselingh, Li and Blessing 1989).

31

1:6:2. Intrinsic adrenal innervation

A small number of intrinsic neurons are found in the adrenal gland and are

believed to be involved with local adrenal innervation. Coupland (1965) described the

relative number of these neurons in comparison to the chromaffin cells in the adrenal

medulla of different mammalian species to be in descending order of guinea pig, man,

cat, dog, rat, mouse, sheep, ox and rabbit. The precise functional role of these neurons

is, however, not totally understood.

1:6:3. Course and distribution o f nerve fibres and terminals within adrenal cortex

Although it has long been shown that nerve fibres traverse the adrenal cortex,

there were a number of studies which failed to show local innervation (Hollinshead

1936; Swinyard 1937; MacFarland and Davenport 1941). Nevertheless, a number of

studies on axonal degeneration following section of adrenal nerve fibres have revealed

the existence of a subcapsular plexus in several species including rat, guinea pig, cat

dog and man (Alpert 1931; Willard 1936; Kiss 1951; Lever 1953; Mikhail and Mahran

1965). From the subcapsular plexus, nerve fibres have been described to innervate

cortical cells of the zonae glomerulosa (Alpert 1931; Willard 1936; Lever 1953) and

reticularis (Mikhail 1961; Mikhail and Mahran 1965). Evidence for the innervation of

adrenal cortex was further provided by ultrastructural studies which demonstrated

nerve terminals containing synaptic vesicles adjacent to the cortical cells in mouse, rat,

guinea pig, golden hamster, pig, sheep, monkey and man (Unsicker 1969, 1971;

Robinson, Perry, Hardy, Coghlan and Scoggins 1977; Uno 1977; Unsicker, Kabura-

Flüh and Zwarg 1978; Migally 1979; Dorovini-Zis and Zis 1991).

1:6:4. Course and distribution o f nerve fibres and terminals within adrenal medulla

Unlike that of adrenal cortex, the innervation of adrenal medulla is rich and has

been described by several investigators (MacFarland and Davenport 1941; Coupland

32

and Holmes 1958; Coupland 1965; Tomlinson and Coupland 1990). Nerve fibres branch

from the subcapsular plexus, and together with nerve bundles which directly cross the

capsule, pass through the cortex alongside the arteries and capillaries inward to the

medulla. Here, nerve fibres with associated Schwann cell cytoplasm branch and

rebranch to form a three dimensional network of nerve fibres around the chromaffin

and ganglion cells. The basement membrane of the Schwann cell fuses with that of the

chromaffin cell and the nerve fibres end as synaptic junctions.

1:7. Neuromediators in adrenal gland

A growing number of neuroactive substances have been demonstrated in the

adrenal chromaffin cells and/or neuronal components. These neuroactive substances

include acetylcholine, several biogenic amines, adenosine 5'-triphosphate (ATP) and

neuropeptides, as reviewed below.

1:7:1. Acetylcholine

The cholinergic nature of the great majority of the nerve fibres innervating the

adrenal medulla and which cause its secretion, was originally shown from functional

studies (Feldberg et al. 1934). Later, a diffuse network of cholinesterase-positive nerve

fibres were demonstrated in the adrenal medulla of cat, rat and rabbit (K ^le 1950;

Coupland and Holmes 1958). Electron microscopic studies have also revealed the

majority of adrenal medullary nerve terminals contain small synaptic electronlucent

vesicles, with positive cholinesterase activity in rat and bird (Lewis and Shute 1969;

Unsicker 1973b).

While the majority of the cholinergic nerve fibres traverse the cortex on their

way towards the medulla, some terminate in the cortex. Nerve fibres displaying

positive cholinesterase activity have been found in the adrenal cortex of rat, cat, rabbit,

sheep and goat (Coupland and Holmes 1958; Robinson et al. 1977; Purwar 1978;

33

Watanabe, Hiramtsu, Ohmori and Paik 1990). Furthermore, muscarinic receptors have

been demonstrated on bovine adrenocortical cells (Hadjian, Ventre and Chambaz

1981). Acetylcholine stimulates secretion of glucocorticoids and mineralocorticoids

from isolated adrenal gland of calf (Rosenfeld 1955) and frog (Benyamina,

Leboulenger, Lirhmann, Delarue, Fcullioley and Vaudry 1987). It also causes such

secretion from calf in vivo (Edwards and Jones 1990) and isolated adrenocortical cells

in cow (Hadjian, Guidicelli and Chambaz 1982).

1:7:2. Biogenic amines

The principal biogenic amines contained in the adrenal gland are adrenaline and

noradrenaline, which are synthesized and released by the adrenal chromaffin cells.

Substantial numbers of intra-adrenal nerves also contain catecholamines. Synaptic

vesicles typical of adrenergic profiles with predominantly small and some large

granular vesicles have been seen in association with adrenal chromaffin cells of toad

(Piezzi 1966). Electron microscopy combined with histochemistry, have shown a close

apposition of catecholaminergic nerve fibres to the adrenal chromaffin cells of bird

(Unsicker 1973c) and cat (Prentice and Wood 1975). Moreover, Omori, Okuno,

Fujisaw and Ono (1991) using TH immunoelectron microscopy have reported the

presence of catecholaminergic nerve fibres in close proximity to the rat adrenal

chromaffin cells. In contrast to such small number of studies which so far managed to

find catecholaminergic nerve fibres in association with the adrenal chromaffin cells,

there is an overwhelming evidence which showed the catecholamine-containing nerve

fibres innervate the capsule, cortex and blood vessels of the gland. Such evidence

comes from studies conducted with catecholamine histofluorescence combined with

electron microscopy in rat, Syrian hamster and sheep (Unsicker 1969; Robinson et al

1977; Kleitman and Holzwarth 1985); TH immunohistochemistry combined with

electron microscopy in rat (Oomori, Okuno, Fujisawa, Ishikawa, Satoh, Matsuda,

Yamano and Ono 1989; Oomori et al. 1991); the chromaffin reaction combined with

34

electron microscopy in rat (Vizi, Toth, Szalay, Windisch, Orso, Szabo and Vinson 1992)

and DBH immunohistochemistry in man (Gilchrist, Leake and Charlton 1993). The

catecholaminergic nerve fibres that are found in the cortex mainly innervate cells of the

zona glomerulosa, although a minor supply to those of the zona fasciculata has also

been found.

The catecholaminergic innervation to the gland is believed to be principally

noradrenergic. There is also evidence which suggested local noradrenergic axon

terminals may take up dopamine from the circulation and release it or convert it to

noradrenaline (Vizi, Toth, Orso, Szalay, Sz, Szabo, Baranyi and Vinson 1993).

Functionally, dopamine has been shown to inhibit aldosterone secretion from the zona

glomerulosa of rat (Pratt, Turner, Bowsher and Henry 1987). However, secretion of

aldosterone by direct /3-adrenergic stimulation has also been shown in cultures of

bovine adrenal cortical cells (De Lean, Racz, McNicoll and Desrosiers 1984).

The other biogenic amines immunohistochemically demonstrated in the adrenal

gland are histamine and serotonin. Histamine has been localized in NA cells of rat

adrenal gland and cultured bovine adrenal chromaffin cells (Hàppôlà, Soinila,

Paivarinta, Joh and Panula 1985; Tuominen, Karhunen, Panula and Yamatodani 1993).

It stimulates the release of catecholamines from isolated bovine adrenal chromaffin

cells (Pender and Burgoyne 1992) and perfused rat adrenals (Borges 1994). Histamine

also causes corticosteroid secretion from the adrenal gland of rat and isolated dog

adrenocortical cells (Mikolajczyk 1965; Hirose, Matsumoto and Aikawa 1978).

Serotonin has been demonstrated in A cells of adrenal gland of rat (Holzwarth,

Sawetawan and Brownfield 1984; Verhofstad and Jonsson 1983). It stimulates

corticosteroid secretion in the rat (Mikolajczyk 1965).

1:7:3. Adenosine 5'-triphosphate

In addition to its role as intracellular energy source, ATP has also extracellular

35

signalling functions and acts as a neurotransmitter and/or modulator (Bumstock 1972,

1975). In the chromaffin cells, ATP is costored and released with the catecholamines

(Douglas, Poisner and Rubin 1965; Stevens, Robinson, Van Dike and Stizel 1975;

Winkler and Westhead 1980). The released ATP has been suggested to exert a

modulatory role on catecholamine secretion, since ATP has been shown to inhibit

(Chem, Herrera, Kao and Westhead 1987) or facilitate (Chem, Kim, Slakey and

Westhead 1988; Kim and Westhead 1989) catecholamine release from isolated bovine

chromaffin cells. More recently, ATP has been shown to induce catecholamine

secretion from perfused bovine adrenal gland (Lin, Bott, Kao and Westhead 1995).

1:7:4. Neuropeptides

1:7:4:1. Enkephalins

Met-enkephalin and leu-enkephalin were the first two opioid peptides

originally isolated from the brain and described as 5 amino acid polypeptides (Hughes,

Smith, Kosterlitz, Forthergill, Morgan and Morris 1975). Subsequently, a number of

other peptides with opiate activity were discovered (Weber, Evans and Barchas 1983).

Enkephalins were also the first peptides to be identified immunohistochemically in the

adrenal chromaffin cells (Schultzberg, Lundberg, Hokfelt, Temius, Brandt, Elde and

Goldstein 1978). They have been demonstrated in the adrenal chromaffin cells of

several species including rat, guinea pig, hamster, cat, rabbit, dog, horse and man

(Schultzberg et at. 1978; Lundberg, Hamberger, Schultzberg, Hokfelt, Granberg,

Efendic, Terenius, Goldstein and Luft 1979; Linnoila, Diaugustine, Hervonen and

Miller 1980; Livett and Dean 1980; Livett, Day, Elde and Howe 1982; Vamdell,

Topia, Demey, Rush, Bloom and Polak 1982; Kobayashi, Ohashi, Fujita, Nakao,

Yoshimasa, Imura, Mochizuki, Yanaihara, Yanaihara and Verhofstad 1983; Kobayashi,

Ohashi, Uchida, Nakao, Imura, Yanaihara and Verhofstad 1984; Kondo, Kuramoto and

Iwanaga 1984; Heym 1985; Kondo 1985; Heym and Kummer 1988; Pelto-Huikko

1989). However, the localization of enkephalins to either A or NA cells is equivocal.

36

Schultzberg et al. (1978) reported as met-enkephalin immunoreactivity is located in the

NA cells of rat, with no such preference being found in the cat. Kobayashi et at. (1983,

1984) and Kondo et at. (1984) reported that met-enkephalin-arg-gly-leu and m et-

enkephalin-arg-phe have no preference of localization to either A or NA cells in rat,

cat, hamster and dog. Pelto-Huikko (1989), in contrast, described that

immunoreactivity to both met-enkephalin and leu-enkephalin was mainly found in A

cells in rat, cat and hamster, and only in NA cells in mouse.

Nerve fibres and terminals immunoreactive for Met-enkephalin have been

found in rat, mouse, guinea pig and cat adrenal medulla (Schltzberg et al. 1978;

Kobayashi et al. 1983; Pelto-Huikko 1989). Such nerve fibres innervate A cells and

some medullary neurons of the gland (Pelto-Huikko 1989).

Enkephalins cause a non-specific inhibition of the nicotinic-evoked, but an

enhancement of the basal, release of catecholamines from cultured bovine adrenal

chromaffin cells (Lemaire, Lemaire, Dean and Livett 1980; Dean, Lemaire and Livett

1982; Saiani and Guidotti 1982; Livett, Boksa, Dean, Mizobe and Lindenbaum 1983).

They reduce the release of acetylcholine and substance P, and thereby inhibit synaptic

transmission (Konishi, Tsunoo and Otsuka 1981). Both met- and leu-enkephalins

stimulate aldosterone secretion from bovine adrenal glomerulosa cells (Bruzzone and

Marusic 1988). They also stimulate aldosterone and corticosterone secretion in

perfused rat adrenal preparation (Hinson, Cameron, Purbick and Kapas 1994b; Hinson,

Purbick, Cameron and Kapas 1994c).

1:7:4:2. Vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) is a 28 amino acid polypeptide originally

isolated from pig small intestine (Said and Mutt 1970). It is part of a pre-prohormone

that is cleaved into VIP and peptide histidine-isoleucine and is structurally related to

secretin, glucagon and gastric inhibitory peptide (Itoh, Obata, Yanaihara and Okamoto

37

1983). Immunoreactivity for VIP occurs in a small number of A cells of rat and human

adrenal medulla and cultured bovine chromaffin cells (Bryant, Bloom, Polak,

Albuquerque, Modlin and Pearse 1976; Kondo 1985; Siegel, Biden and Pruss 1985).

Immunoreactivity for VIP also occurs in a number of intra-adrenal neurons of rat

(Hokfelt, Lundberg, Schultzberg and Fahrenkrug 1981; Holthwarth 1984; Kondo 1985;

Dagerlind, Goldstein and Hokfelt 1990; Oomori, Okuno, Fujisawa, luchi, Ishikawa,

Satoh and Ono 1994).

VIP-immunoreactive nerve fibres forming a plexus with varicosities and nerve

terminals are found in the zona glomerulosa and subcapsular region of the cortex of

adrenal gland of several species, including rat, guinea pig, cat, dog, pig, hamster and

man (Linnoila et al. 1980; Hokfelt et al 1981; Holzwarth 1984; Kondo 1985; Heym

and Kummer 1988). A moderate distribution of VIP-immunoreactive nerve fibres also

occurs in the adrenal medulla.

VIP stimulates catecholamine release from adrenal medullary cells of foetal bird

(Cheung and Holzwarth 1986) and perfused rat adrenal gland (Guo and Wakade 1994).

A rise in the concentration of blood aldosterone and corticosterone levels occurs

following administration of VIP in rat (Nussdorfer and Mazzocchi 1987; Pralong,

Corder and Gaillard 1991). Prolonged (7 days) treatment of rat with VIP stimulates the

growth and steroidogenic capacity of the zona glomerulosa of adrenal cortex

(Mazzocchi, Robba, Malendowicz and Nussdorfer 1987). In perfused adrenal gland of

pig, administration of VIP causes an increase in both aldosterone and cortisol secretion

(Ehrhart-Bomstein, Bomstein, Scherbaum, Pfeiffer and Holst 1991). Similarly, VIP

causes an increase in secretion of both aldosterone and corticosterone in perfused rat

adrenal gland preparation (Hinson, Kapas, Orford and Vinson 1992; Hinson et al

1994b, c). Reports on the effect of VIP on isolated adrenocortical cells are, however,

not coherent. Some studies have found as VIP could stimulate steroidogenesis (Kowal,

Horst, Pensky and Alfonzo 1977; Morera, Cathiard, Lurthe and Saez 1979; Li, Queen

and LaBella 1990), while some others found it with no effect (Hinson et al 1992;

38

Enyedi, Szabo and Spat 1983).

1:7:4:3. Somatostatin

Somatostatin is a 14 amino acid polypeptide originally isolated from bovine

hypothalamus (Brazeau, Vale, Burgus, Ling, Butcher, Rivier and Guillemin 1973).

Singly dispersed somatostatin-immunoreactive chromaffin cells occur in human

adrenal gland (Lundberg et al. 1979). In addition, nerve fibres immunoreactive for

somatostatin are found in the adrenal medulla of guinea pig and cat (Heym and

Kummer 1988).

Somatostatin causes a noncompetitive inhibition of catecholamine secretion

from isolated bovine and pig chromaffin cells (Mizobe, Kozousek, Dean and Livett

1979; Role, Leeman and Perlman 1981; Livett et al. 1983). It also inhibits the growth

of the zona glomerulosa and its steroidogenic capacity in rat (Rebuffat, Robba,

Mazzocchi and Nussdorfer 1984).

1:7:4:4. Neurotensin

Neurotensin was originally isolated from bovine hypothalamus and

characterized as a 13 amino acid polypeptide (Carraway and Leeman 1973, 1975). In

the cat, immunoreactivity for neurotensin occurs in the majority of the NA cells

(Lundberg, Rokaeus, Hokfelt, Rosell, Brown and Goldstein 1982; Terenghi, Polak,

Vamdell, Lee, Wharton and Bloom 1983). However, it is found only in a few adrenal

chromaffin cells in guinea pig, rat and dog (Heym 1985; Reinecke 1985). Neurotensin-

immunoreactive nerve terminals innervate NA cells and intra-adrenal neurons in

hamster and cat (Lundberg et al. 1982; Pelto-Huikko, Salminen, Partnen, Toivanen

and Hervonen 1985).

Administration of neurotensin in the rat causes hypertrophy of the zona

glomerulosa and elevates plasma aldosterone levels (Malendowicz, Nussdorfer,

39

Majchrzak, Nowak and Lesniewska 1992). It has also been shown that infusion of

neurotensin into isolated rat adrenal gland causes an increase in both aldosterone and

corticosterone secretion (Hinson et al. 1994b, c). Moreover, the finding of high levels

of specific binding sites for neurotensin on the cells of zona reticularis of rat adrenal

gland has suggested that circulating neurotensin may also influence steroid secretion

from this region of the gland as well (Goedert, Mantyh, Hunt and Emson 1984).

1:7:4:5. Calcitonin gene-relatedpeptide

Calcitonin gene-related peptide (CGRP) was first isolated from rat adrenal

medullary cells as a 37 amino acid polypeptide which is expressed by the same gene as

that of calcitonin by alternative processing of the mRNA (Rosenfeld, Mermod, Amara,

Swanson, Sawchenko, Rivier, Vale and Evans 1983). Subsequent studies have shown

its localization in a number of A cells of adrenal medulla in mouse, rat, cat, hamster

and man (Kondo 1985; Kuramoto, Kondo and Fujita 1987; Pelto-Huikko 1989).

Several CGRP-immunoreactive nerve fibres are found in the adrenal gland of

rat, cat, mouse, hamster, rabbit and pig (Rosenfeld et al. 1983; Kuramoto et al. 1987;

Heym and Kummer 1988; Pelto-Huikko 1989). Such immunoreactive fibres are

associated with chromaffin cells and ganglion cells in the medulla and some blood

vessels in the cortex.

CGRP has inhibitory effect on aldosterone secretion when administered to dog

and rat in vivo (Murakami, Hiromichi, Nakajima, Nakamoto, Kageyama and Saruta

1989a; Mazzochi, Malendowicz, Meneghelli and Nussdorfer 1992). It also causes

inhibition of steroid biosynthesis in isolated rabbit adrenal glomerulosa cells

(Murakami et al. 1989a). However, in isolated perfused rat adrenal gland, CGRP has

caused vasodilatation accompanied with an increased secretion of aldosterone (Hinson

and Vinson 1990). It has also been shown that CGRP causes cortisol secretion from

adrenal gland of calf in vivo (Bloom, Edwards and Jones 1989).

40

1:7:4:6. Neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acid polypeptide and shares a considerable

sequence homology with various pancreatic polypeptides and peptide YY (Tatemoto,

Carlquist and Mutt 1982). Immunohistochemically, NPY has been demonstrated in

adrenal chromaffin cells in mouse, rat, guinea pig, hamster, cat, pig, dog, horse, cow

and man (Vamdell, Polak, Allen, Terengchi and Bloom 1984; Kuramoto, Kondo and

Fujita 1986; Lundberg, Hokfelt, Hcnscn, Theodorsson-Norheim, Pemow, Hamberger

and Goldstein 1986; Schalling, Seroogy, Hokfelt, Chai, Hallman, Persson, Larhammar,

Ericsson, Terenius, Graffi, Massoulie and Goldstein 1988b; Pelto-Huikko 1989). The

localization of NPY immunoreactivity in A and NA cells is not coherent, with

conflicting reports in the literature. NPY immunoreactivity was originally described in

the NA cells of adrenal medulla of mouse, rat, guinea pig, cat, horse and cow (Vamdell

et al. 1984; Majane, Alho, Kataoka, Lee and Yang 1985). However, subsequent

investigators have reported NPY immunoreactivity is instead located in the A cells of

mouse, rat, hamster, guinea pig, cat, bovine and man adrenal medulla (De Quidt and

Emson 1986; Kuramoto et al. 1986; Lundberg et al. 1986; Pelto-Huikko 1989).

Furthermore, Schalling et al. (1988b) described NPY immunoreactivity in all A and a

small number of NA cells of rat adrenal medulla.

Immunoreactivity for NPY also occurs in the ganglion cells of the adrenal gland

in rat, guinea pig and hamster (Pelto-Huikko 1989; Dagerlind et al. 1990; Oomori et

al. 1994). Several nerve fibres and terminals immunoreactive for NPY are found in the

adrenal gland of rat, mouse, guinea pig, hamster, dog, horse and cow (Vamdell et al.

1984; Majane et al. 1985; De Quidt and Emson 1986; Kuramoto et al. 1986; Pelto-

Huikko 1989; Maubert, Tramu, Croix, Beauvillain and Dupouy 1990). Such

immunoreactive nerve fibres form a plexus in the zona glomemlosa, and make synaptic

contacts with cortical cells, chromaffin and ganglion cells of medulla and some blood

vessels in the gland.

41

Specific binding sites for NPY have been localized on the glomerulosa cells of

bovine adrenal cortex (Torda, Cruciani and Saavedra 1988). In the rat, NPY is shown

to cause aldosterone secretion from adrenal gland in vivo (Mazzocchi and Nussdorfer

1987) and perfused adrenal gland preparation (Hinson et al. 1994b). In addition,

hypertrophy of the rat zona glomerulosa, accompanied with a rise in plasma levels of

aldosterone, were observed following prolonged treatment with NPY (Rebuffat,

Malendowicz, Belloni, Mazzocchi and Nussdorfer 1988a). In contrast, prolonged

treatment of a smaller dose of NPY has resulted in inhibition of aldosterone and

corticosterone levels accompanied with a decrease in the volume of the zona

fasciculata cells of adrenal gland in rat (Lesniewska, Nowak, Miskowak, Nussdorfer

and Malendowicz 1990). NPY has also been found to inhibit corticosterone secretion

from dispersed rat zonae fasciculata and reticularis cells (Malendowicz, Lesniewska

and Miskowiak 1990).

1:7:4:7. Substance P

It was von Euler and Gaddum (1931) who first extracted substance P (SP) from

horse brain and intestine, and observed that it could cause hypotension and tachycardia

when administered intravenously. Later, SP was identified as an 11 amino acid

polypeptide (Chang, Leeman and Niall 1971). Immunoreactivity for SP has been

observed in a number of adrenal chromaffin cells in rat, dog, guinea pig and rabbit

(Heym 1985; Kuramoto, Kondo and Fujita 1985; Heym and Kummer 1988).

SP-immunoreactive nerve fibres have been localized in the capsule and zona

glomerulosa of adrenal cortex in rat, guinea pig and man (Linnoila et al. 1980;

Kuramoto et al. 1985). SP-immunoreactive nerve fibres with varicosities have also

been demonstrated in apposition to chromaffin cells of rat, guinea pig, hamster, dog,

pig, cat and man adrenal medulla (Linnoila et al. 1980; Kuramoto et al. 1985; Heym

and Kummer 1988).

At a relatively high concentration (> 10” ^ mol/1), SP inhibits the nicotinic

42

acetylcholine-mediated catecholamine release from adrenal chromaffin cells (Livett,

Kozousek, Mizobe and Dean 1979; Mizobe, Kozouzek, Dean and Livett 1979; Livett

et aL 1983). At a lower concentration (< 10” ^ mol/1), SP prevents the nicotinic

receptor desensitization and thereby protects and prolongs catecholamine release from

adrenal chromaffin cells (Livett et al. 1983; Boksa and Livett 1984). It has also been

shown that SP facilitates acetylcholine release from the splanchnic nerve terminals

presynaptically (Zhou and Livett 1990). By itself SP has no effect on secretion, but

requires the presence of acetylcholine or a nicotinic agonist for effect (Livett, Marley,

Wan and Xin-Fu 1990). It has therefore been suggested that SP by acting as a

modulator maintains a prolonged output of catecholamines during stress condition

(Livett et at. 1983; Livett et al. 1990).

SP enhances aldosterone secretion from rat adrenal gland in vivo (Nussdorfer,

Malendowicz, Belloni, Mazzocchi and Rebuffat 1988). Prolonged infusion of SP

results in the hypertrophy of zona glomerulosa cells associated with a rise in

aldosterone levels (Nussdorfer et al. 1988). In addition, SP increases both aldosterone

and corticosterone secretion from isolated perfused rat adrenal gland (Hinson et al.

1994b, c).

1:7:4:8. Bombesin

Bombesin was originally isolated from frog skin extracts and characterized as a

14 amino acid polypeptide (Anastasi, Erspamer and Bucci 1971). Later, it was found in

the mammalian central and peripheral nervous system (Panula 1986).

Immunohistochemically, bombesin has been demonstrated in bovine adrenal medullary

cells (Lemaire, Chouinard, Mercier and Day 1986) and guinea pig adrenal medullary

nerve fibres (Heym and Kummer 1988). Bombesin has a direct stimulating action on

catecholamine secretion from rat adrenal gland in vivo (Okubo, Kaku, Kaneko and

Yanaihra 1985). In contrast, it inhibits basal corticosterone secretion from isolated cells

43

of the zonae fasciculata and reticularis of rat adrenal cortex (Malendowicz, Lesniewska,

Baranowska, Nowak and Majchrzak 1991).

1:7:4:9. Galanin

The 29 amino acid polypeptide, galanin was first isolated from pig intestine

(Tatemoto, Rokaeus, Jomvall, McDonald and Mutt 1983). Immunoreactivity for

galanin has been shown in adrenal chromaffin cells of mouse, hamster and cat (Pelto-

Huikko 1989). A few nerve fibres showing galanin immunoreactivity have been found

near intra-adrenal blood vessels in rat, mouse, cat and hamster (Pelto-Huikko 1989).

Galanin stimulates secretion of corticosterone and aldosterone in rat in vivo

(Mazzocchi, Malendowicz, Rebuffat and Nussdorfer 1992). It also causes release of

both corticosterone and aldosterone from isolated perfused porcine adrenal gland

(Holst, Ehrhart-Bomstein, Messell, Poulsen and Harling 1991).

While the above described neuromediators are the most established ones with

functional evidence in the adrenal glandular activities, there are also other additional

neuroactive substances demonstrated in the gland. These include: atrial natriuretic

polypeptide (Inagaki, Kubota, Kito, Kangawa and Matsuo 1986; Heym and Kummer

1988); corticotrophin-releasing hormone (Bruhn, Engeland, Anthony, Gann and

Jackson 1987; Rundle, Benedict, Robinson and Funder, 1988); vasopressin and

oxytocin (Hawthorn, Nussey, Henderson and Jenkins 1987); delta sleep-inducing

peptide (Ekman, Bjartell, Eklblad and Sundler 1987); growth-associated protein

(Grant, Konig, Deloume, Aunis and Langley 1992; Dorsey and Schmidt 1993) and

gamma-aminobutyric acid (Kataoka, Fujmoto, Alho, Guidotti, Geffard, Kelly and

Hambauer 1986; Oomori, luchi, Nakaya, Tanaka, Ishikawa, Satoh and Ono 1993). In

addition, by the time the work presented in this thesis was commenced a few studies

have indicated the presence of the NO-synthesizing enzyme, NOS in bovine and rat

adrenal glands (Palacios et al. 1989; Bredt et al. 1990; Dawson et al. 1991). While this

work was in progress, additional reports on localization of NOS in rat and human

44

adrenal glands have also appeared (Aim, Larsson, Ekblad, Sundler and Andersson 1993;

Dun, Dun, Wu and Forstermann 1993; Heym, Colombo-Benckmann and Mayer 1994).

As the present study is concerned with investigation of the NO-synthesizing cellular

elements in the adrenal gland of rat, relevant literatures regarding NO is reviewed

below.

1:8. Nitric oxide

NO is a colourless gas with an extremely simple chemical structure. It is a free

radical, labile molecule whose activity, when superfused over a tissue, decays by 50 %

in about 4 seconds (Moncada, Palmer and Higgs 1989). It reacts with oxygen to form

nitrogen dioxide, ultimately breaking down into the stable end products nitrite and

nitrate. Recently, it has come apparent from observations by investigators in different

unrelated research areas that NO is a major secretory product of mammalian cells in

host defence and homeostasis.

Mitchell, Shonle and Grindley (1916) first suggested that mammals produced

oxides of nitrogen. This was, however, largely ignored or thought to be due to

intestinal nitrifying bacteria (White 1975; Tannenbaum, Fett, Young, Land and Bruce

1978). Later, Green, Tannenbaum and Goldman (1981), while analyzing the metabolic

balance of nitrate levels observed that germ-free and ordinary rats excreted

comparably more nitrate than they ingested. Therefore, nitrate synthesis in mammals

was suggested to be a normal process, than is due to intestinal nitrifying bacteria.

Furthermore, the observation of an enhanced nitrate excretion in a human with an

episode of fever and diarrhoea instigated investigations and the discovery that rats

injected with Escherichia coli lipopolysaccharides (LPS) could result in an increase in

nitrate excretion (Wagner and Tannenbaum 1982; Wagner, Young and Tannenbaum

1983). Subsequently, Stuehr and Marietta (1985) with studies on LPS-induced nitrate

biosynthesis in LPS-sensitive versus LPS-resistant mice combined with experiments

45

on cells in culture showed that macrophages can account at least, in part, for the increase

in nitrate excretion. In such nitrate biosynthesis pathway of macrophages, L-arginine

was found to be the substrate while L-citrulline was a coproduct (Hibbs, Taintor and

Vavrin 1987a; Iyengar, Stuehr and Marietta 1987). It was subsequently found that NO

was an intermediate coproduct along with L-citrulline (Marietta, Yoon, Iyengar, Leaf

and Wishnok 1988).

In another laboratory, Furchgott and Zawadzki (1980) observed the relaxation of

isolated preparations of rabbit thoracic aorta by acetylcholine, required an intact

endothelium. This led to the postulation that a labile substance, which was termed

endothelium derived relaxing factor (EDRF), was released from stimulated

endothelium to bring about vasodilatation. Subsequently, the effect of EDRF in the

smooth muscle of blood vessels was shown to be acting by increasing the levels of

cGMP (Rapoport and Murad 1983). This indicated the similarity of the mode of action

of EDRF to that of nitrovasodilators (Katsuki and Murad 1977; Katsuki, Arnold and

Murad 1977), which do not require endothelium for their effect (Furchgott and

Zwadzki 1980; Rapoport and Murad 1983). As nitrovasodilators had already been

speculated to exert their effect via NO (Arnold, Mittal, Katsuki and Murad 1977;

Gruetter, Barry, McNamara, Gruetter, Kadowitz and Ignarro 1979) EDRF was

postulated to be NO. Eventually, based on the indistinguishability of EDRF from NO

in terms of biological activity, stability and susceptibility to an inhibitor and to a

potentiator, EDRF was identified to be identical to NO (Ignarro, Buga, Wood, By ms

and Chudhuri 1987; Palmer, Ferrige and Moncada 1987).

Another interesting finding was made by Garthwaite, Charles and Chess-

Williams (1988). Upon stimulation of cerebellar slices with excitatory amino acids,

they observed that there was release of a labile substance which had pharmacological

properties similar to those of NO, including the ability to raise cGMP and to relax

vascular smooth muscle. In retrospect, NO was then considered to be responsible for

the earlier unexplained observations of increased cGMP levels in certain brain regions

46

upon stimulation by neurotransmitters, particularly amino acids (Drumond 1983). In

fact, there had been previous reports where NO was shown to stimulate guanylyl

cyclase activity in the brain (Arnold et al. 1977; Miki, Kawabe and Kuriyama 1977),

although this was considered to be simply a pharmacological phenomenon.

1:8:1. Biosynthesis o f nitric oxide: nitric oxide synthase

In mammalian cells, NO is synthesized from the guanidino nitrogen of L -

arginine by the enzyme NOS (EC 1.14.13.39) through a process that consumes five

electrons and results in the formation of L-citrulline. There are three isoforms of NOS

which have been identified and their cDNAs are isolated. These are: NOS isoform I, in

brain; NOS isoform II, in cytokine and endotoxin-induced cells such as macrophages,

hepatocytes and vascular smooth muscles; and NOS isoform III, in endothelial cells

(see Forstermann et al. 1991; Nathan 1992; Forstermann, Close, Pollock, Nakane,

Schwartz, Gath and Kleinert 1994). In the literature, there are varying nomenclatures

assigned to the three NOS isoforms. Isoform I is also referred to as brain, neuronal or

constitutive NOS. Isoform II is also called inducible or macrophage NOS, while

isoform III is referred to as endothelial or constitutive NOS (Forstermann et al 1994;

Nathan and Xie 1994a).

Each of the NOS isoforms is produced by distinct genes with the deduced NOS

amino acid sequences from the three different cDNAs being approximately 50-60 %

identical with one another. In humans, the genes for NOS isoform I are located on

chromosome 12 (Nakane, Schmidt, Pollock, Forstermann and Murad 1993), whereas

those of isoforms II and III are located on chromosomes 17 and 7, respectively

(Marsden, Heng, Schere, Stewart, Hall, Shi, Tssi and Schappert 1993; Xu, Charles,

Moncada, Gorman, Sheer, Liu and Emson 1994). The molecular mass of the denatured

proteins of NOS isoforms I, II and III are 150-160 kDa, 133-135 kDa and 125-135

kDa, respectively (Bredt and Snyder 1990; Revel, White and Marietta 1991; Schmidt,

47

Pollock, Nakane, Gorsky, Forstermann and Murad 1991; Stuehr, Cho, Kwon, Weise and

Nathan 1991; Lamas, Marsden, Li, Tempt and Michel 1992; Sessa, Harrison, Barber,

Zeng, Durieux, D'Angelo, Lynch and Peach 1992).

All the three isoforms of NOS use L-arginine and molecular oxygen as

substrate and require the cofactors NADPH, tetrahydrobiopterine (BH4 ), flavin adenine

dinucleotide (FAD) and flavin mononucleotide (FMN). They all bind calmodulin and

contain heme (Marietta et al. 1988; Mayer, John, Heinzel, Werner, Wachter, Schultz

and Bohme 1991; Forstermann et at. 1994). The C-terminal half of NOS has similarity

to the cytochrome P-450 reductase where there are binding sites for FMN, FAD and

NADPH (Sessa 1994).

The NOS isoforms I and III are generally constitutively expressed by cells

which contain them, and require calcium and calmodulin for their activity (Knowles,

Palacios, Palmer and Moncada 1989; Bredt and Snyder 1990; Malinski and Taha

1992). Following receptor or physiologically induced increase in the intracellular

calcium level, calcium binds to calmodulin and the calcium-calmodulin complex

activates NOS. This facilitates the formation of NO in small amounts (picomoles) for a

short period of up to several minutes (Malinski and Taha 1992). Such intermittent

productions of NO in small amounts transmit signals. Although, originally NOS

isoform I is thought to be exclusively a cytosolic enzyme (Schmidt et al. 1991), recent

findings show that up to 60 % of its total activity in rat and rabbit cerebella is found in

the particulate fraction (Hecker, Miilsch and Busse 1994). About 95 % of the NOS

isoform III is found as particulate, while the remaining 5 % is cytosolic (Pollock,

Forstermaim, Mitchell, Warner, Schmidt, Nakane and Murad 1991).

The NOS isoform II is normally absent from the cell until it is activated by

cytokines, endotoxins or exotoxins (Moncada, Palmer and Higgs 1991; Nathan 1992).

Upon its activation, the enzyme facilitates the release of large amount of NO

(nanomoles), for a period of up to 5 days, which acts as a cytotoxic molecule against

invading microorganisms and tumour cells (Vodovotz, Kwon, Pospischil, Manning,

48

Paik and Nathan 1994). The NOS isoform II is found as a soluble fraction. Like the

isoforms I and III which are calcium dependent, the NOS isoform II has also binding

site for calmodulin despite the calcium independence of its activity (Cho, Xie,

Calaycay, Mumford, Swiderek, Lee and Nathan 1992; Lowenstein, Glatt, Bredt and

Snyder 1992; Schini and Vanhoutte 1992). However, in NOS isoform II, calmodulin is

very tightly bound and is unaffected by calcium.

NOS isoform I has been localized immunohistochemically in neurons found in

both central and peripheral nervous tissue; astrocytes; epithelial cells of lung, uterus

and stomach; kidney macula densa cells; and pancreatic islet cells (see Stuehr and

Griffith 1992; Henry, Lepoivre, Drapier, Ducrocqu, Boucher and Guissani 1993;

Forstermann et aL 1994). Induced NOS isoform II has been localized

immunohistochemically in a variety of cells which include macrophages; lymphocytes;

neutrophils and eosinophils in red pulp of spleen; kupffer cells; endothelial cells and

hepatocytes of liver; alveolar macrophages in lung; macrophages and endothelial cells

in adrenal gland; eosinophils mast cells and endothelial cells in colon; nerves

supplying mesenteric blood vessels; and epithelium of large air ways (Bandaletova,

Brouet, Bartsch, Sugimura, Esumi and Ohshima 1993; Buttery, Springall, Carpenter,

Riveros-Moreno, Moncada, Cohen and Polak 1993; Kobzik, Bredt, Lowenstein,

Drazen, Gaston, Sugarbaker and Stamler 1993). NOS isoform III has been localized in

endothelial cells of a wide variety of arterial and venous blood vessels (Pollock,

Nakane, Buttery, Martinez, Springall, Polak, Forstermann and Murad 1993).

1:8:2. Functional significance o f nitric oxide

The best characterized molecular target of NO in the cell is iron contained in

certain proteins as a heme group or as an iron-sulphur complex (Henry et al. 1993).

Binding of NO to such iron-containing enzymes leads to either activation or

inactivation of the enzyme. One enzyme which is activated by NO is guanylatc

49

cyclase. Binding of NO to the iron located in the heme group of guanylate cyclase

causes the production of cGMP, which in turn activates a cascade of other cellular

processes leading to activities such as dilatation of blood vessels (Moncada et al. 1991)

and neuronal transmission (Garthwaite 1991; Schuman and Madison 1994). Important

enzymes that are inhibited by the binding of NO to the iron contained in their catalytic

centre include: ribonucleotide reductase, which is rate-limiting in DNA replication;

iron-sulphur cluster-dependent enzymes (complex I and II) involved in mitochondrial

electron transport; and *'cis”-aconitase in the citric acid cycle (Kwon, Stuehr and

Nathan 1991; Lepoivre, Fieschi, Coves, Thelander and Fontecave 1991; Nathan and

Hibbs 1991; Beredeaux 1993; Stamler 1994). Such diversity of the targets of NO

coupled with its production by wide variety of cells make NO to be involved in

multiple functions. So far it has been implicated in the cardiovascular system,

immunity and inflammation and neuronal transmission.

1:8:2:1. Nitric oxide in the cardiovascular system

Small amounts of NO continuously released from the vascular endothelial cells

brings about a basal level of vascular smooth muscle relaxation (Moncada et al. 1991).

Furthermore, NO released from the endothelial cells and also possibly from the

platelets causes inhibition of platelet aggregation and adhesion, and thereby it prevents

blood clot and facilitates blood flow (Radomski and Moncada 1991). Release of NO

along with other possible factors from endocardium have been suggested to be

involved in the regulation of cardiac function (Smith, Shah and Lewis 1991; Smith,

Shah, Fort and Lewis 1992). NO released form perivascular nerves of certain blood

vessels may also contribute to the regulation of blood flow and pressure (Rand 1992).

By such means, NO regulates the systemic blood pressure as well as local blood flow

in specific vascular beds in brain, kidney, lung, heart and gastrointestinal tract

(Moncada and Higgs 1993; Lowenstein, Dinerman and Snyder 1994). Decrease in the

systemic NO production could therefore lead to vasospasm or hypertension, as for

50

example, decreased endothelium-dependent relaxation is found in patients with

essential hypertension (Panza, Quyyami, Brush and Epstein 1990). In contrast,

overproduction of NO is implicated in hypotension of sepsis, and may also be

associated with rare causes of abnormally low blood pressure, such as idiopathic

orthostatic hypotension (Lowenstein et al. 1994).

1:8:2:2. Nitric oxide in immunity and inflammation

Locally released NO in large quantities from activated cells, such as

macrophages and neutrophils, as well as destroying invading pathogenic organisms

may damage normal near by cells. By such a mechanism, NO kills or inhibits the

growth of many pathogens including bacteria, fungi, parasites and tumour cells, and

also normal host cells in autoimmune diseases (Lowenstein et al. 1994). Inhibitors of

NOS have been shown to reduce acute inflammation in rat (lalenti, Ignaro, Moncada

and Di Rosa 1992). In addition, glucocorticoids, in a potency which correlates to that

of their antiinflammatory action have been found to inhibit induction of the calcium-

independent NOS, but not the activity of the induced enzyme (Moncada and Palmer

1991). It has, therefore, been suggested that NO may be involved in the pathogenesis

of inflammation (Moncada and Palmer 1991; lalenti et al. 1992).

1:8:2:3. Nitric oxide in neuronal transmission

In the central nervous system, NO has been suggested to participate in

modulating neuronal transmission. NO released postsynaptically is suggested to diffuse

in retrograde fashion and affect the presynaptic neurons resulting in a stable increase in

synaptic transmission, as a phenomenon of long-term potentiation (LTP) (Bohme,

Bon, Stutzmann, Doble and Blanchard 1991; Schuman and Madison 1994). Such LTP

is thought to be linked to memory function. Indeed, NOS inhibitors have been found to

impair learning and can produce amnesia in chick and rat (Chapman, Atkins, Allen,

51

Haley and Steinmetz 1992; Hoischer and Rose 1992; Bohme, Bon, Lemaire, Reibaud,

Plot, Sutzmann, Doble and Blanchard 1993), The NO released from the postsynaptic

neurons during LTP is, however, found to be generated by the NOS isoform III rather

than the NOS isoform I (O’Dell, Huang, Dawson, Dinerman, Snyder, Kandel and

Fishman 1994).

Another activity where NO has been implicated in the central nervous system

concerns neurotoxicity. Excessive calcium entry during overstimulation with excitatory

amino acids has been associated with high levels of NO production in the central

nervous system leading to convulsion and neurotoxicity (Garthwaite 1991; Moncada

and Higgs 1993; Triggle 1994). In such manner, NO has been implicated in neuronal

damage during a variety of neuropathological conditions including stroke, Parkinson's

disease, multiple sclerosis and the dementia associated with Alzheimer's disease and

acquired immunodeficiency syndrome (Moncada and Higgs 1993; Triggle 1994).

In the peripheral autonomic nervous system, NO is implicated in the non-

adrenergic non-cholinergic inhibitory neurotransmission. NO has been found to

mediate some forms of gastrointestinal relaxation, including dilatation of the stomach

to adapt to increase in intra-gastric pressure (Rand 1992). NO released from pelvic

plexus neurons has been shown to be responsible for penile erection (Ignarro 1990;

Burnett, Lowenstein, Bredt, Chang and Snyder 1992). Additionally, NO released from

nerve fibres innervating adventitia of certain blood vessels may participate in the

regulation of blood flow (Rand 1992). NO has also been found involved in the

relaxation of tracheal muscle (Belvis, Stretton, Yacoub and Barnes 1992) and bladder

(Persson, Igawa, Mattiasson and Andersson 1992). In the adrenal gland, functional

studies have suggested the involvement of NO during secretion of catecholamines

(Dohi, Morita and Tsujimoto 1983; O'Sullivan and Bourgoyne 1990; Uchiyama,

Morita, Kitayama, Suemitsu, Minami, Miyasako and Dohi 1994) and corticosterone

(Adams, Nock, Truong and Cicero 1992; Cameron and Hinson 1993).

NO is, however, atypical transmitter in many ways and therefore differ from

52

hitherto known neurotransmitters. Unlike the classical mediators and the neuropeptides

which are frequently stored in vesicles and released specifically (see Bumstock 1986;

Kupfermann 1991), NO is not stored but diffuses freely from its site of formation

without any specialized release machinery (see Garthwaite 1991; Schuman and

Madison 1994). Being a free radical molecule it permeates membrane and pass normal

signal transduction routes without interaction with synaptic membrane receptors and

forms covalent bonds fairly easily with its target molecules (Stamler et al. 1992;

Stamler 1994), in contrast to the classical transmitters and the neuropeptides which

have complex structures and depend for their action on a complimentary fit to a

specific receptor. The action of conventional neurotransmitter is terminated by

presynaptic reuptake or enzymatic degradation, whereas the action of NO is

presumably terminated by diffusion away from its targets as well as by forming

covalent linkage to the superoxide anion or scavenger proteins (Stamler 1994). NO

acting as a covalently reactive redox type mediator may not be the only one of its type,

and may be the first example of a completely new signalling molecule. It has already

been suggested that CO, another activator of guanylate cyclase could be a neuronal

messenger (Verma, Hirsch, Glatt, Ronnett and Snyder 1993).

1:9. Plasticity in adrenal gland innervation and expression of neuromediators

1:9:1. During development and aging

As shown in the rat, the developmental expression of various mediators found in

the adrenal gland is not synchronous. In the developing adrenal chromaffin cells,

noradrenergic expression, immunoreactivity for TH and DBH, is evident by embryonic

day (E)12.5, while adrenergic expression, immunoreactivity for TH, DBH and PNMT,

is not observed prior to E17 (Bohn, Goldstein and Black 1981). During late foetal life,

all developing adrenal medullary chromaffin cells of the rat achieve adrenergic

expression, and solely noradrenaline expressing cells start reappearing by 2-3 days

53

postnatally (Verhofstad, Coupland, Parker and Goldstein 1985). The relative

concentration of normal adult proportion of about 80 % adrenaline and 2 0 %

noradrenaline is reached by the 4^ ̂ day of postnatal life (Verhofstad et al. 1985).

Immunoreactivity for NPY and Leu-enkephalin in the developing adrenal medullary

cells of rat begin to appear at E15 and E16, respectively (Henion and Landis 1990).

The proportions of adrenal chromaffin cells expressing met-enkephalin, CORF and

neurotensin during the first week are higher than those found in the adult (Holgert,

Dagerlind, Hokfelt and Lagercrantz 1994). NPY and galanin are expressed in

equivalent proportions of chromaffin cells in the glands from developing and adult rats

(Holgert et at. 1994).

The development of adrenal innervation begins during foetal life, although on

birth, functional innervation depends on the state of maturity of the neonate (Slotkin

1986). Ultrastracturally, synaptic profiles associated with the adrenal medullary

chromaffin cells of foetal rats are observed as early as 15.5 days of gestation (Daikoku,

Kinutani and Sato 1977). However, as found during the first postnatal week, most of

the synaptic profiles on the chromaffin cells are immature, although, they account for

about 50 % of those found in the adult rat (Tomlinson and Coupland 1990).

Pharmacological studies have also revealed that the innervation of adrenal medulla of

rat is non-functional until about the end of the first week, and becomes mature by 1 0

days after birth (Slotkin, Smith, Lau and Bareis 1980; Slotkin, Chantry and Bartolomé

1982). However, a neuronal connection between the adrenal medulla and the spinal

cord is already established by 2 days after birth (Parker, Kesse, Tomlinson and

Coupland 1988).

As shown with immunohistochemical studies, bundles of nerve fibres labelled

with neurofilament 160 kDa are visible in the embryonic adrenal medulla as early as 15

days of gestation (Henion and Landis 1990). Immunoreactive nerve terminals

associated with the chromaffin cells are, however, found only postnatally by the 2 ^^

day for neurofilament 160 kDa and choline acetyl transferase (ChAT), and by the 5^^

54

day for leu-enkephalin (Henion and Landis 1990). Intra-adrenal nerve fibres

immunoreactive for CGRP and galanin are found fully developed by day 2 after birth

(Holgert et al. 1994). However, intra-adrenal nerve fibres immunoreactive for ChAT,

leu-enkephalin and acetylcholinesterase (AChE) increase during the early postnatal

period and reach to that of adult levels by days 11-12, 14 and 16, respectively (Henion

and Landis 1990; Holgert et al. 1994). Met-enkephalin-immunoreactive nerve fibres

were found only by the 1̂ ̂ (Holgert et al. 1994) or the 2”^ week (Kent and Coupland

1989) after birth and increased afterwards throughout the developmental period

(Holgert et al. 1994).

There are a few studies carried out on the state of innervation and expression of

neuromediators in the adrenal gland during aging. An increased sympathoadrenal

activity associated with elevated plasma catecholamine levels was reported by Ito, Sato

and Suzuki (1986). Reduced numbers of AChE-positive nerve fibres were described in

the nodular hyperplastic regions that occur in aging Long-Evans rat (Tischler et al.

1985). However, studies by Tomlinson and Coupland (1990) did not reveal any change

in the frequency of synaptic profiles associated with the chromaffin cells of aging

Wistar rat from that observed in young adult rat.

1:9:2. During stress

Varieties of stress stimuli such as immobilization, cold, swimming, electric

shock and insulin administration activate secretion of glucocorticoids and

catecholamines from the adrenal gland. They also produce changes in the activities of

the catecholamine biosynthetic enzymes (Kvetnansky, Weise and Kopin 1970;

Kvetinansky, Sun, Lake, Thoa, Torda and Kopin 1978; Axelrod and Reisine 1984;

Stachwiak, Fluharty, Strieker, Zigmond and Kaplan 1986; Sietzen, Schober, Fischer-

Colbrie, Scherman, Sperk and Winkler 1987). Biosynthesis and secretion of peptides

from the gland have also been found influenced by stress (Sietzen et al. 1987; Fisher-

55

Colbrie, lacangelo and Eiden 1988).

1:9:3. After selective surgical denervations

1:9:3:1. Extrinsic adrenal denervation

As the synthesis and secretion of adrenal medullary hormones are mainly

regulated transynaptically via the splanchnic nerves (Thoenen, Muller and Axelrod

1969; Axelrod 1971; Thoenen 1975; Zigmond 1985), transection of these nerves

causes derangements in the biosynthetic and secretory activities of the gland.

Splanchnic nerve section has been found to cause small but significant decrease in the

levels of mRNAs for TH, PNMT and NPY (Schalling, Franco-Cereceda, Hemsen,

Dagerlind, Seroogy, Persson, Hokfelt and Lundberg 1991). It also causes a decrease in

the catecholamine content of the adrenal medulla in cat and rat (Vogt 1952; Fleminger,

Lahm and Udenfriend 1984). In contrast, splanchnic nerve transection increases the

number of immunoreactive chromaffin cells for enkephalin, CGRP and neurotensin in

the adrenal medulla of rat (Schultzburg et al. 1978; Lewis, Stem, Kilpatrik, Gerber,

Rossier, Stein and Udenfriend 1981; Flemingr et al. 1984; Kilpatrick, Howells,

Fleminger and Udenfriend 1984; Pelto-Huikko 1989; Dagerlind, Pelto-Huikko,

Lundberg, Ubink, Verhofstad, Brimijoin and Hokfelt 1994).

Since the majority of adrenal medullary nerve fibres degenerate upon splanchnic

nerve section (Elliott 1913; Hoshi 1926; Hollinshead 1936; Swinyard 1937; McFarland

and Davenport 1941), the occurrence of changes in the intra-adrenal nerve fibres

immunoreactive for various mediators is also expected. Indeed, a marked loss of nerve

fibres immunoreactive for AChE, and total loss of those immunoreactive for

enkephalin, CGRP and SP from rat adrenal gland have been found after splanchnic

nerve section (Pelto-Huikko 1989; Dagerlind et al. 1994). In contrast, ligation of the

splanchnic nerve in rat, results in an increased intra-adrenal VIP-immunoreactive

nerve fibres and cell bodies (Holzwarth 1984). Splanchnic nerve transection has also

been shown to increase the number of galanin-immunoreactive ganglion cells found in

56

the rat adrenal medulla (Dagerlind et al. 1994).

1:9:3:2. Intrinsic adrenal denervation

Following adrenal demedullation which causes intrinsic denervation, a marked

loss of VIP-immunoreactive nerve fibres occurs from the zona glomerulosa of rat

adrenal cortex (Holzwarth 1984). Reduction in the NPY-immunoreactive nerve fibres

forming a plexus in the capsule and zona glomerulosa of rat adrenal gland also follows

after adrenal demedullation (Maubcrt, Dopouy and Bemet 1993).

1:9:3:3. Hypophysectomy

Hypophysectomy results in a major atrophy of adrenal cortical cells of zonae

fasciculata and reticularis accompanied with diminished glucocorticoids secretion

(Deane and Creep 1946; Sayers 1950). The activities of the catecholamine synthesizing

enzymes TH, DBH and PNMT are also reduced (Wurtman and Axelrod 1966; Mueller,

Thoenen and Axelrod 1970; Weinshilboum and Axelrod 1970; Sietzen et al. 1987).

However, indicating that an intact pituitary-adrenocortical axis is not a prerequisite for

the synthesis of these enzymes, the neurally mediated induction of the enzymes is still

possible in hypophysectomized animals (Thoenen et al. 1969; Ciaranello, Wooten and

Axelrod 1976).

No change occurs in the peptide levels of enkephalins and NPY as well as their

mRNAs after hypophysectomy (Sietzen et al. 1987; Yobum, Franklin, Calvano and

Inturrisi 1987; Fisher-Colbrie et al. 1988). However, it has been shown that the

denervation-induced increase in adrenal enkephalin synthesis is glucocorticoid-

dependent, since the increase in enkephalin is markedly reduced or even totally

abolished by hypophysectomy and can be restored by corticosterone and

dexamethasone treatment (Yobum et al. 1987).

57

1:9:4. During chronic exposure to drugs

1:9:4:1. Reserpine

An enhanced adrenal catecholamine synthesis and secretion occurs after

administration of reserpine (Thoenen et al. 1969, Molinoff, Brimijoin, Weinshiloum

and Axelrod 1970). It increases mRNAs for TH and NPY, but decreases that of PNMT

in the rat adrenal chromaffin cells (Schalling, Dagerlind, Brené, Hallman, Djurfeldt,

Persson, Terenius, Goldstein, Schlesinger and Hokfelt 1988a). Reserpine also causes

an increase in the peptide levels of met-enkephalin, CGRP and NPY in the rat adrenal

medullary cells (Wilson, Abou-Donia, Chang and Viveros 1981; Bohn, Kessler,

Golightly and Black 1983; Seitzen et at. 1987; Hofle, Weiler, Fisher-Colbrie, Humpel,

Laslop, Wohlfarter, Hogue-Angeletti, Saria, Flemong and Winkler 1991).

1:9:4:2. 6 -Hydroxydopamine

Administration of 6 -Hydroxydopamine (6 -OHDA) causes an increased

synthesis and turnover of adrenal catecholamines (Mueller, Thoenen and Axelrod

1969). It causes transynaptic induction of TH and DBH (Muller et at. 1969; Molinoff

et at. 1970). Injection of 6 -OHDA in rat during early foetal and neonatal period has

been shown to cause a substantial reduction in the number of intra-adrenal ganglion

cells, although, the chromaffin cells remained unaffected (Aloe and Levi-Montalcini

1980).

6 -OHDA administered to adult rat causes an increase in the number of CGRP-

immunoreactive adrenal chromaffin cells, but decrease in the staining intensity of those

immunoreactive for NPY (Pelto-Huikko 1989). Furthermore, treatment of neonatal

rats with 6 -OHDA results in elimination of the catecholaminergic nerve fibres from

the capsule and the zona glomerulosa of adrenal gland (Kleitman and Holzwarth 1985).

In contrast, 6 -OHDA does not cause any change in the rat adrenal nerve fibres

immunoreactive for enkephalin, NPY or CGRP (Bohn et at. 1983; Pelto-Huikko and

Salminen 1987; Pelto-Huikko 1989).

58

1:9:4:3. Guanethidine

A marker reduction in the number of adrenal chromaffin and ganglion ceils in

the adrenal medulla of 14 day-old rats which were exposed to guanethidine during

foetal and neonatal period have been reported by Aloe and Levi-Montalcini (1980).

However, no such effect was observed in the adrenal medulla of adult rats treated with

guanethidine as neonate or adult (Angeletti and Levi-Montalchini 1972; Johnson and

O'Brien 1976). Guanethidine administered to neonatal rats has been shown to eliminate

the catecholaminergic nerve fibres from the capsule and the zona glomerulosa of

adrenal gland (Kleitman and Holzwarth 1985).

1:9:4:4. Capsaicin

When capsaicin is administered to both neonatal and adult rat and hamster, the

number of CGRP-immunoreactive chromaffin cells show an early increase followed

by a gradual decrease to the normal levels (Pelto-Huikko 1989). It also causes a

decrease in the staining intensity of enkephalin and NPY immunoreactivity in the

chromaffin cells of rat adrenal medulla (Pelto-Huikko 1989). In addition,

administration of capsaicin into neonatal rat eliminates the few galanin-

immunoreactive chromaffin cells (Pelto-Huikko 1989). Substantial reduction in the rat

adrenal nerve fibres immunoreactive for CGRP occurs after capsaicin treatment to both

neonatal and adult rats (Pelto-Huikko 1989). However, capsaicin does not have any

effect on nerve fibres immunoreactive for neurotensin, NPY and enkephalins (Bohn et

al. 1983; Pelto-Huikko 1989).

1:9:5. In diabetes mellitus

Diabetes mellitus is a disease complication caused by a relative or complete lack

of insulin secretion by the ^6-cells of the pancreas or of defects of the insulin receptors.

59

There are two major forms of diabetes which are known as insulin-dependent diabetes

mellitus (IDDM or type I diabetes) and non-insulin-dependent diabetes mellitus

(NIDDM or type II diabetes) (Keen and Ng Tang Fui 1982). The former is

characterized by an absolute deficiency of insulin and massive ^5-cell lesions and

necrosis. NIDDM is characterized by significant insulin production, varying from less

than normal to above normal but always in quantities insufficient to maintain glucose

homeostasis, and by target-organ resistance to insulin. One of the complications

associated with diabetes mellitus is diabetic neuropathy.

The adrenal gland in diabetic rat is enlarged (Bennett and Knoeff 1946;

Rebuffat, Belloni, Malendowicz, Mazzocchi, Gottardo and Nussdorfer 1988b;

Rebuffat, Belloni, Malendowicz, Mazzocchi, Meneghelli and Nussdorfer 1988c). This

is mainly associated with hypertrophy of the zona fasciculata, although, in contrast,

there is also atrophy of the zona glomerulosa of cortex (Rebuffat et al. 1988b, c).

Furthermore, there is occurrence of adrenal medullary fibrosis in long standing human

patients with IDDM (Brown, Zuckerman, Longway and Rabinowe 1989; Brown,

Smith, Longway and Rabinowe 1990). Reduction in the number of adrenal medullary

preganglionic nerve fibres in diabetic rats has also been reported (Wilke, Riley, Lelkes

and Hillard 1993).

Derangements in the secretion from the adrenal gland occurs in diabetes. There

is an increased levels of plasma cortisol in diabetic man (Lentle and Thomas 1964;

Cameron, Thomas, Tiongco, Hariharan and Greden 1987; Tsigos, Young and White

1993). Similarly, increased adrenal and plasma levels of corticosterone occurs in

diabetic rat (De Nicola, Fridman, Del Castillo and Foglia 1976; Torenello, Coirini and

De Nicola 1981; Rebuffat etal. 1988b). In contrast, adrenaline secretion in response to

insulin-induced hypoglycemia is diminished in diabetic man (Hilsted 1982; Hoeldtke,

Boden, Shuman and Owen 1982; Cryer 1989). Decrease in adrenaline secretion also

occurs in diabetic rat (Patel 1983; Wilke et at. 1993).

Other additional conditions in which a number of reports have indicated the

60

occurrence of changes in the adrenal glandular activity include hypertension (McCarty,

Chiueh and Kopin 1978; Kvetnasky, McCarty, Thoa, Lake and Kopin 1979; Sato, Sato

and Suzuki 1987) and pregnancy (Watanabe, Meeker, Gray, Sims and Solomon 1968;

Rosenthal, Slaunwhite and Sandberg 1969; Ehrlich, Nolten, Oparil and Lindheimer

1976; Natrajan, McGarrigle, Lawrence and Lachelin 1982). It is also apparent that the

adrenal secretory activity is affected during diseases such as Phaeochromocytoma and

Addison's disease, as well as Cushing's, Conn's and adrenogenital syndromes which

directly affect the adrenal gland.

61

SECTION 2

MATERIALS AND METHODS

62

2:1. Animals used in this study

For studies involving the distribution of NOS immunoreactivity in the adult rats

reported in Section 3:1:1 and that of diabetic experiments reported in Section 3:2:2

Wistar rats were used, while all remaining studies were carried out on Sprague-

Dawley rats. All studies on postnatal rats were carried out on male rats, while either

sex of the prenatal rats were used. Sprgue-Dawley rats for the studies on the effects of

reserpine treatment and hypophysectomy, and all Wistar rats were obtained from

Charles River UK Ltd (Morgate, UK). All remaining Sprgue-Dawley rats were

supplied by the animal breeding unit of the Biological Services at the University

College London (London, UK).

2:2. Maintenance of the animals

Animals were maintained in the Animal House Unit of the Biological Services

at the University College London. The animal house had normal conditions of 21 °C

temperature, 12 h light and 12 h darkness. The animals were fed standard rat chow, and

had access to water ad labitum.

2:3. Tissue processing

2:3:1. Preparation o f adrenal sections

Animals were killed by asphyxiation with carbon dioxide or ether. Adrenals

were quickly dissected out, and in order to facilitate fixation, the adrenal glands

(except those from the early developing rats) were bisected. The adrenals from the

early developing rats, which were small in size, were fixed intact. Fixation was done

by immersion of the adrenals in 4 % paraformaldehyde (TAAB, Reading, UK) in 0.1

M phosphate buffered saline (PBS), pH 7.4, for 2 h at room temperature. After fixation,

the adrenal glands were cryoprotected by leaving them in 15 % sucrose in PBS

63

overnight, at 4°C.

The adrenals were then embedded in OCT, Tissue-Tek compound (Miles Inc,

Elhart, USA) and frozen onto specimen blocks by gradually immersing in iso-pentane

precooled at -150°C. Frozen tissue blocks were stored in liquid nitrogen at -190°C

until used. Sections were cut at 14 fjm in a cryostat (Reichert-Jung, Germany) at

cutting temperature of -24°C, and thaw-mounted onto 0.1 % poly L-lysine (sigma,

Poole, UK) coated glass slides, and then stored in the dark at 4°C until processed

within 1 week.

2:3:2. Immunohistochemistry

Introduction

Immunohistochemistry, introduced by Coons and his colleges (Coons, Creech

and Jones 1941; Coons and Kaplan, 1950) is a method by which cell and tissue

constituents are made visible in situ by the application of antigen-antibody reaction.

The tissue constituent acts as an antigen and its site is visualized by the application of

its antibody conjugated to various labels such as fluorescent compounds, enzymes or

ferritin or colloidal gold particles (see Polak and Van Noorden 1987; Beltz and Burd

1989). The original approach was the direct or one step method, where the label is

conjugated straight to the antibody directed against the antigen (Coons et al. 1941).

Subsequently, the indirect method was introduced by the inclusion of additional step(s)

(Coons and Kaplan 1950). When a secondary antibody directed against unlabelled

primary antibody is conjugated with a label and added as a second layer, the method is

called two step method. A three step method involves the addition of a tertiary labelled

antibody as a third layer directed against unlabelled secondary antibody.

The indirect method has been modified to incorporate avidin and biotin into the

system in order to obtain the advantage of the very high affinity of avidin for biotin.

Avidin is a large glycoprotein from egg white and some bacteria. It has four binding

sites per molecule for biotin which is a small vitamin molecule found in egg yolk. In

64

this method, biotin is conjugated to a secondary antibody, directed against the

immunoglobulin G of the host in which the primary antibody was raised. This is

followed by incubation with avidin coupled with a label. Strcptavidin, which is derived

from Streptococcus avidini and behaves in a similar way to avidin could also be used

as a substitute for avidin (Coggi, Dell'Orto and Viale 1986). For its high sensitivity and

efficiency, the biotin-streptavidin method was used in the present study.

Experimental protocols

Sections were rinsed in PBS containing 0.1 % Triton X-100 for 5 min to

facilitate antibody penetration, and arranged in a humidified incubating chamber. In

each chamber sections from experimental and their respective controls were processed

in parallel. Sections were incubated by covering them with the respective primary

antisera at the optimum dilution in PBS (pH 7.4) containing 0.1 % Triton X-100, 0.1

% lysine, 0.01 % bovine serum albumin and 0.1 % sodium azide. After 18 h of

incubation at room temperature, sections were washed 3 times for 5 min each with PBS

and incubated with biotinylated donkey anti-rabbit antibody (Amersham Ltd,

Amersham, UK) at a dilution of 1:250 for 1 h. Sections were washed ( 3 x 5 min) with

PBS and incubated with streptavidin fluorescein (Amersham Ltd) at a dilution of 1:100

for 1 h, washed in PBS and then mounted with citifluor mountant (City University,

London, UK).

Replacements of the primary antibodies with preimmune serum were used as

negative controls to establish the specificity of each of the antiserum. Furthermore,

replacements of the primary antibodies with non-immune serum and/or omissions of

the primary antibodies were served as a routine negative controls each time the

immunohistochemistry was carried out.

65

2:3:3: NADPH-diaphorase histochemistry

Introduction

NADPH-diaphorase histochemistry is an enzyme histochemicai method which

employs the principle of dehydrogenase histochemistry (Farber, Stemberger and

Dunlap 1956; Nachlas, Walker and Seligman 1958; Pearse 1972). Dehydrogenases are

enzyme systems which bring oxidation of organic compounds by transferring the

hydrogen atoms to another organic molecules, generally thought of as the coenzymes

(cofactors) in the system. In dehydrogenase histochemistry the reduced cofactors are

used to reduce in turn a dye which is visible as an indication for the presence of the

enzyme either directly or via a second enzyme known as a diaphorase. NADPH-

diaphorase is one of such enzymes that utilizes the cofactor NADPH. Recently, studies

have found that neuronal NADPH-diaphorase is NOS (Dawson et al. 1991; Hope,

Michael, Knigge and Vincent 1991).

In NADPH-diaphorase histochemistry, as in dehydrogenase histochemistry,

various tétrazolium salts whose redox potentials are suitable to be easily reduced by

certain enzymes may be used as final substrate (i.e., dye) for localization of the site of

the enzyme. In their oxidized form tétrazolium salts are colourless, while upon

reduction they precipitate to give coloured water insoluble compounds called

formazans. Nitroblue tétrazolium chloride is the most commonly used tétrazolium salt

employing the light microscopic study (Tsou, Cheng, Nachlas and Seligman 1956). It

readily accepts electrons to be reduced and give formazan of good pigment quality as

amorphous red-blue coloured dye. The formazan is insoluble in aqueous and common

organic solvents. In addition, it is resistant to oxidation by molecular oxygen back to

tétrazolium. It is therefore, this tétrazolium salt that has been utilized as a marker for

the present NADPH-diaphorase histochemicai study.

Experimental protocols

Cryostat sections, were rinsed in 0.1 M TRIS HCl buffer, pH 8 for 5 min and

66

incubated in a freshly prepared incubating medium for 30 min at 37°C, in a humidified

chamber. The incubating medium contained: 1 mg/ml ^-NADPH (Sigma), 0.2 mg/ml

nitroblue tétrazolium (Sigma), 2.7 mg/ml L-malic acid (Sigma) and 0.1 % Triton X -

100 in 0.1 M TRIS HCL buffer, pH 8 . The reaction was stopped by rinsing in 0.1 M

TRIS HCl buffer followed by several washes with distilled water. Sections were then

mounted with citifluor mountant.

In control experiments, in order to test for endogenous reduction in the tissue

which may convert the nitroblue tétrazolium salt to the formazan product, one set of

section was incubated in media devoid of ^-NADPH. Another set of section was

incubated with 0.1 M TRIS HCl buffer heated at 75°C for 10 min before incubation in

order to test the heat stability of the NADPH-diaphorase activity. Such procedures

should give negative reaction (Hope and Vincent 1989).

2:4. Microscopy and computer assisted image analysis

Immunoprocessed and NADPH-diaphorase stained tissue sections were

analyzed with a Zeiss photomicroscope (Carl Zeiss, Inc., Thomwood, New York)

equipped with both epifluorescence and appropriate filters as well as ordinary light

sources. Immunolabelled cells and nerve fibres were studied and photographed with

Kodak, T-max black-and-white film (ASA 3200; Kodak, Rochester, New York).

Similarly sections processed for NADPH-diaphorase histochemistry were studied and

labelled cellular elements were photographed with Kodak T-max black-and-white

film (ASA 100; Kodak, England).

When required, measuring of the labelled cell bodies was achieved by aligning

the long and short axes of the labelled cell bodies with clear nuclear outlines

perpendicular to each other to calibration bars fitted to an eye-piece. From the long (1)

and short (s) axes a mean diameter (d) of the labelled cells was calculated as d = VI x s

(see Neuhber, Sandoz and Fryscak 1986). Indication to the shape of the labelled cell

67

bodies were obtained from the ratio of the long to the short axes.

Quantitative analysis of the immunoreactive nerve fibres for the studies reported

in Section 3:2:1 was made using a computer image analyzer (Seescan Imaging Ltd.,

Cambridge, UK) connected to a Zeiss photomicroscope via a video camera (Sony,

Japan). With a x l 6 objective regions of adrenal gland in every 10^^ consecutive

sections were randomly selected and analyzed in both the medulla as well as the

subcapsular region and zona glomerulosa of the cortex. The frame area of measured

region in the medulla was always kept constant to the maximum possible view on the

screen monitor, and the analyzed regions had comparable medullary tissue and were

free of labelled neuron. In the adrenal cortex, a smaller standard measuring area was

set to include only the subcapsular region and zona glomerulosa, and no region of zona

fasciculata or reticularis was included.

The levels of sensitivity and light intensity were adjusted until optimum at the

beginning of each set of the analysis and then kept constant throughout the subsequent

measurements. Values have been expressed as mean ± standard deviation of the

percentage of the ratio of positive to negative immunofluorescence for each

experiment.

2:5. Biochemical assay of nitric oxide synthase activity

Introduction

A radiometric assay has been developed for measuring the activity of NOS in

the neurons of both central and peripheral neural tissue (Bredt and Snyder 1990;

Schmidt et al. 1991; Lincoln and Messersmith 1995). The assay is based on the

conversion of [^H]-labelled arginine to [^H]-labelled citrulline, in the presence of

NADPH, BH4 , FAD, CaClo and calmodulin at optimum concentrations and

conditions. In order to permit optimal and reproducible enzyme activity the

composition of the buffer utilized for homoginization and/or incubation includes

EDTA, dithiothreitol, leupeptin, soybean trypsin inhibitor and aprotonin. EDTA binds

68

divalent cations which may inhibit the enzyme activity. Dithiothreitol protects against

oxidative damage of the enzyme. Leupeptin, soybean trypsin inhibitor and aprotonin

inhibit proteinase activity, thereby protect the enzyme.

The reaction is terminated by the addition of Dowex ion exchange resin which

binds any residual arginine leaving the newly formed citrulline in the supernatant

which is transferred into liquid scintilants for counting the radioactivity and then

corrected to dpm. In order to remove any activity due to artifacts, blanks such as the

assay of the homogenizing medium alone are also carried out in identical process. The

radioactivity of the blanks converted into dpm is subtracted from that of the samples

and the residual dpm is then converted into enzyme activity using the specific activity

of the L-arginine used.

Experimental protocols

The biochemical assay of NOS activity was used for the studies reported in

Section 3:2:2. Adrenal glands were dissected and stored in liquid nitrogen until assay.

When required, the adrenal glands were weighed and homogenized in 10 mM HEPES

buffer (pH 7.4) containing 0.32 M sucrose, 0.1 mM EDTA, 1 mM dithiothreitol, 10

pg/ml leupeptin, 10 ^g/ml soybean trypsin inhibitor and 2 jug/m\ aprotonin to give a

tissue concentration of 50 mg/ml. The homogenate was centrifuged at 12,000 g for 10

min and NOS activity was measured in the supernatant.

The control incubating medium was 50 mM HEPES (pH 7.4) containing 1 mM

EDTA, 1 mM dithiothreitol, 1 mM NADPH, 1.25 mM CaClo, 10 yt/g/ml calmodulin, 10

pM FAD and 100 pM BH4 . The [^H]-arginine solution was prepared by diluting

[^H]-arginine with non-radioactive arginine to give a final arginine concentration of

10 pM with a specific activity of 4.05 Ci/mmol. 25 p\ of each supernatant was

equilibrated with 100 p[ of the incubating medium at 37®C. The reaction was started by

the addition of 25 p\ of [^H]-arginine solution. After 10 min at 37®C, the reaction was

69

Liquid scintilation counting was carried out using a Beckman LS-600IC liquid

scintiiation counter. This equipment is fitted with automatic quench calibration using

the H number method. The percentage efficiency of counting was therefore measured

for each sample individually and the cpm corrected to dpm.

stopped by the addition of 2 ml of Dowex ion-exchange resin in water (1:1) followed

by vortex mixing. The tubes were stored on ice and the resin allowed to settle. The

supernatants were transferred to scintillation vials containing 5 ml Optiphase "HiSafe”

3 for liquid scintillation counting. ; :. ̂ Blanks

consisted of the homogenizing medium, which was taken through an identical process.

NOS activity was further characterized by carrying out the assay in the presence of the

following inhibitors: L-NAME (1 mM); BGTA (2 mM). All samples were assayed in

triplicate and corrected for the blanks. NOS activity was calculated as nmol citrulline

formed/h/gland and expressed as total activity, L-NAME-sensitive activity, and

EGTA-sensitive activity.

2:6. Surgical procedures

2:6:1. Extrinsic adrenal denervation

Introduction

In the rat, the extrinsic nerve fibres to the adrenal gland come through the

greater and lesser splanchnic nerves which join at the suprarenal ganglion, found as a

lateral extension of the coeliac plexus (Baljet and Drukker 1979). Several nerve trunks

branch from the suprarenal ganglion and enter the adrenal gland. Denervation of the

extrinsic nerve fibres to the gland is therefore, best achieved by impairing the nerve at

the suprarenal ganglion or proximal to it before it gives off the small adrenal branches.

Experimental protocols

Rats were anaesthetized by intramuscular injection of fentanyl-fluanisone

(Hypnorm; Janssen, Oxford, UK; 3 mg/kg body wt) and subcutaneous injection of

diazepam (Valium; Phoenix Pharmaceutical Ltd, Gloucester, UK; 2 mg/kg body wt).

The loss of the stretch reflex in the hind limb was taken as indicative of the level of

anaesthesia.

Anaesthetized rats were placed in the supine position over an operating board.

70

The skin over the left flank was shaved and swabbed with 70 % ethanol. An L-shaped

incision was then made through the skin and muscle separately and reflected laterally

and held with artery forceps. The stomach, intestine and spleen were gently moved

laterally onto warm saline soaked cotton gauze placed over the reflected muscle flap,

and covered with similar saline soaked cotton gauze. Under an operating binocular

microscope the left splanchnic nerve along with the suprarenal ganglion was transected

using a pair of microscissors and removed. The stomach, intestine and spleen were

carefully placed back to their original positions, and the muscle and the skin incisions

were separately sutured with continuous suture using 3/0 chromic catgut, 16 mm

cutting (Ethicon Ltd, Edinburgh, UK). Animals were then resuscitated and kept in

cages bedded with shredded tissue papers.

For sham control studies, in age-matched rats the left splanchnic nerve along

with the suprarenal ganglion were exposed in a similar manner as above, but no nerve

was cut.

2:6:2. Intrinsic adrenal denervation

Introduction

As the intrinsic adrenal neurons are found intermingled with the other cells of

the gland, intrinsic surgical denervation of the gland is difficult and is impossible to

perform without damaging the adrenal medulla as well. A method utilized for such

denervation takes advantages of the fact that if the adrenal gland is bilaterally

enucleated, the cortex will regenerate while the medulla and the intrinsic neurons

remain unregenerated (Ingle and Higgins 1938; Creep and Deane 1949). Such method

of intrinsic adrenal denervation in association with studies from extrinsic adrenal

denervation is a valuable approach to determine source of the nerve fibres found in the

gland with regard to their extra-adrenal or intra-adrenal origin (see for example,

Holzwarth 1984).

71

Experimental protocols

Rats were anaesthetized as described in Section 2:6:1, and placed in the prone

position over an operating board. The skin of the back around the midline at the region

of the lower throracic and upper lumbar vertebrae was shaved and swabbed with 70 %

ethanol. A midline incision was made through the skin from the level of the eighth

thoracic to the fifth lumbar vertebrae. The left skin flap was reflected laterally and held

with artery forceps. A small vertical parasagittal incision was made on the muscle

below the last rib and retracted laterally with an artery forceps. With the help of a

cotton bud, the left adrenal gland was gently manipulated from the surrounding adipose

tissue found in the retroperitoneal space and gently grasped with small forceps. The

capsule at the posterior aspect of the gland was slit with a pair of microscissors, and by

a gentle squeeze with small curved forceps the content of the gland was extruded

intact. The capsule of the enucleated gland, with the pedicle of the gland untouched

was then restored to position. The muscle incision was closed by a continuous suture

using 3/0 chromic catgut, 16 mm cutting.

A similar procedure was repeated on the right side to expose and enucleate the

right adrenal gland. Finally, the skin incision was sutured using 3/0 chromic cutgut, 16

mm cutting. Throughout the operation the exposed tissue was kept moist by warm

physiological saline solution. Animals were resuscitated and kept in a cage bedded

with shredded paper.

For sham operation, the adrenal glands in age-matched control rats were

exposed and manipulated in a similar manner as above, except that the capsules were

not slit nor the adrenals were enucleated.

2:6:3. Hypophysectomy

Hypophysectomized, sham-hypophysectomized and age-matched non­

operated rats were obtained from Charles River UK Ltd. Hypophysectomized rats

72

gained less body weight as compared to their age-matched unoperated and sham

operated rats.

2:7. Drug treatment

2:7:1. Reserpine

Introduction

Administration of reserpine into animals causes depletion of catecholamines

from neurons and adrenal chromaffin cells (Brodie, Pletscher and Shore 1955; Carlsson

1965; Thoenen et al. 1969). It also activates the splanchnic nerves (Haeusler 1974;

Pemow, Thorén, Millberg and Lundberg 1988). Reserpine can therefore be used as an

agent in studies involving depletion of adrenal catecholamines and/or splanchnic nerve

stimulation (see fore example, Sietzen et al. 1987; Schalling et al. 1991).

Experimental protocols

Rats were subcutaneously injected with 2.5 mg/kg body wt of reserpine (Sigma;

10 mg dissolved in 30 p\ glacial acetic acid, 0.25 ml propylene glycol, 0.25 ml ethanol,

1.47 ml HoO [Sietzen et al. 1987]) on day 1, 3, 5 and 7, and killed on day 8 . Age-

matched control rats were injected with the solvent alone.

2:7:2. Guanethidine and 6 -hydroxydopamine

Introduction

Prolonged administration of guanethidine both to newborn and adult rats

produces an irreversible destruction of the cell bodies of sympathetic neurons (Jensen-

Holm and Juul 1970; Bumstock, Evans, Gannon, Heath and James 1971; Eranko and

Eranko 1971; Angeletti and Levi-Montalcini 1972; Johnson and O'Brien 1976).

Similarly, injection of 6 -hydroxydopamine (6 -OHDA) into the newborn animals

results in selective and permanent destruction of cell bodies in sympathetic ganglia

73

(Angeletti and Levi-Montalcini 1970). However, administration of 6 -OHDA into

adult animals results i n destruction of adrenergic nerve terminals while cell bodies in

the sympathetic ganglia are resistant, and therefore regeneration of the adrenergic

terminals follows after sometime (Thoenen and Tranzer 1968; Tranzer and Thoenen

1968; Bennett, Bumstock, Cobb and Malmfors 1970; Furness, Campbell, Gillard,

Malmfors, Cobb and Bumstock 1970; De Champlain 1971; Jonsson and Sachs 1972).

For these reasons, guanethidine is usually utilized for studies involving chronic

sympathetic denervation, while 6 -OHDA is employed for studies involving acute

denervation of the sympathetic nerve terminals in adult animals.

Experimental protocols

Litter rats were injected subcutaneously with 50 mg/kg body wt of guanethidine

sulphate (Ismelin; Ciba Laboratories, Horsham, UK) from day 8 after birth for 3 weeks,

5 days per week, and killed at 12-14 weeks of age (Aberdeen, Corr, Milner, Lincoln

and Bumstock 1990). Age-matched control rats were injected with saline alone.

Another rats which were 13 weeks old were subcutaneously injected with 6 -OHDA

(Sigma, Poole, UK) dissolved in sterile saline containing 1 mg/ml ascorbic acid, at

doses of 100 mg/kg body wt on day 1 and 250 mg/kg body wt on day 2, and killed on

day 8 (Aberdeen et al. 1990). Age-matched control rats were injected with the solvent

alone.

2:7:3. Capsaicin

Introduction

Administration of capsaicin into adult animals causes desensitization for

noxious chemical stimuli which lasts for weeks or months (Jancso 1968). In newbom

rats, administration of capsaicin brings about destmction of the jS-type peptide

containing primary afferent neurons with loss of specific sensory functions (Jancso,

Kiraly and Jancso-Gabor 1977; Gamse, Holzer and Lembeck 1980; Nagy, Hunt,

74

Iversen and Emson 1981; Jancso, Hokfelt, Lundberg, Kiraly, Halasz, Nilsson,

Terenius, Rehfeld, Steinbush, Veihofstad, Hide, Said and Brown 1981; Lawson 1987).

Neonatal treatment of capsaicin is therefore utilized for studies aimed at denervation of

such sensory nerves.

Experimental protocols

Neonatal rats were subcutaneously treated with 50 mg/kg body wt of capsaicin

(Sigma, Poole, UK) dissolved in saline containing 10 % Tween 80 and 10 % ethanol at

1 , 2, 3, 5, 7 and 14 days of age, and killed at 14 weeks (Mione, Cavanagh, Kirkpatrik

and Bumstock 19992). For control study litter-matched neonates were injected with

same volume of vehicle. Ice anaesthesia was used for injections of the 1 and 2 days old

rats.

2:8. Induction of diabetes

Introduction

There are various experimental as well as spontaneous animal diabetic models

which are utilized to study diabetes (Rerup 1970; Grodsky, Anderson, Coleman,

Craighead, Gerritsen, Hansen, Herberg, Howard, Lemmark, Matschinsky, Rayfield,

Riley and Rossini 1982; Mordes and Rossini 1985). In the experimental animal models

the most successful method of provoking diabetes is by use of chemical agents, such as

alloxan and streptozotocin which exert toxicity on the ^-cells of pancreas. In rat, the

specificity of streptozotocin on ^-cells of pancreas is superior to that of alloxan

(Junod, Lambert, Orci, Pictet, Gonet and Renold 1967), where a single injection of 25-

100 mg/kg body wt streptozotocin induces diabetes (Rakieten, Rakieten and Nadkami

1963; Junod, Lambert, Stauffacher and Renold 1969). Such diabetic rats show rapid

body weight loss, hyperglycemia, intermittent to continuous diarrhoea, polydipsia,

polyuria and glycosuria (Junod et al. 1969; Ganda, Rossini and Like 1976).

75

Although diabetic neuropathy is known as a major clinical problem, there has

not been a satisfactory treatment so far available. One of the various therapeutic

modalities investigated to prevent and/or cure diabetic neuropathy involves

ganglioside. Ganglioside is a diverse group of sialic acid-containing glycosphingolipid

that is present in the plasma membrane of mammalian cells, being particularly

abundant in neurons and associated glial cells (Wiegandt 1971; Ledeen 1978; Ledeen

and Yu 1982; Ando 1983; Stults, Sweeley and Macher 1989).

A ganglioside molecule is composed of a hydrophilic portion called syalosyl-

oligosaccharide and a hydrophobic part called ceramide. The ceramide is formed of

sphingosine and fatty acid and is inserted into the outer leaflet of the lipid bilayer of

the plasma membrane, whereas the negatively hydrophilic sugar moiety, the syalosyl-

oligosaccharide, protrudes towards the extracellular environment (see Cuello 1990).

The asymmetrical structure of ganglioside and its location in the plasma membrane

makes it best placed to interact with other constituents of the plasma membrane of the

same or neighbouring cells and other exogenous molecules coming in contact with the

cell. Treatment of ganglioside, both in vivo and in vitro stimulates nurite outgrowth,

axonal sprouting, synapse formation and regeneration. Furthermore, ganglioside has

been shown to prevent or facilitate recovery from diabetic-induced neuropathic

changes (see Cuello 1990). In the present study, if ganglioside treatment has any effect

on results obtained in the streptozotocin-induced diabetic rats was also investigated.

Experimental protocols

Diabetes was induced by a single intraperitoneal injection of streptozotocin (65

mg/kg body wt; Upjohn, Kalamazoo, USA) in saline. Onset of diabetes was established

by rapid body wt loss, polyuria and glycosuria. To investigate the effect of ganglioside

upon changes occurring in diabetic rats, starting one week after induction of diabetes, a

group of diabetic rats were given intraperitoneal injections of 30 mg/kg body wt bovine

brain ganglioside (AGFj^; Fidia, Abano Terme, Italy) twice a week for 7 weeks.

76

2:9. Sources of Antibodies, Chemicals and Reagents

Antibodies

Calretinin..........................Gift from Dr J. Rogers, Cambridge, UK

CGRP................................Cambridge Research Biochemicals, UK

N O S..................................Gift from Dr U. Forstermann, Abbot Laboratories, USA

(For studies reported in Sections 3:1:1-2 and 3:2:2.

....................................... Euro-Diagnostica, Maimo, Sweden

(For studies reported in Sections 3:1:3 and 3:2.1.

N PY .................................. UCB, Belgium

PGP 9 .5 ............................ Ultraclon, UK

S P ......................................Cambridge Research Biochemicals

T H .....................................Affinity, UK

V IP ....................................Incstar, UK

Biotinylated donkey anti-rabbit antibody.....................Amersham Ltd, Amersham, UK

Biotinylated sheep anti-mouse antibody...................... Amersham

Rhodamine conjugated goat anti-rabbit antibody Nordic Immunological

Laboratories, Maidenhead Berks, UK

Streptavidin fluorescein..................................................Amersham

Chemicals and Reagents

Aprotonin...........................Sigma, Poole, UK

L-arginine........................ Dupont/New Research Products, Stevenage, UK

BH4 ................................... Research Biochemicals Inc, St Albans, UK

Bovine serum albumin Sigma

CaClo.................................. Sigma

Calmodulin..........................Sigma

77

Capsaicin......................... Sigma

Citifluor........................... City University, London, UK

Colchicine........................ Sigma

Diastix..............................Ames, Slough, UK

Diazepam (Valium) Phonix Pharmaceutical Ltd, Gloster, UK

Dithiothreitol...................Sigma

Dowex 50X8-200 (H form) ion-exchange resin Aldrich Chemical Co Ltd,

Gillingham, UK

EDTA............................... Sigma

EGTA............................... Sigma

FA D .................................. Sigma

Fentanyl-fluanisone (Hypnorm)........... Janssen, Oxford, UK

Glacial acetic ac id .................................. BDH, Poole, UK

Ganglioside.............................................Fidia, Abano Terme, Italy

Guanethidine sulphate (Ismelin)...........Ciba Laboratories, Horsham, UK

H C l........................................................... BDH

Iso-pentane..............................................BDH

Leupeptin..................................................Sigma

Lysine.......................................................Sigma

L-malic ac id ............................................Sigma

^-N A D PH ...............................................Sigma

L-N A M E................................................Sigma

N aC l........................................................ DBH

NaOH...................................................... Sigma

Nitroblue tétrazolium.............................Sigma

OCT (Tissue-tek).................................. Miles Inc., Elhart, USA

6 -O H D A ............................................... Sigma

78

Optiphase "HiSafe" 3 .......................... Fisons, Loughbonigh, UK

Paraformaldehyde................................TAAB, Reading, UK

Phosphate-buffered saline..................BDH

Physiological saline..............................Antigen Pharmaceuticals Ltd., Roscrea, Ireland

Poly L -lysine .......................................Sigma

Propylene glycol..................................Sigma

Reserpine.............................................Sigma

Sodium azide...................................... Sigma

Soybean trypsin inhibitor...................Sigma

Streptozotocin.....................................Upjohn, Kalamazoo, USA

Sucrose................................................BDH

T R IS .................................................... Sigma

Triton X -1 0 0 ......................................BDH

Tween 8 0 ............................................ Sigma

79

SECTION 3

RESULTS

80

SECTION 3:1

DISTRIBUTION AND CHARACTERISTICS OF NITRIC OXIDE-

SYNTHESIZING NERVES IN THE RAT ADRENAL GLAND

81

SECTION 3:1:1

COLOCALIZATION OF NITRIC OXIDE SYNTHASE AND

NADPH-DIAPHORASE IN RAT ADRENAL GLAND

82

Abstract

The distribution and colocalization of nitric oxide synthase and NADPH-

diaphorase was studied in the adrenal gland of rat. Ganglion cells and many nerve

fibres in the gland showed both nitric oxide synthase immunoreactivity and NADPH-

diaphorase staining. The adrenal cortical cells showed NADPH-diaphorase staining

but were not immunoreactive for nitric oxide synthase.

Positive labelling for both NADPH-diaphorase and nitric oxide synthase was

found in bundles and in single fibres with varicosities, preferentially located around the

noradrenaline-storing cells. Adrenaline-storing cells and ganglion cells in the medulla,

along with the cortical cells and blood vessels in the zona glomerulosa received

relatively fewer positive fibres.

83

Introduction

There are recent reports that refer briefly to the occurrence of NOS-

immunoreactive and NADPH-diaphorase-reactive ganglion cells and nerve fibres in

the adrenal gland of the rat (Bredt et al. 1990; Dawson et al. 1991). The ganglion cells

of the adrenal gland are poorly understood with regard to their transmitters and

function, although there is some evidence for the existence of both cholinergic

(Coupland and Holmes 1958) and noradrenergic (Dagerlind et al. 1990) neurons in the

gland.

The present study, utilizing NOS immunohistochemistry and NADPH-

diaphorase histochemistry, was carried out in order to give a fuller understanding of the

nature and distribution of NOS-containing cells and nerve fibres in the adrenal gland

of rat. This study demonstrates for the first time the differential distribution of NOS-

containing fibres within different regions of the gland and characterizes the NOS-

containing neurons.

84

Materials and methods

Five adult male Wistar rats were killed by an overdose inhalation of carbon

dioxide. The adrenal glands were then removed, bisected and fixed in 4 %

paraformaldehyde in 0.1 M PBS and cryostat sections were prepared as described in

Section 2:3:1. Fixation with 4 % paraformaldehyde enables NA cells to be

distinguished from A cells by fluorescence microscopy (Eranko 1952, 1955).

Therefore, prior to processing for NOS immunohistochemistry, the adrenal sections

were mounted in PBS and examined under fluorescence microscope and photographed.

The objective of this process was to examine if there is any correlation between the

distribution of NOS-immunoreactive elements and either NA or A cells. Coverslips

were then removed and adrenal sections were rinsed in PBS and

immunohistochemistry for NOS was carried out as described in Section 2:3:2. The

NOS antibody used was raised in rabbit against purified soluble NOS extracted from

rat cerebellum (Schmidt et al. 1991), and was used at a dilution of 1:2500.

Immunoprocessed adrenal sections were examined under a fluorescence microscope

and labelled cellular elements were studied and photographed. Coverslips were

removed and adrenal sections were rinsed in PBS.

NADPH-diaphorase histochemistry as described in Section 2:3:3 was carried

out on adrenal gland sections previously immunoreacted for NOS and their non-

immunoreacted consecutive sections. NADPH-diaphorase stained adrenal sections- t u e .

were then inspected under ordinary light illumination using^same microscope as for

NOS immunoreacted ones.

85

Results

Control experiments in which the NOS antiserum was omitted or substituted

with non-immune serum and preimmune serum failed to show labelling. Similarly, no

NADPH-diaphorase staining was seen in the control sections incubated in media

devoid of )3-NADPH or in those sections pretreated with TRIS HCl heated at 75°C.

Examination of the sections immunostained with NOS antisera revealed that

several ganglion cells and many nerve fibres were positively immunoreactive, while

chromaffin and cortical cells were non-reactive. The majority of the immunoreactive

ganglion cells were large with an average diameter of 32 fim and were round in shape

with a circular nucleus devoid of reaction product (Fig. la). A small number of

immunoreactive neurons appeared to contain two nuclei (Fig. Ic). The NOS-positive

neurons generally occurred as small clusters of cells ranging up to 6 in number,

although occasionally they were found singly or in pairs. Apart from a few

immunoreactive ganglion cells which appeared in the juxtamedullary region of the

zona reticularis, no reactive cell was observed in the cortex. The majority of the

immunoreactive neurons showed similar bright, homogeneous fluorescence. A small

number of ganglion cells showed very light fluorescence.

Occasional NOS-immunoreactive nerve fibres were rarely observed in the

capsule of the gland. However, NOS-positive fibres with varicosities formed a plexus

in the subcapsular region and zona glomerulosa of the cortex (Fig. 2a). Single nerve

fibres with varicosities were seen to arise from this plexus, branch and run among the

cortical cells and blood vessels in the zona glomerulosa.

In the zonae fasciculata and reticularis occasional NOS-immunoreactive nerve

bundles without varicosities were encountered running among blood vessels and cords

of cortical cells (Fig. 2b).

In the adrenal medulla several NOS-immunoreactive nerve bundles were

observed, in addition to many single nerve fibres with varicosities. The single nerve

86

fibres appeared to form a closely associated network particularly around the NA cells

(Figs. 2c, d). Relatively less positive nerve fibres were observed around the A cells and

immunoreactive ganglion cells.

Examination of sections processed for the NADPH-diaphorase histochemistry

showed that all cortical cells forming the parenchyma of the cortex and those

occasionally found dispersed in the medulla as cortical islands, along with several

ganglion cells and nerve fibres, were positively stained (Fig. lb). The reactive ganglion

cells were similar in morphology to the NOS-immunoreactive cells described above. A

number of the NADPH-diaphorase stained neurons displayed a cytoplasmic process

extending from one pole of the cell.

When consecutive sections were compared, almost all NOS-immunoreactive

ganglion cells were found to be stained for NADPH-diaphorase. In double labelling

studies all NOS-immunopositive neurons were also found to be stained for NADPH-

diaphorase (Figs. la, b). As for NOS immunoreactivity, a small number of ganglion

cells showed very light staining. These neurons accounted for not more than 5 % as

compared to the strongly positive reactive neurons counted, which ranged in number

from 87 to 114, in 5 animals. The intensity of NADPH-diaphorase staining in the

double labelling experiment was reduced as compared to the NADPH-diaphorase

staining alone in consecutive sections. This was particularly noticeable for cortical

cells.

NADPH-diaphorase stained nerve fibres in both adrenal medulla (Fig. lb) and

cortex showed a similar pattern of distribution to that observed and described with the

NOS immunoreaction. However, the intensity of the staining and the frequency of the

NADPH-diaphorase stained fibres appeared relatively less in comparison with those

immunopositive for NOS, and no NADPH-diaphorase-positive fibre was seen in the

zona glomerulosa of the adrenal glands of 2 of the 5 animals.

87

Fig. 1. Micrographs of rat adrenal gland sections demonstrating:

a, b. double labelled ganglion cells and nerve fibres with NOS

immunoreactivity (a) and NADPH-diaphorase staining (b); arrowheads point to

labelled nerve fibres. Note in b the cortical cells (Co) are stained for NADPH-

diaphorase. M: Medulla. Bars = 35 ywm.

c. NOS-immunoreactive binucleated ganglion cell {arrowhead)^ as well as

mononucleated ganglion cell {open arrow)\ arrow points to NOS-

immunoreactive nerve bundle. Co: Cortex; M: Medulla. Bar = 35 /rni.

88

- -

- - . f ' .1

f

Co

IM

Fig. 2. Fluorescence micrographs of rat adrenal gland demonstrating:

a,b. NOS-immunoreactive nerve fibres in the subcapsular region and zona

glomerulosa (ZG; a), and zonae fasciculata (ZF) and reticularis (ZR; b); Ca:

capsule. Bars = 60 fjm.

c,d. catecholamine fluorescence (c) and the same section immunostained for

NOS (d). Note that the noradrenaline-storing cells (NA) are more densely

innervated than the adrenaline-storing cells (A). Bars = 6 0 ^ .

89

^Bl

Discussion

The failure of immunoiabelling in the control experiments in which the NOS

antiserum was omitted or substituted with preimmune serum and non-immune serum

showed that the NOS-immunoreactivity observed with the NOS antibody was specific.

In addition, the NOS antisera used was tested on other tissues, such as cultured

myenteric plexus of the guinea-pig (Saffrey, Hassall, Hoyle, Belai, Moss, Schmidt,

Forstermann, Murad and Bumstock 1992) and whole mount preparations of rat

myenteric plexus (Belai, Schmidt, Hoyle, Hassall, Saffrey, Moss, Forstermann, Murad

and Bumstock 1992), and its specificity has also been established. Therefore, the NOS

immunoreaction observed in this study was due to NOS present in the tissue.

Adrenal cortical cells are involved in steroid synthesis and contain NADPH-

requiring steroidogenic enzymes (Sweat and Lipscomb 1955). It may be because of

these enzymes other than NOS that the cortical cells gave staining for NADPH-

diaphorase but failed to immunoreact for NOS antisera. Altematively, as NOS is

known to exist in different isoforms (see Forstermann et al. 1991), the cortical

NADPH-diaphorase may be related to another isoform, which could not be recognized

by the neuronal NOS antibody used in this study. This possibility cannot be ruled out,

and awaits further investigation.

The labelling of the ganglion cells and the nerve fibres of the adrenal gland with

both NOS and NADPH-diaphorase confirms earlier observations by other workers

(Bredt et al. 1990; Dawson et al. 1991) and supports the suggestion that NOS and

neuronal NADPH-diaphorase label similar sites (Dawson et al. 1991; Hope et al.

1991). The reduction in the intensity of the NADPH-diaphorase staining in neurons

and the lack of staining of some of the nerve fibres during double labelling may be due

to the weakening of the reactivity as a result of one or more of the prior

immunoreaction procedures.

The occasional immunoreactive fibres observed in the capsule suggest that some

90

NOS fibres in the gland may have an extrinsic origin rather than originating from

intrinsic reactive neurons. The sympathoadrenal preganglionic neurons located in the

intermediolateral cell column of the spinal cord (Kesse et al. 1988) show NADPH-

diaphorase staining (Blottner and Baumgarten 1992). In addition, neurons in the dorsal

root ganglia and the vagal ganglia from where the adrenal gland gets its additional

nerve supply (Parker et al. 1990a; Coupland et al. 1989) also show NADPH-

diaphorase activity (Aimi, Fujimura, Vincent and Kimura 1991). Denervation

experiments are carried out in order to explore the possible existence of an extrinsic

innervation and its contribution in comparison to the intrinsic one (see Section 3:1:3).

NO from both pre- and postsynaptic neural sources has been implicated with

various functions (see Garthwaite 1991). Administration of L-nitroarginine

methylester, one of the NOS inhibitor drugs, has been shown to increase mean arterial

blood pressure and decrease the adrenal blood flow from the adrenal gland of dog

(Breslow, Tobin, Bredt, Ferris, Snyder and Traystman 1992). Similarly, the NOS-

immunoreactive fibres in the adrenal gland of rat may modulate vasodilation and

increase the adrenal blood flow during the secretory activities of the gland.

The close association of a plexus of NOS-positive varicose fibres with the

cellular elements of the gland suggests that NO may have a role concerning cellular

function. Catecholamine secretion from the adrenal medulla of rat is mediated by

cholinergic and non-cholinergic mechanisms (Malhotra and Wakade 1987a). While

the cholinergic component comes from the preganglionic nerve fibres in the spinal cord

(Kesse et al. 1988), the non-cholinergic component is not well understood in terms of

the type and origin of transmitters, although some peptidergic nerve fibres found in the

gland have been suggested for their possible involvement (see Livett et al. 1990). The

morphological observations of the NOS-positive fibres presented here provide support

for the view that NO may be an additional transmitter involved in the control of

catecholamine secretion from the gland. As reported here the NOS-positive fibres are

91

more densely associated with NA than A cells. The functional implication of these

observations needs resolution.

While the adrenal cortex has traditionally been considered to be principally

controlled by ACTH, there is now increasing morphological and functional evidence

for neural control of such functions as steroidogenesis (Holzwarth, Cunningham and

Kleitman 1987), compensatory adrenal growth (Dallman, Engeland and Shinsako

1976) and adrenal blood flow (Engeland, Lilly and Gann 1985). The NOS-positive

fibres found in a varicose plexus in the zona glomerulosa may therefore imply that NO

in this region of the cortex exerts its effect in either one or a combination of the above

activities.

In conclusion, the present immunohistochemical and histochemical study shows

that NOS and NADPH-diaphorase are colocalized in the adrenal ganglion cells. This

further confirms that neuronal NADPH-diaphorase is identical to NOS in the nervous

system and provides morphological evidence for the synthesis of NO in nerves

supplying the adrenal gland. Several NOS-positive fibres arising from the intrinsic

neurons and possibly also from another extrinsic source, innervate the adrenal medulla

and zona glomerulosa of the adrenal cortex. In the adrenal medulla the NA cells

receive greater proportion of the NOS-positive nerve fibres than the A cells or the

ganglion cells. It seems that NO may have a regulatory and/or modulatory role in the

cellular function of the adrenal medulla and zona glomerulosa of the adrenal cortex

and/or blood flow of the gland.

92

SECTION 3:1:2

DISTRIBUTION AND COLOCALIZATION OF NITRIC OXIDE SYNTHASE

AND NADPH-DIAPHORASE IN ADRENAL GLAND OF DEVELOPING,

ADULT AND AGING SPRAGUE-DAWLEY RATS

93

Abstract

The distribution and colocalization of nitric oxide synthase and NADPH-

diaphorase was investigated in the adrenal gland of developing, adult and aging rats

with the use of immunohistochemical and histochemical techniques. Nitric oxide

synthase-immunoreactive neurons within the adrenal gland were found from the 20^^

day of gestation onwards. During early development the neurons were found as small

clusters of smaller-size cells compared to those observed in the adult gland. Their

number reached that of adult level by the 4^ ̂ day after birth, and in the glands from

aging rats a 28.6 % increase was observed.

Whilst no immunofluorescence was seen in chromaffin cells during early

development, some cells from glands of aging rats showed nitric oxide synthase-

immunoreactivity with varying intensity. The immunoreactive neurons from postnatal

rat adrenals were also positive for NADPH-diaphorase, whilst those in prenatal rats

were negative or lightly stained.

Nitric oxide synthase-immunoreactive nerve fibres were present in all adrenal

glands examined from the 16^ ̂ day of gestation onwards. A considerable degree of

variation in the distribution of immunoreactive fibres both in medulla and outer region

of cortex at the different age groups was observed and described. Most, but not all,

nitric oxide synthase-immunoreactive nerve fibres also showed NADPH-diaphorase

staining.

94

Introduction

Innervation of the adrenal gland of the rat shows developmental plasticity.

Although synaptic elements can be identified as early as 15.5 days of gestation

(Daikoku et al. 1977), functional splanchnic innervation of the medulla develops

during the first week of postnatal life reaching maturity by day 10 (Slotkin et al. 1980,

1982). During development, there is also evidence of transitional changes in various

messenger molecules such as acetylcholine and catecholamines (Tomlinson and

Coupland 1990), and neuropeptides (Henion and Landis 1990) contained in the gland.

In aging, the adrenal medullary cells show evidence of proliferative lesions and

structural changes (Tischler et al. 1985; Tischler and DeLellis 1988; Coupland and

Tomlinson 1989). An increase in adrenal sympathetic neural activity associated with an

increase in catecholamine secretion is another phenomenon observed during aging (Ito

et al. 1986). Therefore, the distribution and colocalization of NOS and NADPH-

diaphorase in the developing, adult and aging rat adrenal gland warrants examination.

Using immunohistochemical and histochemical techniques, this study demonstrates

evidence of plasticity in the distribution of NOS in the adrenal gland of the rat during

development and aging.

95

Materials and methods

The study was conducted on male (postnatal) and either sex (prenatal) of

Sprague-Dawley rats. The age of the animals was determined by counting from the

appearance of a cervical mucous plug as day 0 of embryonic life (EO) and the day of

delivery as day 0 of postnatal life. Adrenal glands from 5 rats of each of the following

age were studied: developing rats of E16, E18, E20, 1, 4, 7, 14 and 21 days of age;

adult rats of 16 weeks of age and aging rats of 24 months.

Pregnant rats were with carbon dioxide and foetuses were

removed and their adrenals quickly dissected out and immersion fixed in 4 %

paraformaldehyde in 0.1 M PBS for 2 h at room temperature. Similarly, all other rats

were sacrificed by carbon dioxide asphyxiation, their adrenals removed and immersion

fixed for 2 h. Cryostat sections were prepared as described in Section 2:3:1 and

immunohistochemistry for NOS antibody was carried out as described in Section 2:3:2.

The NOS antibody was raised in rabbit against soluble NOS extracted from rat

cerebellum (Schmidt et al. 1991), and used at a dilution of 1:2500. Immunoprocessed

adrenal sections were examined under fluorescence microscope and labelled cellular

elements were studied and photographed. Coverslips were removed and sections were

rinsed in PBS.

Adrenal gland sections that were previously immunostained with NOS

antiserum, their consecutive sections and several other consecutive sections were

processed for NADPH-diaphorase histochemistry as described in Section 2:3:3.

Adrenal sections processed for NADPH-diaphorase histochemistry were inspected

using same microscope under ordinary light illuminations.

All labelled neurons were counted and the diameter of representative neurons

were measured. To determine whether significant differences existed among the mean

number of labelled neurons at the different age groups, statistical tests with one way

analysis of variance were performed using Fisher's least significant difference

procedure.

96

Results

Adult rats

The NOS immunoreactivity and NADPH-diaphorase staining in the adrenal

glands of the adult Sprague-Dawley rats were comparable to that of Wistar rats

described in Section 3:1:1. NOS-immunoreactivity was found in several ganglion cells

of the adrenal gland (Fig. 3a). A small number of these neurons were located in the

zona reticularis, whereas the remainder was found in the adrenal medulla. Within the

medulla the majority of the NOS-immunoreactive neurons appeared to be peripherally

localized in comparison to a variable number of larger sized, closely packed non-

immunoreactive ganglion cells seen mainly towards the centre of the medulla.

Several NOS-immunoreactive nerve fibres, running in bundles and as single

nerve fibres with varicosities, were found in both adrenal medulla and cortex. In the

medulla, the single nerve fibres were closely associated with chromaffin cells (Fig. 3c).

As reported in Section 3:1:1, paraformaldehyde-induced fluorescence showed that NA

cells were more densely innervated than the A cells. Ganglion cells in the gland were

also innervated, with the large non-reactive neurons being more densely innervated

than those immunoreactive for NOS.

In the adrenal cortex the NOS-immunoreactive nerve fibres were observed in

the subcapsular region and zona glomerulosa (Fig. 6g). In the inner two zonae of the

gland, immunoreactive nerve bundles were seen occasionally running along blood

vessels and cords of cortical cells. In addition, in the zona reticularis and also the

medulla, a small number of blood vessels was seen closely surrounded by

immunoreactive nerve fibres. Some of the fibres were seen projecting from nearby

reactive neurons (Fig. 3d).

NADPH-diaphorase histochemistry revealed that all cortical cells, occasional

cellular elements dispersed in the medulla and several ganglion cells as well as nerve

97

fibres were positively stained (Figs. 3b, e, f). The majority of the dispersed positively

stained cells found in the medulla were morphologically similar to cortical cells.

However, a smaller number of the cells, some of which were elongated with a tapering

end observed between groups of chromaffin cells (Fig. 3f) and apposed to non-reactive

neurons, appeared to be morphologically different. These cells were also obvious due

to their intensity of staining, which was similar to other cells at the corticomedullary

junction (Fig. 3f).

In double-labelling experiments, all neurons and most of the nerve fibres that

were immunoreactive for NOS were also found to be positive for NADPH-diaphorase.

However, reduction in staining intensity of the cortical cells was noticeable on NOS-

preimmunolabelled sections as compared to sections stained for NADPH-diaphorase

alone.

Developing rats

While a weak fluorescence was observed in most cells of the adrenal cortical

primordium of prenatal rats, no clearly labelled NOS-immunoreactive cell was

observed in the adrenal glands at E l6 and E l8. However, in the extra-adrenal

medullary blastema, accumulated at the medial aspect of the developing adrenal glands

of E18 rats, a small number of small-sized immunoreactive neural cells (10-13 pm in

diameter) and nerve fibres were observed (Fig. 4a).

Intra-adrenal NOS-immunoreactive neurons were found in glands from rats of

E20 and older. At E2Ü the immunoreaction in the majority of neurons and nerve fibres

was of lower intensity than that observed in postnatal rats. The neurons, in the

developing rat adrenals, were small in diameter (15-20 pm) and found as small

compact clusters located in the cortex at E20 and day 1 (Figs. 4d, g) and in the outer

regions of the medulla in all other age groups of developing rats (Fig. 4i). Cytoplasmic

processes were seen arising from some of the neurons found in the developing rat

adrenals. In addition to the NOS-immunofluorescence, the cytoplasmic processes

98

helped to ascertain the neural identity of the labelled cells in glands from early stages of

development where NADPH-diaphorase staining intensity of the labelled neurons was

relatively indistinguishable from the surrounding cortical ceils.

NOS-immunoreactive nerve fibres with varying intensity and distribution were

seen in all age groups of developing rat adrenals studied. At E l 6 and E l8 a few fibres

were found as faintly labelled nerve bundles running through the cortical primordium.

In the medulla of E20 and older rats, an increase in frequency of the immunoreactive

fibres was found with increasing age of the animal (Figs. 4c, f, i), reaching at 21 days

the level seen in the adult rats. In the outer cortex, the immunoreactive fibres

progressively increased in frequency up to 7 days, followed by a progressive decrease

in 14 and 21 day-old rats (Figs. 6a-f).

NADPH-diaphorase histochemistry of adrenal sections from prenatal rats

showed staining in a few nerve fibres and most of the cortical cells with varying

intensity. In double-labelling experiments, nerve fibres and neurons that were

immunoreactive for NOS antiserum were negative or stained weakly for NADPH-

diaphorase (Figs. 4b, e). In postnatal developing adrenals, NADPH-diaphorase

staining was found in the same cellular elements, and also was colocalized with NOS-

immunoreactivity in a similar pattern as described for the adult gland (Figs. 4h, j).

Aging rats

Neurons comparable in morphology and size (20-35 /^m) to those seen in the

adult gland, found occasionally in small groups but mainly occurring singly in the

medulla, exhibited bright NOS-immunofluorescence (Fig. 5a). Occasionally, regions

of adrenal medullary chromaffin cells showed a weak heterogeneous

immunofluorescence (Fig 5c). Furthermore, in two specimens, an occasional group of

chromaffin cells displayed bright homogeneous fluorescence (Fig. 5d).

In the regions of adrenal medulla showing weak heterogeneous

99

immunofluorescence, an increase in NOS-immunoreactive nerve fibres was observed,

A dense innervation, similar to that of adult rats, was also seen in the region of the

non-reactive large ganglion cells (Fig. 5c). Alternatively, the immunoreactive nerve

fibres in the remaining regions of the adrenal medulla (Fig. 5a) and cortex (Fig. 6h)

were reduced in comparison with those in the adult rats. The NADPH-diaphorase

staining was localized in the same cellular elements as described in the adult gland and

was colocalized with NOS-immunoreactivity in the same pattern (Figs. 5a, b), except

for the immunoreactive chromaffin cells which failed to stain for NADPH-diaphorase

(Figs. 5d, e).

100

Fig. 3. Photomicrographs of adrenal gland sections demonstrating:

a, b. NOS-immunoreacted (a) and NADPH-diaphorase double-stained (b)

ganglion cells in adrenal medulla of 16 week-old rat. Arrows in b point to

NADPH-diaphorase stained cortical cells located in medulla. Bars = 33 /am.

c. NOS-immunoreactive nerve fibres in adrenal medulla of 16 week-old rat

associated to medullary cells and also surrounding the outlines of large-size

non-immunoreactive neurons (arrows). Bar = 33 /am.

d,e. NOS-immunoreactive (d) and NADPH-diaphorase-stained (e) nerve

processes projecting from nearby reactive neurons to encircle a blood vessel in

zona reticularis of cortex (Co) of 16 week-old rat. Co: cortex; M: medulla. Bars

= 33 /am.

f. NADPH-diaphorase-staining in 16 week-old rat. Arrows point to NADPH-

diaphorase-positive elongated cells located between groups of medullary cells.

Note at corticomedullary junction a group of cells (arrowhead) displaying a

similar intensity of staining as those found scattered in the medulla, but heavier

than cortical cells. Co: cortex; M: medulla. Bar = 33 /cm.

101

M

Fig. 4. Photomicrographs of:

a, b. NOS-immunoreactive neurons and nerve fibres in the extra-adrenal

medullary blastema of E18 rat (a), and same section double-stained for

NADPH-diaphorase (b). Note NOS-immunoreactive neurons and nerve fibres

are negative or weakly stained for NADPH-diaphorase. Bars = 33 f/m.

c-e. Sections of adrenal gland from E20 rat showing NOS-immunoreactive

nerve fibres (c) and neurons (d), and same section as Figure d double-stained

for NADPH-diaphorase (e). Note that the NOS-immunoreactive neurons are

weakly stained for NADPH-diaphorase. Arrows in Figure c point to

immunoreactive nerve fibres. Ca: capsule; Co: cortex; M: medulla. Bars = 33

jum,

f-h. Sections of adrenal gland firom 1 day-old rat showing NOS-

immunoreactive nerve fibres (f) and neurons (g), and same section as g double­

labelled with NADPH-diaphorase (h). Co: cortex; M: medulla. Bars = 33 jum.

i, j. NOS-immunoreactive (i) and NADPH-diaphorase double-stained (j)

neurons and nerve fibres in sections of adrenal medulla from 7 day-old rat. Bars

= 33 jum.

102

' ' 'A

•

j

ca

Co

Co

* - m

Fig. 5. Photomicrographs of:

a, b. Section of adrenal gland from 24 month-old rat showing labelled neurons

and nerve fibres for NOS-immunoreactivity (a) and the same section double­

stained for NADPH-diaphorase (b). Note occasional reactive neuron adjacent to

sinusoid (S). Co: cortex. Bars = 33 fim.

c. NOS-immunoreactive neurons and nerve fibres in section of adrenal medulla

from 24 month-old rat. Note dispersed immunoreactive granules in chromaffin

cells. point to large-sized non-immunoreactive neurons. Bar = 5 2 ^ .

d, e. NOS-immunoreactive chromaffin cells of adrenal medulla (d) and same

section double processed for NADPH-diaphorase (e) in 24 month-old rat. Note

the NOS-positive chromaffin cells are negative for NADPH-diaphorase. Bars =

33ywm.

103

0 3

i

V

r 4 "\#l/\l

Fig. 6a-h. Photomicrographs of NOS-immunoreactive nerve fibres in the

subcapsular and outer cortical regions of adrenal sections from E20 (a), 1 day-

(b), 4 day- (c), 7 day- (d), 2 week- (e), 3 week- (f), 16 week- (g) and 24

month- (h) old rats. Note that the frequency of NOS-immunoreactive nerve

fibres increased with increasing age up to 7 days (d) and showed reduction in 14

days (e) and 21 days (f), to increase again in the adult (g), which then slightly

reduced during aging (h). Ca: capsule; Co: cortex; PC: periadrenal connective

tissue containing NOS-immunoreactive nerve fibres approaching the adrenal

gland of 4 day-old rat. Bars = 52 fim.

104

-s®

r ■'~yzr

Table 1. Number of NOS-containing neurons per adrenal gland of the different age

groups, as counted from NOS-immunoreactive and/or NADPH-diaphorase-stained,

14 ̂ m consecutive sections. Values are mean ± standard deviation, n = 5.

Age E20 1 day 4 days 7 days 14 days 21 days 16 weeks 24 months

No o fNOS-\-ve 6±3 20±8.7 108±16.8 92±6.5 104±19.6 107±9.2 112±10.7 144±27.8neurons

105

Discussion

Chromaffin cells and neurons of the adrenal medulla develop from neural crest

cells, which migrate and invade the medial aspect of the cortical analage during foetal

life (Le Douarin 1980; Landis and Patterson 1981; Coupland 1989). Before entering

the adrenal cortical primordium, the migrating cells accumulate at the medial aspect of

the gland and are identifiable as extra-adrenal medullary blastema (Verhofstad et al.

1985). The NOS-immunoreactive neural cells observed in the present study at E18,

near the medial aspect of the adrenals, most likely therefore represent NOS-containing

neurons amongst migrating cells. With increasing age, the progressive inward

localization of the immunoreactive neurons towards the medulla, away from the

periphery of the cortex, followed the developmental pathway taken by the cells.

During early development, the NOS-containing neurons in the adrenal glands

were found to be fewer in number and occurred as clusters of smaller-size cells as

compared to those found in the other age groups. Statistical analysis revealed that with

a level of probability of 0.05 or less the number of neurons found in the gland showed

significant increase at two age levels (see Table 1). The first was between the 1̂ ̂ and

4^ ̂ day after birth where the mean number of neurons increased by 5.4 fold and

reached a number not significantly different from that found in the adult. The second

was in aging rats where a 28.6 % increase above that of adult was observed. An

increase in the number of neurons during postnatal development in the rat is

documented in both the central (Altman and Das 1966) and peripheral (Gabella 1971)

nervous system. In addition, increase in average neural size and reduction in package

density is also a characteristic of developing visceral neurons (Gabella 1971). The

increase in number of neurons may be due to cell division, migration and

transformation (Altman and Das 1966) or subsequent development of expression of a

neuronal marker used in previously negative neurons. It is not known at present which

one or combinations of these factors may be responsible for the observed increase in

106

NOS-positive neurons during development and aging.

The increase in size of NOS-positive neurons during development may be

attributed to an increase in the neuroplasm associated with an increase in size of the

gland. Such a correlated increase of the size of neurons in relation to the size of the

innervated target tissue has been reported in the myenteric ganglia (Gabella and Trigg

1984). In addition, elimination of the small-size neurons as a result of naturally

occurring neural death during postnatal development (see Oppenheim 1991) and their

substitution by larger-size, newly appearing cells may also be possible.

The colocalization of NADPH-diaphorase staining in all NOS-immunoreactive

neurons in postnatal rats is consistent with previous observations in adult rats that

neuronal NADPH-diaphorase is identical to NOS labelling (Dawson et al. 1991; Hope

et al. 1991; Bêlai et al. 1992; Hassall, Saffrey, Bêlai, Hoyle, Moules, Moss, Schmidt,

Murad, Fôrstermann and Bumstock 1992; Saffrey et al. 1992; also Section 3:1:1).

However, the dramatic reduction or failure of the NOS-reactive cells to stain for

NADPH-diaphorase in prenatal rats was interesting. This may indicate that although

NOS is present in a number of neural cells before birth its activity may not be fully

developed to the extent of detection by NADPH-diaphorase histochemistry.

Alternatively, the amount of the NOS present in the prenatal adrenal gland may be

smaller than in glands from postnatal rats and may be below the level of detection with

NADPH-diaphorase histochemistry. The weak immunofluorescence in most NOS-

positive intra-adrenal neurons and nerve fibres before birth may be similarly

accounted by the smaller quantity of the available enzyme. Nevertheless, the lack of

correlation between NADPH-diaphorase activity and NOS immunoreaction in the

adrenal neurons during early development may be an example of the non-functional

nature of adrenal innervation during this period (Slotkin et al. 1980,1982).

There is increasing frequency of spontaneous change in adrenal chromaffin cells

during aging. This change occurs as proliferative lesions of the adrenal medullary cells

107

from hyperplasia, phaeochromocytoma and/or hypertrophy of chromaffin ceils (Tischler

et al. 1985; Tischler and DeLellis 1988; Coupland and Tomlinson 1989). A number of

noradrenaline-storing cells also shows synaptic type vesicles with electron-dense

content indicating differentiation towards the neural phenotype (Coupland and

Tomlinson 1989). It is debatable whether such chromaffin cells displaying

morphological plasticity with age actually present NOS immunoreaction.

The NOS-immunoreactive nerve fibres seen in the subcapsular region and zona

glomerulosa are of interest. Those found as single nerve fibres with varicosities may

indicate a local role for NO in the region, in addition to the possible passage of

extrinsic fibres destined for the medulla. Innervation of this region of the adrenal

cortex with nerve fibres containing several different neurotransmitters with possible

functional implication has been described (see Holzwarth et al. 1987; Charlton 1990;

Hinson 1990). Neural mechanism appears to modulate steroidogenesis in adrenal

cortical cells, as electrical stimulation of the splanchnic nerve has been shown to

enhance the sensitivity of adrenal cortex to ACTH in hypophysectomized animal

(Edwards and Jones 1987). It is a matter to be clarified if the NOS-containing nerve

fibres observed in the zona glomerulosa may innervate cortical cells in the region and

modulate steroidogensis. Furthermore, noradrenaline and adrenaline have been

demonstrated to stimulate release of cortisol and aldosterone from isolated perfused pig

adrenal preparation (Bomstein, Ehrhart-Bomstein, Scherbaum, Pfeiffer and Holst

1990). Since there is evidence for the presence of islets of chromaffin cells in the zona

glomerulosa of rat adrenals (Palacios and Lafarga 1975; Bomstein, Ehrhart-Bomstein,

Usadel, Bockmann and Scherbaum, 1991) influence of locally released catecholamines

from the chromaffin cells and local adrenergic nerve terminals on steroidogenesis has

been suggested (Bomstein et al. 1990; Vizi et al. 1992). Further study is needed to

investigate if NOS-containing nerve fibres innervate chromaffin cells in outer cortex,

similar to those found in adrenal medulla, and play a secondary role in steroidogenesis.

Steroid secretion from adrenal cortex follows an increase in the rate of adrenal blood

108

flow (see Vinson and Hinson 1992). It may therefore be possible for NO, released from

the NOS-containing nerve fibres found in the subcapsular region, to facilitate

steroidogenesis by regulating blood flow to the gland. Nevertheless, such speculation,

on the possible functional implication of the distribution of NOS-containing fibres in

relation to the observed developmental plasticity needs further resolution.

109

SECTION 3:1:3

THE INTRA-ADRENAL DISTRIBUTION OF INTRINSIC AND EXTRINSIC

NITRERGIC NERVE FIBRES IN THE RAT

110

Abstract

The intra-adrenal distribution of nitric oxide synthase-immunoreactive nerve

fibres was studied in rats subjected to various denervations. Splanchnic nerve section

eliminated the nitric oxide synthase-immunoreactive nerve fibres which innervate

adrenal chromaffin and neuronal cells. It did not affect those innervating blood vessels

and zona glomerulosa, which instead were affected by adrenal demedullation.

Guanethidine, 6-hydroxydopamine and capsaicin treatments, however, did not produce

any change.

These results suggest that nitrergic nerves which innervate adrenal medullary

cells are extrinsic (largely preganglionic sympathetic), whilst those innervating zona

glomerulosa and the majority of adrenal blood vessels are intrinsic, and that they do

not belong to nerves sensitive to the sympathetic nerve neurotoxins, guanethidine and

6-hydroxydopamine, or the sensory neurotoxin, capsaicin.

I l l

Introduction

In the rat, NOS-immunoreactive nerve fibres are closely associated with adrenal

chromaffin and ganglion cells and cortical cells of zona glomerulosa as well as with

intra-adrenal blood vessels (Bredt et al. 1990; Sections 3:1:1-2). Such ample

distribution of the nitrergic nerve fibres in the gland suggests the possible presence of

an extrinsic input, since the number of local NOS-immunoreactive neurons, which

would account for intrinsic innervation, is relatively small (Sections 3 :l:l-2 ). In

addition, NOS-positive preganglionic neurons found in the intermediolateral cell

column of the spinal cord have been shown to have their terminals in the gland

(Blottner and Baumgarten 1992). The intra-adrenal distribution of extrinsic with

respect to intrinsic NOS-immunoreactive nerve fibres is, however, not known. Thus,

this study examined the intra-adrenal distribution of NOS-immunoreactive nerve

fibres following either section of the splanchnic nerve or demedullation. In addition, to

investigate the nature of NOS-immunoreactive adrenal nerves, the distribution of

NOS-immunoreactive nerves in the gland was also studied following chemical

denervation of sympathetic nerves with guanethidine or 6-OHDA and denervation of

primary sensory neurons with capsaicin.

112

Materials and Methods

Studies were carried out on male Sprague-Dawley rats. In each of six 15 week-

old rats, the left splanchnic nerve along with the suprarenal ganglion was removed as

described in Section 2:6:1. The rats were allowed to survive for 5 days postoperation.

In each of another six rats, which were 10 week-old, bilateral adrenal enucleations

were performed as described in Section 2:6:2. These rats were given physiological

saline solution instead of water for 10 days in order to maintain their electrolyte

balance in the absence of normal aldosterone secretion (Holzwarth 1984). The rats

were allowed to survive for 6 weeks postoperation.

Six other rats for each of guanethidine, 6-OHDA and capsaicin treatments were

also included in the study. Guanethidine and 6-OHDA treatments were carried out as

described in Section 2:7:2, while capsaicin treatment was done as described in Section

2:7:3. Guanethidine and capsaicin were treated neonatal, whereas 6-OHDA was

administered into adult rats.

As controls, adrenal glands from the following rats were studied: 6 normal non-

denervated rats; 3 sham-operated rats for each of the two surgical adrenal

denervations; and 6 rats of 3 groups, each group treated with either one of the solvents

in which guanethidine, 6-OHDA or capsaicin was dissolved. The adrenal glands from

the contralateral side of the splanchnic nerve section also served as controls.

Animals were sacrificed by ether asphyxiation and the adrenals were quickly

dissected out, bisected and immersion fixed in 4 % paraformaldehyde in 0.1 M PBS,

and cryostat sections were prepared as described in Section 2:3:1,

Immunohistochemistry for rabbit primary antibody against neuronal NOS (Euro-

Diagnostica, Malmo, Sweden) at a dilution of 1:2500 was carried out as described in

Section 2:3:2. On consecutive adrenal sections of the NOS-immunoreacted ones,

NADPH-diaphorase histochemistry was carried out as described in Section 2:3:3.

113

Results

In the adrenal glands from all groups of control rats NOS immunoreactivity was

observed in several intrinsic neurons located in the medulla and nerve fibres found in

both medulla and cortex, as described in Sections 3 :l: l-2 . In the medulla, the nerve

fibres were closely associated with chromaffin and neuronal cells and blood vessels

(Fig. 7a). However, in the adrenal medulla of the glands from the contralateral side of

the splanchnic nerve section, the NOS-immunoreactive nerve fibres associated with

the large NOS-immunonegative ganglion cells were found to be increased (Fig. 7b). In

the adrenal cortex, immunoreactive nerve fibres formed a plexus in the subcapsular

region and zona glomerulosa (Fig. 7c).

In the adrenal glands ipsilateral to the splanchnic nerve section side, only a few

immunoreactive nerve fibres were found near the chromaffin cells (Fig. 8a). The vast

majority of the nerve fibresjseen innervating the chromaffin and neuronal cells in the

medulla of control rat^ were absent. There were, however, occasional NOS-

immunoreactive nerve fibres found in association with the blood vessels (Fig. 8a).

Some of such nerve fibres were seen projecting from nearby reactive neurons, which

did not show any apparent change from that of the controls. Occasional

immunoreactive nerve fibres and moderate size nerve bundles were seen running

radially across the cortex (Fig. 8b). The immunoreactive nerve fibre plexus in the

subcapsular region and the zona glomerulosa of cortex was similar to that in controls

(Fig. 8c).

In sections of the demedullated adrenal glands a small number of

immunoreactive nerve fibres were seen in the connective tissue which replaced the

region of the enucleated medulla of the gland (Fig. 9a). Some of these nerve fibres

were closely associated with a few large blood vessels found in the region. A few large

immunoreactive nerve bundles were also seen running radially in the superficial region

of the cortex, some crossing the capsule (Fig. 9b). However, the type of the nerve fibre

114

plexus found in the subcapsular region and zona glomerulosa of the control rat adrenals

was not present.

No apparent change was observed in NOS immunoreactivity in adrenal gland

sections from rats treated with guanethidine, 6-OHDA or capsaicin with respect to the

controls.

NADPH-diaphorase-staining of the adrenal gland sections from all groups of

rats, both experimental and controls, closely matched their respective NOS

immunoreactivity.

115

Fig. 7. Fluorescence photomicrographs of NOS immunoreactivity in control

sections from: (a) adrenal medulla of normal non-denervated rat; (b) adrenal

medulla of the contralateral side of the splanchnic nerve section; and (c) adrenal

cortex of normal non-denervated rats. Note in b the high density of NOS-

immunoreactive nerve fibres around large non-immunoreactive ganglion cells

(arrows). In a and b arrowheads and arrows point to NOS-immunopositive and

immunonegative neuronal cell bodies, respectively. V: blood vessel; ZG: zona

glomerulosa; ZF: zona fasciculata. Bars = 52 fim.

116

Fig. 8. Fluorescence photomicrographs of NOS immunoreactivity in sections of

adrenal gland ipsilateral to the splanchnic nerve section side showing NOS-

immunoreactive nerve fibres are: (a) absent from medulla (M), but present in

relation to a blood vessel (V); (b) traversing radially the adrenal cortex; (c)

present as plexus in the zona glomerulosa (ZG) of cortex. Small arrows in a and

b point to immunoreactive nerve fibres, while arrowheads and large arrow in a

indicate NOS-immunopositive and immunonegative neurons, respectively. Co:

cortex; ZF: zona fasciculata; ZR: zona reticularis. Bars = 52//m.

117

Fig. 9. Fluorescence photomicrographs of NOS immunoreactivity in sections of

demedullated adrenal gland, (a) NOS-immunoreactive nerve fibres (small

arrows) are seen in the connective tissue occupying the region of enucleated

medulla, and some of such fibres are associated with large blood vessels (V). (b)

NOS-immunoreactive nerve bundles (small arrows) are seen in the periadrenal

connective tissue, and one of such bundle is seen crossing the adrenal capsule

(Ca). Note the absence of immunoreactive nerve plexus from the subcapsular

region and the zona glomerulosa (ZG) of cortex. Open arrows in a point to

adrenal cortical cells. ZF: zona fasciculata. Bars = 52 pm.

118

4 '

4..' .. '.f

Discussion

The increase in NOS-immunoreactive nerve fibres observed innervating the

large ganglion cells in the adrenal gland from the contralateral side of section of the

splanchnic nerve is intriguing. It may be related to increased secretory activity of the

gland occurring to compensate for a reduced secretion from the denervated gland.

However, it is not clear at present as to the significance of such nitrergic innervation of

the ganglion cells in the glandular activity.

Both intrinsic as well as extrinsic origins for the NOS-immunoreactive intra­

adrenal nerve fibres could be inferred from the present study. Those that innervate

chromaffin and neuronal cells appear to be extrinsic as shown by the disappearance of

these fibres following section of the splanchnic nerve. In contrast, the NOS-

immunoreactive nerve fibres found as plexus in the subcapsular region and the zona

glomerulosa of the cortex seem to arise from the intrinsic neurons. This is implied

since these fibres were unaffected by splanchnic nerve section, but disappeared upon

demedullation.

The nitrergic innervation of the adrenal vasculature also appears to originate

largely from the intrinsic NOS-immunoreactive neurons; indeed some immunoreactive

neurons have been seen to project to nearby vessels. This is also in agreement with the

finding that no apparent change in the vascular nitrergic innervation occurred

following section of the splanchnic nerve. In contrast, the existence of a small number

of immunoreactive nerve fibres associated with the vasculature in the fibrous

connective tissue occupying the central region of the demedullated gland suggests that

there may be an additional minor extrinsic innervation as well. It is possible that the

loss of such minor extrinsic nerve fibres may not be detected by splanchnic nerve

section alone. However, it is also possible that these fibres may not pass via the

splanchnic nerve. Instead, they may use an alternative route, probably running along

with the adrenal blood vessels, and therefore may have been missed during splanchnic

119

nerve section. Alternatively, such innervations of the blood vessels observed in the

demedullated gland may be a compensatory development for the loss of innervation

following removal of the intrinsic nerves.

The absence of any detectable change in the distribution of NOS

immunoreactivity in the adrenal gland of rats treated with any of guanethidine, 6-

OHDA or capsaicin indicate that the NOS-immunoreactive nerves in the gland are not

components of the sympathetic or primary sensory nerves which are affected by the

above drugs (Fumuss et al. 1970; Bumstock et al. 1971; Jancsô et al. 1977). It is also

evident from the present study that the loss of nerves sensitive to these drugs and found

within or outside the gland do not appear to have any secondary effect on the

distribution of NO-synthesizing nerves in the gland. The majority of the extrinsic

NOS-immunoreactive nerve fibres may be sympathetic preganglionic fibres as shown

previously with tracer injection into the gland and colocalizing NOS in the retrogradely

labelled neurons located in the intermediolateral cell column of the spinal cord

(Blottner and Baumgarten 1992). Further similar studies which also include the other

neural projection sites such as the sympathetic chain ganglia, dorsal root ganglia and

vagal ganglia (Parker et al. 1993) may be needed to fully elucidate the origin and

relative proportions of the extrinsic nitrergic nerve fibres to the gland.

120

SECTION 3:1:4

COLOCALIZATION OF NEUROPEPTIDES AND NADPH-DIAPHORASE

IN THE INTRA-ADRENAL NEURONAL CELL BODIES AND FIBRES OF

THE RAT

121

Abstract

Colocalization of vasoactive intestinal peptide, neuropeptide Y, calcitonin

gene-related peptide, substance P, and tyrosine hydroxylase, respectively, with

NADPH-diaphorase staining in rat adrenal gland was investigated using the double

labelling technique.

All vasoactive intestinal peptide- and some neuropeptide Y-immunoreactive

intrinsic neuronal cell bodies seen in the gland were double stained with NADPH-

diaphorase. Double labelling also occurred in some nerve fibres immunoreactive to

vasoactive intestinal peptide and neuropeptide Y in the medulla and cortex.

No colocalization of calcitonin gene-related peptide, substance P or tyrosine

hydroxylase immunoreactivity with NADPH-diaphorase staining was observed.

However, nerve fibres with varicosities immunoreactive for all the neuropeptides

examined were closely associated with some of the NADPH-diaphorase-stained

neuronal cell bodies. Thus, in rat adrenal gland, nitric oxide is synthesized in all

ganglion cells containing vasoactive intestinal peptide and in some containing

neuropeptide Y, but not in those containing calcitonin gene-related peptide, substance

P or tyrosine hydroxylase.

122

Introduction

In rat adrenal gland, NOS immunoreactivity is found in some intrinsic ganglion

cell bodies and nerve fibres. Immunoreactivity to TH, NPY and VIP has also been

demonstrated in the intra-adrenal ganglion cell bodies and nerve fibres of the rat

(Kondo 1985; Pelto-Huikko 1989; Dagerlind et al. 1990; Oomori et al. 1994).

However, it is not known whether NOS is coexpressed with either of these neuroactive

substances or whether it is found in other distinct population of nerves. Elsewhere,

neural NOS has been found colocalized with VIP, NPY, SP or enkephalin (Costa,

Furness, Pompolo, Brookes, Bomstein, Bredt and Snyder 1992; Kummer, Fisher,

Mundel, Mayer, Hoba, Philippin and Preissler 1992; Yamamoto, Bredt, Snyder and

Stone 1993; Heym et al. 1994; Tay, Moules and Bumstock 1994).

To investigate the situation in the rat adrenal gland, colocalization of neuronal

NADPH-diaphorase, which marks the site of NOS (Dawson et al. 1991; Hope et al.

1991; Bêlai et al. 1992; Hassall et al. 1992; Saffrey et al. 1992; also Sections 3 :l:l-2 ),

with the neuropeptides VIP, NPY, CGRP and SP, and the rate limiting enzyme in the

catecholamine synthesis, TH were carried out by a double labelling technique.

123

Materials and methods

A total of 40 adult Sprague-Dawley rats were used; 15 rats were

intraperitoneally injected with colchicine (Sigma, Poole, UK; Img/lOOg body wt, in

saline) whereas 10 other rats were injected with saline alone, 6 h prior to sacrifice. Rats

were killed t'f carbon dioxide and the adrenals were quickly dissected

out, bisected and immersion-fixed in 4 % paraformaldehyde in 0.1 M PBS, and

cryostat sections were prepared as described in Section 2:3:1. Immunohistochemistry

on every other consecutive adrenal section for rabbit polyclonal primary antibodies

against VIP (1:2000; Incstar, UK), NPY (1:3000; UCB, Belgium), CGRP (1:2000;

Cambridge Research Biochemicals, UK), SP (1:2000; Cambridge Research

Biochemicals) and TH (1:1000; Affinity, UK) was carried out as described in Section

2:3:2. Adrenal gland sections from 3 untreated, 3 colchicine-treated and 2 saline-

treated rats were studied for each of the antibody. Immunoprocessed adrenal sections

were inspected under fluorescence microscope and labelled cellular elements were

studied and photographed. Coverslips were removed and sections were rinsed in PBS.

All immunoreacted sections together with their respective fresh consecutive

sections were then processed for NADPH-diaphorase histochemistry as described in

Section 2:3:3.

124

Results

Examination of the immunoprocessed sections of the gland showed labelling in

cellular elements with the respective antibodies as described by previous investigators

(Kondo 1985; Pelto-Huikko 1989; Dagerlind et al. 1990; Oomori et al. 1994), whereas

control experiments in which the primary antibodies were omitted or substituted with

preimmune and non-immune sera resulted in no labelling. Immunoreactivity was

enhanced in the adrenal gland sections of rats pretreated with colchicine compared with

that of untreated glands.

Application of NADPH-diaphorase histochemistry to the VIP-immunoreacted

sections revealed that all VIP-immunoreactive intrinsic neuronal cell bodies observed

in the gland were also labelled positively for NADPH-diaphorase (Figs. lOa-b, d-e).

Such neuronal cell bodies were only occasionally observed and formed a minority of

the NADPH-diaphorase-stained neuronal cell bodies in the gland. Double labelling

was also observed in some VIP-immunoreactive nerve fibres seen in medulla and

cortex (Figs. 10b, e) and a few of those forming a plexus in the zona glomerulosa and

subcapsular region. However, the VIP-immunoreactive nerve fibres observed around a

small number of double labelled neuronal cell bodies did not stain for NADPH-

diaphorase (Figs. 10b, e). In addition, VIP-immunoreactive chromaffin cells were

negative for NADPH-diaphorase (Figs. 10c, f). Alternatively, some nerve fibres in the

medulla and cortex were negative for the VIP immunoreaction but positive for

NADPH-diaphorase staining (Figs. 10c, f).

Double labelling with NADPH-diaphorase was also observed in some NPY-

immunoreactive neuronal perikarya (Figs. 11a, d). The intensity of the NADPH-

diaphorase staining in these neurons was moderate compared with the intense staining

seen in most of the other NADPH-diaphorase-positive neurons. In addition, NADPH-

diaphorase staining and NPY immunoreactivity were seen in some nerve bundles found

radially traversing the cortex (Figs. 11b, e). However, colocalization occurred only in

125

occasional nerve fibres and varicosities within the bundles. A few nerve fibres in the

zona glomerulosa and subcapsular region were also double labelled for NPY and

NADPH-diaphorase. Conversely, no NADPH-diaphorase staining was found in NPY-

immunoreactive chromaffin cells (Figs. l la -f ) , or the majority of NPY-

immunoreactive ganglion cells (Figs. 11c, f) and nerve fibres. The NPY and NADPH-

diaphorase double labelled neuronal cell bodies appeared to be smaller than the NPY-

immunoreactive non-NADPH-diaphorase-stained neuronal cell bodies. Moreover,

NPY immunoreactivity in the former neuronal cell bodies was seen as homogeneous

immunofluorescence throughout the cytoplasm except the nucleus (Fig. 11a), in

contrast to the granular immunofluorescence mainly located at the perinuclear region

in the latter neurons (Fig. 11c). A small number of NPY-immunoreactive nerve fibres

were seen around some NADPH-diaphorase-stained neuronal cell bodies. The

occurrence of NADPH-diaphorase labelling in some NPY-immunonegative neuronal

cell bodies and nerve fibres was also observed.

Immunoreactivity to CGRP, SP or TH was not seen together with staining for

NADPH-diaphorase (Figs. 12a-b, c-d, e-f, respectively). However, nerve fibres with

varicosities immunoreactive for either CGRP or SP were found closely associated with

some NADPH-diaphorase-stained neuronal cell bodies (Figs. 12a-b, c-d,

respectively). The relative density of such innervation and the number of NADPH-

diaphorase-labelled neuronal cell bodies being innervated appeared to be higher for

CGRP than SP.

Similar observations to the above described results were also made by

comparison of the observations from studies carried out on consecutive sections

processed separately for NADPH-diaphorase and neuropeptides or TH.

126

Fig. 10. Photomicrographs of adrenal gland sections processed for VIP

immunoreaction (a-c) and NADPH-diaphorase double staining (d-f,

respectively). VIP-immunoreactive neuronal cell bodies (large arrowheads in

a, b) are double stained for NADPH-diaphorase (large arrowheads in d, e,

respectively), whereas an immunoreactive chromaffin cell (large arrow in c) is

NADPH-diaphorase-negative (large arrow in f). Some VIP-immunoreactive

nerve fibres seen in the cortex (small arrowheads in b) are double labelled with

NADPH-diaphorase (small arrowheads in e) but some others found around VIP

and NADPH-diaphorase double positive neuronal cell bodies (small arrow in b)

are not stained for NADPH-diaphorase (small arrow in e). On the other hand,

some nerve fibres that are VIP-immunonegative (small arrowheads in c) are

NADPH-diaphorase-positive (small arrowheads in f). Co: cortex; M: medulla.

Bar = 33 fjm.

127

%

Fig. 11. Photomicrographs of adrenal gland sections immunoreacted for NPY

(a-c) and double stained for NADPH-diaphorase (d-f, respectively). NPY-

immunoreactive neuronal cell bodies (large arrowheads in a) and nerve bundles

(small arrowheads in b) are NADPH-diaphorase double positive (large

arrowheads in d and small arrowheads in e, respectively). Other NPY-

immunoreactive neuronal cell bodies (large arrows in c) are NADPH-

diaphorase-negative (large arrows in f). Note that NPY-immunoreacted

chromaffin cells found in medulla (a-c) are NADPH-diaphorase-negative (d-f,

respectively), whereas NADPH-diaphorase-positive cortical cells (e) are NPY-

immunonegative (b). Co: cortex; M: medulla. Bar = 33 ^m.

128

Fig. 12. Photomicrographs of:

a, b. Adrenal gland section immunoreacted for CGRP (a) and double stained for

NADPH-diaphorase (b) showing CGRP-immunoreactive but NADPH-

diaphorase-negative nerve fibres with varicosities {small arrowheads in a, b,

respectively) are associated with neuronal cell bodies that are CGRP-

immunonegative but NADPH-diaphorase-positive (large arrowheads in a, b,

respectively). A CGRP-immunoreactive chromaffin cell (large arrow in a) is

NADPH-diaphorase-negative (large arrow in b). M: medulla. Bar = 33 ywm.

c, d. SP immunoreactivity (c) and NADPH-diaphorase double staining (d) of an

adrenal gland section showing an SP-immunoreactive but

NADPH-diaphorase-negative nerve fibre with varicosities (small arrowheads

in c, d, respectively). This fibre approaches neuronal cell bodies that are

negative for SP but positive for NADPH-diaphorase (large arrowheads in c, d,

respectively). M: medulla. Bar = 33 /mi.

e, f. TH immunoreactivity (e) and NADPH-diaphorase double staining (f) of an

adrenal gland section. Non-TH-immunoreactive neuronal cell bodies (large

arrowheads in e) and nerve fibres (small arrowheads in e) are NADPH-

diaphorase-positive (large and small arrowheads in f, respectively). Note that

TH-immunoreactive chromaffin cells in the medulla are NADPH-diaphorase-

negative. The moderately NADPH-diaphorase-positive islands of cells seen

near the intensely NADPH-diaphorase-positive neuronal cells and fibres in f

are cortical cells, and note that they are TH-immunonegative in e. M: medulla.

Bar = 33 /mi.

129

1

%

Discussion

Neuronal NADPH-diaphorase staining and NOS immunoreactivity have been

shown to be identical and mark similar sites in various regions of central and peripheral

nervous tissue, including those found in the adrenal gland (Dawson et al. 1991; Hope et

al. 1991; Bêlai et al. 1992; Hassall et al. 1992; Saffrey et al. 1992; also Sections 3:1:1-

2). It is therefore evident from the present study that some intrinsic adrenal neurons

that synthesize NO are VIP-immunoreactive. A small number of these neurons are

innervated by VIP-immunoreactive NADPH-diaphorase-negative nerve fibres. This

suggests that VIP-containing fibres, most probably arising from non-NO-synthesizing

extra-adrenal neurons, influence some of the NO-producing neurons in the gland. In

this connection, stimulation of NO production by VIP has been shown during

relaxation of the gut (Grider 1993) and lung (Lilly, Stamles, Gaston, Meckel, Loscalzo

and Drazen 1993).

The other neuropeptide found to be colocalized in NO-synthesizing neurons of

the gland is NPY. There is no evidence from previous investigations that NPY and VIP

are colocalized in ganglion cells of the gland (Pelto-Huikko 1989; Maubert et al. 1990;

Oomori et al. 1994); therefore, it appears that these neuropeptides are contained in

separate subpopulations of NO-producing intrinsic neurons. At least some of the nerve

fibres double labelled for NPY and NADPH-diaphorase, and those double labelled for

VIP and NADPH-diaphorase, may arise from intrinsic double labelled neuronal cell

bodies with the respective markers. However, an extrinsic input cannot be ruled out.

Unlike in the intra-adrenal neuronal cell bodies, NPY and VIP have been found to be

colocalized in some nerve fibres that are regarded as extrinsic and that are mainly

found in the superficial region of the cortex (Maubert et al. 1990; Oomori et al. 1994).

Some of these fibres also show TH immunoreactivity (Oomori et al. 1994). Although it

is possible that NADPH-diaphorase may also coexist in some of the nerve fibres

containing VIP and NPY, the lack of NADPH-diaphorase staining in the TH-

130

immunoreactive nerve fibres implies that it is not found in those fibres that also contain

TH.

The absence of CGRP or SP immunoreactivity in the rat adrenal ganglion cells

suggests that intra-adrenal nerve fibres immunoreactive to either of these

neuropeptides are extrinsic. As there are a large number of CGRP-immunoreactive and

SP-immunoreactive neurons in the dorsal root ganglia (Gibson, Polak, Bloom, Sabate,

Mulderry, Ghatei, McGregor, Morrison, Kelly, Evans and Rosefeld 1984), and because

most of the intra-adrenal immunoreactive nerve fibres for these neuropeptides

disappear following capsaicin treatment (Pelto-Huikko 1989), nerve fibres containing

CGRP and SP in the gland are likely to be sensory fibres. It has recently been

demonstrated that the adrenal gland of rat receives a substantial sensory innervation

from dorsal root and sensory vagal ganglia (Parker et al. 1993); some of the neurons in

the former ganglia contain SP (Zhou, Oldfield and Livett 1991). The observation of the

CGRP-containing or SP-containing nerve fibres in association with the NO-

synthesizing neurons may therefore indicate their sensory innervation.

The absence of colocalization of TH immunoreactivity and NADPH-diaphorase

staining suggests that NO is not synthesized by noradrenaline-containing neurons in

the gland. Furthermore, the large-sized ganglion cells that have been shown to be

noradrenergic (Dagerlind et al. 1990) are negative for NADPH-diaphorase staining.

Thus, NO and noradrenaline appear to be produced in different intra-adrenal neurons.

The colocalization of NADPH-diaphorase with VIP and NPY immunoreactivity

in the intra-adrenal nerves of the rat demonstrated in the present study is different from

that in the human gland (Heym et al. 1994) where immunoreactivity to SP in the

nerves, and VIP and enkephalin in the chromaffin cells are coexpressed with NOS. The

distribution of NOS and its colocalization with other neuroactive substances in adrenal

gland thus show species variation.

In conclusion, NO-synthesizing intra-adrenal nerves of the rat appear to

131

contain VIP or NPY, but not SP or CGRP, and are not noradrenergic. The evidence that

only subpopulations of the NADPH-diaphorase-stained nerves immunoreact for NPY

or VIP, or receive distinct peptidergic innervation indicates the diversity of the NO-

synthesizing nerves found in the gland.

132

SECTION 3:1:5

CALRETININ IMMUNOREACTIVITY IN ADRENAL GLAND OF

DEVELOPING, ADULT AND AGING SPRAGUE-DAWLEY RATS

133

Abstract

Localization of cairetinin immunoreactivity in the rat adrenal gland was studied

using immunohistochemical methods. Calretinin-immunoreactive adrenal chromaffin

cells and nerve fibres were found at all of the ages examined from embryonic day 16

up to 2 year-old. Immunoreactive chromaffin cells showed a decrease in number with

increasing age, and in the adult and aging rats they were found among the adrenaline-

storing chromaffin cells. Cairetinin immunofluorescence was also observed in a few

ganglion cells of the adult and aging rats, and in most of the cortical cells at embryonic

day 16. The density of immunoreactive nerve fibres in the gland showed a progressive

increase with age to a peak by day 4 after birth and then a gradual decrease afterwards;

they became associated mainly with some of the ganglion cells in the adult rat. As

revealed by double labelling of cairetinin immunoreactivity in adrenal sections from

the adult rat with NADPH-diaphorase histochemistry, no colocalization existed

between cairetinin and NADPH-diaphorase; however, a small number of neuronal cell

bodies which contained nitric oxide synthase were found heavily surrounded with

calretinin-immunoreactive nerve fibres. In aging rats, an increased density of

calretinin-immunoreactive nerve fibres was seen associated largely with the

chromaffin cells.

It is concluded that cairetinin is found in the rat adrenal gland; the high degree

of variability in its expression with age suggests that cairetinin may be concerned with

age-related activities in the gland.

134

Introduction

Cairetinin is a 29 kDa, more recently discovered member of a family of calcium

binding proteins (Rogers 1987; Winsky, Nakata, Martin and Jacobowitz 1989). It is

mainly found in the nervous tissue and structurally related to the 28 kDa calbindin,

with 58 % amino acid sequence homology (Rogers 1987). Immunohistochemically,

cairetinin has been localized in neurons with different features found in several regions

of both central (Rogers 1987, 1989; Winsky et al. 1989; Résibois and Rogers 1992;

Ren, Ruda and Jacobowitz 1993) and peripheral nervous tissue (Rogers 1989; Brookes,

Steele and Costa 1991; Siou, Belai and Bumstock 1992). The precise function of

cairetinin has not yet been recognized, although it is thought that it may have

additional cellular function apart from just buffering intracellular calcium

concentration (Rogers 1991; Anderssen, Bliimcke and Celio 1993).

Identification of its occurrence in further tissues and examining changes in its

distribution in development and aging may give a clue to its function. Accordingly, this

study investigated immunohistochemically its localization in adrenal glands of adult

rats, and examined the changes that occur during development and aging. Furthermore,

by applying NADPH-diaphorase histochemistry, which demonstrates the NO-

synthesizing neurons (Dawson et al. 1991; Hope et al. 1991; Belai et al. 1992; Hassall

et al. 1992; Saffrey et al. 1992; also Sections 3 :l:l-2 ) , onto the cairetinin

immunoreacted sections, this study also has examined if there is any structural

relationship between calretinin-immunoreactive elements and the NOS-containing

nerves recently described in the rat adrenal gland (Bredt et al. 1990; Dawson et al.

1991; also Sections 3 :l:l-2 ).

135

Materials and methods

Male (postnatal) and either sex (prenatal) of Sprague-Dawley rats were used.

Five rats from each of the following ages were studied: developing rats of E l 6, E18,

E20, 1, 4, 7, 14, and 21 days; adult rats of 16 weeks and aging rats of 24 months. The

age of the developing rats was determined by counting the appearance of the cervical

mucous plug as EO and the day of delivery as day 0 of postnatal life. Animals were

sacrificed by carbon dioxide asphyxiation. Foetuses from the pregnant rats were

removed and their adrenals along with those from rats at the other ages were dissected

out and immersion fixed in 4 % paraformaldehyde in 0.1 M PBS and cryostat sections

were cut as described in Section 2:3:1. Immunohistochemistry for rabbit primary

antibody against cairetinin (gift from J. Rogers, Cambridge, UK) at a dilution of

1:5000 was carried out as described in Section 2:3:2.

In the adrenal gland sections at E l6, where the chromaffin and cortical cells are

found intermingled with each other, in order to identify between these cell types some

sections were also incubated as above in a mixture of the antibody against cairetinin

and a mouse monoclonal antibody against protein gene product 9.5 (PGP 9.5; 1:2000;

Ultraclone, UK). PGP 9.5 is a cytosolic neuronal and neuroendocrine ubiquitin and

used as a general marker for neuronal and neuroendocrine cells (Thompson, Doran,

Jackson, Dhillon and Rode 1983; Wilson, Barber, Hamid, Power, Dhillon, Rode, Day,

Thompson and Polak 1988; Wilkinson, Lee, Deshpande, Duerksen-Hughes, Boss and

Pohl 1989). Previously, PGP 9.5 has been used for a similar study in the adrenal gland

of rat where it has been found labelling the chromaffin but not the cortical cells (Kent

and Coupland 1989). In the latter preparations the sites of antibody-antigen reaction

were revealed by incubation with biotinylated sheep anti-mouse antibody (1:250;

Amersham) followed by streptavidin fluorescein (1:100) for PGP 9.5 and rhodamine

conjugated goat anti-rabbit antibody (1:40; Nordic Immunological Laboratories, UK)

for cairetinin.

136

Immunoreacted sections were inspected using a fluorescence Zeiss microscope.

In order to examine whether calretinin-immunoreactive chromaffin cells have any

correlation with either A or NA cells, regions of adrenal medulla with labelled

chromaffin cells were examined with a filter for paraformaldehyde-induced

catecholamine fluorescence.

To investigate any structural relationship that exists between calretinin-

immunoreactive cellular elements and the NO-synthesizing nerves in the gland,

coverslips were removed from immunoprocessed sections and NADPH-diaphorase

histochemistry was applied onto the calretinin-immunoreacted sections from the adult

rats as described in Section 2:3:3.

137

Results

No labelling was found in control experiments in which the primary antibodies

were omitted.

Developing rat

In adrenal glands from rats at E l 6 most of the chromaffin and cortical cells gave

positive calretinin immunoreactivity (Fig. 13a, b). The fluorescence intensity among

the labelled cells varied, with a smaller proportion being intensely stained than the rest

of the cells. As revealed by the colocalization studies with the PGP 9.5, the intensely

labelled cells were cortical cells (Figs. 13a, b). In the intensely labelled cells the

immunofluorescence was homogeneously located throughout the cell cytoplasm,

including the nucleus. In the adrenal gland sections from rats at the remaining ages of

development the occurrence of immunoreactive chromaffin cells progressively

declined with increasing age and no labelled cortical cell was found.

A small number of weakly reactive nerve fibres was already present at E16. The

frequency of labelled nerve fibres progressively increased with age reaching a peak by

the 4^ ̂day after birth (Fig. 13c) and then decreasing with age afterwards. The reactive

fibres were seen running in bundles and single nerve fibres within the gland, and a few

were found associated with chromaffin and ganglion cells.

Adult rat

Calretinin-immunoreactive chromaffin cells were also found in sections of

adrenal glands from the adult rats (Fig. 14a, c, g). In most of the adrenal medullary

sections up to 5 labelled chromaffin cells were found singly dispersed or in a small

group. As evidenced from paraformaldehyde-induced catecholamine fluorescence, the

immunoreactive cells were always found among the A cells (Figs. 14a, b). A few

ganglion cells positively labelled with calretinin immunoreactivity were also observed

138

(Fig. 14c). However, unlike in the reactive chromaffin cells, the intensity of the

immunofluorescence in the neuronal perikarya was weak and the outline of the nucleus

was not labelled.

Occasional calretinin-immunoreactive nerve bundles were seen traversing

radially across the cortex towards the medulla (Fig. 14d). In the medulla, calretinin-

immunoreactive nerve fibres with varicosities and terminals of varying density were

seen accumulated around immunoreactive (Fig. 14c) and non-immunoreactive intra­

adrenal ganglion cell bodies. A small number of the non-immunoreactive ganglion

cells which were densely surrounded by calretinin-immunoreactive nerve fibres were

found to be NADPH-diaphorase-positive (Figs. 14e, f), while the majority of the

ganglion cells which received a moderate degree of innervation were NADPH-

diaphorase-negative (Figs 14g, h). Calretinin-immunoreactive neuronal perikarya

were not NADPH-diaphorase reactive. Only a few calretinin-immunoreactive nerve

fibres were seen in relation to the chromaffin cells of the adrenal medulla of the adult

rats.

Aging rat

The occurrence of calretinin-immunoreactive chromaffin cells in the adrenals of

aged rats was reduced compared to that in the adult rat, and observed only

occasionally. However, similar to those in the adult rat, such labelled cells occurred

among the A cells, and a few ganglion cells were seen with faint immunofluorescence.

Conversely, an increased number of immunoreactive nerve fibres with varicosities and

endings were found in close association with some adrenal chromaffin cells (Fig. 15),

in addition to those found in relation to ganglion cells.

139

Fig. 13a-c. Photomicrographs of adrenal gland sections of developing rats at (a,

b) E l 6 and (C) 4 day-old. Figures a and c show calretinin immunoreactivity,

while Figure b shows immunoreactivity for PGP 9.5 on same section as a. Note

the occurrence of calretinin immunoreactivity in cells of both developing

adrenal medulla (M) and cortex (Co), and that some cortical cells (arrows) are

intensely calretinin-immunoreactive (a, b). Note also in c the calretinin-

immunoreactive nerve fibres run singly as well as in bundles (arrows). Bar = 33

fjm.

140

Fig. 13a-c. Photomicrographs of adrenal gland sections of developing rats at (a,

b) E l6 and (C) 4 day-old. Figures a and c show calretinin immunoreactivity,

while Figure b shows immunoreactivity for PGP 9.5 on same section as a. Note

the occurrence of calretinin immunoreactivity in cells of both developing

adrenal medulla (M) and cortex (Co), and that some cortical cells (arrows) are

intensely calretinin-immunoreactive (a, b). Note also in c the calretinin-

immunoreactive nerve fibres run singly as well as in bundles (arrows). Bar = 33

140

Fig. 14a-h. Photomicrographs of sections of adrenal medulla (a-c, e-h) and

cortex (d) from 16 week-old rats. A group of calretinin-immunoreactive

chromaffin cells (arrow in a) are seen among the adrenaline-storing chromaffin

cells (A in b) as shown with paraformaldehyde-induced catecholamine

fluorescence on the same section as a. An immunoreactive ganglion cell

(arrowhead in c) is shown which is approached by immunoreactive nerve fibres

(small arrows in c). Calretinin-immunoreactive nerve bundles (small arrows in

d) are seen radially crossing the outer region of the adrenal cortex. Figures e-h

show calretinin immunoreactivity (e, g) and the same sections double stained for

NADPH-diaphorase (f, h, respectively). Note the calretinin-negative cells

(arrowheads in e) surrounded by dense calretinin-immunoreactive nerve fibres

(small arrows in e) are NADPH-diaphorase-positive neurons (arrowheads in f).

However, the calretinin-negative cells (arrowheads in g) moderately

surrounded by calretinin-immunoreactive nerve fibres (small arrows in g) are

NADPH-diaphorase-negative neurons (arrowheads in h). Note also that

calretinin-immunoreactive chromaffin cell (large arrow in g) is not stained for

NADPH-diaphorase (large arrow in h). Large and small arrows show

immunoreactive chromaffin cells and nerve fibres, respectively, while

arrowheads point to ganglion cells. A: adrenaline-storing cells; N:

noradrenaline-storing cells; Ca: capsule; Co: cortex. Bar = 33 fjm.

I4l

- V '

T é.

Fig. 15. Photomicrograph of calretinin immunoreactivity in section of adrenal

medulla from 24 month-old rat showing immunofluorescent nerve endings and

varicosities (arrows) associated with chromaffin cells. Bar = 33 yum.

142

Discussion

The present study has demonstrated that calretinin-immunoreactive cell bodies

and nerve fibres are present in the adrenal gland of the rat. Fixation with 4 %

paraformaldehyde allows, under the appropriate filter, the identification of fluorescing

NA cells from non-fluorescing A cells (Eranko 1992, 1955). This procedure enabled

us to note that the calretinin-immunoreactive chromaffin cells in the adrenal glands of

the adult and aging rats occurred among the A cells. However, since the

paraformaldehyde-induced fluorescence was so weak in glands from the developing

rats, such a correlation of the labelled cells to either type of the chromaffin cell was not

possible. It is interesting that the occurrence of calretinin in a subpopulation of A cells

in the adult and aging rats is different fi"om its closely related calcium binding protein,

calbindin, which exclusively occurs in the NA cells of rat adrenal gland (Buffa, Mare,

Salvadore, Solcia, Furness and Lawson 1989).

As bundles of calretinin-immunoreactive nerve fibres were seen running across

the cortex towards the medulla, and since only a small number of neurons showed

weak calretinin immunoreactivity, it seems likely that intra-adrenal reactive nerve

fibres are mainly extrinsic in origin. Although the adrenal medulla receives its major

innervation from sympathetic preganglionic neurons in the intermediolateral cell

column (Kesse et al, 1988), there are additional nerve fibres projecting to the gland

from postganglionic sympathetic, as well as parasympathetic and sensory, ganglia

located at various sites (Parker et al. 1993). The intra-adrenal calretinin-

immunoreactive nerve fibres may thus originate from either, or combinations of, these

regions. In fact, calretinin immunoreactivity has been demonstrated in neurons found

in the lateral cell column of the spinal cord as well as in dorsal root ganglia of the rat

(Ren et al. 1993). Retrograde fibre tracing combined with colocalization studies may

be required to determine the source and the relative proportion of the projections of the

calretinin-immunoreactive fibres in the gland.

143

Although at the early period of development calretinin-immunoreactive nerve

fibres in the gland were also associated with chromaffin cells, at later stages of

development and in the adult they were mainly confined to some ganglion cells. A

smaller number of such ganglion cells receiving a higher level of innervation are NO-

synthesizing neurons as compared to the moderately innervated ones which do not

synthesize NO. The functional implication of such specificity in the innervation of the

intrinsic neurons of the adrenal gland is not known at present and is a matter of future

investigation. On the other hand, in the aged rat adrenals in addition to the ganglion

cells, some chromaffin cells also received immunoreactive nerve fibres. The adrenal

medulla of aging rats shows signs of proliferative lesions and differentiation of the

chromaffin cells towards a neuronal phenotype (Tischler et al. 1985; Coupland and

Tomlinson 1989) as well as changes in its pattern of cholinergic (Tischler et al. 1985)

and nitrergic (Section 3:1:2) innervation. It is interesting that such changes in

innervation also occur with calretinin-immunoreactive nerve fibres. However, the

cause and effect of the change in the innervation with calretinin-containing nerves, and

whether such a change of innervation is associated with those chromaffin cells affected

in aging remains to be established.

The function of calretinin in the adrenal gland, as in other regions, is not yet

known. Nevertheless, several possible roles in cellular activities may be considered.

One suggestion is that calretinin is involved in activities such as short term intracellular

calcium buffering. Changes in intracellular levels of calcium are known to modulate

several important processes including cell metabolism, cell division, cell movement,

nerve impulse conduction and exocytosis (Campbell 1983; Carafoli and Penniston

1985; Miller 1988). However, in excess, calcium is toxic and causes cell death

(Schanne, Kane, Young and Farber 1979). In a protective role against such damage,

calretinin may be one of the calcium binding proteins involved in calcium buffering.

Indeed, recently it has been shown that calretinin-immunoreactive cultured rat brain

1#

cortical neurons are more resistant to calcium overload and excitotoxicity as compared

to those which are not calretinin-immunoreactive (Lukas and Jones 1994). The

increase in calretinin-immunoreactive adrenal cells and nerve fibres during early

development as observed in this study may be related to an increase in calcium levels

as required by the cells for developmental activities such as cell division and cell

migration occurring during this stage. The observation of calretinin immunoreactivity

in the adrenal cortical cells at E l6 may also indicate involvement of calretinin in early

developmental activities. During development the temporary occurrence of calretinin

has also been described in several neurons as well as in non-neuronal cells including

photoreceptors and ependymal cells of the brain (Ellis, Richards and Rogers 1991;

Anderssen et al. 1993; Bastianelli and Pochet 1993). However, as calretinin

immunoreactivity is found in distinct subpopulations of cells and nerve fibres,

calretinin may alternatively have additional unknown functions. In such cases, the

variability in the occurrence of calretinin immunoreactivity in cell bodies and nerve

fibres of the adrenal gland during different ages may be related to the appearance and

disappearance of cells due to cell division and cell death, respectively.

In conclusion, the present study reveals that calretinin-containing cell bodies

and nerve fibres show dramatic changes of expression in development and aging in the

adrenal gland of the rat. The plasticity in the distribution of calretinin with age may be

indicative of age-related functions in the gland.

145

SECTION 3:2

CHANGES IN THE EXPRESSION OF NITRIC OXIDE SYNTHASE

AND NADPH-DIAPHORASE IN THE ADRENAL GLAND OF RATS IN

DEVELOPMENT, AGING AND ALTERED ADRENAL GLAND ACTIVITY

146

SECTION 3:2:1

EFFECT OF RESERPINE TREATMENT AND HYPOPHYSECTOMY ON THE

NITRIC OXIDE SYNTHASE IMMUNOREACTIVITY AND NADPH-

DIAPHORASE STAINING IN THE RAT ADRENAL GLAND

147

Abstract

Nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in the

adrenal gland of rats subjected to either reserpine treatment or hypophysectomy as

compared with the controls were studied. Reserpine treatment caused a significant

decrease in immunoreactivity to the neuronal isoform of Nitric oxide synthase in the

nerve fibres that innervate adrenal medullary cells. Hypophysectomy did not cause any

change in the Nitric oxide synthase immunoreactivity, although it eliminated most of

the NADPH-diaphorase staining non-NOS-immunoreactive cortical cells. The

reduction in Nitric oxide synthase, and hence decreased production of Nitric oxide in

the adrenal gland of reserpine-treated rats may be related to the increased biosynthesis

and secretion of catecholamines following treatment of the drug.

148

Introduction

The distribution and age related changes of the NO-synthesizing nerves in the

adrenal gland of rat has been described (Sections 3:1:2). Changes in the expression of

the NOS isoform has also been detected in sensory ganglia and central nervous system

neurons under various conditions (Verge, Xu, Xu, Weisenfeld-Hallin and Hokfelt

1992; Wu 1993; Bredt and Snyder 1994; Sessa 1994). It is not known if the nitrergic

innervation of the adrenal gland is also changed in different conditions that cause

changes in the activity of its innervation and glandular secretion. In the study reported

here the state of neuronal NOS immunoreactivity in the adrenal gland of rats was

assessed following reserpine treatment or hypophysectomy. Reserpine causes depletion

of adrenal catecholamine and increases its biosynthesis via a transynaptic mechanism

(Thoenen et al. 1969). Hypophysectomy is known to cause a major atrophy of adrenal

cortical cells, and thereby diminishes the secretion of glucocorticoids (Sayers 1950)

which have a regulatory influence on adrenal medullary catecholamines and peptide

synthesis and secretion (Wurtman and Axelrod 1966; Youbum et at. 1987).

149

Materials and Methods

15 week-old Sprague-Dawley rats were injected subcutaneousiy with 2.5

mg/kg reserpine as described in Section 2:7:1 on day 1, 3, 5, and 7 and killed on day 8.

14 week-old hypophysectomized, sham-hypophysectomized and non-operated

control Sprague-Dawley rats were obtained from Charles River Ltd, and allowed to

survive for two weeks. Six rats were studied for each of the experiments.

Animals were sacrificed by ether asphyxiation and the adrenal glands were

quickly dissected out, bisected and immersion fixed in 4 % paraformaldehyde in 0.1 M

PBS for 2 h and cryostat sections were prepared as described in Section 2:3:1.

NOS immunohistochemistry against the neuronal NOS (1:2500; Euro-

Diagnostica, Sweden) and NADPH-diaphorase histochemistry were carried out as

described in Sections 2:3:2 and 2:3:3, respectively. Quantitative analysis of the NOS-

immunoreactive nerve fibres in every 10^ ̂ consecutive sections of the adrenal glands,

in both the medulla and the zona glomerulosa and subcapsular region of the cortex,

was made using a computer image analyzer as described in Section 2:4. To determine

whether significant differences existed among the means from each groups, statistical

analysis was performed using one way analysis of variance with Fisher's least

significant difference procedure.

150

Results

In the adrenal glands from all groups of control rats NOS immunoreactivity was

observed in several intrinsic neuronal cell bodies located in medulla and in nerve fibres

found in both medulla and cortex, as described in Sections 3 :l: l-2 . In the medulla, the

nerve fibres were closely associated with chromaffin cells (Fig. 16a), neuronal cells

and blood vessels. In addition, the immunoreactive nerve fibres formed a plexus in the

subcapsular region and the zona glomerulosa of cortex.

In sections of the adrenal gland of the reserpine-treated rats, NOS-

immunoreactive nerve fibres that innervate the adrenal medullary chromaffin cells and

also those associated with the neuronal cell bodies were significantly reduced (Fig.

16b; Table 2) as compared with that of the controls (Fig. 16a). However, no change

was found in the NOS-immunoreactive nerve fibres that form a plexus in the

subcapsular region and zona glomerulosa (Table 2). The NOS-immunoreactive nerve

fibres found in association with blood vessels and the NOS-positive intrinsic neuronal

cell bodies were also unaffected in the reserpine-treated rats.

The weight of adrenal glands from hypophysectomized rats was reduced by

about 76 % as compared with the controls. However, there was no significant change

in the NOS immunoreactivity in the adrenal gland of the hypophysectomized rats

(Table 2).

NADPH-diaphorase-staining of the neuronal elements in the sections of

adrenal gland from all groups of rats both experimental and controls, closely matched

the NOS immunoreactivity. The adrenal cortical cells were also positively stained.

NADPH-diaphorase stained and non-NOS-immunoreactive cells that are found as

islands or singly dispersed in the medulla of the glands from the control rats were not

present in the medulla of the hypophysectomized rats (Figs. 17a, b). There was also a

substantial loss of the cortical cells of the zonae fasciculata and reticularis of the

adrenal cortex of the hypophysectomized rats.

151

Fig. 16. Fluorescence photomicrographs of NOS immunoreactivity in sections

of adrenal medulla of (a) control and (b) reserpine-treated rats. Arrows and

arrowheads indicate NOS-immunoreactive neuronal cell bodies and nerve

fibres, respectively. Note the absence of the large majority of NOS-

immunoreactive nerve fibres in the medulla of the reserpine-treated rat (b) as

compared with that of the control rat (a). Bar: 33 fm .

152

Fig. 17. Photomicrographs of NADPH-diaphorase stained adrenal gland

sections of (a) control and (b) hypophysectomized rats. Note that NADPH-

diaphorase stained cells found dispersed in the medulla of control ratot*

(arrowheads in a) are absent^^reduced from that of the hypophysectomized rat

(b). Co, cortex; M, medulla. Bar: 75 fjm.

153

i

^ *

5 - ' i

>

M />

• Po% ♦>c

f " »>

Table 2. Image analysis showing density of NOS-immunoreactive nerve fibres in the

adrenal gland of reserpine-treated and hypophysectomized rats with respect to the

controls.

Medulla Subcapsular region &zona glomerulosa

Control 4.58 ± 0.18 1.46 ± 0.09

Solvent-treated 4.70 ± 0.27 1.42 ± 0.09

Reserpine - treated 2.14 ± 0.29* 1.45 ± 0.07

Sham -operated 4.48 ± 0.23 1.43 ± 0.06

Hypophysectomized 4.64 ± 0.13 1.46 ± 0.10

Values are mean ± standard deviation of the percentage of the ratio of positive to negative immunofluorescence per standard frame area, indicates a significant effect of reserpine treatment (P < 0.001). n = 6.

154

Discussion

Neuronal and endothelial NOS isoforms are thought to be only expressed

constitutively and to be regulated by calcium and calmodulin. However, recently a

number of conditions have been found to cause changes in the expression and/or

activity of these isoforms (Verge et al. 1992; Wu 1993; Bredt and Snyder 1994; Sessa

1994; also Section 3:1:2). In the present study, the expression of NOS in the adrenal

gland of rats histochemically detected by an antibody against the neuronal isoform also

showed a significant change after reserpine treatment. The reduction in the NOS

immunoreactivity was found in the nerve fibres that innervate adrenal medullary cells.

As found from studies reported in Section 3:1:3, these nerve fibres are extrinsic and

that they appear to be preganglionic cholinergic nerve terminals (Blottner and

Baumgarten 1992). The mechanism(s) involved in the changes caused by reserpine in

these nerves is not clear at present and is a matter of speculation. Reserpine is known

to cause depletion of catecholamines and coexisting transmitters, such as NPY, from

adrenergic nerves (Thoenen et al. 1969; Lundberg, Saria, Franco-Cerceda and

Theodorsson-Northeim 1985). However, since reserpine has not been claimed to affect

the preganglionic cholinergic nerves directly, the change we observed in the present

study may have not occurred as a direct consequence of reserpine treatment. Instead,

indirect effects of the drug may be the possible cause of the change. Such an indirect

effect may be related to a retrograde feedback mechanism from the gland associated

with the increased adrenal catecholamine biosynthesis and secretion that are caused by

reserpine treatment (Thoenen et al. 1969).

NO appears to be a non-cholinergic transmitter and/or modulator in adrenal

medullary catecholamine secretory activity. However, since NO has been implicated in

both inhibition (Torres, Ceballos and Rubio 1994) and potentiation (Oset-Gasque,

Parramon, Hortelano, Bosca and Gonzalez 1994) of catecholamine secretion from

adrenal chromaffin cells, it also appears that its involvement is complex. In view of

155

this, and the enhanced biosynthesis and secretion of catecholamines from the adrenal

chromaffin cells occurring after reserpine treatment, the reduction in the level of NOS

observed in this study may be related to suppression of the production of NO from the

preganglionic nerve fibres which consequently reduces any possible inhibitory effect

that NO may otherwise have in the biosynthesis and secretion of catecholamines in the

gland. Nevertheless, although further functional study is needed to clarify such

speculation, the reduction of the levels of NOS in the cholinergic preganglionic nerve

fibres at the time when their activity is supposedly increased (Thoenen et al. 1969)

may indicate that the production of NO is regulated differently from that of

acetylcholine in these nerves.

Although the adrenal medullary secretion is affected by adrenal glucocorticoids,

and hypophysectomy has been shown to affect adrenal medullary catecholamine and

peptide biosynthesis (Wurtman and Axelrod 1966; Yobum et at. 1987),

hypophysectomy did not cause any change in the NOS immunoreactivity in the gland

in the present study. However, the disappearance of the NADPH-diaphorase stained

cells, which are found dispersed in the medulla of control rats, further supports the

suggestion that these cells are cortical cells. It is a matter for further investigation if

such cortical cells located in the medulla could be, at least partly, the source for local

glucocorticoids needed in the regulation of adrenal medullary secretory activity in the

gland.

156

SECTION 3:2:2

INCREASE IN NITRIC OXIDE SYNTHASE AND NADPH-DIAPHORASE IN

THE ADRENAL GLAND OF STREPTOZOTOCIN-DIABETIC WISTAR RATS

AND ITS PREVENTION BY GANGLIOSIDE

157

A bstract

Levels of nitric oxide synthase and NADPH-diaphorase in adrenal glands of

streptozotocin-diabetic rats of 8 and 12 weeks duration compared with control rats

were assessed with histochemical and biochemical techniques. Adrenal glands from

streptozotocin-diabetic rats of 8 weeks duration treated with ganglioside were also

examined. In the adrenal medulla of 8 weeks and 12 weeks diabetic rats nitric oxide

synthase-immunoreactive nerve fibres were increased and decreased, respectively;

additional nitric oxide synthase-immunoreactive and NADPH-diaphorase stained

cells, which appeared to be cortical cells, were located in medulla and cortex as

compared with controls. Increased intensity in NADPH-diaphorase staining of the

cortical cells of diabetic rats was also observed. Ganglioside treatment of the 8 weeks

diabetic rats prevented the diabetic-induced increase in nitric oxide synthase-

immunoreactive nerve fibres. It also reduced most of the diabetic-induced increase in

the nitric oxide synthase-immunoreactive and NADPH-diaphorase stained cells and

the intensity of NADPH-diaphorase staining of cortical cells. With biochemical assay

a significant increase in nitric oxide synthase activity was found in the adrenal glands

from 8 weeks diabetic rats, and this increase was reduced by ganglioside treatment in

4 out of 6 diabetic rats.

In summary, streptozotocin-induced diabetes causes an initial increase in the

levels of nitric oxide synthase and NADPH-diaphorase in adrenal gland of rat, which

was prevented by ganglioside treatment.

158

Introduction

Chromaffin cells of adrenal medulla synthesize and secrete adrenaline which is

involved in glucose counteyegulation next to glucagon (Cryer 1981). In long standing

diabetes in humans, the glucagon response to insulin-induced hypoglycemia is reduced

(Gerich, Langlois, Noacco, Karam and Forsham 1973) and is absent in diabetes with

autonomic neuropathy (Maher, Tanenberg, Greenberg, Hoffman, Doe and Goetz

1977). Reduction in glucagon response to insulin-induced hypoglycemia has also been

observed in alloxan-diabetic (Lykkelund and Lund 1979), streptozotocin-diabetic

(Patel 1983) and B B -W istar diabetic (Wilke et al. 1993) rats. In the absence of

glucagon, maintenance of glucose homeostasis depends on adrenaline secreted from

adrenal medulla (Cryer 1981; Bolli, De Feo, Campagnucci, Cartechini, Angeletti,

Santeucanio and Brunett 1982), the other glucose counte^egulatory hormones: cortisol

and growth hormone not being critical. However, an eventual reduction of adrenaline

release in response to insulin-induced hypoglycemia has also been found in some

patients with IDDM (Hoeldtkeer at. 1982) as well as in streptozotocin-diabetic (Patel

1983) and B B -W istar diabetic (Wilke et al. 1993) rats. The cause for such

complications in diabetes is not known conclusively, although a correlation with

autonomic neuropathy has been found in studies of clinical (Hoeldtk et al. 1982; Cryer

1989) and experimental diabetes in rat (Wilke et al. 1993). However, other clinical

studies did not report such a correlation (Ryder, Owens Hayes, Ghatei and Bloom

1990).

In this study, the distribution of NOS and NA DPH-diaphorase in the adrenal

glands of streptozotocin-diabetic rats was investigated. Ganglioside is a sialic ac id -

containing glycolipid that is found in the plasma membrane of mammalian cells, being

especially abundant in neurons and associated glial cells (Ledeen and Yu 1982; Ando

1983). It has been claimed to increase neural survival in a wide variety of

neuropathological conditions (Cuello 1990; Rodden, W iegant and Bauer 1991). In this

159

laboratory, ganglioside treatment has been shown to prevent diabetes-induced changes

in the innervation of the pyloric sphincter of rat (Soediono, Belai and Bumstock 1993).

Thus, in this study, if ganglioside can affect changes in the level of NOS and NADPH-

diaphorase in adrenal glands of streptozotocin-diabetic rats was also investigated.

160

Materials and methods

Male Wistar rats with initial body weight of 400-450 g were used. Diabetes was

induced in 27 rats by a single intraperitoneal injection of streptozotocin (65 mg/kg

body wt; Upjohn, Kalamazoo, USA) in saline. Onset of diabetes was established by

rapid body weight loss, polyuria and glycosuria (Diastix; Ames, Slough, UK).

Starting one week after induction of diabetes, 11 diabetic rats were given

intraperitoneal injections of mixtures of bovine brain ganglioside (AGF^, 30 mg/kg

body wt; Fidia, Abano Terme, Italy) twice a week for 7 weeks. AGF^ is a mixture of

ganglioside whose carboxyl group of the sialic acids of GMj ,̂ GD^^, GD^y and GT^y

are esterified with the hydroxyl groups of the neighbouring saccharides.

Two groups of age-matched controls, which consisted of 16 untreated non­

diabetic and 11 ganglioside-treated non-diabetic rats similar to ganglioside-treated

diabetic rats were used. Most of the rats were killed 8 weeks after injection with

streptozotocin, while 5 untreated diabetic and 5 untreated non-diabetic rats were killed

12 weeks after induction of diabetes. Animals were killed with an overdose inhalation

of ether and blood samples (3-5 ml) were quickly withdrawn from the posterior vena

cava into heparinized syringes for plasma-glucose level determination by the

hexokinase method, using Sigma diagnostics kit (Sigma, Poole, UK). The adrenal

glands were then dissected out.

Histochemistry

Adrenal glands were immersion fixed in 4 % paraformaldehyde in 0.1 M PBS

and cryostat sections were prepared as described in Section 2:3:1. Every other

consecutive sections were processed for NOS immunohistochemistry as described in

Section 2:3:2. The NOS antibody used was raised in rabbit against purified soluble

NOS extracted from rat cerebellum (Schmidt et al. 1991), and was at a dilution of

1:2500.

161

After studying the immunoprocessed sections under fluorescence microscope,

coverslips were removed and sections were rinsed in PBS. NADPH-diaphorase

histochemistry, as described in Section 2:3:3, was carried out on adrenal sections

which were previously immunoreacted with NOS antiserum and other consecutive

sections.

Biochemical Assay o f NOS Activity

More than one type of NADPH-diaphorase exists in the adrenal gland. In

addition, NADPH-diaphorase activity measured biochemically does not correlate with

NOS activity (Tracey, Nakane, Pollock and Forstermann 1993). Therefore, the

biochemical study was restricted to the measurement of NOS activity.

Adrenal glands from diabetic and ganglioside-treated diabetic rats of 8 weeks

duration and that of ganglioside-untreated and treated non-diabetic controls were

assayed for NOS enzyme activity as described in Section 2:5. Statistical analysis of the

biochemical data was carried out by two way analysis of variance with diabetes and

ganglioside treatment as factors by the general linear model for unbalanced data using

the Minitab 10 programme. Such statistical analysis also revealed any outlying values

within each group that were significantly outside normal distribution. In such cases the

data was calculated and analyzed both with and without the outlying values as

suggested by Stevens (1990).

162

RESULTS

Upon death both diabetic and ganglioside-treated diabetic rats had lower body

weight, increased adrenal gland weight and higher blood glucose level as compared

with ganglioside-untreated and treated non-diabetic control rats (Table 3).

NOS immunoreactivity in medulla o f control rats

Control experiments in which the NOS antibody was omitted did not give any

staining. A comparable distribution of NOS immunoreactivity was observed in the

adrenal medulla from the untreated non-diabetic (Fig. 18a) and ganglioside-treated

non-diabetic control rats, and was as reported previously (Sections 3:1:1-2). Briefly,

NOS immunoreactivity was localized in several intrinsic neuronal cell bodies, and

nerve fibres which were associated with chromaffin cells, ganglion cells and blood

vessels.

NOS immunoreactivity in medulla o f 8 weeks diabetic rats

No apparent change was observed in the NOS immunoreactivity of the intrinsic

neuronal cell bodies. However, increased NOS-immunoreactive nerve fibres were seen

in the adrenal medulla from the diabetic rats of 8 weeks duration (Fig. 18b). The

increased nerve fibres were found accumulated in some regions of medulla near

chromaffin cells and large non-immunoreactive ganglion cells. Furthermore, several

additional cells occasionally found as columns of cells in between groups of

chromaffin cells and non-immunoreactive ganglion cells displayed homogeneous NOS

immunofluorescence (Fig. 18b). Most of these cells were small in diameter (6-10 ytrni)

and had a small central nucleus.

NOS immunoreactivity in medulla o f ganglioside-treated 8 weeks diabetic rats

In the ganglioside-treated diabetic rat adrenal medulla the diabetic-induced

163

increase in NOS-immunoreactive nerve fibres was not observed, and the pattern of

innervation was similar to that of the non-diabetic controls (Fig. 18c). In addition, the

majority of NOS-positive cells that appeared in the medulla of diabetic rats were not

observed with ganglioside treatment. However, several cells found dispersed in the

outer region of medulla persistently gave NOS immunoreactivity.

NOS immunoreactivity in cortex o f control rats

In the adrenal cortex of ganglioside-untreated and treated non-diabetic control

rats, there were NOS-immunoreactive nerve fibres in the zona glomerulosa and

subcapsular region (Fig. 19a), and around some blood vessels.

NOS immunoreactivity in cortex o f 8 weeks diabetic and ganglioside-treated diabetic

rats

Immunoreactive nerve fibres similar to that of controls were present in the

adrenal cortex of both diabetic and ganglioside-treated diabetic rats of 8 weeks

duration. However, a small number of cortical cells located in some regions of the

zonae glomerulosa and fasciculata of the diabetic rats gave positive NOS

immunoreactivity (Fig. 19b). Such immunoreactive cortical cells were not found in the

glands from ganglioside-treated diabetic rats (Fig. 19c).

NOS immunoreactivity in medulla and cortex o f 12 weeks diabetic rats

In the diabetic rats which were left for 12 weeks after induction of diabetes, the

occurrence of NOS-immunoreactive nerve fibres in medulla was reduced as compared

with age-matched non-diabetic controls (Figs. 20a, b). In addition, a weaker

immunofluorescence was seen in the chromaffin cells of some regions of the medulla.

However, similar to the 8 weeks diabetic rat gland, NOS immunoreactivity in

occasional small sized cells was found in the medulla (Fig. 20b). Furthermore, in the

164

12 weeks diabetic rats, the NOS-immunoreactive nerve fibres found in the outer cortex

were reduced as compared with controls, while the occurrence of NOS-

immunoreactive cortical cells in the region was increased (Figs. 21a, b).

NADPH-diaphorase staining in control rats

No NADPH-diaphorase staining was seen in the control sections incubated in

media devoid of ^-NADPH or in those sections pretreated with TRIS HCl heated at

75°C. A similar pattern and intensity of NADPH-diaphorase staining was seen in the

adrenal glands from ganglioside-untreated and treated non-diabetic control rats.

NOS-immunoreactive cellular elements were double positive for NADPH-diaphorase.

Cortical cells forming the parenchyma of adrenal cortex and those found as occasional

dispersed cells in medulla were also positively stained for NADPH-diaphorase (Fig.

22a). The intensity of NADPH-diaphorase staining in the cortical cells varied between

regions of the gland, being weaker in those found in zona glomerulosa than those

found in the zonae fasciculata and reticularis. Small groups of cells located at the inner

aspect of zona reticularis and at the corticomedullary junction gave a heavier NADPH-

diaphorase staining than the remaining regions. The staining intensity of these cells

matched those of cortical cells occasionally found dispersed in medulla.

NADPH-diaphorase staining in diabetic rats

NOS-immunoreactive cellular elements in the glands from all groups of the

diabetic rats were also NADPH-diaphorase double positive. However, cortical cells of

adrenal glands from diabetic rats of both duration gave a heavier intensity of NADPH-

diaphorase staining compared with that of non-diabetic control rats (Figs. 22a, b). The

increase in staining was more significant in cells of zonae fasciculata and reticularis,

and those cells found dispersed in medulla. In glands of ganglioside-treated 8 weeks

diabetic rats, the intensity of the NADPH-diaphorase staining of the cortical cells (Fig.

22c) was lower than that of diabetic rats, but was higher than that found in the non­

165

diabetic controls.

Biochemical assay o f NOS activity

In the assay for NOS activity in the soluble fraction from adrenal glands of

untreated non-diabetic control rats, the production of citrulline was totally inhibited

(99.6 ± 0.3 %, mean ± standard error of mean, n = 6) by the arginine analogue, (N ^ -

nitro-L-arginine methyl ester (L-NAME). L-NAME inhibits both constitutive and

inducible NOS activity. The percentage inhibition by EGTA, a calcium chelator, was

89.7 ± 3.1 % (n = 6), indicating that virtually all the NOS activity measured was

calcium-dependent and thus of the constitutive isoform of the enzyme. Neither

diabetes nor ganglioside treatment had any significant effect on the degree of inhibition

by L-NAME or EGTA. The levels of NOS activity in the adrenal glands from the four

groups of animals are given in Table 4.

Total NOS activity/gland was significantly increased (p < 0.001) in the adrenal

glands from diabetic rats. Ganglioside treatment did not have any significant effect on

NOS activity in control rats. However, it was apparent that ganglioside treatment did

result in increased variability in the levels of NOS activity measured in diabetic rats.

Two outlying values of NOS activity, were revealed in the ganglioside-treated diabetic

group during two way analysis of variance. When these values were removed

ganglioside treatment did have a significant effect on NOS activity in diabetes (p =

0.025). Thus there was a significant interaction (P < 0.05) between the effect of

diabetes and the effect of ganglioside treatment. A similar pattern of change was

observed when results were expressed as activity inhibited by L-NAME and EGTA

(Table 4) indicating that the increase in NOS activity in diabetes was due to an increase

in constitutive NOS activity. The increase in NOS activity/gland in diabetes was not

simply due to the increased weight of adrenal glands induced by diabetes. When NOS

activity was expressed per gram tissue there was still a significant increase in diabetic

166

tissue (Total NOS activity, nmoi/h/g tissue; Controls = 73.14 ± 4.42, n = 6; Diabetics =

141.16 ± 8.51, n = 5; P < 0.001). Furthermore, the decrease in NOS activity/gland in 4

out of the 6 ganglioside-treated diabetics was observed despite the fact that

ganglioside treatment did not significantly affect the diabetic-induced increase in

adrenal gland weight (Table 3).

167

Fig. 18a-c. Photomicrographs of NOS immunoreactivity in sections of adrenal

medulla of (a) non-diabetic control, (b) 8 weeks diabetic and (c) ganglioside-

treated 8 weeks diabetic rats. Arrowheads in a indicate NOS-immunoreactive

neurons, while small arrows show NOS-immunoreactive nerve fibres in this

and subsequent figures. Inset in b shows a group of small size immunoreactive

cells (large arrow) found in between groups of chromaffin cells. Note the

increased immunoreactive nerve fibres that occurred in the medulla of the

diabetic rat (b). Co: cortex. Bar = 33 fnn.

168

; ’ Î

• Ît - .

Fig. 19a-c. Photomicrograph of NOS immunoreactivity in sections of adrenal

cortex of (a) non-diabetic control, (b) 8 weeks diabetic and (c) ganglioside-

treated 8 weeks diabetic rats. Note the presence of immunoreactive cells {arrows

in b) in a region of cortex of the adrenal gland from diabetic rat. ZG: zona

glomerulosa; ZF: zona fasciculata. Bar = 33 ̂ m.

169

' ZF

-■ * * r t ^ .rC ' • . * • “̂ ,. , T / ' ZG

' ' \ > \ ; -

Fig. 20a, b Photomicrographs of NOS immunoreactivity in sections of adrenal

medulla from (a) non-diabetic control rat and (b) diabetic rat of 12 weeks

duration. Inset in b shows a group of small size NOS-immunoreactive cells

{large arrow) found in between groups of chromaffin cells. Note the absence of

the large majority of immunoreactive nerve fibres in the gland from diabetic rat

(b) as compared with that of the non-diabetic controls (a). Small arrows in a

show immunoreactive nerve fibres. Arrowhead in b points to an

immunoreactive neuron. Bar = 33 ^m.

170

Fig. 21a, b Photomicrographs of NOS immunoreactivity in sections of adrenal

cortex from (a) non-diabetic control and (b) 12 weeks diabetic rats. Note in b

the presence of several immunoreactive cells {arrows) and the absence of

immunoreactive nerve fibres. ZG: zona glomerulosa, ZF: zona fasciculata. Bar =

33y«m.

171

Fig. 22a-c. Photomicrographs of sections of adrenal cortex stained black for

NADPH-diaphorase of (a) non-diabetic control, (b) 8 weeks diabetic and (c)

ganglioside-treated 8 weeks diabetic rats. Note the high intensity of NADPH-

diaphorase staining in adrenal cortex of the diabetic rat in b which is reduced by

ganglioside treatment (c), although not to the level of that of the non-diabetic

control rat (a). Black arrowheads in a show groups of intensely stained cells

occasionally found in some regions of zona reticularis and at corticomedullary

junction. ZG; zona glomerulosa; ZF: zona fasciculata; ZR: zona reticularis. Bar

= 64 /Mm.

172

m

Table 3. Comparison of body weight, adrenal gland weight and blood-glucose level of

diabetic and ganglioside-treated diabetic rats of 8 weeks duration with respect to the

non-diabetic controls. Values represent mean ± standard error of mean. ̂ indicates p <

0.001, and ^ indicates p < 0.05 with respect to both ganglioside-untreated and treated

non-diabetic controls.

Controls Ganglioside'treated

Diabetics Ganglioside- treated

Bodywt(g)(n = 11)

609±14.48 594±9.91 382±16.16^ 399±9.35^

Adrenal wt (mg) (n = 6)

25±0.89 23.8±0.92 32.6±2.73^ 29.811.18'’

B lood- glucose (mM) (n = 11)

10±0.55 11.5±0.47 44.5±2.27^ 45±1.60^

173

Table 4. Effects of diabetes and ganglioside treatment on NOS activity (nmol/h/gland)

in the adrenal gland of diabetic rats of 8 weeks duration.

Total L -N A M E - EGTA-sensitive sensitive

Controls (n = 6) 1.95±0.20 1.94±0.19 1.73±0.14

Ganglioside-treated 1.80±0.33 1.80±0.33 1.64±0.33controls (n = 6)

Diabetics (n = 5) 4.61±0.52* 4.59+0.51* 4.05+0.40*

Ganglioside-treateddiabeticsa) Whole group (n = 6) 3.94±0.81 3.92±0.81 3.45±0.69

b) Outliers removed 2.74±0.47'*^ 2.74±0.47‘*'̂ 2.49±0.45'*‘̂(n = 4)

NOS activity was measured by the conversion of arginine to citrulline. Total activity represents activity corrected for a reagent blank. L-NAME-sensitive and EGTA- sensitive activity represent the difference in NOS activity measured in the presence and absence of L-NAME and EGTA, respectively. Analysis of the data was carried out using two way analysis of variance. Where such analysis revealed the presence of values in individual groups significantly outside the normal limits of distribution the data has been calculated both with and without the outliers. Results have been given as the mean ± standard error of mean. * indicates a significant effect of diabetes (p < 0.001), + indicates a significant effect of ganglioside treatment (p < 0.025), # indicates a significant interaction between the effects of diabetes and of ganglioside treatment (p < 0.05).

174

Table 5. Summary of the changes observed in the relative levels of NOS

immunoreactivity in diabetic and ganglioside-treated diabetic rats with respect to the

controls.

Medulla Cortex

Controls +++ ++

8 weeks diabetics

++++++++

+++

Ganglioside- a treated b8 weeks diabetics

++++

++

12 weeks diabetics

++++

+++

a: relative density of nerve fibres. Rated as (+) sparse to dense (+++++). b: relative number of additional cells to the NOS-immunoreactive neurons. Such cells are presumed to be adrenal cortical cells (see text for discussion). Rated as absent (-), a few (+) to several (+++).

175

Discussion

Effect o f diabetes

The increase in the weight of the adrenal glands of diabetic rats observed in the

present study is in agreement with earlier reports that the adrenal gland of diabetic rat

is enlarged (Bennett and Koneff 1946; Rebuffat et al. 1988b, c). As has been

summarized in Table 5, the changes on the NOS immunoreactivity caused by diabetes

and the effect of ganglioside treatment on such diabetic-induced changes are marked.

In the adrenal glands from the diabetic rats of 8 weeks duration, the occurrence of NOS

immunoreactivity in additional cells and nerve fibres when compared with the glands

from the non-diabetic control rats was observed immunohistochemically. Biochemical

assay also demonstrated a significant increase in the NOS activity in the adrenal glands

from the 8 weeks diabetic rats with respect to the non-diabetic controls, confirming the

immunohistochemical results.

The NOS activity measured biochemically by the conversion of arginine to

citrulline was calcium-dependent, indicating that it was of the constitutive isoform of

the enzyme. Both endothelial and neuronal NOS are of the constitutive isoform;

however, more than 90 % of the endothelial NOS is membrane bound whereas

neuronal NOS is mainly found free in the cytoplasm (Forstermann et at. 1994). Since

no detergent was added to the homogenizing medium and only the supernatant

(cytoplasmic fraction) was analyzed for NOS activity, it is unlikely that endothelial

NOS contributed to the results reported here. The conclusion that NOS activity in the

cytoplasmic fraction reflects neuronal rather than endothelial NOS activity has been

reported previously (Bush, Gonzalez and Ignarro 1992). Since the increased NOS

activity in the adrenal glands from the diabetic rat was also calcium-dependent the

increase was due to constitutive NOS, as found immunohistochemically using antibody

against the constitutive NOS type I isoform (Schmidt et at. 1991), rather than induction

of the inducible isoform of the enzyme.

176

As shown histochcmically, in the diabetic rat adrenals there were NOS-

immunoreactive and NADPH-diaphorase stained small sized cells found in between

the chromaffin cells and ganglion cells of the medulla. As found in Sections 3 :l:l-2 ,

there are some cells found scattered in the adrenal medulla in between groups of

chromaffin cells as well as ganglion cells that display NADPH-diaphorase staining,

but not NOS immunoreaction. These cells are negative for the chromaffin cell marker

TH (Section 3:1:4) and the general neuronal marker PGP 9.5 (unpublished

observation), and are thought to be cortical cells that are found dispersed as islands in

the medulla of rat (Coupland 1965). The NOS-immunoreactive cells observed in

diabetic rats may be of these cortical cell type where diabetes has increased their

number and induced the expression of NOS. This is further indicated by the finding of

similar NOS-immunoreactive cells in the adrenal cortex of the diabetic rats. However,

it is not known as what could be the cause and effect of expression of NOS in such

cortical cells during diabetes. It has been shown that in streptozotocin-induced diabetic

rats there is atrophy and hypertrophy of zona glomerulosa and fasciculata cells,

respectively (Rebuffat et al. 1988b, c). It may be either of such affected cortical cells

that expressed NOS in diabetes.

This study has also demonstrated an increase in the density of NOS-

immunoreactive nerve fibres found in the adrenal medulla of diabetic rats of 8 weeks

duration and the dramatic disappearance of such nerve fibres with increasing duration

of diabetes at 12 weeks. Previous studies have investigated changes in neurotransmitter

content of autonomic nerves in diabetes (Lincoln, Bokor, Crowe, Griffith, Haven and

Bumstock 1984; Belai, Lincoln, Milner, Crowe, Loesch and Bumstock 1985; Belai,

Lincoln, Milner and Bumstock 1988; Bumstock 1990; Soediono et al. 1993). In a

similar pattem to the present observation on NOS-immunoreactive nerve fibres in

adrenals of the diabetic rats, in rat proximal colon an initial increase in tissue level and

immunoreactivity of VIP and noradrenaline at 8 weeks has been found to be followed

177

by a subsequent decrease at 16 and 25 weeks after induction of diabetes with

streptozotocin (Belai et al. 1988). In addition, an early increase of VIP and PGP 9.5

immunoreactivity in the skin of human IDDM patient has been found to precede a

subsequent depletion (Properzi, Francavilla, Poccia, Aloisi, Gu, Terenghi and Polak

1993). Increased activities of NOS and NADPH-diaphorase have also been found in

myenteric nerves of 8 weeks diabetic rat ileum (Lincoln, Messersmith, Belai and

Bumstock 1993). NO has been implicated in neural death in cerebral infarction

(Nowicki, Duval, Poignet and Scatton 1991) and in activated microglia in culture (Boje

and Arora 1992). It is possible that the increase in the NOS immunoreactivity in the

nerve fibres and the appearance of NOS-containing cellular elements in the glands of 8

weeks diabetic rats may be indicative of enhanced production of NO as an event

leading to neuropathy and cellular degeneration. In this connection, reduction in the

amount of preganglionic nerve terminals innervating the chromaffin cells in addition to

changes in adrenal cellular structure has been found in the diabetic rats of long duration

(Wilke et al. 1993). It has also been reported that there is adrenal medullary fibrosis in

human IDDM patients of long duration (Brown et al. 1989,1990).

Although NOS is generally colocalized with NADPH-diaphorase, the reverse is

not always true, since the latter can exist independently of the former in some tissues.

In adrenal cortex, where NADPH is utilized by several steroidogenic enzymes (Sweat

and Lipscomb 1955), NADPH-diaphorase labelling of the cells with a characteristic

zonal intensity of staining distribution occurs. Comparison of such staining of adrenal

glands from experimental animals with that of controls has been utilized in the past to

assess the state of adrenal cortical activities. Accordingly, the increase in NADPH-

diaphorase staining that we observed in the adrenal cortex of the diabetic rats over that

of the non-diabetic controls may indicate increased enzymatic adrenocortical activities

with increased corticosteroid production. In this respect, in streptozotocin-treated rats

a rise in the intra-adrenal (Tomello et al. 1981) and plasma (De Nicola et al. 1976;

Rebuffat et al. 1988c) corticosterone level have been reported. As indicated by the

178

increased plasma level of ACTH, increased activity of the hypothalamic-pituitary-

adrenal axis has been implicated in the increase in corticosterone level during diabetes

(Rebuffat et al. 1988b, c). In clinical studies the cause for such an increase in

hypothalamic-pituitary-adrenal axis has been suggested to be diabetic neuropathy

(Tsigos et at. 1993). It is therefore a matter to be investigated if the increased

NADPH-diaphorase staining in the cortical cells of the diabetic rats reflects diabetic

neuropathy somewhere along the hypothalamic-pituitary-adrenal axis.

Effect o f ganglioside treatment

The immunohistochemical results indicate that ganglioside treatment tends to

prevent the diabetic-induced increase in NOS and NADPH-diaphorase reactivity in

the 8 weeks diabetic rat adrenal glands. The biochemical assay results for the effect of

ganglioside treatment on NOS activity are somewhat equivocal due to the fact that

ganglioside treatment per se appeared to result in increased variability in the data.

According to the results from assay of NOS activity with ganglioside treatment, there

was a significant reduction in the diabetes-induced increase in NOS activity in the

adrenal glands from four out of the six diabetic animals studied, although such

reduction did not occur in the remaining two rats. The reason for the inconsistency

between the biochemical and histochemical approaches is unknown at present. The

persistency of some reactive cells in the ganglioside -treated diabetic rats, as observed

histochemically, may in part be accounted for by the observed variability in the NOS

activity.

Ganglioside has been shown to prevent diabetic-induced neuropathic changes

(Soediono et al. 1993) and facilitate recovery from diabetic-induced neural

dysfunction (Norido, Canella, Zanoni and Gorio 1984; Triban, Guidolin, Fabris,

Marini, Schiauinato, Dona, Bortolami, Giamberardino and Fiori 1989; Figliomeni,

Bacci, Panozzo, Fogarolo, Triban and Fiori 1992). It is therefore possible that

179

ganglioside treatment, by preventing an increase in NO production in the adrenal glands

from diabetic rats may protect against neuropathy. Furthermore, it remains to be

established if the observed inhibition of the increase in adrenal cortical NADPH-

diaphorase staining intensity in the diabetic rats by ganglioside is secondary to possible

protection from neuropathy. However, the possibility that ganglioside may have a

direct parallel influence on the cortical cells similar to that on neural tissue can not be

ruled out and awaits investigation.

180

SECTION 4

GENERAL DISCUSSION

181

Each of the separate studies presented in this thesis has been discussed

individually in detail in Section 3. In this Section of the thesis a general discussion with

regard to the feature of adrenal NOS-synthesizing nerves and their possible functional

involvement in the adrenal gland activity is presented. Plasticity in the expression of

the nitrergic nerves and its possible cause and mechanism has also been discussed here.

4:1. Adrenal nitric oxide-synthesizing nerves

The NOS immunoreactivity in the adrenal gland from the adult rats was found

only in the neuronal elements. This is also in agreement with the results obtained by

others (Bredt et al. 1990; Dawson et al. 1991; Aim et al. 1993), except one group (Dun

et al. 1993) who claimed the occurrence of the immunoreactivity in all of the

chromaffin cells as well. The lack of NADPH-diaphorase staining in the chromaffin

cells also further confirmed the absence of NOS from the adrenal chromaffin cells of

the adult rats. However, indicating possible species difference, NOS immunoreactivity

has been reported in a small number of chromaffin cells in human adrenal gland (Heym

et al 1994).

The adrenal nitrergic nerves are heterogeneous, since they costore different

neuromediators (Section 3:1:4) and the nerve fibres originate from extrinsic as well as

intrinsic adrenal neuronal sources (Section 3:1:3). The source for the extrinsic NOS-

containing nerve fibres that innervate adrenal medullary cells has not been totally

demonstrated, although sympathetic preganglionic cell bodies in the intermediolateral

cell column of the spinal cord have been shown to have their terminals in the gland

(Blottner and Baumgarten 1992). However, the nitrergic nerve fibres found as a plexus

in the subcapsular region and the zona glomerulosa of the adrenal cortex, as well as

those associated with some intra-adrenal blood vessels, come from the NOS-

containing intrinsic adrenal neurons (Section 3:1:3).

The nature and function of the adrenal intrinsic neurons have been a mystery for

182

many years. Two principal types of intra-adrenal neurons have been described (Unsicker

et al. 1978). One group of neurons are large multipolar, which are typical of ganglion

cells. These neurons express TH, NA and NPY, and do not have mRNA for choline

acetyltransferase and arc therefore regarded to be sympathetic postganglionic neurons

producing noradrenaline (Dagerlind et al. 1990; Oomori et al. 1994). They are

surrounded by Schwann cells and are contacted by dense nerve terminals. In the guinea

pig, such terminals contain both small clear and large dense cored vesicles, and as

neither of them show uptake of 5-OHDA are considered to be cholinergic (Unsicker et

al. 1978). These type of neurons do not contain NOS, but are heavily innervated with

nerve fibres containing NOS and calretinin (Sections 3:l:4-5). The second group of

intrinsic neurons are smaller in size (Unsicker et al. 1978). From their morphology and

the colocalization studies, it could be inferred that it is this second type of neurons

which demonstrate the NOS immunoreaction and NADPH-diaphorase staining

(Section 3:1:4). Some of these neurons also contain VIP and NPY, and are distinctly

innervated by nerve fibres containing SP, CGRP, VIP and NOS. A few of these

neurons which contain NOS are densely surrounded by calretinin-containing nerve

fibres (Section 3:1:5).

Because of the poor accessibility of the adrenal intrinsic neurons no evidence is

available which shows their functional role. However, based on the frequent

observations of their processes contacting nearby blood vessels (Kondo 1985; Oomori

et al. 1994; also present study) and the present selective surgical denervation studies

(Section 3:1:3), their possible involvement in the innervation of intra-adrenal blood

vessels could be suggested. Furthermore, previous studies which used extrinsic and/or

intrinsic surgical adrenal denervations have shown VIP and NPY-containing adrenal

intrinsic neurons innervate the outer region of adrenal cortex (Hokfelt et al. 1981;

Holzwarth 1984; Maubert et al. 1993). The present study for the NOS-containing

nerves has also supported the suggestion that adrenal intrinsic neurons innervate

183

adrenal cortex (Section 3:1:3).

4:2. Nitric oxide in the regulation of adrenal medullary secretion

The adrenal chromaffin cells continuously secrete catecholamines to maintain

the homeostatic balance of the body. There are also occasions of extreme activation of

these cells with increased secretion of catecholamines during stress conditions. The

chromaffin cells of the adrenal medulla also synthesize several other substances, such

as the neuropeptides. In order to achieve such secretory activities in a harmonious

manner, the adrenal chromaffin cells may be regulated by a variety of

neurotransmitters and hormones. Although acetylcholine released from the

preganglionic nerve terminals is the main regulator of these cells, several studies have

shown evidence for a non-cholinergic transmission as well. Stimulation of adrenal

medulla with subcutaneous injection of nicotine in the rat produced induction of TH

expression which could only be partially blocked with the nicotine receptor

antagonists, hexaméthonium and mecamylamine (Fossom, Carlson and Tank 1991a).

In addition, in the denervated adrenal gland, the induction of TH by nicotine could be

prevented by the above nicotinic receptor antagonists (Fossom, Sterling and Tank

1991b). These observations led to the suggestion that nicotine activates the adrenal

chromaffin cell nicotinic receptors directly as well as activating the central nervous

system pathway resulting in the release of substances from the splanchnic nerves which

interact with adrenal chromaffin cell receptors other than the cholinergic nicotinic

receptors (Fossom et al. 1991a, b). It has also been found that denervation, but not the

nicotinic receptor antagonist chlorisondamine, could completely block the reserpine-

induced increase in adrenal TH levels (Mueller et at. 1970; Schalling et al. 1991). In

addition, the cold-induced increase in the level of PNMT mRNA could not be

inhibited by chlorisondamine or atropine (Baruchin, Vollmer, Miner, Sell, Strieker and

Kapllan 1993), suggesting possible presence of non-cholinergic neuronal transmission

in the regulation of PNMT expression. Studies in isolated perfused rat and bovine

184

adrenal glands have also provided evidence for a significant contribution of non-

cholinergic, secretomotor nerve fibres in the catecholamine secretion elicited by field

stimulation of adrenal nerve fibres (Malhotra and Wakade 1987a; Wakade 1988;

Wakade, Blank, Malhotra, Pourcho and Wakade 1991; Marley, Thomson and

Smardencas 1993).

Several neuromediators that are contained in the splanchnic nerves, intrinsic

neurons and/or the chromaffin cells themselves may be involved in the non-

cholinergic mechanism of regulation of adrenal medullary hormone biosynthesis and

secretion. In rat, VIP has been shown to be involved in non-cholinergic transmission

(Malhotra and Wakade 1987a, b; Wakade 1991; Wakade et al. 1991; Guo and Wakade

1994). Pituitary adenylate cyclase-activating polypeptide which is structurally related

to VIP has also been shown to elicit catecholamine secretion fi’om rat adrenal

chromaffin cells (Wakade, Guo, Strong, Arimura and Haycock 1992).

The occurrence of NO in the preganglionic nerve terminals that innervate

adrenal chromaffin cells (Bredt et al. 1990; Blottner and Baumgarten 1992; also

present study) suggests that NO as a non-cholinergic transmitter may anterogradely

regulate adrenal medullary hormone biosynthesis and secretion. Being an easily

diffusible molecule, NO could also translocate from the preganglionic nerve terminals

to adjacent nerve terminals for a collateral modulation. In addition, the occurrence of

NO in the intrinsic adrenal neurons (Dawson et al. 1991; Aim et al. 1993; also presents

study) suggest that NO may also serve in the gland as a signal arising from

postganglionic neurons for possible modulation of adrenal medullary secretion.

As shown in this study, the nitrergic nerve fibres are found closely associated

with the chromaffin cells, and the NA cells are innervated with a higher proportion

than the A cells (Section 3:1:1). There are other studies that also show that adrenal NA

cells generally receive more nerve endings than the A cells (Allen, ErankO and Hunter

1958; Eranko 1959; Grynszpan-Winograd 1974). Such differential innervation may

185

suggest distinct anatomical pathways controlling A and NA cells. In fact, selective

secretion of adrenaline or noradrenaline can be achieved by stimulating specific

regions in the central nervous system in rat and cat (Folkow and von Euler 1954;

Matsui 1979, 1984). However, the proportion of catecholamines released may also

vary with different conditions such as stress and the state of stimulation. In stress-

induced catecholamine secretion, for example, the proportion of adrenaline and

noradrenaline secreted depends upon the type of the stress experienced (Feuerstcin and

Gutman 1971; Khalil, Livett and Marley 1986). Differential secretion of adrenaline or

noradrenaline can also be achieved by varying the pattem of electrical stimulation of

the splanchnic nerve alone (Mirkin 1961; Edwards 1982).

The involvement of NO in adrenal catecholamine secretory activity has been

suggested by several authors, although their findings are not coherent with regard to

the precise role of NO in causing secretion. Effluents from the endothelial cells and the

chromaffin cells were found to inhibit potassium or the nicotinic agonist

l,l-dimethyl-4-phenylpiperazinium-induced catecholamine secretion from cultured

bovine chromaffin cells (Torres et al, 1994). Since the NOS inhibitors L-NAME or

N^-monoethyl-L-arginine (L-NMMA) and the guanylate cyclase inhibitor

methylene blue could prevent such inhibition of catecholamine secretion caused by the

effluents, but augment the catecholamine secretion induced by the secretagogues, it

was suggested that NO is involved in inhibition of catecholamine secretion via

activation of guanylate cyclase (Torres et al. 1994). In fact, cGMP which is produced

by activated guanylate cyclase and whose cellular level is known to be regulated by

NO (see Garthwaite 1991) has previously been found to inhibit catecholamine

secretion from bovine chromaffin cells (Schneider et al. 1979; Derome et al. 1981) and

PC 12 cells (Fiscus, Robles, Waldman and Murad 1987; Drewett, Marchand, Ziegler

and Trachte 1988). However, a biphasic catecholamine secretion has also been caused

by 8 -bromo-cGMP from cultured bovine chromaffin cells (O'Sullivan and Burgoyne

1990). At low doses, 8 -bromo-cGMP caused potentiation, while at supramaximal

186

doses it caused inhibition of catecholamine release (O'Sullivan and Burgoyne 1990).

Moreover, NO and other NO generating agents have been shown to stimulate

catecholamine release accompanied with increased output of cGMP from perfused dog

adrenal gland (Dohi et al, 1983). In addition, NO has produced calcium and

concentration-dependent stimulation of basal catecholamine secretion from bovine

chromaffin cells (Oset-Gasque et at. 1994). It has also been found that NO stimulates

the catecholamine secretion evoked by small doses of nicotine, but inhibits at higher

doses of nicotine (Oset-Gasque et al. 1994). Furthermore, N ^-nitro-L-arginine (L-

NNA), an inhibitor of NOS, has been shown to reduce the acetylcholine-induced

increase in intracellular calcium level and catecholamine release which could be

restored by L-arginine (Uchiyama et al. 1994).

There are other studies which do not implicate NO and/or cGMP in

catecholamine secretion. The splanchnic nerve stimulation-induced catecholamine

secretion in pentobaribitol-anaesthetized dog was not affected by L-NAME (Breslow

et al. 1992). Certain stimuli that evoke catecholamine secretion, such as electrical

stimulation of splanchnic nerves, methacholine, dimethyl-4-phenylpiperazinium

iodide, high levels of potassium and alamethicin have caused a rise in cGMP from

perfused cat adrenal medulla (Moro, Michelena, Sanchez-Garcia, Palmer, Moncada

and Garcia 1993). However, although, L-NAME could abolish such rise in cGMP, it

did not cause any change in the catecholamine release (Moro et al. 1993). It is not

known at present as to what could be the reason for the discrepancies on the effect of

NO in the catecholamine secretion as inferred from the above mentioned studies. It is,

however, possible that differences in the methods and preparations used to elicit

catecholamine release, in the type of secretagogues used, and in their concentration and

duration of the stimuli as suggested by Torres et al. (1994), may partly account for the

discrepancies.

NO in the adrenal medulla may also be involved in the synthesis of other

187

substances such as the neuropeptides whose biosynthesis may not directly correlate with

that of the catecholamines. Although, adrenal peptides are costored and coreleased

with the catecholamines, their biosynthesis may be differently regulated, as is found for

enkephalins (see Naranjo, Mocchetti, Schwartz and Costa 1987). Elsewhere, NO has

already been found to modulate the release of corticotrophin-rcleasing hormone from

rat hypothalamus in vitro (Costa, Trainer, Besser and Grossman 1993) and inhibit

vasopressin and oxytocin secretion from isolated neural lobe of the rat pituitary gland

(Lutz-Bucher and Koch 1994). There is also evidence to suggest that NO modulates

the transcriptional induction of a number of genes, since it has been found to activate

the immediate early genes c-fos and jun-B in the PC 12 cells (Peunova and

Enikolopov 1993; Haby, Lisovoski, Aunis and Zwiller 1994).

4:3. Nitric oxide in the regulation of adrenal cortical secretion

It is apparent that adrenal cortical activity is influenced by neuronal

mechanisms, in addition to hormonal regulation. Electrical stimulation of the

splanchnic nerves in calf and dog causes an increase in the rate of ACTH-induced

cortisol secretion (Edwards and Jones 1987; Engeland and Gann 1989). Splanchnic

nerve stimulation has also been found to enhance the activity of 1 1 ^-hydroxylase,

which in turn increases the synthesis of cortisol from 11 deoxycortisol (Engeland and

Gann 1989). In contrast, transection of the splanchnic nerve in lamb and calf causes a

decrease in adrenocortical sensitivity to ACTH (Edwards and Jones 1987; Edwards,

Jones and Bloom 1986). Moreover, several mediators, including NO, which exist in the

intra-adrenal nerves and/or the chromaffin cells have been shown to influence

corticosteroid secretions from the gland (see Section 1:7).

Treatment of rats with the NOS inhibitor L-NAME causes a dose-dependent

increase in serum corticosterone levels, suggesting endogenous NO may negatively

regulate steroidogenesis (Adams et al. 1992). Perfusion of isolated rat adrenal gland

preparation with a medium which contains L-arginine at a higher dose above 500

188

fimo\/\ results in a decrease in adrenal corticosterone secretion (Cameron and Hinson

1993). However, at a lower dose of L-arginine upto 500 /^mol/1 an increase in the

corticosterone secretion occurs in a dose dependent manner (Cameron and Hinson

1993). It therefore appears that NO could influence adrenal cortical secretory activity.

With the majority of NOS-containing nerve fibres located in the subcapsular region

and the zona glomerulosa of the cortex, NO liberated from these nerve fibres may

reach the cortical cells in the other region by diffusion. Since the NOS-containing

intrinsic neurons are the source for the NO-containing nerve fibres which innervate the

cortex (Section 3:1:3), adrenal medullary neurons which synthesize NO therefore

appear to regulate the activity of adrenal cortical cells. Such local regulation has also

been suggested for adrenal neurons that contain the neuropeptides VIP and NPY

(Hokfelt et al. 1981; Holzwarth 1984; Maubert et al. 1993). Furthermore, there are a

population of chromaffin cells located in the cortex which are thought to exert a

paracrine influence on adrenal cortical activity (Palacios and Lafarga 1975; Bomstein

etal. 1990,1991; Vizi etal. 1992).

The adrenal cortex in turn is known to influence the secretion of adrenal

medullary chromaffin cells. Following hypophysectomy roughly 100 times more

glucocorticoids are required to restore the activity of adrenal PNMT than required for

those of extra-adrenal enzymes regulated by glucocorticoids (Pohorecky and Wurtman

1971; Wurtman, Pohorecky and Baliga 1972). Adrenaline synthesis by the adrenal

chromaffin cells has therefore been suggested to be regulated by very high

concentrations of glucocorticoids compared to the level found in the circulation

(Pohorecky and Wurtman 1971; Wurtman et al. 1972). Glucocorticoids also regulate

the expression of other adrenal chromaffin cell products, such as chromogranin A

(Fischer-Colbrie et al. 1988) and Leu-enkephalin (Henion and Landis 1992).

Furthermore, in the rat, the initial expression of PNMT and Leu-enkephalin occurs

after the precursor cells have arrived in the adrenal gland, and is temporarily correlated

189

with the large increase in the production of glucocorticoids by adrenal cortical cells at

E16 (Seidl and Unsicker 1989; Henion and Landis 1992). It therefore appears that there

is inter-regulation between medulla and cortex of the gland. However, although the

influence of adrenal medulla over cortex appears to be accomplished by the projections

of adrenal intrinsic neurons located in the medulla and the paracrine action of adrenal

chromaffin cells located in the cortex, it has not been unequivocally resolved as to how

the secretion from the adrenal cortex could reach the medulla in the high concentration

needed. The hypothesis of a portal circulation to facilitate such influence is unlikely,

since it is not supported by anatomical evidence (Coupland and Selby 1976; Murakami

et al. 1989a). Hamaji, Miyata and Kawashima (1985) have suggested that

glucocorticoids may reach the medullary cells either via the interstitial fluid of the

cortex or by free diffusion firom the adrenal cortical veins into the medullary interstitial

fluid. Moreover, there are islands of cortical cells found in the medulla of the gland

(Coupland 1965). As found in the present study, significant numbers of NADPH-

diaphorase positive but NOS-negative cells and which appear to be cortical cells are

seen in groups or singly dispersed throughout the medulla. These cells are negative for

the chromaffin cell marker TH (Section 3:1:4) and the general neuronal marker PGP

9.5 (unpublished observation). In addition, the disappearance of these cells following

hypophysectomy further suggested their cortical cell identity (Section 3:2:1). There is

therefore a possibility that such cortical cells found in the medulla could, at least partly,

be the source of local glucocorticoids needed by the chromaffin cells, and that further

work needs to be performed.

4:4. Nitric oxide in the regulation of adrenal blood flow

The mammalian adrenal gland is a highly vascular organ and receives high

levels of blood flow compared with other organs (see Vinson and Hinson 1992). The

blood flow through the gland is actively regulated and does not simply follow changes

in systemic blood pressure. However, a number of physiological conditions which

190

cause secretion from either medulla or cortex also result in increased adrenal blood

flow.

As found in the dog, blood flow between medulla and cortex may be regulated

independently. Splanchnic nerve stimulation is shown to cause an increase in the rate

of adrenal medullary blood flow without altering that of the adrenal cortex (Breslow,

Jordan, Thellman and Traystman 1987). The increase in adrenal medullary blood flow

in response to haemorrhagic hypotension is only accompanied by a transient decrease

in cortical blood flow (Breslow, Mennen, Koehler and Traystman 1986). However, in

the rat, indicating possible species difference, the proportion of blood flow to medulla

and cortex is conserved following haemorrhagic hypotension (Sparrow and Coupland

1987).

Although adrenal blood flow accompanies adrenal catecholamine secretion, it

appears that these two phenomena are regulated independently. As found from analysis

of the levels of catecholamine secretions with respect to the changes in the rate of

delivery of perfusion medium in isolated perfused rat adrenal gland, increase in adrenal

flow rate by itself has no effect on catecholamine secretion (Bouloux, Perrett, Sopwith

and Besser 1986). Furthermore, in spite of a 95 % abolition of the splanchnic nerve-

induced increase in catecholamine secretion that could be caused by administration of a

nicotinic antagonist hexaméthonium, the increase in medullary vasodilatation could

only be abolished by a combination of nicotinic and muscarinic antagonists but not by

either one alone (Kennedy, Breslow, Tobin and Traystman 1991). In contrast, adrenal

steroid secretion appears to be closely regulated with adrenal blood flow. At

submaximal concentration of ACTH, enhancement of adrenal steroid secretion in rat

and dog could be achieved by mechanically increasing the rate of blood flow in the

gland (Porter and Klaiber 1965; Urquhart 1965). In the calf, changes in the adrenal

flow rate is shown to cause changes in the rate of presentation of ACTH which in turn

correlates with the rate of adrenal steroid secretion (Jones, Edwards and Bloom 1990).

191

The regulation of blood flow in the gland may be effected through various

agents. ACTH causes a large increase in the rate of blood flow in the adrenal gland of

rat and dog (Hartman, Brownell and Liu 1955; Holzbauer and Vogt 1957). Certain

neuropeptides, which are known to exist in the splanchnic nerves, chromaffin cells

and/or adrenal intrinsic neurons, also appear to regulate blood flow as shown in

isolated perfused rat adrenal gland preparations (Hinson, Cameron, Purbic and Kapas

1994a). VIP was found to have the greatest effect causing 136 % increase in adrenal

flow rate. Met-enkephalin caused a 50 % increase, while leu-enkephalin, neurotensin

and substance P had up to 35 % increase in perfusion medium flow rate. In contrast,

NPY caused a 30 % decrease in the perfusion medium flow rate. Since NO is a

powerful vasodilator in several regions of the body (see Moncada et al. 1991;

Lowenstein et al. 1994), it may also be involved in the regulation of blood flow in the

adrenal gland. Indeed, as found in the isolated perfused rat adrenal gland preparation,

infusion with medium containing L-arginine increases adrenal flow rate in a direct

dose dependent manner, and such an increase is inhibited by L-NAME (Cameron and

Hinson 1993). In the anaesthetized dog, a substantial reduction in adrenal cortical

blood flow occurs following administration of L-NAME (Breslow, Tobin, Bredt,

Ferris, Snyder and Traysman 1993). Moreover, the increase in the adrenal blood flow

which accompanies the catecholamine secretion induced by splanchnic nerve

stimulation is associated with an increase in NOS activity, and that inhibition of NOS

by L-NAME abolishes the vasodilation response to splanchnic nerve stimulation

(Breslow et al. 1992,1993).

The NOS-positive nerve fibres found closely associated with adrenal blood

vessels and in the subcapsular region, may be the source of NO involved in the

regulation of blood flow in the gland. NADPH-diaphorase staining has been shown to

mark the presence of NOS in the endothelial cells of some blood vessels (Loesch, Belai

and Bumstock 1993, 1994; Tomimoto, Nishimura, Suenaga, Nakamura, Akiguchi,

Wakita, Kimura and Mayer 1994). However, the absence of NADPH-diaphorase

192

staining in the endothelial cells within the adrenal gland may suggest that the adrenal

nerves are the only source of the NO needed in normal physiological condition in the

gland. Nevertheless, further studies with specific antibody which recognizes

endothelial NOS may be needed to fully investigate the possibility of the presence of

NO-synthesizing endothelial cells which may have not been detected by NADPH-

diaphorase histochemistry in the gland.

4:5. Plasticity in nitrergic innervation

The production of NO is thought to be regulated in at least two ways. The NO

produced by the NOS isoforms I and III is mainly regulated by calcium and

calmodulin, where increase in the intracellular calcium concentration binds to

calmodulin and activates the enzyme (Knowles et al. 1989; Bredt and Snyder 1990;

Malinski and Taha 1992). The NO produced by the NOS isoform II, on the other hand,

is caused by an induction of the synthesis of the enzyme after challenge with a variety

of immunologic or inflammatory stimuli (Hibbs et al. 1987b; Vodovotz et al. 1994).

However, as found in the present study, the level of NOS immunohistochemically

recognized with antibody specific to isoform I, shows changes during development and

aging (Section 3:1:2), after reserpine treatment (Section 3:2:1) and in streptozotocin-

induced diabetes (Section 3:2:2). This suggests that the NOS isoform I may also be

induced. Other recent investigations have also shown the protein levels and/or

activities of the NOS isoforms I and III can be affected under various conditions such

as during development, pregnancy, neuronal lesions, neuronal activity, mechanical

stimuli and exercise, and by several agents such as NGF, estrogens, testosterone and

cytokines (Verge et a/.1992; Fiallos-Estrada, Kummer, Mayer, Bravo, Zimmermann

and Herdegen 1993; Hirsch, Steiner, Dawson, Mammen, Hayek and Snyder 1993;

Matsumoto, Pollock, Nakane and Forstermann 1993; Weiner, Lizasoan, Bayliz,

Knowles, Charles and Moncada 1993; Wu 1993; Wu and Li 1993; Bredt and Snyder

193

1994; Roskams, Bredt, Dawson and Ronnett 1994; Sessa et al. 1994).

The changes in the expression of NOS isoform I during development and after

neuronal lesions has thrown light onto possible additional functions, in addition to

neurotransmission. During early development, several regions of the brain c«idc the

cortical plate, sensory ganglia and olfactory epithelium express NOS (Bredt and

Snyder 1994; Roskams et at. 1994). The expression of NOS in these regions appears

when the cells have completed cell division and begun to extend processes. In some of

the regions NOS is expressed transiently and lost later in development (Bredt and

Snyder 1994). Furthermore, corticothalamic fibres express NOS when cortical neurons

cease dividing and extend processes (Bredt and Snyder 1994). Treatment of PC 12

cells with NGF, which also causes neurite extension (Greene and Tischler 1976), elicits

expression of the NOS isoform I (Hirsch et al. 1993). Based on such observations a

role for NO in modification of synaptic efficacy in the developing nervous system has

been suggested (Bredt and Snyder 1994; Roskams et al. 1994).

Several studies have shown the occurrence of changes in NOS expression

immediately following neuronal lesions. In adult rat, after olfactory bulboctomy the

newly developing neuronal cells become NOS positive as they extend processes to

replace those lost (Roskams et al. 1994). The number of NOS-positive neurons in

sensory ganglia increases after peripheral axotomy (Verge et al. 1992; Fiallos-Estrada

et al. 1993). Ventral root avulsion has resulted in expression of NOS staining in motor

neurons which are negative in unlesioned animals (Wu 1993). However, the increase in

expression of NOS, and thus enhanced production of NO following neuronal lesions is

not known as to whether it is related to trophic action or involved in the degeneration

process of the lesioned nerves. There is evidence where lesions of medial forebrain

bundle and mammilothalamic tract causes NOS staining in cell bodies of these

bundles, which persists for up to 150 days, implying a possible neuroprotective action

for NO (Herdegen, Brecht, Mayer, Leah, Kummer, Bravo and Zimmermann 1993). In

contrast, the NOS positive motor neurons expressed in the spinal cord following

194

ventral root avulsion have been found to die ultimately, whereas treatment of such

animals with the NOS inhibitor nitroarginine prior to ventral root avulsion protects the

cells from death (Wu and Li 1993). Furthermore, NO-generating agents have been

shown to cause a collapse of neuronal growth cones in regenerating dorsal root

ganglion cells (Hess, Patterson, Smith and Skene 1993).

Various factors and mechanisms may be responsible for the changes in the

expression of NOS. Increase in NOS expression in a tissue may be due to an increase

in number of cells that express the enzyme as a result of induction of its synthesis in

cells which previously did not contain the enzyme. It could also be due to proliferation

of cells with the capacity of expressing the enzyme. In contrast, decrease in the levels

of expression of the enzyme in a tissue may reflect a cessation of its synthesis from

cells, or may be due to a disappearance of the cells containing the enzyme as a result of

degeneration or cell death.

The induction and disappearance of transmitters and their synthesizing enzymes,

such as the NOS, in intact nerves may be responsible for causing changes in neuronal

phenotype and hence plasticity that occur in response to different stimuli. The basis for

the changes in the levels of transmitters and their synthesizing enzymes appears to be

effected mainly at the gene level, although up or down regulation of the activities of

preexisting enzymes may also be possible. It appears that all neurons carry the genes

responsible for the neurotransmitters and their synthesizing enzymes. Some of the

genes are silenced in certain subpopulations of neurons, while some other genes that

are appropriate to a given differentiated neurons are expressed by an active control

mechanism. Such processes enables neurotransmitter choice that is neither

precommitted nor stable, but is continuously influenced by environmental conditions.

Several factors such as the state of membrane electrical activity, and the levels of

neurotrophic factors, neurotransmitters and hormones could influence the expression of

the genes.

195

The mechanisms of change for the expression of NOS are not fully understood

at present, although most likely are mediated through alterations in NOS gene

expression, mRNA stability or protein biosynthesis (see Morris and Billiar 1994;

Nathan and Xie 1994b; Sessa 1994). In many instances the changes in the activity of

NOS isoform II have been correlated with similar changes in its mRNA abundance

which suggested the major part of the regulation of this enzyme is at a pretranslational

step such as transcription or mRNA stability (Xie, Cho, Calaycay, Mumford, Swiderck,

Lie, Ding, Troso and Nathan 1992; Nunokawa, Ishida and Tanaka 1993). Such

correlation of enzyme levels and/or activities with mRNA levels have also been

observed in studies involving the NOS isoforms I and III (Yoshizumi, Perrella, Burnett

and Lee 1993; Roskams et aL 1994; Sessa, Pritchard, Seyedi, Wang and Hintze 1994).

The levels of substrates, cofactors and products may also influence the state of

enzyme activity and could play an important role in determining rates of cellular NO

production. GTP-cyclohydrolase I, the first and rate-limiting enzyme in the synthesis

of BH4 from GTP, has been shown to be responsible for tumour necrosis factor-a,

interferon-r or LPS-induced increases in endothelial cell NOS activity (Wemer-

Felmayer, Werner, Fuchs, Hausen, Reibnegger, Schmidt, Weis and Wachter 1993).

Moreover, it has been shown that NO can inhibit the production of NO in cerebellum,

macrophages and central nervous system glial cells (Rogers and Ignarro 1992;

Assreuy, Cunha, Liew and Moncada 1993; Griscavage, Rogers, Shermann and Ignarro

1993; Park, Lin and Morphy 1994). Such inhibition of the production of NO by NO

itself is effected through the regulation of NOS catalytic activity by interaction with the

heme moiety of the enzyme (Rogers and Ignarro 1992; Griscavage et al. 1993) and

through the regulation of NOS expression by limiting transcriptional induction of the

gene (Park et al. 1994).

The expression of NOS and/or production of NO therefore appear to be complex

and is subject to regulation at various cellular levels. It is likely that some of the

changes in the expression of NOS in the adrenal gland which were observed in the

196

present study are correlated with changes at the molecular levels, such as gene

expression, mRNA stability and/or derangements in the enzyme biosynthesis or

activity. They may also be correlated with cellular and nerve fibre degeneration or

proliferation. Such changes and their effect may have not been fully revealed by the

present histochemical studies at the light microscopic level. Future investigations

which employ electron microscopy and techniques such as molecular biology and

biochemical methods may reveal more changes and throw light onto the mechanisms

of the plasticity.

197

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