RAT ADRENOCORTICAL CELL DIFFERENTIATION: EFFECTS OF SIGNAL

TRANSDUCTION ALTERATIONS, rasONCOGENE EXPRESSION,

AND PARENCHYMAL/STROMAL INTERACTIONS

by

CALVIN ROSKELLEY

B.Sc., The University of Victoria, 1983

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR ThE DEGREE OF DOCTOR OF PHILOSOPHY

in

THE FACULTY OF GRADUATE STUDIES

(Faculty of Medicine, Department of Anatomy)

We accept this thesis as conforming to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA

May 1992

© Calvin D. Roskelley

Signature(s) removed to protect privacy

In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives, It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.

(Signature)

_____________________

Department of ANATOMY

The University of British ColumbiaVancouver, Canada

Date June 1, 1992

DE-6 (2/88)

Signature(s) removed to protect privacy

II

ABSTRACT

The zones of the adrenal cortex contain distinct subpopulations of cells which

share a common mesodermal origin and steroidogenic template. How zonal specializations

are overlaid upon this template is unknown. During adrenocortical cytogenesis zonal

phenotypes are expressed transiently as a cell moves inward from a subcapsular

position toward the medulla. In this study, homogeneous populations of rat adrenocortical

parenchymal cells were maintained and manipulated in vitro. This was done in an effort

to determine the mechanism responsible for zone-specific differential responsiveness to

the trophic hormone angiotensin II (Ang II). In addition, the effects of ras oncogene

expression and parenchymal/stromal interactions on overall steroidogenic

differentiation were assessed.

Initially, a method was devised to separate rat adrenocortical cells by density

into populations which retain zone specific properties in primary culture. Two different

parenchymal populations were obtained. They were designated FASC (1 .034g/mI, 1 8.Oj.i.

cell diameter) and GLOM (1.069g1m1, 11.711. cell diameter). In freshly isolated cell

suspensions the physical characteristics and differential steroidogenic responses to

adrenocorticotropin and angiotensin II suggested that FASC cells originated

predominantly from the zona fasciculata and GLOM cells from the zona glomerulosa. In

primary culture, the two populations exhibited different morphologies. FASC cells

retained lipid and formed cohesive epithelial monolayers that remained stationary for

three weeks. GLOM cells were initially epithelial but rapidly lost lipid, spread, assumed

fibroblastic shapes and expressed vimentin. Both cell types were positive for the

steroidogenic cytochrome P-450 side chain cleavage enzyme (P-45Oscc). Therefore,

the morphological changes observed in GLOM cultures were due to modulation, not

fibroblastic overgrowth.

III

Ang II increased steroidogenesis in GLOM cells but not in FASC cells. To determine

the mechanism responsible for this zonal specialization, Ang Il-mediated signal

transduction was closely examined. Ang II significantly increased the production of

inositol 1,4,5 triphosphate in both cell types. In contrast, the two cell types exhibited

very different intracellular release dependent increases in free intracellular calcium

([Ca2]i). Ang II induced [Ca2]i increases of >5OnM in 90% of individual GLOM cells,

but in only 28% of FASC cells. Also, in populations, Ang II induced dose dependent

[Ca2]i increases in GLOM but not FASC cells. Importantly, calcium ionophore

treatment increased [Ca2+]i and steroidogenesis in both cell types. These results

suggest that FASC cells lack a steroidogenic response to Ang II, at least in part, because of

an interruption of the signalling pathway at the level of intracellular calcium release.

Therefore, alterations within a specific signal transduction pathway are responsible for

one of the cellular phenotypes associated with zonal specialization in the rat adrenal

cortex.

GLOM and FASC cells were infected with Kirsten murine sarcoma virus, which

contains an activated form of the ras oncogene. FASC cells exhibited no discernable

morphologic change after infection. In GLOM cultures, discrete foci did not appear.

Instead, transient cellular multilayers formed from which rounded cells emerged. After

selective passaging these cells (designated KGLOM) acquired a transformed morphology,

proliferated rapidly in both 10% and 1% serum, grew to high saturation densities, and

they were tumorigenic. They also expressed P-45Oscc and the viral ras protein, p21.

In addition, KGLOM cells produced endpoint steroids under conditions in which GLOM

cells did not. KGLOM steroidogenesis could be inhibited by lovastatin, a pharmacological

inhibitor of p21 ras function. Therefore, expression of the ras oncogene induces

transformation and enhances the steroidogenic differentiation of rat glomerulosa cells.

This dual function of ras p21 likely reflects the state of the signal transduction pathways

present within steroidogenic cells.

iv

In longterm culture, FASC and GLOM cells de-differentiated and lost their

steroidogenic characteristics. This did not occur in a third density isolated population

that consisted of a mixture of cell types (designated MIX). In primary culture, MIX

populations formed highly proliferative cellular multilayers that were composed of

stromal fibroblasts, endothelial cells and parenchymal cells. From these multilayers

rounded cells emerged that contained lipid and the steroidogenic enzymes A5,3B-

hydroxysteroid dehydrogenase (A5,313-HSD) and P-45Oscc. This phenomenom was

associated with a 10 fold increase in the number of z5,3I3-HSD containing cells during

the first 21 days in culture. Rounded cells were selectively passaged into secondary

culture where they continued to proliferate and remained steroidogenic. Thus, primary

MIX cultures provide a microenvironment that produces proliferating, steroidogenic

cells independently of trophic hormone treatment. This unique model may prove useful

in the quest to further identify specific inducers that are responsible for adrenocortical

cytogenesis in general and steroidogenic differentiation in particular.

V

TABLE OF CONTENTS

ABSTRACT ii

TABLE OF CONTENTS v

LIST OF TABLES x

LiST OF FIGURES xi

LIST OF APPENDICES xv

LIST OF ABBREVIATIONS xvi

ACKNOWLEDGEMENTS x i x

I. INTRODUCTION 1

1.Rationale 1

2. The Rat Adrenal Cortex 2

i) Morphology 2

ii) Development 4

iii) Steroidogenesis 6

iv) Steroidogenic Regulation 7

a)ACTH 7

b)Angll 8

C) The ras Oricogene 9

3. Rat Adrenocortical Cells in vitro 13

II. MATERIALS AND METHODS 1 9

1. Production of Rat Adrenocortical Cells 19

i) Animals 19

ii) Adrenocortical Fibroblast Migration from Explants 19

vi

iii)Collagenase Dissociation of Rat Adrenocortical Cells 20

2. Separation of Rat Adrenocortical Cell Types 21

i) Differerential Substratum Adhesion Separation 21

ii) Differential Morphological Response to cAMP 22

iii) Dissection of Adrenocortical Capsules from the Inner Portion of the Gland 22

iv) Discontinuous Percoll Density Gradient Separation 23

a) Gradient Preparation 23

b) Cell Separation and Density Estimation 24

3. Tissue Culture 24

4. Differentiation Markers: Histochemistry 26

i) Cytoplasmic Lipid 26

ii) A5,3r3-hydroxysteroid dehydrogenase (z\5,313-HSD) 26

iii) Catecholamines 27

5. Differentiation Markers: Immunofluorescence 27

i) Cytochrome P-450 side chain cleavage enzyme (P-45Oscc) 28

ii) Keratin 28

iii) Double Staining for Vimentin and P-45Oscc 29

iv) Factor VIII 30

6. Time Lapse Microscopy 30

7. Electron Microscopy 31

8. Steroidogenesis 31

i) Acid-ethanol fluorescence 31

ii) Radloimmunoassay (RIA) 32

9. Inositol Phosphate Determination 33

i) Analysis of inostol phosphates 33

ii) Analysis of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 34

vii

10. Free Intracellular Calcium ([Ca2]i) Determination 35

i) Culture of cells 35

ii) Fura-2 loading 36

iii) Fluorescence measurement 36

iv) Experimental Protocol 37

v) Calculation of [Ca2]i 38

11. Kirsten Murine Sarcoma Virus (KiMSV) Isolation, Infection and Oncogenic

Transformation 39

i) KiMSV isolation and titration 39

ii) KiMSV infection 40

iii) Selective passaging of morphologically altered cells 40

iv) Serum independence 41

v) Tumorigenicity 41

12. p21 ras Expression 42

i) Immunofluorescence 42

ii) Immunoprecipitation 42

13. Lovastatin Treatment of KGLOM cells 43

Ill. Results 44

1. Preliminary Attempts at Rat Adrenocortical Cell Separation 44

i) Whole Gland Dissociation of the Rat CorLex 44

ii) Differential Substratum Adhesion of Collagenase DissociatedAdrenocorLical Cells in Primary Culture 46

iii) cAMP-Induced Rounding and Detachment of Parenchymal Cells fromCollagenase Dissociated Adrenocortical Cells in Primary Culture 47

iv) Dissection of the Adrenal Cortex 50

a) Capsular Glands (containing zona glomerulosa) 50

b) De-capsulated Glands (containing zonae fasciculata and reticularis) 52

viii

2. Percoll Density Gradient Separation of Rat Adrenocortical Cells 52

i) Cell Separation into 7 Visible Bands 53

ii) Preliminary characterization of separated cells 56

3. Characterization of FASC (fasciculata) and GLOM (glomerulosa) Populations 61

i) Steroidogenesis 61

a) In Suspension 61

b) In Primary Culture 63

c) In Secondary Culture

ii) Primary Culture Morphology 66

iii) Phenotypic Markers in Primary Culture 70

a) cAMP Induced Retraction 70

b) Cytoplasmic Lipid 70

C) Ultrastructure 70

d) P-4SOscc Expression 71

e) Cytokeratin Expression 71

f) Vimentin Expression 72

g) Catecholamine Activity 80

4. Angiotensin Il-mediated Signal Transduction in GLOM and FASC Cells 82

i) Ang Il-Mediated Steroidogenesis 82

ii) Inositol Phosphate Accumulation 83

a) InsPi, lnsP2, lnsP3 83

b) lns(1,4,5)P3 83

iii) [Ca2]i in Cell Groups 88

iv) [Ca2]i in Single Cells 92

v) Calcium lonophore-Mediated Steroidogenesis 93

ix

5. ras Oncoprotein Induced Transformation and Differentiation of FASC and GLOM cells 99

i) KiMSV Infection and Selection of Morphologically Altered Cells 99

ii) Transformation Associated Parameters of KGLOM(KiMSV infected glomerulosa) Cells 101

a) Transformation in vitro 101

b)Tumourigenicity in vivo 102

iii) Steroidogenic Differentiation of KGLOM Cells 110

iv) Inhibition of p21 ras function in KGLOM Cells 113

6. Steroidogenic Differentiation of a Mixed Parenchymal-Stromal (MIX) Cell

Population in Culture 119

i) Characterization 119

ii) Primary Culture Morphology 120

iii) Steroidogenic Differentiation in Primary Culture 124

iv) Secondary Culture Morphology 130

v) Steroidogenic Differentiation in Secondary Culture 130

IV. DISCUSSION 136

1. Preliminary Attempts at Parenchymal Cell Separation 136

2. Percoll Density Gradient Parenchymal Cell Separation 138

3. in vitro Characterization of FASC and GLOM Cells 141

4. Ang Il-Mediated Signal Transduction in FASC and GLOM Cells 143

5. Expression of the ras Oncogene in GLOM Cells 150

6. Parenchymal Cell Proliferation and Differentiation in MIX Cultures 156

7. Conclusions 159

V. REFERENCES 162

x

LIST OF TABLES

Table 1: Characteristics of Rat Zona Glomerulosa and Zona Fasciculata Cells 1 8

Table 2: Density Gradient Separation 55

Table 3: Intermediate Filament Expression in FASC and GLOM Cells 79

Table 4: KGLOM Tumour Histopathology 1 08

xi

LIST OF FIGURES

Fig 1: Collagenase dissociated cells from whole adrenal cortices 45

Fig 2: Differential adhesion and cAMP treatment in primary culture 4 8

Fig 3: Factor VIII immunofluorescence of adrenocortical endothelial cell

colonies obtained by differential adhesion 4 9

Fig 4: Adrenocortical cells derived from decapsulated (a,c) or capsular (b,d)

glands 51

Fig 5: Representative experiment of separated cell bands obtained from the

percoll density gradient 54

Fig 6: t5,313-hydroxysteroid dehydrogenase (A5,313-HSD) staining of cells

derived from bands separated on percoll gradient 5 9

Fig 7: In vivo origin of FASC and GLOM cells 6 0

Fig 8: Steroidogenesis in freshly isolated cell FASC and GLOM suspensions 62

Fig 9: FASC and GLOM corticosterone production in primary culture 64

Fig 10: FASC and GLOM cell steroidogenesis before and after passaging in

culture 65

Fig 11: FASC and GLOM morphology in primary culture 6 7

Fig 12: Seeding efficiencies and growth in primary culture of FASC and GLOM

cells 68

Fig 13: cAMP mediated morphological response of FASC and GLOM cells in

primary culture 69

Fig 14: Lipid content of FASC and GLOM cells in primary culture 73

Fig 15: Transmission electron microscopy of FASC (a,c) and GLOM (b,d)cells

in primary culture 74

Fig 16: P-45Oscc expression of FASC and GLOM cells in primary culture 75

xii

Fig 17: Cytokeratin expression of FASC and GLOM cells in primary culture 76

Fig 18: P-45Oscc and vimentin expression in FASC and GLOM cells 77

Fig 19: P-45Oscc and vimentin expression in GLOM cells and rat lung

fibroblasts (RLF) 78

Fig 20: Comparison of morphology and catecholamine expression in adrenal

medullary cells (a,c,e) and FASC cells (b,d,f) in primary culture 8 1

Fig 21: Ang Il-mediated steroidogenesis in freshly isolated suspensions of

GLOM and FASC cells 84

Fig 22: Ang Il-mediated steroidogenesis of GLOM and FASC cells in primary

culture 85

Fig 23: Ang fl-mediated inositol phosphate production 8 6

Fig 24: Ang Il-mediated lns(1,4,5)P3 production 87

Fig 25: Ang Il-mediated changes in [Ca2]i in groups of FASC and GLOM cells

in primary culture 89

Fig 26: Examples of [Ca2]i changes in GLOM and FASC cell groups 9 0

Fig 27: Comparison of Ang II and Thapsigargin induced [Ca 2]i changes in cell

groups 91

Fig 28: Examples of [Ca2]i changes in individual GLOM and FASC cells 94

Fig 29: Ang Il-mediated [Ca2]i changes in individual GLOM cells in the

absence of extracellular calcium (A) and in the presence of

verapamil (B) 95

Fig 30: Frequency distribution of increases in [Ca2]i in single GLOM and

FASC cells after 1 OnM Ang II treatment 9 6

Fig 31: Ang Il-mediated [Ca2]i responses in an individual GLOM cell (A) and

a Yl mouse adrenal cortical tumour cell (B) 9 7

Fig 32: Calcium ionophore-mediated steroidogenesis of GLOM and FASC cells in

primary culture 98

xiii

Fig 33: Morphology of KiMSV infected GLOM cells 1 00

Fig 34: p21 ras expression in KGLOM cells (p2) 1 03

Fig 35: p21 ras expression in uninfected GLOM cells (p2) 1 04

Fig 36: p21 ras extraction in KGLOM cells (p5) 1 05

Fig 37: p21 ras immunoprecipitation in GLOM and KGLOM cells (p5) 1 06

Fig 38: Proliferation of GLOM and KGLOM cells (p5) 1 07

Fig 39: KGLOM tumour histopathology 1 09

Fig 40: P-45Oscc expression in KGLOM cells 111

Fig 41: Corticosterone production by GLOM and KGLOM cells (p2) 11 2

Fig 42: Corticosterone production in lovastatin treated early primary GLOM

cultures 11 5

Fig 43: Corticosterone production in lovastatin treated late primary GLOM

cultures 11 6

Fig 44: Corticosterone production in lovastatin treated KGLOM cultures 11 7

Fig 45: p21 localization in lovastatin treated KGLOM cells 11 8

Fig 46: Morphology of MIX adrencocortical cell population in primary

culture 121

Fig 47: Growth of FASC, GLOM and MIX cells in primary culture 1 22

Fig 48: Time lapse microscopy of proliferating MIX cells in primary

culture 123

Fig 49: Corticosterone production by FASC, GLOM and MIX cells in primary

culture 126

Fig 50: Fluorogenic steroid production by FASC, GLOM and MIX cells in

primary culture 1 27

Fig 51: A5,313-Hydroxysteroid dehydrogenase (A5,3I3-HSD) activity of FASC,

GLOM and MIX cells in primary culture 1 28

xiv

Fig 52: Differentiation marker expression of multilayered MIX primary

cultures in situ 1 29

Fig 53: Morphology of MIX adrencocortical cell population in secondary

culture 132

Fig 54: P-45Oscc expression in MIX (p1) cells 133

Fig 55: Response to cAMP and P-45Oscc expression in heterogeneous MIX

(p1) cultures 134

Fig 56: Steroidogenesis of passage 2 GLOM and MIX cultures 1 35

xv

LIST OF APPENDICES

Appendix A: Percoll Gradient Preparation 1 77

Appendix B: Ins(1,4,5)P3 Determination 179

Appendix C: p21 ras Immunoprecipitation 1 82

xvi

LIST OF ABBREVIATIONS

CTh adrenocorticotropic hormone

Aldo aldosterone

AMCA 7-amino-4 methylcoumarin-3-acetic acid

Ang II angiotensin II

CAMP 3’-5’ cyclic adenosine monophosphate

FE cAMP response element

cs corticosterone

DG diacylglcerol

DHEA dehydroepiandrostenedione

DMEM/F12 Dulbecco’s modified essential medium/Ham’s F12 medium (1:1)

DMSO dimethylsulfoxide

EOM extracellular matrix

epidermal growth factor

RTA ethylenebis(oxyethelenenitrilo)-tetraacetic acid

FASC percoll separated cells derived from the rat zona fasciculata

FBS fetal bovine serum

GTPase activating protein

GLY.4 percoll separated cells derived from the rat zona glomerulosa

GscL G protein a-subunit that stimulates adenylate cyclase

GTP guanosine trisphosphate

HBSS Hanks’ balanced salt solution

HEPES N-2-hydroxyethylpiperazine-N’-2-ehanesulfonic acid

HMG CoA reductase 3-hydroxy-3-methytglutaryl-co-enzyme A

HS horse serum

IGF-1 insulin-like growth factor-I

xvii

lgG immunoglobulin

I ns( 1 ,4,5) P3 inositol (1 ,4,5)-trisphosphate

lnsP1 inositol-monophosphate

lnsP2 inositol-bisphosphate

lnsP3 inositol-trisphosphate

IP ionophore

KGLOM KiMSV infected/transformed GLOM cells

KiMSV Kirsten murine sarcoma virus

KNRK K1MSV infected/transformed NRK cells

MIX percoll separated cells containing a mixture of parenchymal,

stromal and endothelial cells

NAD nicotinamide adenine dinucleotide

NE-i neurofibromatosis gene #1

I%12F nerve growth factor

NRK normal rat kidney cells

P-450C2 1 cytochrome p-450 C21 -hydroxylase

P-45O cytochrome P-450 side chain cleavage enzyme

P-4501 1 B cytochrome P-450 11f3-hydroxylase

45017a cytochrome P-450 l7cx-hydroxylase

PAS protein A sepharose

PBS phosphate buffered saline

PC-i 2 adrenomedullary tumour cell line

PDGF platelet derived growth factor

PKC protein kinase C

PLC phosphoilpase C

p(n) passage number in pre-senescent culture

RIA radioimmunoassay

RIPA Buffer radiolabeled immunoprecipitation assay buffer

SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis

TCA trichioroacetic acid

TPA 1 2-O-tetradecanoylphorbol-13-acetate (phorbol ester)

TRE TPA (PKC) response element

Yl adrenocortical tumour cell line

A5,3 13-H SD A5,313-hydroxysteroid dehydrogenase

FITC fluoroscein isothiocyanate

xviii

xix

ACKNOWLEDGEMENTS

I would like to thank the members of my research committee for their expert guidance and

assistance throughout the lifespan of this project. These include Drs. Nelly Auersperg,

Bruce Crawford, Keith Humphries and Peter Leung. Also, the help of Dr. Ken

Baimbridge was instrumental in carrying out the calcium studies. Special thanks go to

Nelly for her wisdom, enthusiasm and friendship from start to finish. Nelly, I’ve never once

regretted sending you that pleuripotent letter, not for a minute.

The faculty, staff and graduate students of the UBC department of Anatomy were a great

reservoir of help and support. It was an exciting and stimulating place to be, and I will look

back with great fondness on my days and nights spent in the Freidman building.

Specifically, I would like to express gratitude to department head Dr. Charles Slonecker

and student advisors Drs. Bill Ovalle and Joanne Emmerman. In addition, Drs. Wayne yogI

and Carl Friz have made sure I will always remain a gross anatomist in structure, if not

function. The technical assistance of Judith Black, Carolyn Burr and Sarah Maines was

greatly appreciated. Also, both Dr. Jie Pan and Mr. lan MacLaren were experts with ideas

as well as experiments.

Heartfelt thanks go to my family, especially my Mom and Dad. They made things possible

in a million different ways, from Jimmy Cricket right through graduate school. And finally,

to Christine Williams, whose patience and understanding were never ending. Chris, I’ll

never be able to repay you completely, but I’ve got a lifetime to try. Maybe if I could find

you Zuzu’s petals, or Allie’s mit, that just might do it.

—1—

I. INTRODUCTION

1. Rationale

Vertebrate development is continuously driven by three processes: growth,

differentiation and morphogenesis (Steinberg, 1963>. Utilizing arbitrary, often

terminal, endpoints these processes have been successfully studied in a number of cell

lineages hi However, there are few well defined models available for the detailed

examination of the ways in which these processes act to alter celluar phenotypes at

specific stages within a particular developmental lineage.

In the rat, the ‘adrenocyte’ develops along a spatial and temporal continuum as it

leaves the region of the capsule and migrates centripitally through the adrenal cortex

(Gottschau, 1883; lannacone and Weinberg, 1987). Therefore, cells at different stages

within this developmental lineage are found within separate anatomical compartments of

the endocrine gland. In the present study, 2 homogeneous adrenocortical cell populations

from different anatomical compartments were isolated and maintained in culture. The

proliferative potential, steroidogen ic differentiation, and morphologic characteristics of

the two parenchymal cell types were then compared to each other in an effort to

determine how, and by what means, subtle differences in developmental staging affect the

cellular phenotype. For example, as an adrenocyte moves inward from the zona

glomerulosa to the zona fasciculata it no longer responds to the trophic hormone

angiotensin II (Ang II; Haning et al., 1970; Tait et al., 1974; Douglas et al., 1978;

Braley et al., 1986; Whitley et al., 1987). Utilizing the 2 isolated populations, which

are derived from these zones, an attempt was made to define the molecular mechanism

responsible for this developmentally driven alteration in the differentiation state.

With increasing time in primary culture, particularly in cells derived from the

rat adrenal cortex, overt steroidogenic function is lost (O’Hare and Neville, 1 973a;

-2-

Ryback and Ramachandran, 1981a; Payet et al.,1984). Efforts were made here to

prolong this tissue-specific differentiative state in both primary and secondary culture.

In one situation, expression of the ras oncogene, which also transforms the cells to

tumorigenicity, was used. In another, steroidogenic differentiation in mixed

parenchymal/stromal cultures was assayed. Interestingly, in each case, the phenotypes

observed appeared to be associated with specific stages of adrenocortical development.

2. The Rat Adrenal Cortex

i Morphology

The adrenal gland is located retroperitoneally in a fat pad just superior to the

kidney. This small endocrine gland consists of two parts, the mesodermally derived

cortex and the medulla, which is derived from neural crest (Nussdorfer, 1986). The

cortex is a major producer of steroid hormones, while the medulla secretes

catecholamines. The adrenal is highly vascularized, receiving branches from the

inferior phrenic, celiac and renal arteries. After passing through the connective tissue

capsule of the gland these branches give rise to a subcapsular plexus of sinusoidal

capillaries that sends longtitudinal branches plunging through the cortex. The latter

empty into venous sinusoids within the medulla. Thus the blood supply flows from the

outside inward.

As is the case in most mammalian species, the parenchymal cells of the rat

adrenal cortex are arranged into 3 morphologically distinct zonae, the glomerulosa,

fasciculata and reticularis (ldleman, 1970).

The outer zona glomerulosa occupies about 10-15% of the gland volume

(Nickerson, 1976; Mazzocchi et al., 1977). The parenchymal cells are small (600-

-3-

700 jim3), have a high nuclear to cytoplasmic ratio and they are grouped into spherical

clusters that are in intimate contact with the subcapsular capillary plexus. Glomerulosa

cells contain an abundant smooth endoplasmic reticulum (SER), well developed Golgi

apparatus, elongated mitochondria with tubular or tubulovescicular cristae, and small

lipid droplets that occupy about 5% of the cytoplasmic volume. The rat zona glomerulosa

is the principal source of the mineralocorticoid aldosterone.

The broad, centrally located zona fasciculata contains large cells (1300-2000

jim3) that are arranged in radial columns around the longtitudinal capillary network

(Nussdorfer et aL, 1974). These columns are approximately 30-40 cells long and often

only 2-3 cells wide. Fasciculata cells contain an abundant SER, well developed Golgi,

many round or oval mitochondria of varying size with tubulovescicular or vescicular

christae, and numerous lipid vesicles that occupy up to 25% of the cytoplasmic volume.

The rat zona fasciculata is the principal source of the glucocorticoid corticosterone.

The zona reticularis arises gradually from the fasciculata and ends abruptly at

the adrenal medulla. The cells are intermediate in size (800-1350 jim3) and are

arranged in a haphazard array (Malendowicz, 1974; Conran and Nickerson, 1979).

Reticularis cells contain a very abundant SER that occupies up to 50% of the cytoplasmic

volume, a small and sparse Golgi apparatus, round or ovoid mitochondria with vescicular

cristae, and lipid vesicles that occupy about 10% of the cytoplasmic volume. Throughout

this zone a small percentage of cells are constantly deleted by a process that involves an

initial separation from neighbours, followed by severe nuclear/cytoplasmic

condensation, and finally ingestion by perisinusoidal histiocytes (Wyllie et al., 1973).

Functionally, it is difficult to separate the rat reticularis from the fasciculata as both

zones produce corticosterone. In some mammalian species the reticularis produces

adrenal androgens such as androstenedione, although this activity is limited in the rat

(Bell et al., 1978).

-4-

ii) Development

At about mid-gestation in the developing rat embryo the cells of the ceolomic

epithelium located between the gastric mesentery and the urogenital ridge undergo a

wave of proliferation and begin to migrate into the underlying stroma just superior to

the developing mesonephros (Idleman, 1970). These mesodermal cells condense to form

the adrenocortical primordia. At day 15 post-conception (PC), there is the evidence of

steroidogenic differentiation as inner and outer cortical zones begin to develop (Daikoku

et al., 1976). At day 17 PC, the outer adrenocortical zone begins to morphologically

resemble the zona glomerulosa, consisting of small cells arranged in clusters that

contain numerous mitoses. The inner zone consists of irregularly grouped cords of large

polygonal cells whose mitochondria have tubulovescicular christae. This is the early

zonae fasciculata/reticularis (Nussdorfer 1986). Also at day 17 PC, a connective tissue

capsule is discernable, the cells of which have steroidogenic characteristics as they

express the steroidogenic enzyme z5,3l3-hydroxysteroid dehydrogenase (A5,313-HSD)

(Farcnik and Auersperg, 1984). It is only at this point, day 17 or 18 PC, that the

pituitary begins to secrete ACTH and true zonation begins in earnest (Jost, 1975). At

birth (Day 22-23 PC), all three adrenocortical zones are present although the zona

reticularis is scanty and intermingled with the neual crest-derived adrenal medulla.

There is no fetal adrenal cortex in the rat as there is in a number of other mammalian

species, including human. Three days after birth the steroidogenic properties of the

connective tissue capsule begin to diminish. Six weeks after birth, when the three zonae

are fully formed and functional, the capsule is no longer steroidogenic.

Scanning electron microscopy suggests that the parenchymal cells of the adrenal

cortex are arranged as a tunneled continuum within and across each of the zones (Motta

et al., 1979). In chimeric rats, individual clones form radial patterns that span the

entire cortex, from the connective tissue capsule to the medulla (lannacone and

-5-

Weinberg, 1987). Thus, parenchymal cells from all three zones must have a common

cellular origin. Most mitoses occur in the outer regions of the cortex and 1 hour after

3H thymidine injection 95% of the labelled cells are found in the subcacapsular stroma

or the zona glomerulosa (Wright and Voncina, 1977; Zajicek et al., 1986). These

labelled cells move slowly inward at the rate of approximately 0.25 cell diameters per

day until they reach the inner region of the zona reticularis where they degenerate. This

directed movement may be driven by the cytogenesis taking place in the outer portion of

the gland, and the cytolysis taking place in the inner portion. Taken together these data

indicate that the Gottschau’s archetypical cell migration hypothesis (1883) accurately

depicts adrenocortical cytogenesis. This theory was deduced purely from an examination

of adrenocortical histology. It states that the adrenocortical zonae represent 3 separate

but related states of the “adrenocyte”, and that each of these states is expressed

transiently as a cell moves inward from a subcapsular position towards the medulla. In

addition to the observational evidence cited above, this hypothesis has also been

supported by experiment. For example, after an enucleation procedure that removes the

whole cortex except for a thin layer of sub-capsular cells, a functional three-zoned

gland regenerates after an initial period of proliferation followed by differentiation

(Ingle and Higgins, 1938; Taki and Nickerson, 1985).

Therefore, it is very likely that cells of the zona glomerulosa give rise to zona

fasciculata cells. There may even be a spatial and developmental midpoint between these

two cell types within the zona intermedia. The zona intermedia is a very thin layer of 3-

5 layers of small cells located between the glomerulosa and the fasciculata. The cells

have mitochondria similar to glomerulosa cells and SER similar to fasciculata cells

(Nickerson, 1976).

-6-

iii) Steroidogenesis (Waterman and Simpson, 1985; Simpson and Waterman, 1988)

All steroids originate with a single precursor, cholesterol. This is either

synthesized de novo within adrenal cells from acetate or it is derived from the plasma

via lipoproteins (Hall, 1984). Cholesterol, in either a free or esterified form, is often

stored in adrenocortical lipid inclusions. All steroidogenic pathways are initiated by the

side chain cleavage reaction that takes place in the inner mitochondrial membrane and

yields pregnenelone by cleaving a 6 carbon aliphatic tail from the cholesterol molecule.

This reaction is catalyzed by the mixed function oxidase, cholesterol side chain cleavage

cytochrome P-450 (P-4SOscc), which obtains its reduction equivalents from NADPH

via a flavoprotein (adrenodoxin reductase) and an iron-sulfur protein (adrenodoxin).

The major steroidogenic pathways in the rat adrenal cortex are as follows.

Pregenelone leaves the mitochondria and travels to the SER where it is converted to

progesterone by the combination dehydrogenase/isomerase iX5,3 13-hydroxysteroid

dehydrogenase (A5,3f3-HSD). Progesterone remains in the SER and is hydroxylated at

position 21 by a C21 hydroxylase (P-450C21) to produce deoxycorticosterone. This

weak glucocorticoid then moves to the mitochondria where it is converted into the

powerful glucocorticoid, corticosterone, by a second hydroxylase that acts at the 1113

postition (P-450i 1 ). Corticosterone production occurs in all 3 zones of the cortex but

production is highest in the fasciculata. Aldosterone, the major endpoint

mineralocorticoid is then produced in the mitochondria of glomerulosa cells by an 18-

hydroxylation of corticosterone. This reaction may also be catalyzed by P-450i 1 13.

Corticosterone is the major endpoint glucocorticoid in this species. Cortisol is

not produced due to the low activity of17 hydroxylase (P-45O17)in the rat adrenal

cortex. This decreased activity also limits the production of adrenal androgens.

When all of the above steroidogenic enzymes are introduced into a non-descript

fibroblast line by multiple transfection, and are expressed constituitively under the

-7

influence of strong heterologous promoters, the cells produce endpoint steroids (Mathew

et al. 1990). When cholesterol is the substrate, steroid production is limited as the

cholesterol is not efficiently transported to the mitochondria and P-45Oscc. In contrast,

when pregnenelone is the substrate steroid, production is greatly increased. These

findings indicate that tissue specific adrenocortical function is dependent upon

steroiciogenic gene expression and cholesterol transport into the mitochondria. Other

aspects of steroid synthesis, such as the movement of steroid intermediates between the

mitochondria and the SER, are not tissue specific as they occurred independently in the

fibroblast line.

iv) Steroidogenic Regulation

Trophic hormones which regulate steroidogenic differentiation act at the cell

surface, thereby activating receptor specific signal transduction pathways. The ras

oncogene, which also appears to regulate steroidogenic differentiation, likely bypasses

the cell surface receptor and impinges directly upon signal pathways.

a ACTH

One of the major regulators of overall adrenal steroidogenesis is the pituitary

hormone ACTH (Simpson et al., 1990). This peptide binds to a cell surface receptor that

activates adenylate cyclase via the cholera toxin sensitive stimulatory G protein (Gs).

This results in the production of the second messenger cAMP and the subequent activation

of the cAMP dependent protein kinase A. There are two temporally separable

steroidogenic responses to this signal pathway activation. The acute response occurs

rapidly within minutes and involves the transport of cholesterol from cytoplasmic lipid

inclusions to the mitochondria (Jefcoate et al., 1987). This transport is dependent on

-8-

adrenal-specific cholesterol carrier protein(s) present in the cytoplasm. The movement

of cholesterol into the mitochondria brings it into the vicinity of P-45Oscc and thereby

leads to the production of pregenelone and the subsequent production of endpoint steroids.

Chronically, over a period of hours or days, ACTH-mediated cAMP production

leads to increased expression of steroidogenic enzymes. This was first observed in

hyphosectomized rats where the levels of steroid hydroxylases decreased unless they

were restored by the administration of exogenous ACTH (Levy et al., 1959; Purvis et

al., 1973). ACTH stimulation of steroid gene expression occurs principally at the

transcriptional level. The genes for steroid hydroxylases contain a variety of cAMP

response elements (CRE) in their 5’ regulatory regions. These CREs bind adrenal

specific transcription factors in response to elevated cAMP. This process is crucial for

increased transcription (Simpson et al., 1990). cAMP-mediated upregulation of gene

expression is also enhanced, and over the longterm may be dependent upon, activation of

the insulin-like growth factor-I (IGF-1) receptor, which is a tyrosine kinase

(Hornsby et al., 1985; Veldhuis et aI., 1986; Naseerudin and Hornsby, 1990).

b) AnQ II

Another major adrenocortical secretagogue is angiotensin II (Ang II). In the rat,

this octapeptide acts principally to increase aldosterone production in the zona

glomerulosa (Haning et al., 1970; Tait et al., 1974; Douglas et al., 1978; Braley et aL,

1986; Whitley et al.; 1987). Ang II binds to a cell surface receptor and activates a G

protein complex (Barrett et al., 1989). This stimulates phosphatidyl inositol specific

phospholipase C (PLC), thereby producing the second messengers diacylglycerol (DAG)

and inositol 1,4,5-trisphosphate (lns(1,4,5)P3). A rapid lns(1,4,5)P3-mediated

intracellular calcium release is critical in initiating the steroidogenic response (Kojima

et al., 1984), while a subsequent influx of calcium across the plasma membrane along

-9-

with the DAG-mediated stimulation of protein kinase C (PKC) are important in

maintaining this response (Kojima et al., 1985; Alkon and Rasmussen, 1988).

Therefore, when this signal transduction pathway is activated, an increase in

intracellular calcium ([Ca2+]i) is required for both the acute and chronic steroidogenic

responses. In addition, the activation of PKC is needed for the maintenance of the chronic

response. It is not yet clear if this kinase always acts directly to upregulate steroid

enzyme gene expression, or if it impinges upon the cAMP pathway via signal pathway

crosstalk (Brami et al., 1987; Woodcock, 1989). For example, while only the P

45Oscc gene has been shown to contain a classical PKC responsive element (TRE),

transcription of the P-450c21 and P-450i 1 B genes are altered after PKC stimulation

by 12-O-tetradecanolyphorbol-13-acetate (TPA; Deutsch et at., 1988; Chang et al.,

1991).

C) The ras Oncoçiene

Expression of the ras oncogene appears to effect steroidogenic differentiation

positively (Auersperg et al., 1981,1990; Amsterdam et at., 1988). There are 3

mammalian ras genes, H, K and N-ras. They are highly homologous and encode for

proteins of approximately 180 amino acids that have a molecular weight of 21 kD (p21

ras; Barbacid, 1987). Like G protein a subunits, p21 ras proteins bind GTP, and have

intrinsic GTPase activity (Shih et al., 1980, Willingham et al., 1983; McGrath et at.,

1984). They also cycle between the cytoplasm and inner face of the plasma membrane.

The biologically active form of p21 ras molecules are bound to GTP and associated with

the plasma membrane (Trahey and McCormick, 1987). Retroviral ras oncogenes have

point mutations at amino acid #12 which is located in one of the domains involved in GTP

binding. The result of this mutation is decreased GTPase activity. It is this characteristic

that most often correlates with the gene’s transforming ability. (Gibbs et a)., 1984).

- 10 -

Similar dominant, activating mutations have been found in a wide variety of human

tumours (Barbacid, 1987). A second point mutation is found in the v-Kras gene of

Kirsten Murine Sarcoma Virus (KiMSV). This mutation is at amino acid #59 and allows

for phosphorylation of the molecule (Shih et al., 1982). All of this information suggests

that p21 ras molecules are involved in signal transduction and that the viral forms may

act constitutively because of their reduced ability to hydrolyze GTP (Macara, 1991).

Unlike the situation in yeast, ras proteins do not act directly as G proteins for

adenylate cyclase in mammalian cells (Toda et al., 1984; Beckner et al., 1985).

However, adenylate cyclase activity is altered in ras-transformed mammalian cell lines.

The nature of these alterations is highly variable depending on the cell type. In a number

of fibroblast lines, ras-transformation reduces basal and agonist stimulated adenylate

cyclase activities (Beckner, 1984; Saltorelli et al., 1985; Levitski et al., 1986).

However, the physiologic state of the cell can alter the nature of the ras effect. For

example, in serum deprived fibroblasts, activated ras expression increases cAMP

production via inactivation of the inhibitory G protein (Franks et al., 1987). Activated

p21 ras expression can also have variable effects on c-AMP production in differentiated

cells. For example, in one thyroid epithelial cell line ras transformation decreases

adenylate cyclase activity (Colletta et al., 1988), while in another line cAMP production

is increased (Spina et al., 1987).

ras p21 also impinges upon other signalling pathways, including those initiated

by PLC. In a number of fibroblast lines, basal and agonist stimulated levels of lnsP3 and

DAG are increased after ras transformation (Chiarugi et al., 1985; Fleischman et al.,

1986; Hancock et al., 1988). Y13-259, a neutralizing ras antibody, is able to block

this increase, suggesting that it is p21 ras specific rather than associated with

tranformation in general (Chiarugi et al., 1986). As with adenylate cyclase, the effects

of ras p21 on PLC activity are not straightforward. Wakelam and co-workers found that

ras-transformation increases agonist stimulated PLC activity in sub-confluent, but not

— 11

confluent, 3T3 cultures (Wakelam et al., 1986; Wakelam, 1988). They suggest the

coupling of p21 ras to PLC may be reduced by the increased accumulation of autocrine

growth factors that occurs when the cultures are crowded. In certain situations,

activated ras expression increases DAG production with no change in lnsP3 levels,

suggesting an effect upon PLC subtypes with specificities for substrates other than

phosphatidyl inositol, perhaps phosphatidyl ethanolamine or phosphatidyl choline (Lacal

et al., 1987a; Wolfman and Macara, 1987; Morris et al., 1989). There is also evidence

that p21 functions both proximally and distally to PLC activation (Yu et al., 1988;

Smith et al., 1990). Regardless, stimulation of this pathway leads to the activation of

PKC which has been implicated in a number of cellular activities including mitogenesis,

determination, differentiation, secretion and cytoskeletal re-arrangements (N ishizuka,

1988). Functional PKC is required for ras-mediated transformation (Lacal et al.,

1 987b), and fibroblast lines that overexpress PKC have an increased susceptibility to

that transformation (Hsio et al., 1989). PKC also influences G protein activity, thereby

stimulating or inhibiting receptor mediated adenylate cyclase, depending on the cell type

(Katada et al., 1985; Zick et al., 1986; Rozengurt et al., 1987). Thus, ras-mediated

activation of PKC could lead to altered cAMP levels via signal pathway crosstalk. This

might help explain many of the ras associated effects on adenylate cyclase described

above.

In certain situations, tyrosine kinases and PLC are linked in normal cells. For

example, the platelet derived growth factor (PDGF) and epidermal growth factor (EGF)

receptors are both transmembrane tyrosine kinases that phosphorylate and stimulate

PLC after binding their respective ligands (Meisenhelder et al., 1989; Nishibe, 1990).

Interestingly, microinjection of neutralizing antibodies to either ras p21 or PLC

prevents tyrosine kinase induced mitogenesis (Smith et al., 1986,1990). This suggests

that ras p21 may act to link tyrosine kinases to PLC. The interaction between p21 and

receptor tyrosine kinases may occur indirectly via the GTPase activating protein (GAP).

- 12 -

GAP associates with the GTP bound form of p21, and in so doing regulates ras function by

increasing GTPase activity more than 100 fold (Trahey and McCormick, 1987). GAP

also physically associates with, and may regulate the function of, tyrosine kinase growth

factor receptors (Molloy et al., 1989; Anderson et al., 1990; Ellis et al., 1990;

Kazlauskas et al., 1990). It appears to do so via a regulatory domain known as SH2,

which is found in a number of other proteins that act within signal transduction

pathways (Heldin, 1991; Koch et al., 1991). Thus, GAP could act as a lateral effector

within the signal transduction network, regulating both the signal and its transduction.

There are actually a number of proteins with GAP like activity. These include alternately

spliced forms and genic homologues such as NF-1, which is mutated in the disease

neurofibromatosis (Bollag and McCormick et al., 1991). Also, there is a group of

guanine nucleotide exchange factors that associate with ras p21 (Downward et al., 1990;

Wolfman and Macara, 1990), as well as an inhibitor of GTPase activity (GIP, Tsai et al.,

1990). Consequently, the ability of ras to act upon numerous transduction pathways

may be enhanced by the multiplicity of proteins that regulate its biochemical state. How

these regulatory proteins are affected by the oncogenic activation of p21 ras, which

eliminates its GTP/GDP switching, may be important. For example, the association

between GAP and activated p21/GTP is long lived, presumably because GTPase activity

cannot be increased (Macara 1991). Thus, activated ras could act as a sink for GAP,

which would diminish the regulatory interactions between GAP and receptor tyrosine

kinases.

Most investigators have used proliferation or oncogenic transformation as

endpoints when studying ras function. However, ras also has profound effects on

differentiation. These effects are not always negative. For example, v-ras enhances

tissue specific differentiation in adrenomedullary tumour cells (PC12), thyroid

medullary carcinoma cells, and small cell lung carcinoma cells (Noda et al., 1985;

Nakagawa et al., 1987; Mabry et al., 1988). In the case of PC12 cells, this

- 13 -

differentiative effect occurs via a PLC stimulation that is normally initiated by nerve

growth factor (Sassone-Corsi et al., 1989; Kremer et al., 1991). Activated ras can also

enhance differentiation in some non-tumour cell types. For example activated ras

expression induces adipocyte differentiation in pre-adipocytes by activating the signal

pathway that is normally initiated by insulin, whose receptor is a tyrosine kinase

(Benito et al., 1991). These tissue specific effects suggest that ras p21 can act upon

differentiation associated signal transduction pathway templatesH that are constructed

during the process of normal development. If this is indeed the case, ras expression

should induce a common set of changes in cells with a similar differentiated phenotype

and developmental history . This appears to be the case with steroidogenic cells of the

rat.

in 3jy, cellular ras is often expressed in differentiated, non-proliferative

tissues. For example, ovarian and adrenocortical cells contain c-ras p21 (Chesa et al.,

1987; Furth et al., 1987). nyj.trn, expression of oncogenic ras is associated with

steroid production in immortalized ovarian granulosa cells (Amsterdam et al., 1988;

Suh and Amsterdam, 1990) and in normally non-steroidogenic ovarian surface

epithelial cells (Pan et al., 1991). Oncogenic ras also enhances the steroidogenic

characteristics of explant-derived adrenocortical fibroblasts (Auersperg et al., 1981,

1990; Wiebe et al., 1987). Presumably, the ras protein elicits these effects by acting

within those signal transduction pathways that are associated with normal steroidogenic

differentiation.

3. Rat Adrenocortical Cells in vitro

Adrenocortical explants produce large numbers of outwardly migrating

fibroblasts. In FBS supplemented medium these cells are myofibroblastic in growth

pattern, ultrastructure and extracellular matrix production (Slavinski et al., 1 974,

- 14 -

1976; Slavinski-Turley and Auersperg, 1978; Turley, 1980). They do however, have a

very limited steroidogenic potential that is increased after ACTH treatment. When they

are maintained in HS-supplemented medium these cells modulate to a more epithelial

form and acquire parenchymal characteristics which include: the accumulation of

cytoplasmic lipid; expression of the steroidogenic enzyme A5,3f3-HSD; and increased

steroid production both in the absence and presence of ACTH. Thus, these adrenal

fibroblasts have a dual stromal/parenchymal potential. These cells likely originate from

the connective tissue capsule of the gland (Bressler, 1973; Ryback and Ramachandran,

1981a). capsular fibroblasts express 5,36-HSD during fetal development but

not in the adult animal, suggesting that capsular cells have the potential to express

parenchymal characteristics early in development but that this potential is suppressed

after birth (Farcnik and Auersperg, 1984). Therefore, the expression of parenchymal

characteristics by adrenal fibroblasts in culture may actually be representative of an

early developmental state of the cells.

The ras oncogene is able to transform explant derived adrenocortical fibroblasts

(Auersperg et al., 1977; Auersperg 1978). These transformed cells grow to high

saturation densities, are serum and anchorage independent, and form locally invasive

tumours in immunosuppressed rats. Depending on the particular transformed line, these

tumours are either sarcomas or carcinomas, or sometimes show elements of both

(Auersperg et al., 1981). This ability to express two tumour phenotypes may reflect

the bipotential nature of the target cells prior to transformation. In culture, the ras

transformed fibroblasts contain lipid, constitutively convert pregnenelone to

progesterone (E5,3f3-HSD activity), and express a number of other steroidogenic

enzymes (Auersperg et al., 1981, 1990; Wiebe et al.1 987). These cells also respond

morphologically to treatment with cAMP in a manner similar to that observed in a

number of steroidogenic cell types. In addition, the production and elaboration of an

extracellular matrix is reduced (Auersperg et al., 1990). Thus, in adrenocortical

- 15 -

fibroblasts, ras oncogene expression is associated with the enhancement of parenchymal

characteristics, and the reduction of stromal ones. As has been noted, this phenotype may

be representative of an early developmental state of these cells.

During development, individual clones move from the adrenal capsule inward

across the entire cortex until they degenerate in the zona reticularis near the medulla

(I. 2. ii). This suggests that as an ‘adrenocyte’ moves inward, its stromal potential

becomes gradually restricted as its parenchymal potential becomes fully expressed.

Therefore, unlike explant-derived fibroblasts, there may be a population of

adrenocortical cells with the potential to exhibit the full complement of stromal and

parenchymal characteristics at the same time. Presumably these cells would be located

closer to the capsule than the medulla. A good candidate would be a population of cells

derived from the sub-capsular zona glomerulosa.

The zona glomerulosa can be separated from the zonae fasciculata/reticularis by

dissection (for summary of cellular characteristics, see Table 1). This involves

removing the capsule with the adherent glomerulosa from the rest of the cortex (Haning

et al., 1970). This is the most common method for separating adrenocortical zones in the

rat. Single cell suspensions can then be obtained by subjecting the separated tissues to

collagenase digestion. Ninety-five percent of zona fasciculata/reticularis cells obtained

by this method contained numerous lipid droplets. In primary culture they produce

corticosterone and responded strongly to ACTH stimulation, both by increasing their

steroid production and retracting from the substratum (O’Hare and Neville, 1973a,b;

Ramachandran and Suyama, 1975; Ryback and Ramachandran,1981a). Nevertheless, the

steroidogenic differentiation of these cells decreases rapidly with increasing time in

culture (Ryback and Ramachandran, 1981b). Initially, they form confluent epithelial

monolayers that exhibit very little growth (Neville and O’Hare, 1973b). However, after

1-2 weeks fibroblasts begin to proliferate which eventually overgrow the entire

culture. Addition of the fibroblastic inhibitor d-valine (Ryback and Ramachandran,

- 16 -

198Th), or addition of a mixture of HS and FBS on a fibronectin substratum reduces the

fibroblast growth in these cultures (McAllister and Hornsby, 1987), but they do not

eliminate the decrease in steroidogenic differentiation. Therefore, decapsulation

provides a population of fasciculata/reticularis cells that are well suited to short term

examinations of parenchymal growth and differentiation. For long term study, at least in

the rat, this method may not be so useful. In bovine adrenocortical cells steroidogenic

differentiation can be maintained for much longer periods (Hornsby et al., 1980, 1987,

Hornsby, 1991).

Collagenase dissociation of adrenocortical capsules produces a population of cells

the majority of which are small and contain scanty amounts of cytoplasmic lipid (Haning

et al., 1970). These cells are also steroidogenic and respond to ACTH with increased

corticosterone and aldosterone production. Unlike in rat fasciculatata/reticularis

populations these cells also respond to Ang II (Barrett et al., 1989). In culture, the

cells spread, flatten and proliferate rapidly (Hornsby et al., 1974; Ryback and

Ramachanciran, 1981a; McAllister and Hornsby, 1987). These authors refer to the

proliferating cells in capsular preparations as adrenal “fibroblasts”. Prior to the

present study it had not been determined whether these cells are true stromal cells, such

as those that migrate from adrenocortical explants (Slavinski et al., 1974, 1976), or if

they are actually modulating parenchymal cells derived from the definitive zona

glomerulosa.

In this study, morphologically homogeneous parenchymal cell populations devoid

of stromal or endothelial cell contaminants have been isolated by density gradient

centrifugation. In particular, cellular sub-populations derived from the zona

glomerulosa (GLOM) and zona fasciculata (FASC) were used to determine whether

stromal and parenchymal characteristics could be expressed simultaneously in culture.

These cells were also used to carry out an unambiguous investigation of the mechanism

responsible for differential zonal responses to Ang II. In addition, these density isolated

- 17

sub-populations were used in mass transformation experiments with the ras oncogene,

as they ensured an unambiguous pool of parenchymally derived target cells.

- 18 -

TABLE 1

Characteristics of Rat Zona Glomerulosa and Zona Fasciculata Cells

Characteristic Glomerulosa Fasciculata

position in gland subcapsular central

cell size 7-12 urn 14-24 urn

lipid content scanty abundant

cell density ‘1.O7 g/ml -1.O4 g/rnl

major steroid aldosterone corticosterone

ACTH response + + + + + +

Ang II response ++++ -

- 19 -

II. MATERIALS AND METHODS

1. Production of Rat Adrenocortical Cells.

i) Animals

Throughout these experiments 8-16 wk. old male Fischer rats (housebred,

strain 344), were used to obtain adrenocortical cells for experimentation. Males were

chosen to eliminate steroidogenic fluctuations that occur during the female eostrous

cycle. Animals were sacrificed at approximately the same time each day (10 am. to 12

noon) to minimize daily fluctuations in steroid output. At no time were the animals kept

on a high or low salt diet as such changes can also effect steroid production, particularly

of mineralocorticoids (Taki and Nickerson, 1985). The animals were kept on a l2hr

light dark cycle and fed a diet of purina rat chow and water without restrictions.

ii) Adrenocortical Fibroblast Migration From Explants (Slavinskl et al., 1974, 1976;

Slavinski-Turley and Auersperg, 1978)

Animals were lightly anaesthetized with either chloroform or halothane (MTC

Pharmaceuticals, Caimbridge Ont.) and killed by cervical dislocation. Adrenal glands

were then removed, trimmed of adherent fat, minced into pieces of approximately 1

mm3 and placed in 35mm tissue culture dishes (1 gland per dish) whose bottoms had

been scratched to provide an adherent surface. In certain experiments, adrenocortical

capsules and de-capsulated glands were minced separately . The minced tissue was left

- 20 -

for 5-10 mm and then tissue culture medium was added, consisting of Waymouth’s 752

(GIBCO) supplemented with 25% Fetal Bovine Serum (FBS, Hyclone).

The cultures were incubated in a humidified atmosphere containing 5%C02 at

37°C. They were left undisturbed for 3-5 days at which time approximately 40-60% of

the explants had adhered to the the dish. The culture medium was then changed to

Waymouth’s supplemented with 10% FBS and changed as needed thereafter. During the

first week in culture fibroblastic cells began to migrate from the explants.

iii) Collagenase dissociation of Rat Adrenocortical Cells (Haning et al.,1970)

Whole adrenal glands were hemisected and the medulla was squeezed out using

blunt ended forceps. The tissue was then minced to 1 mm3. In certain experiments either

adrenal capsules or inner glands were used. The minced tissue was then placed in a

collagenase digestion solution. Typically, tissue from 8 glands was placed in 30 ml of

HBSS (pH 7.2) containing 5 mg/mI BSA (Fraction V, Sigma), 3 mg/mI collagenase type

II (Sigma), 0.05 g/ml DNAse type I (Sigma) and 0.5 mg/mI soybean trypsin inhibitor.

Collagenase type II was first developed for the isolation of adipocytes (Rodbell, 1964).

This enzyme has very little tryptic activity which is harmful to lipid-containing cells

such as those derived from the adrenocortical parenchyma. As an additional precaution

trypsin inhibitor was also added to the digestion solution. DNAse was added to prevent

cell clumping due to DNA that was released from damaged and dead cells during the

procedure.

The tissue supension in digestion medium was gassed for 1 mm with 95% 02/5%

C02 and incubated in a lOOmI spinner flask (Bellco, Vineland NJ) at 50 rpm for 1 hour

at 37°C. This produced friable tissue pieces that were then dissociated by pipetting 50-

75X in a 5 ml glass serological pipette. Dissociated cells were removed and the

remaining tissue was further incubated in digestion solution for 15 mm followed by

- 21

pipetting. This procedure was continued until all of the tissue was dissociated. The

resulting cell suspensions were pooled, filtered through multilayered nitex cloth

(lOOjim mesh size) to remove cell clumps, centrifuged twice (200X g, 5 mm), and re

suspended in tissue culture medium which consisted of Dulbecco’s modified essential

medium mixed 1:1 v:v with Ham’s F12 medium (DMEM/F12, Sigma or GIBCO).

Typically the majority of the cells were single (>80%) or in small aggregates of 2-4

cells.

2. Separation of Rat Adrenocortical Cell Types

A number of methods were used in an effort to separate the steroidogenic cells of

the adrenocortical parenchyma from contaminating stromal and endothelial cells.

i) Differential Substratum Adhesion Separation

Different cell types have different capabilities for attaching to tissue culture

plastic. These differences can be exploited to separate specific cell types from mixed

populations (Folkman et al., 1979).

Cell suspensions obtained by collagenase dissociation of whole adrenocortices

were seeded into 35 mm tissue culture dishes (approximately 1 Xl cells per dish)

for 15-180 minutes. At the end of the prescribed period, the medium was removed and

the unattached cells were re-seeded into new tissue culture dishes. The two populations

were then cultured and observed morphologically for one week.

- 22 -

ii) Differential Morphological Response to cAMP.

Many steroidogenic cell types in culture respond to cAMP treatment by

retracting from the substratum and rounding up (Gill et al., 1980; Amsterdam et al.,

1989; Hornsby et al., 1989). Therefore, we treated primary cultures derived from

collagenase dissociation of whole adrenocortices with 8Br-cAMP (0.5 mM, 72 hrs,

Sigma) to induce rounding of the parenchymal cell population.

cAMP caused many of the cells in 1 week old primary cultures to round severely.

These rounded cells were detached from the substratum by gentle flushing with medium

using a pasteur pipette. These cells were then centrifuged (200 Xg, 5 mm) and re

plated into secondary culture utilizing medium that did not contain cAMP.

iii) Dissection of Adrenocortical Capsules (zona glomerulosa from the Inner Portion of

the Gland (zonae fasciculata and reticularis.

This procedure has been described previously and is the most commonly used

method for separating rat adrenocortical cell types (Haning et al., 1970; Tait et al.,

1974; Hornsby et al., 1974, Barrett et al. 1989).

Whole adrenal glands were trimmed of fat and placed on HBSS soaked filter paper.

Under a dissecting microscope an incision through the capsule was then made along one

face of the gland from pole to pole. The capsule was then peeled off of the rest of the gland.

This provided two fractions, the adrenal capsule with the adherent zona glomerulosa and

the inner portion of the gland which predominantly contains the zona fasciculata and the

zona reticularis. These glandular fractions were utilized in explant, collagenase

dissociation, and percoll gradient experiments.

- 23 -

iv) Discontinuous Percoll Density Gradient Separation

Utilizing serum albumin gradients, the densities of rat fasciculata cells and zona

glomerulosa cells were estimated previously to be 1 .040 g/ml and 1 .072 g/ml

respectively (Tait et al., 1974). However, since fasciculata cells are large and

glomerulosa cells are small it is very difficult to separate these cell types in albumin

gradients because the rate of cell movement depends on both cell density and cell size.

Thus, cells with a low density and large size (ie. fasciculata) move through the gradient

at a similar rate as cells with a high density and small size (ie. glomerulosa).

In the following experiments we have therefore utilized percoll gradients which

separate fasciculata and glomerulosa cells solely on the basis of density. In addition,

discontinuous gradients were utilized so that density interfaces could be constructed with

values similar to those determined previously in the serum albumin gradients.

a) Gradient Preparation

Stock isosmotic percoll (SIP) was prepared by mixing 9 parts percoll

(Pharmacia, Uppsala Sweden, density- 1.128 g/ml) with 1 part lox DME/F12

(Appendix A). This was diluted with 1X DME/F12 containing 0.5% BSA to produce

solutions of 15,25,30,35,40,45,50, and 65% SIP. These solutions were layered upon

one another from heaviest to lightest in a round bottom plastic centrifuge tube to produce

an eight step discontinuous density gradient with seven density interfaces that was stable

for several hours at room temperature.

- 24 -

b) Cell Separation and Density Estimation

Collagenase dissociated cell suspensions, derived from either whole

adrenocortices, glandular capsules, de-capsulated glands or supra-renal fatpads were

placed atop the gradient and the tube centrifuged at 1000 Xg for 20 minutes. Typically, a

cell suspension derived from 8-20 glands (approximately 4-10 X 106 cells) was added

to the gradient in a maximum volume of 1.5 ml. Separated cells, visible as 7 bands,

were collected by pipetting, diluted in DME/F1 2 medium (1:5 v:v) to reduce the density

of the medium, and centrifuged (200 Xg 5 mm) to remove the percoll. All

manipulations involving percoll were carried out at room temperature.

The density of each cell band was estimated by measuring its distance from the

meniscus and then extrapolating back to a standard curve that was constructed using

colour coded density marker beads (Pharmacia) which were separated in parallel

gradients. Cells isolated from the 25-30% Percoll interface (Band 2, 1 .034 g/ml)

were designated FASC (derived from the zona fasciculata). Cells isolated from the 50-

65% interface (Band 7, 1 .069 g/ml) were designated GLOM (derived from the zona

glomerulosa). Cells isolated from the 40-45% interface (Band 5, 1.057 g/ml) were

designated MIX (contained a mixture of stromal, endothelial and parenchymal cells).

In addition, rat adrenal medullary cells isolated using the method of Livett

(1984) were subjected to percoll gradient separation. These cells were used as a

catecholamine positive control for comparison with FASC and GLOM cells.

3. Tissue Culture.

Collagenase dissociated cells were routinely cultured in DME/F12 medium

supplemented with 15% HS and 2.5% FBS (Hyclone, Logan UT, USA) at 37°C in a

humidified atmosphere of 5% C02 in air. Medium was changed 48 hrs after plating and

- 25 -

subsequently on every third or fourth day. When these culture conditions were varied

for specific experiments it is so noted in the text. Keeping in mind the mesodermal

origin of the adrenal cortex, we attempted to provide an environment conducive to the

maintenance of parenchymal function while minimizing the possibility of stromal

overgrowth. Therefore, the high HS/low FBS supplement combination used in these

experiments was chosen for three reasons: (1) it allows for the maintainence of

steroidogenesis by Yl adrenocortical tumour cells (Yasumura et al., 1966) and primary

cultures of collagenase dissociated cells from whole rat adrenal cortices (Leonard et al.

1983); (2) pre-senescent stromal adrenocortical cells derived from explants modulate

from a myofibroblastic state in FBS to a steroidogenic, epithelial state in HS (Slavinski

et al., 1974, 1976); (3) HS and FBS together have been reported to inhibit

adrenocortical fibroblast overgrowth in culture (McAllister and Hornsby, 1987).

Cells were passaged either with trypsin (1:250 in Ca2/Mg2free HBSS), or

with collagenase (1 mg/mI in Ca2/Mg2containing HBSS), or by gentle agitation

using a pasteur pipette when rounded, loosely adherent cells were removed selectively

from the substratum.

Cells were counted using a hemocytometer or an Artek semi-automated cell

counter (Artek Corp. East Farmingdale NY, USA). In certain experiments the cell

number was estimated by determining the amount of DNA per culture using the

fluorescent DNA-binding dye bisbenzimide (Mates et al., 1986; Sigma). Fluorescence

was measured using an Aminco-Bowmann Spectrophotometer. After assaying cultures of

known cell number, it was determined that rat adrenocortical cells contained

approximately 9 pg DNA per cell.

- 26 -

4. Differentiation Markers: Histochemistry

Adrenocortical cells separated on percoll gradients were assayed histochemically

for a number of differentiation markers, both in suspension and in primary culture.

i) Cytoplasmic Lipid

Cytoplasmic lipid could be seen as areas of refractility within percoll separated

adrenocortical cells, especially when viewed with phase microscopy. The presence of

cytoplasmic lipid was confirmed by staining primary cultures with the lipophilic dye

Oil Red 0 (Culling, 1974). Cultures were rinsed with HBSS, rapidly frozen and thawed

3 times on dry ice and then stained with 0.5% oil red 0 in 60% isopropyl alcohol for 15

minutes. The cultures were then rinsed in dH2O and the nuclei counterstained with

hemotoxylin. Lipid then appears as red cytoplasmic vesicles.

ii) A5.3B-hydroxysteroid dehydrogenase (5.3B-HSD)

A5,313-HSD activity was assayed in percoll separated adrenocortical cells using

dehydroepiandrostenedione (DHEA) as substrate (Levy et al., 1959; Farcnik and

Auersperg, 1984).

Cells on glass coverslips were rapidly freeze-thawed on dry ice and incubated in

PBS (pH 7.2) containing 0.05 mg/mI DHEA, 0.13 mg/mI nitroblue tetrazolium dye, and

0.38 mg/mI nicotinamide adenine dinucleotide (NAD, all Sigma) for 1 hour at 37°C. The

DHEA is converted to androstenedione by z5,3l3-HSD activity. This produces a reduction

equivalent that is carried to nitroblue tetrazolium by the NAD. The result is the

reduction of the dye which produces insoluble, blue fomazan deposits within the cells.

- 27

Cells were scored as z5,3I3-HSD positive if the formazan deposits obscured the nucleus.

Yl mouse adrenocortical tumour cells (Yasamura et al., 1966), pre-senescent rat lung

fibroblasts, and crude rat testicular cell suspensions were used as tissue specific

positive (Yl and Leydig cells) and negative (fibroblasts and developing germ cells)

controls.

iii) Catecholamines

To determine if adrenal medullary cells were present as contaminants, primary

cultures were stained for the presence of catecholamines using the sucrose-phosphate

glyoxylic (SPG) method (de Ia Torre and Surgeon, 1976).

Cells were grown on coverslips for 4-7 days, rinsed in PBS, and dipped rapidly

3 times in the SPG solution which contained 1% w:v glyoxylic acid (Sigma) and 0.200 M

sucrose in PBS (pH of 7.4). The coverslips were then air dried, heated to 80°C for 5

mm, and mounted in mineral oil. Catecholamine staining was viewed by epifluorescence.

5. Differentiation Markers: Immunofluorescence

Percoll separated adrenocortical cells in culture were fixed and stained for a

number of differentiation markers that were identified by immunofluorescence. After

staining the coverslips were mounted in gelvatol, (pH 7.2, Rodriques and Kienhardt,

1960), viewed by epifluorescence and photographed with Kodak Tn X Pan film (for

black and white, ASA 800), or with Kodak Ektachrome 400 (for colour, ASA 800).

- 28 -

i) Cytochrome P-450 side chain cleavage enzyme (P-45Oscc).

P-45Oscc is a mitochondrial enzyme that converts cholesterol to pregnenelone.

It is found only in steroidogenic cells (Goldring et al., 1986).

Cells on glass coverslips were fixed in formalin or 3.6% paraformaldehyde,

blocked with 0.1M glycine and permeabilized in 1% triton X-100 (Sigma) for 4

minutes; next the cells were incubated with a rabbit polyclonal antibody raised against

rat P-45Oscc (1:20 dilution, courtesy of J. Orly, Jerusalem, Israel) for 15 mm. at

room temperature, washed thoroughly in PBS and incubated with the second antibody, a

rhodamine labelled goat anti-rabbit IgG (1:20, Heavy and Light chain specific, Cappel,

West Chester PA, USA). Normal rabbit serum was used as an immunological control and

full antibody staining of rat lung fibroblasts was used as a tissue specific negative

control. The mitochondrial location of P-4SOscc aided in determining specificity as this

resulted in small definitive fluorescent bodies in positive cells rather than a diffuse

staining in negative cells. The exception was in rounded cells where cell thickness made

it difficult to pick out individual positive mitochondria.

ii) Keratin

Keratins are a class of intermediate filament proteins that are expressed in a

number of epithelial cell types (Steinert and Liem, 1990). The presence of keratins has

been noted in the adrenal cortex of some species, including human (Henzen-Logmans et

al., 1988), but not in the rat.

FASC and GLOM cells were cultured on glass coverslips for either 4 or 10 days.

Three different antibodies were used in an effort to determine if any keratin was present

in these cells. Two antibodies (CK-49, CK-55) were mouse monoclonals raised against

low molecular weight rat keratins found in many glandular epithelia (Germain et al.,

- 29 -

1988; courtesy N. Marceau, Laval Que.). A third antibody (RAK), was a rabbit

polyclonal that reacts with a number of different rat keratins (O’Guin et al., 1985;

courtesy T.T. Sun, New York, USA, REF).

The cells were fixed in methanol (-20°C), permeabilized with MeOH:acetone

(1:1, -20°C) for 5 mm., air dried and then rehydrated in PBS; next the cells were

incubated with one of either CK-49 (1:100), CK-55 (1:200) or RAK (1:50) for 30

mm at 37°C, washed in PBS and incubated with the appropriate second antibody,

rhodamine labelled goat anti-mouse lgG (1:20, Jackson Labs, for CK-49 and CK-55),

or goat anti-rabbit lgG (1:20, Cappel, for RAK) for 30 mm at 37°C.

Mouse lgG (for CK-49, CK-55) and normal rabbit serum (for RAK) were used

as immunological controls. The rat ovarian surface epithelial cell line ROSE199

(Hornby and Auersperg 1992) was used as a species and tissue specific positive control

cell line.

IH) Double Staining for Vimentin and P-45Oscc

Vimentin is an intermediate filament protein that is expressed in many

mesodermally derived cells including stromal fibroblasts (Steinert and Liem, 1990).

FASC and GLOM cells were cultured on glass coverslips for 3 or 9 days, fixed in

methanol (-20°C), permeabilized with MeOH:acetone (1:1, -20°C), air dried and

rehydrated in PBS; next they were incubated with the rabbit anti-rat P-45Oscc

polyclonal antibody (1:20 dilution, courtesy J. Orly, Jerusalem, Israel) and a mouse

anti-rat vimentin monoclonal antibody (1:50, courtesy N. Marceau, Laval Que.) for 30

mm at 37°C; finally, they were washed with PBS and incubated with the second

antibodies, rhodamine labelled goat anti-rabbit lgG (1:20, Cappel) and AMCA labelled

swine anti-mouse lgG (1:20, heavy and light chain specific, Jackson lmmunoresearch,

West Grove PA, USA).

- 30 -

Normal rabbit serum (for anti P-45Oscc) and mouse IgG (for anti-vimentin)

were used as cytochemical controls. Full antibody staining of rat lung fibroblasts was

used as a tissue specific positive control for vimentin, and a simultaneous negative

control for P-45Oscc.

iv) Factor VIII

Factor VIII, one of the components of the blood clotting cascade is found in many

endothelial cell types (Folkman et al. 1979).

Rapidly adherent cells from whole adrenocortices were cultured on glass

coverslips for 14 days, at which time endothelial cell colonies were readily visible. The

cells were fixed in methanol (-20°C), air dried and rehydrated in PBS; next they were

incubated with a rabbit polyclonal antibody raised against human factor VIII (1:25,

Calbiochem) for 60 mins at room temperature, washed in PBS and incubated with

rhodamine labelled goat anti-rabbit lgG (1:20, Cappel) for 45 mins at room

temperature.

Normal rabbit serum was used as an immunological control. Human umbilical

vein endothelial cells were used as a tissue specific positive control.

6. Time Lapse Microscopy

Mixed parenchymal-stromal populations (MIX) in primary and secondary

culture were kept in sealed flasks, mounted on the stage of an inverted phase contrast

microscope (Leitz, Fluorovert), and observed through the camera port using a video

camera equipped with mechanical focus (Panasonic, model #VW-1 850). The image was

then collected and recorded on a time lapse video recorder at 1/120 real time

(Panasonic, model #TL-6050). The temperature was maintained at 37°C by using a

- 31

thermo-regulatable air curtain (Sage Instruments, White Plains NY). Routinely,

individual fields were recorded for periods of 48-72 h. For detailed morphological

examination, single fields were photographed periodically for periods of up to 72 hours.

7. Electron Microscopy

The ultrastructure of FASC and GLOM cells was examined by transmission

electron microscopy.

After 14 days in primary culture, the cells were scraped from the substratum,

fixed with 2.5% gluteraldehyde in Millonig’s Buffer (pH 7.2) for 60 mm at 4°C,

washed, and post fixed with 1% 0s04 for 30 mm; next they were dehydrated in a graded

ethanol series and embedded in epon. Sections were stained with uranyl acetate/lead

citrate and examined on a Zeiss EM-i 0 electron microscope.

8. Steroidogenesis

Steroidogenesis was assayed by two methods: (1) fluorescence after treatment

with acid-ethanol was used to give an overall indication of steroid production; (2)

radioimmunoassay was used to specifically measure corticosterone (CS) and aldosterone

(Aldo).

i) Acid-ethanol fluorescence

A number of 3-keto, 4-ene steroids, including 20a-dihydroprogesterone and

corticosterone, fluoresce after treatment with acid-ethanol (Kowal and Fiedler 1968;

Neville and O’Hare 1973a). The steroid present in conditioned tissue culture medium

was extracted into 10 volumes of dichloromethane and then mixed 1:5 (v:v) with cold

- 32 -

ethanol/sulphuric acid (35:65 v:v). The mixture was shaken vigorously and left to stand

for 60 mm. The fluorescence in the acqueous layer was then measured using an Amino-

Bowman spectrofluorometer (activating wavelength-468 jim, emitting wavelength-525

nm). Corticosterone (Calbiochem, La Jolla CA, USA) in tissue culture medium was used

as the standard. Results were expressed as amount of fluorogenic steroid/time/culture

dish or cell number, depending on the experiment.

ii) Radioimmunoassay (RIA)

Competitive RIA was used to specifically measure the production of CS and Aldo.

To assay steroidogenesis in suspension prior to culture, percoll-separated cells

were counted and immediately suspended in 1.0 ml aliquots of 0.5% BSA in DME/F1 2

(4.2mM Kj. Parallel tubes were supplemented with or without trophic hormones at

the indicated concentrations. The cell suspensions were then placed in 1.5 ml plastic

microfuge tubes and rotated at 30 rpm at 37°C for the indicated time. Cells were then

removed by centrifugation (800 Xg, 10 minutes, 4°C) and the medium frozen at -30°C

until assayed. In culture, cells were treated with or without trophic hormones for the

time indicated. The conditioned medium was then collected and frozen at -30°C until

assay. The cells were then counted or their number was estimated by DNA assay.

CS was ethanol-extracted to remove serum associated CS binding proteins and

assayed by RIA using an antiserum raised against corticosterone-3-carboxymethyl-

oxime:BSA (ICN, Costa Mesa CA, USA). Aldo was assayed by RIA directly from the medium

using antiserum raised against aldosterone-18,21-dihemisuccinate (ICN).

Steroid standard or experimental samples were diluted to 500 il in PBS (pH

7.0) containing 1mg/mi gelatin (Sigma) . 3H steroid tracer (NEN, Boston MA, USA)

and steroid antibody were then mixed 1:1, and 200 jil was added to the steroid/PBS

solution. 12-15,000 c.p.m. of 3H tracer was used per sample and a concentration of

- 33 -

antibody was used that was capable of binding 30-50% of the tracer in the absence of

cold steroid. The steroid/tracer/antibody mixture was incubated for 4 hours at 4°C.

Unbound steroid was then removed from the solution by adding 200 jil of charcoal-coated

dextran (charcoal:dextran, 10:1), mixing for 20 mm., and then clearing by

centrifugation (2000 Xg, 10 mm). Antibody bound steroid left in the aqueous phase was

then mixed with ACS scintillation fluid (Amersham, Oakville Orit.) and counted for 5 miii

in a 6-counter.

The amount of steroid per sample was determined by extrapolating the percent of

tracer bound back to a standard curve that was constructed using known quantities of re

crystallized corticosterone and aldosterone (Calbiochem). Fresh tissue culture medium

was always assayed twice per assay and subtracted as a blank.

9. Inositol Phosphate Determination

Angiotensin II (Ang II) binds to a cell surface receptor and stimulates the activity

of phosphatidyl inositol specific phospholipase C resulting in the increased production of

inositol phosphates (Barrett et al., 1989).

I) Analysis of Inositol Phosphates

Two day old GLOM and FASC cultures (2.5 X 1 cells/i 6mm well) were labelled

with myo-2-3H-inositol (5j.tCi/well, Dupont-NEN, Mississauga Canada) for 24 hrs,

washed and pre-incubated for 30 minutes in serum-free medium with 10mM LiCI2.

HBSS (200 III, with 10 mM HEPES, pH 7.2), with or without 100 nM angiotensin II

(Ang II), was then added to cells and incubated at 37°C for either 3 or 10 mm.

hnciibations were terminated with 1 .Oml cold (-20°C) methanol. Radiolabelled inositol

- 34 -

phosphates were then extracted into the aqueous phase of acidified

chloroform/methanol/water (10:5:3, v:v:v). The extracts were neutralized and the

inositol phosphates were separated by ion exchange chromatography on 1 .Oml Dowex

columns (AG1-X8 resin, 200-400 mesh, formate form, Bio-Rad, Richmond CA) using

sequential washes of 0.1M formic acid containing 0.2 M (InsPi), 0.4 M (lnsP2), and

1.0 M (lnsP3) ammonium formate (Downes and Mitchell, 1981; Leung et al., 1986).

The relative amount of inositol phosphate per fraction was then determined by by liquid

scintillation counting.

ii) Analysis of Inositol 1 .4.5-trisphosphate (lns(1 .4.5)Pp).

Freshly isolated suspensions or 2 days old cultures of GLOM and FASC cells (2-4

X 105 cells/i 6mm well) were rinsed and maintained in serum free medium for 4 hrs

prior to experiment to reduce baseline variation. The cells were then incubated in HBSS

(125 iii, with 10 mM HEPES, pH 7.2> with or without lOOnM Ang II or 25 pM calcium

ionophore (Br-A23187) for the indicated times at 37°C.

lncubations were terminated and the intracellular inositol phosphates were

released into the medium by treatment with percloric acid (Appendix B). The samples

were cooled, centrifuged and neutralized with KOH. lns(i,4,5)P3 was then assayed

utilizing a commercially available, competitive, isomer specific, radioligand binding

assay (Palmer et al., 1989; Amersham, Arlington Heights IL).

lns(1 ,4,5)P3 standards (0.19-25 pm), or aliquots from experimental

samples, were incubated in Tris buffer (pH 9.0) containing 3H lns(1 ,4,5)P3 tracer

and a slurry of lns(1,4,5)P3 binding protein. The binding protein was then centrifuged

out of this mixture, resuspended in dH2O and the amount of tracer bound determined by

liquid scintillation counting. The binding protein bound 25-40% of the tracer in the

- 35 -

absence of cold standard or sample. The amount of lns(1 ,4,5)P3 in the sample was

estimated by extrapolating back to a standard curve constructed utilizing the % binding

obtained in the presence of known quantities of lns(1 ,4,5)P3 standards. 20 pM

Ins(1,4,5)P3 in HBSS was extracted, assayed and then used to estimate the percent

recovery. In 4 of 5 experiments this value was >80%.

10. Free Intracellular ([Ca2]i) Determination

Ang Il-induced lns(1,4,5)P3 production triggers a release of calcium from

intracellular stores in rat zona glomerulosa cells, causing an increase in [Ca2+]i that is

critical for the stimulation of steroidogenesis (Barrett et al. 1989).

GLOM and FASC cells were assayed for levels of [Ca2]i in primary culture using

an optical method that involved loading the cells with the fluorescent calcium binding dye

Fura-2 (Grynkewicz et aL, 1985, Wang et al., 1989). Specifically, at excitation

wavelengths of 350 and 380 nm, the fluorescence emissions vary inversely depending

on the amount of calcium bound to the dye. Therefore, by recording emission levels at

these wavelengths in Fura-2 loaded cells, [Ca2]i can be estimated.

i) Culture of Cells

GLOM and FASC cells (approximately 1 X cells) were maintained in primary

culture on round 18 mm glass coverslips for 4-12 days. It was necessary to culture the

cells for at least 4 days as this ensured that they were sufficiently attached to the

coverslip to prevent washing away during the assay. We found no difference in [Ca2]i

responses at either extreme of this time period. However, after two weeks or more in

culture the [Ca2]i responses, particularly to Ang II, began to decline.

- 36 -

ii) Fura-2 Loading

The cultures were loaded with the aceto-methyl ester derivative of Fura-2

(Fura-2AM). In this form the dye is lipophilic, and is thus able to diffuse freely into the

cells. Fura-2AM in DMSO was added to HBSS (with 10mM HEPES, pH 7.2) to a final

concentration of 5jiM. The non-ionic detergent plurionic F-127 (0.25% v:v, Molecular

Probes) and fetal bovine serum (5%) were added as dispersing agents. Cells on

coverslips were then loaded in this mixture for 90 mm at room temperature.

Cytoplasmic esterase conversion of the Fura-2AM to the free, Ca2+ responsive, form of

the dye (Fura-2) occurred during this period. Examination of the cells with 360nM

excitation showed even, diffuse cytoplasmic fluorescence. The use of this loading regimen

was critical in preventing non-cytoplasmic compartmentalization of the dye, which

could be seen in lipid vesicles and mitochondria if the cells were loaded at 37°C or

without dispersing agents. Cells exhibiting this compartmentalization were not used for

the assay as they did not respond to calcium ionophore treatment.

iii’ Fluorescence Measurement

The coverslips were mounted cells down atop a laminar flow-through chamber.

The chamber was inserted into a stainless steel holder and mounted onto the stage of a

Zeiss Jena Jenalumar microscope equipped for epifluorescence. Light from a 200 watt

mercury arc lamp was passed first through one of three diffential interference filters

(350, 363 or 380 nm ÷/-5nm) inserted in a rotating computer operated turret, then

through a 400 nm dichroic mirror, and finally through a bOX apochromat oil

immersion lens before reaching the cells. A circular field diaphragm in the light path

was used to restrict the area of illumination to colonies of 10-30 cells or to a single

cell. The fluorescent light emitted by the cell(s) was passed back through the dichroic

- 37

mirror, an interference filter (490 nm cutoff), and then deflected to a camera port

mounted with a photomultiplier. At the photomultiplier the emitted light was converted

to DC voltage, and digitized. The ratio of 350/380 nm fluorescence was obtained on a 1 .8

or 5.0 sec. time base, stored on a computer, and later converted to [Ca2+]i (Il.10.v.).

iv) Experimental Protocol

A 400 iI chamber and holder were designed to provide a warmed (36 +/-0.5°C)

and continuous laminar flow at a rate of 2m1/min. HBSS (1 .2 mM calcium) was used as

the perfusate. Ang II was delivered directly into the chamber in 75 il aliquots through a

short port via a cannulated syringe. Use of a non-toxic coloured dye indicated that the

bolus reached the cells on the coverslip within a few seconds and then was washed out of

the chamber within 1 minute. Therefore, Ang II treatment was rapid and transient. All

single cells which did not respond to Ang II with [Ca2]i increases of >5OnM were

treated with the non-fluorescent calcium ionophore Br-A231 87 (25j.tM) to show that

the cells had been properly loaded with Fura-2 and that the dye had not become

compartmentalized into non-cytoplasmic pools.

For calcium free experiments, HBSS without calcium and 1mM EGTA was

perfused through the chamber. Ang II was added in aliquots made up in calcium free

medium. In other experiments, the calcium channel blocker verapamil (50 liM, Sigma)

was added to calcium containing HBSS and perfused through the chamber. Also,

thapsigargin (Thastrup et al., 1990; Calbiochem), which induces intracellular calcium

release independently of lns(1 ,4,5)P3,was added to calcium containing and calcium free

HBSS. It was flooded into the chamber and then the flow was stopped, so that a static

concentration of 200nM was reached.

- 38 -

v) Calculation of [Ca2]j

[Ca2]i was calculated using the following formula (Grynkiewicz et al., 1985).

[Ca2ji= kd(Beta)(R-Rmjn)/(R-Rmax)

Where:

kd = the rate constant for the association of fura-2 with cytosolic free calcium: 224 nM.

B = ratio of the values: 380 nm fluorescence in calcium free HBSS /380 nm

fluorescence in calcium containing HBSS.

R = experimentally determined ratio of the values 350 nm/380 nm fluorescence.

Rmin = ratio of the values 350 nm1380 nm in calcium free HBSS.

Rmax = ratio of the values 350 nm/380 nm in calcium containing HBSS.

These values were determined by first perfusing the chamber with calcium free

medium, stopping the flow and adding calcium ionophore (Br-A23187, 25ii.M). 350

nM/380 nM ratios of cell groups or individual cells recorded at this time represented

Rmin. HBSS with calcium (1 .3mM) was then perfused into the chamber and 350

nM/380 nM ratios recorded taken again which now represented Rmax. The 380 nm

readings in each medium were then used to obtain B.

Typical calibration values obtained in these experiments were:

GLOM: 13=7.65, Rmin=0.63, Rmax=2.5.

FASC: 3=8.20, Rmin=0.45, Rmax=2.7.

- 39 -

11. Kirsten Murine Sarcoma Virus (KIMSV) Isolation. Infection and Oncogenic

Transformation.

FASC and GLOM cells in primary culture were infected with KiMSV, which

contains a virally activated Ki-ras gene (Shih et al., 1982). The cells were then

monitored for morphologic changes. Morphologically altered cells were selected,

passaged into secondary culture and assayed for changes in growth, serum independence,

tumourigenicity and differentiation.

i) KiMSV Isolation and Titration

K1MSV transformed normal rat kidney cells (KNRK) served as the virus

producer line (Roy-Burman and Klement, 1975). This virus is a replication defective C

type oncogenic retrovirus (Teich et al., 1982). The non-acutely transforming Maloney

murine leukemia virus is present as the helper virus.

KNRK cells were grown to subconfluence in T-75 flasks in 10% FBS containing

Waymouth’s medium. Medium was conditioned for 24 hr, chilled to 4°C and centrifuged

at 3000 Xg for 30 mm to remove cells and debris. The supernatant was further

centrifuged at 50,000 Xg for 60 mm to pellet the virus. The pellet was then resuspended

in tissue culture medium at one tenth the original volume to give a 10 X virus

concentration. The concentrated viral isolates were pooled and frozen in liquid N2.

Focus forming efficiency was evaluated in cultures of normal rat kidney cells

(NRK). Subconfluent NRK cultures were infected with serial dilutions of K1MSV in 5%

heat inactivated calf serum (56°C, 30 mm) in Waymouth’s medium for 24 hours. The

medium was then changed and after 6 days the cultures were fixed and the number of foci

counted. Foci appeared as discrete areas of piled up, disorganized, spindle shaped cells

- 40 -

against a monolayered background of flattened, morphologically normal NRK cells. Using

this assay it was estimated that the virus pool contained 4.4 X105 FFU/ml.

ii K1MSV Infection

Freshly isolated FASC and GLOM cells were seeded into 35 mm dishes at 3

different cell densities (3 X 1.2 X105, 5 X i0 cells per dish). To these dishes

1.0 ml KiMSV containing medium (10 X virus concentration) was added for 2 hours.

This procedure was repeated daily for the first 3 days in culture. Polybrene, a fusogen

which has been used to facilitate viral infection of explant adrenocortical fibroblasts

(MacAuley et al., 1986), was not used here because in preliminary experiments it

appeared to be toxic to primary FASC and GLOM cultures.

iii) Selective Passaging of Morphologically Altered Cells

Duplicate FASC cultures from 3 experiments and GLOM cultures from 2

experiments were infected with KiMSV. In no instance did KiMSV infection result in any

morphologic changes in FASC cultures.

KiMSV infected GLOM cultures did not give rise to discrete foci. Instead, after 1-

2 weeks, diffuse areas of transient cellular multilayering appeared. In these multilayed

areas refractile cells often emerged which were very loosely adherent to the underlying

substratum. These cells were detached from the multilayer by gentle flushing with

medium using a pasteur pipette. The medium was then collected, centrifuged (200 X g, 5

mm) and the cells were resuspended in fresh medium and replated into secondary

culture. These cells were designated KGLOM cells.

41 -

lv) Snnsn Indnpendwme

KGLOM and unlfected GLOM cells were maintained until passage 5 by

subculturlng at a ratio of 1t The cells were then plated In 16mm wells (1X104cellsl

well) and cultured In DMEMIFI2 medium supplemented with eIther 10% or 1% FBS.

Cell numbers were then estimated by determining the amount of DNA per culture at days

2, 4, 6, 8 and 10.

v) Tumorlçienldty

Tumorigeniclty testing was carried out essentIally as described previously

(Auersperg at al., 1977, 1981). Male Fischer rats (aged 6 weeks) were

Immunosuppressed by exposure to a Cobalt-60 radiation source (400 rads) 36 hours

prior to acinilnlsntlon of KGLOM cells. KGLOM cells In peseagelO were trypsin

dissocIated and suspended In serum free DMEWdF12 medIum. 2 X106 KGLOM cells were

then Injected subcutaneously or Intraperltoneally. In addition, 1X106cells were

injected Into the adrenal fat —, the adrenal gland, or under the kidney capsule. After 7-

12 days the animals were killed and the tumour masses were excised. At this time the

sizes of the tumours were recorded. The tumoure were then fixed In phosphate buffered

formalin (pH 72), embedded In paraffin, sectioned and stained with hematoxylln and

eosln for histopathologlcal examination.

- 42 -

12. p21 ras Expression

i) Immunofluorescence

GLOM and KGLOM cells were grown to subconfluence on glass coverslips, fixed in

3.6 % paraformaldehyde and washed in PBS. The cells were then incubated with a rat

monoclonal antibody raised against p21 ras (30 gig/mI, Y13-259, Oncogene Science,

Manhasset NY, USA) for 30 mins at room temperature, washed in PBS, and incubated

with FITC labelled goat anti-rat lgG (1:80, heavy and light chain specific, Cappel) for

45 mm at room temperature.

Rat lgG was used as an immunological control. KNRK cells were used as a v-Ki

ras expressing positive control (Myrdal et al., 1985). KGLOM cells were also treated

with 1% triton X-100 (Sigma) for 30 sec to 4 mm, prior to immunfluorescence assay

to determine if this detergent treatment extracted the membrane associated p21 ras.

ii) Immunoprecipitation

This procedure was carried out essentially as described previously (Shih et al.,

1982; Auersperg et al., 1990).

GLOM and KGLOM cells (p5) were grown to subconfluence in 60 mm dishes,

labelled for 24 hours with 35 methionine, and lysed (Appendix C). Lysates were

incubated with protein A coated sepharose (Pharmacia) linked to rabbit anti-rat lgG

(Cappel) in the presence and absence of the anti-ras antibody Y13-259. The

immunoprecipitated p21 ras was then separated by SDS-PAGE, and visualized by

autoradiography.

KNRK and NRK cells were utilized as v-Ki-ras positive and negative controls

respectively.

- 43 -

13. Lovastatin Treatment of KGLOM cells

In an attempt to determine if the maintenance of steroidogenic differentiation in

KGLOM cells was v-ras specific, we treated the cells with lovastatin, a pharmacologic

inhibitor of p21 function.

Lovastatin is a fungal metabolite derived from Aspergillus terreus (Alberts et

al., 1980). It inhibits the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG

CoA reductase). Therefore, it blocks cholesterol biosynthesis at the level of mevalonate

production and is used clinically for treating hypercholesterolemia. This inhibition of

mevalonate production also blocks the synthesis of isoprenoids such as farnesyl. As

farnesylation of the ras protein is essential for its localization to the cytoplasmic face of

the plasma membrane, treatment of cells with lovastatin blocks the biologic function of

p21 ras (Schafer et al., 1989).

GLOM and KiGLOM cells were treated with lovastatin (.5-50 jiM, Merck Frosst,

Missasauga Orit.) for 8 or 26hr in the presence or absence of ACTH (10 nM) or 8Br-

cAMP (0.5 mM). Pregnenolone (100 ng/ml) was also added to the medium to overcome

the lovastatin associated inhibition of cholesterol synthesis. At the end of the incubation

period the medium was assayed for corticosterone content by RIA. The cells were then

trypsin dissociated and the cell number estimated by DNA assay.

- 44 -

Ill. RESULTS

1. Preliminary Attempts at Rat Adrenocortical Cell Separation

Our initial goal was to produce homogeneous populations of steroidogenic rat

adrenocortical cells that were free of capsule derived fibroblasts, other stromal cells

and endothelial cell contamination. In so doing a pool of steroidogenic cells would be

acquired that could be utilized for assays of in ijfl differentiation and oncogenic

transformation without resorting to cloning. Cloning was not possible here due to the

short lifespan of the differentiated phenotype of rodent derived steroidogenic cells in

ii.trn (O’Hare and Neville, 1 973a,b; Hornsby et al., 1974; Ryback and Ramachandran,

1981a,b; Payet et al., 1984).

i) Whole Gland Dissociation of the Rat Adrenal Cortex

Collagenase dissociation of the whole adrenal cortex followed by filtration

through 100 jtm filters produced approximately 5 X cells per gland. These cells

were examined microscopically in suspension just prior to culture. The majority of the

cells were single and contained lipid, which is a characteristic of steroidogenic

adrenocortical parenchymal cells (Haning et al., 1970; Tait et al., 1974). However,

the amount of lipid per cell and the size of the cell varied widely and there were always a

few cells present which did not contain lipid (Fig la).

- 45 -

Fig 1: Collagenase dissociated cells from whole adrenal cortices.

a) Cells at the time of dissociation. The cells are spherical, of varying, diameter and most

contain refractile cytoplasmic lipid (phase microscopy; bar=1 00 jim).

b) Primary culture at day 7. A central epithelial island of adrenocortical cells is shown.

It is not readily apparent at this magnification, but at higher power cells with this

epithelial morphology often can be seen to contain retractile lipid inclusions. At the

periphery of the field the cells are spread and fibroblastic. (phase microscopy;

bar=100 jim).

c) Fluorogenic steroid production in primary and passage 1 culture with or without 24

hr ACTH stimulation. The cultures were assayed at confluence in 35 mm dishes.

Ill Basal, EIl 108M, 107M, • 106M ACTH. The data represent the amount of

steroid per dish per 24 hr (each in triplicate +1- SEM).

F.:

r&

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- 46 -

Primary cultures derived from these suspensions contained a mixture of

morphologically distinct cell types. These included: epithelial regions made up of

retractile lipid containing cells; large areas of flattened fibroblastic cells; and

occasional endothelial cell colonies. Often the borders between the epithelial islands and

fibroblastic areas were not sharp (Fig 1 b). Instead, at the periphery of the epithelial

island the cells gradually spread and lost some of their lipid. This suggested that some

phenotypic modulation between an epithelial and fibroblastic morphology had taken place

in primary culture.

Primary cultures of whole gland-derived adrenocortical cells were

steroidogenic. They produced fluorogenic steroid basally and responded to ACTH

treatment by greatly increasing their steroid production (Fig lc). However, after

enzymatic dissociation and passaging the cells produced little steroid and no longer

responded to ACTH.

ii) Differential Substratum Adhesion of Collagenase Dissociated AdrenocorLical Cells in

Primary Culture.

We attempted to separate stromal and endothelial cells from parenchymal

adrenocortical cells by differential substratum adhesion (Folkman et al., 1979). Cell

suspensions derived from the whole adrenal cortex were plated into tissue culture dishes

for varying time periods up to 180 mm. Unattached cells were then transferred to new

dishes. In this way we attempted to leave behind rapidly attaching fibroblasts and

endothelial cells in the first dish and transfer only the lipid containing steroidogenic

cells to the second dish.

Significant numbers of endothelial and fibroblastic cells were left behind in the

first dish (Fig 2 a,b). The endothelial cells had a cobblestone, epithelial morphology and

the cells did not contain lipid. These cells expressed the clotting factor VIII (Fig 3 a,b),

- 47 -

while the fibroblastic cells did not (Fig 3 e,f). There were also cells left behind in the

first dish which contained lipid. However, the lipid was present in scanty amounts such

that the nucleus could be seen.

The great majority of the cells transferred to the second dish contained large

amounts of lipid that obscured the nucleus (Fig 2c). In primary culture these cells

formed epithelial areas in which the cells retained there lipid. However, a small

percentage of fibroblastic cells were always carried over to these second dishes, thereby

contaminating the lipid containing parenchymal cells (Fig 2d).

iii) cAMP-Induced Rounding and Detachment of Parenchymal Cells from Collagenase

Dissociated Adrenocortical Cells in Primary Culture

Primary cultures derived from collagenase dissociated whole adrenocortices

were treated with 8Br-cAMP (0.5 mM, 72 hrs). The majority of the cells in these

cultures responded by retracting from the substratum and rounding up (Fig 2e). These

rounded cells could be easily detached from the culture dish by gentle agitation. This left

behind an underlying bed of fibroblastic cells still firmly attached to the substratum

(Fig 2f).

cAMP-induced retraction and rounding is a characteristic of most steroidogenic

cell types in culture (Gill et al., 1980; Amsterdam et al., 1989; Hornsby et al., 1989).

Thus, we reasoned that the rounded, detached cells in our cAMP treated whole gland

cultures represented an enriched population of adrenocortical parenchymal cells.

However, upon re-plating into secondary culture these cells. did not re-attach to a

plastic substratum, either in the presence or absence of cAMP, which suggested that a

fibroblastic feeder layer may be required for attachment.

- 48 -

Fig 2: Differential adhesion and cAMP treatment in primary culture.

(a-d) Differential Adhesion: (a.cl day 2: (b.d) day 10.

Cells from whole glands were collagenase dissociated and then seeded into tissue

culture dishes for 3 hrs (first dish). The non-adherent cells were then passaged to a

second dish. The cells were then cultured for 10 days.

Cells which adhered rapidly in the first dish are seen in a) & b). At day 2,

endothelial cell colonies are visible as well as cells with varying amounts of refractile

cytoplasmic lipid (a). At day 10, an endothelial colony formed of lipid-free epithelial

cells is present, along with numeruous elongated fibroblasts and some lipid containing

cells (b).

Cells which were transferred to a second dish are seen in c) & d). At day 2, the

great majority of the cells contained large amounts of lipid that obscured the nucleus.

The dark unucleated cells are red blood cells (C). At Day 10, most cells are epithelial and

contain lipid although there are some fibroblastic cells present (d).

e. Whole gland derived cells in primary culture treated with cAMP.

In (e), cells were cultured for one week and then treated for 4 days with 8Br-

cAMP (0,5 mM). Many of the cells are rounded and refractile (compare with Fig lb). In

(f), cAMP treated cultures have been flushed with a pasteur pipette to remove the

loosely attached rounded cells, leaving behind an underlying fibroblastic cells which did

not round in response to cAMP treatment.

Phase microscopy; bar=50 jim.

Yb

- 49 -

Fig 3: Factor VIII immunofluorescence of adrenocortical endothelial cell colonies

obtained by differential adhesion.

Cells in primary culture (Day 10) were stained with a polyclonal antibody for the

surface antigen factor VIII which is found on adrencortical endothelial cells (Folkman et

al., 1979).

An endothelial colony stained with antibody against factor VIII. The cells are

positive.

An endothelial colony stained with normal rabbit serum. The cells are negative.

fl Fibroblastic adrenocortical cells were stained with the antibody against factor VIII.

The cells are negative (a,c,e immunofluorescence; b,d,f phase; bar=20 jim).

Lfl

9 rw:

I

- 50 -

iv) Dissection of the Adrenal Cortex

The capsule of the rat adrenal cortex can be separated from the inner portion of

the gland by dissection. The capsular portions of the gland have been shown to contain

most of the zona glomerulosa and the inner de-capsulated portion of the gland contains

the zonae fasciculata and reticularis (Hanirig et al., 1970).

a) Capsular Glands (containing zona glomerulosa)

When adrenocortical capsules were minced and cultured as explants they

produced outgrowths of outwardly migrating fibroblastic cells that lined up in parallel

arrays (Fig 4b). These explant derived cells have been characterized previously and

they are not parenchymally derived (Slavinski et al., 1974, 1976) Instead, they

appear to be stromal cells with some parenchymal potential depending on the culture

conditions (Turley, 1980, Auersperg et al., 1990).

When adrenocortical capsules were collagenase dissociated they yielded

approximately 1.8 X cells per gland. Presumably, the bulk of these cells were

derived from the parenchyma of the zona glomerulosa. In primary culture most of the

cells were fibroblastic (Fig 4d). Whether these fibroblastic cells represented stromal

cell overgrowth or parenchymal cell modulation was not clear.

- 51 -

Fig 4: Adrenocortical cells derived from decapsulated (a.c) or capsular (b.d glands.

Adrenocortices were dissected to separate the connective tissue capsules (with adherent

zona glomerulosa) from the inner decapsulated portion of the gland that contains the

zonae fasciculata and reticularis.

a.b) Dissected tissue cultured as explants for 14 days.

In (a), decapsulated glands produced little fibroblastic outgrowth from the lipid laden

explant in top left corner of figure. In (b) there was vigorous fibroblastic outgrowth

from the capsular explant.

c.d Dissected tissue collagenase dissociated and resulting cells cultured for 14 days.

In (c), the cells from decapsulated glands are predominantly epithelial and contain

cytoplsamic lipid, although a fibroblastic area is visible. In (d), most of the cells are

fibroblastic and they do not contain cytoplasmic lipid. (phase microscopy; bar=50 jim).

p

- 52 -

b. De-capsulated Glands (containing zonae fasciculata and reticularis)

When the inner, de-capsulated portions of the adrenal cortex were cultured as

explants they formed slightly adherent clumps of lipid filled tissue. Very few

fibroblastic cells migrated out from these explants (Fig 4a).

When de-capsulated adrenocortices were collagenase dissociated, they yielded

approximately 2.5 X i05 cells per gland. Presumably, the bulk of these cells were

derived from the parenchyma of the zonae fasciculata and reticularis. In primary

culture, many of these cells did not attach to the tissue culture plastic. Most of the cells

that did attach contained large amounts of lipid and formed epithelial monolayers during

the first few days in primary culture. However, after 2 weeks occasional endothelial cell

and fibroblastic colonies appeared (Fig 4c). After 3-4 weeks the cultures were often

overgrown with fibroblasts.

It was readily apparent that our preliminary attempts at adrenocortical

parenchymal cell separation had not been wholly successful. Thus, we attempted to

separate these cells on the basis of density.

2. Percoll Density Gradient Separation of Rat Adrenocortical Cells

Due to the wide variation in lipid content and cell size, adrenocortical

parenchymal cells derived from the different adrenocortical zona have different

densities. Therefore, we attempted to separate these cell types by density using

discontinuous percoll gradients.

- 53 -

i) Cell Separation into 7 Visible Bands

Whole adrenocortices were collagenase dissociated and filtered through

multilayered nitex cloth (100 jim mesh) to remove undigested tissue and cell clumps.

The resulting suspension, which consisted of single cells with occasional doublets and

triplets, was placed atop an eight step discontinuous percoll density gradient (4-10

Xl 06 cells per gradient). The parameters of the percoll gradient were chosen to give

maximum separation of fasciculata and glomerulosa cells based on the estimated cell

densities obtained using serum albumin gradients (Tait et al., 1974).

After centrifugation, 7 cell bands were visible at the density interfaces within

the gradient. The bands were numbered 1 through 7 relative to their position from top

to bottom of the tube. These numbered bands did not include an opaque region that

remained atop the gradient and a reddish sediment that formed below the gradient at the

bottom of the tube.

Cell band density was determined by extrapolating back to a standard curve that

was obtained from a parallel tube containing colour coded marker beads of known

density. An example density determination from a representative experiment is provided

(Fig 5). The density of each cell band was highly reproducible between experiments

(Table 2).

- 54 -

Fig 5: Representative experiment of separated cell bands obtained from the percoll

density gradient.

A: Standard curve

The distance travelled by density marker beads in the percoll gradient was plotted

against the known density of the beads (n). Cell band distance was then plotted on the

graph to determine the density by extrapolation (a). Cell bands are numbered 1

through 7 in order of increasing density.

B. Summary of marker bead migration and density.

Six different colour coded marker beads of known density were added atop the percoll

gradient and centrifuged into it. Each bead type settled at its isopycnic point, and the

distance from the top of the gradient (meniscus) was determined. These values were

utilized to construct the standard curve in (A).

C. Summary of cell band migration and density.

The distance travelled by each cell band from the meniscus was determined. This distance

was then used to determine cell band density by extrapolation from the standard curve in

(A).

-54a-

1.10

1.08

1.06

1.04

1.02

1.00

A

BDensity Marker Beads

80

Bead Distance Density# (mm) (g/mI)1 4 1.0162 18 1.0333 28 1.0484 39 1.0625 50 1.0776 65 1.087

CEstimated Cell Band Density

Band Distance Density# (mm) (g/mI)1 10 1.0252 16 1.0323 22 1.0404 28 1.0475 35 1.0556 42 1.0647 47 1.070

EC)

>

Cl)

ci)U

0 20 40 60Distance from meniscus (mm)

- 55 -

TABLE 2

Density Gradient Separation

Banda Density Cells#/Band Cell Diameterb t\5,3,f3HSDc# (gIml) % of Total (urn) % positive

(step) n=4 exp n=4exp n=3 exp >20 Fields

1 1.021 +/-0.003 7.2 +/-2.0 19.9 +1-0.6 51 +/-292 1.034 +/-0.002 10.6 +1-3.9 18.0 +1-0.6 86 +1- 7

3 1.044 +/-0.002 15.1 +1-5.1 16.6 +1-0.9 65 +/-154 1.051 +/-0.001 20.9 +1-9.0 N.D.d N.D.5 1.057 +/-0.001 23.3 +/-4.3 14.0 +1-1.1 60 +/-156 1.061 +/-0.001 15.1 +1-7.4 N.D. N.D.7 1 .069 +/-0.002 7.8 +/-2.5 1 1 .7 +1-0.7 80 +1- 5

Characterization of collagenase-dissociated adrenocortical cellsfrom whole glands after separation on an 8-step discontinuousdensity gradient. Cells were present in visible bands at each of the 7density interfaces between steps (means +1- SD).

a A few cells and debris were found atop the gradient, erythrocytes were found belowthe gradient.

b Each experiment 20-50 cells/band were evaluated.C Each field examined contained 50 or more cells.d Not Done

- 56 -

ii) Preliminary Characterization of Separated Cells in Suspension

The visible cell bands and the material atop and below the gradient were washed

free of percoll, counted for nucleated cells, and examined microscopically. In the

opaque, un-numbered fraction that sat atop the gradient there was cellular debris, free

lipid and a few lipid containing cells. The sediment from the bottom of the tube consisted

exclusively of red blood cells. Each of the 7 numbered bands located within the gradient

consisted almost entirely of nucleated cells. The upper bands (#1-2) consisted mostly

of single cells. The middle bands (#3-6) contained the greatest number of cells as a

percentage of the total, and contained a wide variety of single cells, small aggregates, and

clumps of up to 20-30 cells. The lowest band (#7) contained single cells and small

aggregates of less than 6-8 cells. The cell aggregates found in bands 3-7 were not

present when the cell suspension was originally loaded onto the percoll gradient.

Moving from top to bottom in the gradient, the density of each numbered band

increased. In general, as the band density increased the average cell size decreased

(Table 2).

Cytoplasmic lipid is often used to store cholesterol esters and cholesterol

precursors which are crucial for the production of pregnenelone (Hall 1984). The

microsomal enzyme z5,3t3-hydroxysteroid dehydrogenase (A5,313-HSD) then converts

pregnenelone to progesterone in steroidogenic cells. Thus, the presence of cytoplasmic

lipid and A5,313-HSD activity are two general markers of adrenocortical parenchymal

cells. All the populations separated on the percoll gradient contained individual cells that

were both lipid and 5,3I3-HSD positive. However, there were vast differences in the

relative number of doubly positive cells in each population.

The population with the lowest density, Band 1 (1.021g/ml), contained

predominantly large, lipid filled cells. Suprisingly, only 51% of these cells were

- 57

A5,313-HSD positive (Table 2, Fig 6a). We reasoned that the non-steroidogenic lipid

containing cells in this band might be contaminating adipocytes, which often adhere to

the capsule when the adrenal is dissected out from the supra-renal fat pad. This was

confirmed by collagenase dissociation of adipose tissue from the fat pad followed by

percoll gradient separation. Most of the adipose cells remained atop the gradient (ie.

density less than 1 .021 g/ml). However, of those fat pad derived cells that did enter the

gradient, greater than 90% separated in Band #1 (1.021 g/ml).

Mid-density adrenocortical populations, such as Band 5 (1 .057g/ml), contained

slightly higher proportions of A5,313-HSD positive cells than Band 1 (60%). However,

morphologically the cells of Band 5 were extremely heterogeneous and the15,36-HSD

negative cells did not contain lipid (Fig 6c). Based on cell suspension observations and

primary culture results (III. 6.), we believe that most of these negative cells were

contaminating stromal and endothelial cells. Therefore, band #5 was designated “MIX”

because it contains a mixture of cell types. All of the mid-range bands (bands #3-6)

were heterogeneous and appeared to be contaminated to varying degrees with stromal

cells. Therefore, they were not characterized fully as to size and z\5,313-HSD staining.

The density gradient parameters used in this study provided two populations

which contained predominantly z\5,313-HSD positive cells. These populations, band #2

and band #7, were found at opposite ends of the density gradient and thus differed

drastically in size and lipid content.

Band 2 contained large, low density cells (18.Ojim mean diameter, 1.034g/ml)

that were packed with lipid inclusions. 86% of these cells were t5,313-HSD positive

(Fig 6b). Band 7 contained small cells with a high nuclear to cytoplasmic ratio and

density (11 .7i.tm mean diameter, 1 .069g/ml). Band 7 cells also contained small

cytoplasmic lipid vesicles. 80% of these cells were 5,3B-HSD positive (Fig 6d).

Adrenocortical capsules were dissected from inner portions of the gland,

collagenase dissociated and separated on the percoll gradients. Band 2 originated

- 58 -

predominantly from the inner glands (zonae fasciculata/reticularis), and band 7

originated predominantly from the capsules (zona glornerulosa) (Fig 7). Therefore,

Band #2 was designated as “FASC” (fasciculata) and band #7 was designated as “GLOM”

(glomerulosa).

- 59 -

Fig 6: z\5.313-hydroxysteroid dehydrogenase (&5.313-HSD) staining of cells derived

from bands separated on the percoll gradient

Cells obtained from various bands on the percoll gradient were plated onto coverslips and

stained histochemically for A5,38-HSD. Enzymatic activity is represented by dark

formazan deposits.

a) Band 1: The cells are large and lipid filled; The upper cells are A5,313-HSD positive

while the lower cells are negative.

b) Band 2 (FASC): The cells are large and lipid filled. All are positive for A5,313-HSD.

c) Band 5 (MIX): The cells are variable in size and15,3i3-HSD staining.

d) Band 7 (GLOM): The cells are small and they are A5,313-HSD positive.

e) Rat lung fibroblasts: All of the cells are z5,3l3-HSD negative (negative control).

f) Collagenase dispersed whole testis: Most of the cells are negative including the long

tailed spermatic cells. Occasional steroidogenic leydig cells are positive (positive

control).

Light microscopy; bar=15 pPm.

-o-o0

- 60 -

Fig 7: In vivo origin of FASC and GLOM cells

Whole adrenal cortices were dissected into two parts: inner fractions with zonae

fasciculata/reticularis; and capsular fractions with zona glomerulosa and connective

tissue capsule. Each fraction was dissociated and cells were loaded onto separate density

gradients. After centrifugation cell bands were collected and counted. Inner fractions

() contained approximately 9X as many FASC cells as the capsular fractions (n).

Capsular fractions contained approximately 8X as many GLOM cells as the inner

fractions. Data represent means +1- SEM from 3 experiments.

%C

ells

/Ban

d

0- 0

00

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(-I) 0

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- 61 -

3. Characterization of FASC (fasciculata) and GLOM (glomerulosa) Populations.

Due to their homogeneous nature and abundance of z5,313-HSD positive cells

FASC and GLOM cells were chosen for in depth characterization both in suspension and in

culture.

i) Steroidogenesis

a) In Suspension

In suspension, immediately after percoll density separation, both FASC and GLOM

cells were steroidogenic. They produced the rat endpoint glucocorticoid corticosterone

(CS) and mineralocorticoid aldosterone (Aldo).

FASC cell suspensions produced 12.4 +1-4.7 ng CS and 142 +1- 42 pg Aldo

iiO5 cellsl2hrs. GLOM cell suspensions produced 9.7 +1- 1.3 ng CS and 291 +1- 87

pg Aldo/1 cellsl2hrs (means +1- SEM from 6 experiments each done in duplicate or

triplicate).

A 2hr incubation with a high dose of ACTH (1 OnM) resulted in increased CS and

Aldo production by both FASC and GLOM cells (Fig 8). ACTH-mediated CS stimulation was

much higher in FASC than in GLOM. In contrast, a 2hr incubation with AnglI (lOOnM)

resulted in a much higher stimulation of steroid production by GLOM than FASC (Fig 8).

Therefore, while both cell types were responsive to ACTH, FASC cells were considerably

more so. In contrast, only GLOM cells responded strongly to Ang II.

- 62 -

Fig 8: Steroidogenesis in freshly isolated cell FASC and GLOM suspensions.

Cells were treated in suspension with (a), or without (la) trophic hormones (10 nM

ACTH, or 100 nM Ang II) for 2 hrs. Corticosterone (CS) and aldosterone (Aldo) were

assayed by RIA. As the basal values varied considerably between experiments, data are

expressed as a ratio of treated vs. basal levels. Therefore, basal values are expressed as

1. Absolute values are noted in the text. Data represent means +1.. SEM from 3

experiments, each in duplicate or triplicate).

Ang II and high basal production of Aldo exhibited by GLOM cells is characteristic of cells

derived from the zona glomerulosa.

Rat

ioIn

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- 63 -

b In Primary Culture

Both FASC and GLOM cells remained basally and inducibly steroidogenic in

primary culture. Cells were treated acutely (2hr), and chronically (24 hr), with ACTH

10 nM to determine if their response was altered after long term trophic hormone

exposure. This dose of ACTH was previosly found to be maximal for cells in primary

culture (Fig ic). For Ang II responses in primary culture see Ill. 4.

At day 4 in primary culture, ACTH treatment increased CS production

dramatically in both FASC and GLOM cultures (Fig 9). In FASC cultures, acute ACTH

stimulation was maximal, and chronic stimulation did not result in a further increase.

This was not the case in GLOM cultures where chronic ACTH treatment resulted in a

greater production of CS than did acute treatment. In addition, after chronic ACTH

treatment FASC and GLOM cells now produced similar amounts of corticosterone.

At day 14 in culture, FASC and GLOM cultures remained steroidogenic. However,

acute ACTH treatment produced much smaller increases in CS production than at day 4,

and chronic ACTH treatment further increased CS production in both cell types. This

decreased acute steroidogenic potential suggested that some de-differentiation was

occuring in both cell types with increasing time in culture.

c) In Secondary Culture

After passaging, both FASC and GLOM cells no longer responded to ACTH treatment

with increased fluorogenic steroid production (Fig 10).

- 64 -

Fig 9: FASC and GLOM corticosterone production in primary culture.

FASC and GLOM cells were seeded at approximately 1 X 1 cells in 16mm wells.

Cultures at day 4 and day 14 were not treated with trophic hormone ( basal, ED) for

2hrs, or with lOnM ACTH for 2hrs () or 24hrs (•). Media were collected and

assayed by RIA for corticosterone (CS). Cultures were then trypsinized and the cells

were counted. All data were normalized for 2hr time periods. Data represent CS/i O

cells /2hr; means +1- SEM; each in triplicate. All ACTH treated cultures produced

significantly greater amounts of CS (p<.05) compared to untreated cultures.

-64a-

0)C

Cl)0

400

300

200

100

0!

FASC GLOM FASC GLOM

- 65 -

Fig 10: FASC and GLOM cell steroidogenesis before and after passaging in culture.

FASC and GLOM cells were seeded in 35 mm dishes and assayed for fluorogenic steroid

production in primary culture (passage 0) or after trypsinization (passages 1-3). In

each passage, medium was collected and assayed after 24 hrs in the absence (IE or

presence of lOnM ACTH (s). Data represent fluorogenic steroid/dish/24hrs; means

+/-SEM; each in triplicate.

Flu

orog

enic

Ste

roid

(ng)

Flu

orog

enic

Ste

roid

(ng)

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- 66 -

ii) Primary Culture Morphology

In the first few days after seeding into culture, both FASC and GLOM cells had

morphologies which have been previously described for adrenocortical parenchymal

cells (O’Hare and Neville, 1973 a,b; Hornsby et al., 1974; Ryback and Ramachandran

1981 a,b). The cells were round and contained a central nucleus surrounded by

cytoplasmic lipid (Fig 11). FASC cells were larger and contained much more lipid than

GLOM cells. In addition, FASC cells adhered less efficiently to the substratum than GLOM

cells (Fig 12a). Through the first 3 days in culture, both FASC and GLOM cultures

remained as homogeneous, parenchymal cell clusters, and there was no evidence of

stromal or endothelial cell contamination (Fig 11).

With increasing time in primary culture, FASC and GLOM cells exhibited

different morphologies. During the first week in culture, FASC cells spread slightly but

maintained much of their lipid and formed cohesive, epithelial monolayers. GLOM cells

were initially epithelial but after one week in culture they had lost much of their lipid,

flattened, and took on bipolar fibroblastic shapes. The difference in morphology of the

two cell types at day 9 in culture was striking (Fig 11). Through 21 days in primary

culture, FASC cells remained epithelial and were quite stationary, while GLOM cells lost

contact and began to divide (Fig 12b).

- 67 -

Fig 11: FASC and GLOM morphology in primary culture.

FASC (b,d) and GLOM (a,c) cells were photographed live at day 2 (a,b) and day 10 (c,d)

in primary culture. At day 2, FASC cells were large and filled with lipid that often

obscured the nucleus (b), while GLOM cells were much smaller and contained less lipid

(a). At this time there was no evidence of fibroblast contamination in either population.

At day 10, FASC cells formed epithelial monolayers and the cells retained much of their

lipid (d), while GLOM cells flattened, spread, lost their lipid and became fibroblastic in

morphology (b)

Phase microscopy, bar=50 urn.

‘44

- 68 -

Fig 12: Seeding efficiencies and growth in primary culture of FASC and GLOM cells.

A. Seeding efficiency

1 X FASC (EJ ) and GLOM cells () were seeded in 35mm dishes. After 48hr,

unattached cells were collected and counted. By subtraction, the cells remaining were

converted to % attached. Means +1- SEM of three experiments, each in triplicate.

B. Growth in primary culture. 1 X FASC (C) or GLOM () cells were seeded in

16mm wells. At the times indicated, triplicate cultures were trypsinized and counted.

Data represent means, all SEM < 10%, each time point is in triplicate. The results are

representative of 3 experiments.

co

peqo

s,ieo%

coCo

c\J0

0

>U

c’J

00

00

00

0)

coCD

Lf)

oi.x)0N

iieo

- 69 -

Fig 13: cAMP mediated morpholoQical responses of FASC and GLOM cells in primary

culture.

Sub-confluent primary cultures (day 7) of density separated cells were treated with

8Br-cAMP (0.5mM) for 48 hours. FASC cells (a) showed prominent retraction from

the substratum (compare with Fig lid). GLOM cells (b) showed very little, if any,

retraction (compare with Fig lic).

Phase contrast microscopy (x200).

- 70 -

iii) Phenotypic Markers in Primary Culture

a) cAMP Retraction

In culture, many steroidogenic cells respond to treatment with ACTH, or its

second messenger cAMP, by retracting from the substratum (Gill et al., 1980). 8Br-

cAMP (0.5mM) treatment resulted in a marked retraction of FASC cells (Fig 13a).

However, 8Br-cAMP treatment resulted in very little retraction of GLOM cells (Fig

13b).

b) Cytoplasmic Lipid

Cytoplasmic lipid, which is found in many steroidogenic cell types, was

visualized by Oil Red 0 staining of FASC and GLOM cells at day 14 in culture.

Epithelial FASC cells contained numerous, large cytoplasmic lipid vescicles (Fig

14). Fibroblastic GLOM cells contained only scanty numbers of small lipid vescicles.

c) Ultrastructure

The ultrastructure of FASC and GLOM cells at day 14 in primary culture was

examined by electron microscopy. FASC cells contained numerous large lipid droplets in

the cytoplasm, a well developed smooth endoplasmic reticulum and numerous globlular

mitochondria with tubulo-vescicular christae (Fig 15). All of these features have been

described previously in steroidogenic cells (Nussdorfer, 1986).

GLOM cells contained large elongated mitochondria with tubulo-vescicular

christae along with promiment smooth endoplasmic reticulum, and golgi complexes (Fig

15). These features are not found in adrenocortical stromal fibroblasts derived from

- 71

explant cultures (Slavinski-Turley and Auersperg, 1978). In addition, GLOM cells

lacked features associated with stromal fibroblasts including: prominent distended rough

endoplasmic reticulum; filament bundles with focal thickenings; and large amounts of

extracellular matrix.

d) P-45Oscc Expression

To determine definitively whether or not the morphological changes seen in GLOM

cultures were due to adrenocorticaf parenchymal cell plasticity or to stromal

overgrowth, we probed the cultures at day 9 for expression of P-45Oscc by

immunofluorescence. This mitochondrial enzyme converts cholesterol to pregnenelone

and is only expressed in steroidogenic cells (Goldring et al., 1986).

Both the epithelial FASC cells and the fibroblastic GLOM cells expressed P

45Oscc, indicating a parenchymal origin for both cell types (Fig 16). In epithelial FASC

cells, P-45Oscc positive mitochondria were compact and globular (Fig 1 6g). In

contrast, the P-45Oscc positive mitochondria of fibroblastic GLOM cells were elongated

and attenuated (Fig 16h).

e) Cytokeratin Expression

Cytokeratins (CK) make up a family of intermediate filaments that are expressed

in a number of epithelial cell types (Steinert and Liem, 1990). We used 3 different

antibodies to probe FASC and GLOM cells in primary culture for CK expression by

immunofluorescence. These experiments were carried out both before (day 4), and after

(daylO), phenotypic modulation of GLOM cells. Two antibodies were monoclonal in

origin and were directed against CK 49 kD, and CK 55 kD, both of which are expressed in

- 72 -

a number of glandular epithelia (Germain et al., 1988). The other antibody, RAK was

polyclonal in origin and reacts with several different rat CKs (O’Guin et at., 1985).

There was no evidence of keratin positivity in either FASC or GLOM cells at

either time in culture using all three of these antibodies (Table 3, Fig 17). In order to

ensure that these results were truly negative we utilized rat ovarian surface epithelial

cells as a tissue specific positive control in these experiments.

f) Vimentin Expression

Vimentin is an intermediate filament protein that is expressed in many

mesodermal and stromal cell types (Steinert and Liem, 1990). Utilizing antibodies

directed against rat vimentin and rat P-45Oscc, we probed FASC and GLOM cells

simultaneously for the expression of these antigens by immunfluorescence. These

experiments were carried out before (day 3), and after (day 9), the phenotypic

modulation of GLOM cells.

Epithelial FASC cells expressed P-45Oscc but not vimentin at both day 3 and day

9 in primary culture (Table 3, Fig 18). When GLOM cells were still epithelial at day 3

in primary culture they expressed P-45Oscc but not vimentin. However, after

phenotypic modulation to a fibroblastic form at day 9, GLOM cells expressed both P

45Oscc and vimentin (Fig 18,19). Vimentin was found in filaments that traversed the

length of the fibroblastic GLOM cells. The expression of P-45Oscc and vimentin in the

same fibroblastic cells of GLOM cultures indicated that these cells were of parenchymal

origin but that they had the potential to express at least one feature of

mesodermal/stromal cells. Stromal lung fibroblasts expressed vimentin but not P

45Oscc (Fig 19).

- 73 -

Fig 14: Lipid content of FASC and GLOM cells in primary culture.

Subconfluent primary cultures (day 10) were stained with oil red 0 to show

cytoplasmic lipid. FASC cells (a) contained many large lipid vesicles while the vesicles

in GLOM cells (b) were small and scanty.

Light microscopy; bar=50 lim.

p

- 74 -

Fig 15: Transmission electron microscopy of FASC (a.c) and GLOM (b.d)cells in

primary culture.

Subconfluent primary cultures (day 14) were scraped from the tissue culture dish

fixed and processed for electron microscopy. In FASC cells, large cytoplasmic lipid

inclusions are visible (a) as well as globular mitochondria with tubulovescicular

cristae (a,b). In GLOM cells, a prominent Golgi apparatus and smooth endoplasmic

reticulum can be seen (b) as well as an elongated mitochondrion with tubular christae

(d). Bar=O.5 Itm.

I’

1•’.

7e__

’cJ

- 75 -

Fig 16: P-45Oscc expression of FASC and GLOM cells in primary culture.

Subconfluent cells in primary culture (day 9) were treated with ACTH (lOnM) for 24

hrs. Cells were then fixed and stained for P-45Oscc by immunofluorescence.

a.b FASC: The cells are epithelial in morphology and positive for P-45Oscc which

appears as punctate regions in the cytoplasm as the antigen is present in the

mitochondria. The large negative areas are nuclei, while the smaller negative areas are

cytoplasmic lipid.

c.d GLOM: The cells are fibroblastic and all of the cells are positive for P-45Oscc.

e.f FASC: Normal rabbit serum (no first antibody) control. There is no mitochondrial

staining.

y.h FASC and GLOM: High power view of FASC (g) and GLOM (h) cells showing P-45Oscc

positive mitochondria. Notice that the mitochondria in the epithelial GLOM cells tend to

be globular while those in the fibroblastic GLOM cells are elongated (compare with

figure 15 c,d).

a,c,e,g,h fluorescence; b,d,f phase microscopy; bar=20 jim.

4-

4-

p

- 76 -

Fig 17: Cytokeratin in FASC and GLOM cells in primary culture.

Subconfluent cultures (day 10) were fixed and stained for keratin using a polyclonal

antibody (RAK) which reacts with several rat keratins.

a.b GLOM: The cells are fibroblastic and there is no evidence of intermediate filament

staining in the cytoplasm. However, staining of the nucleoli was first antibody specific

as normal rabbit serum staining was negative (not shown).

c.d FASC: The cells are epithelial and there is no evidence of intermediate filament

staining.

e.f ROSE 199: This is a keratin positive cell line which was derived from the rat ovarian

surface epithelium (Hornby et al. 1992). The cells are epithelial and the keratin

positive intermediate filaments form a cytoplasmic cage around the nucleus.

a,c,e fluorescence microscopy; b,d,f phase microscopy; bar=25 jim.

ta.cir

- 77 -

Fig 18: P-45Oscc and vimentin expression in FASC and GLOM cells.

Subconfluent cultures (Day 9) were treated for 24 hours with lOnM ACTH and then

fixed and double stained for P-45Oscc (a,b) and the intermediate filament vimentin

(c,d). Photography consisted of individual exposures for each fluorochrome.

a.c.e GLOM: The cells are fibroblastic and positive for both P-45Oscc (a) and

filamentous vimentin (b).

b.d.f FASC: The cells are epithelial and are positive for P-45Oscc (b) but not for

vimentin (d). The slight punctate staining in (d) is fluorescent bleed through from the

P-45Oscc wavelength (rhodamine) to the vimentin wavelength (AMCA).

a-d fluorescence microscopy; e,f phase microscopy; bar=25 jim.

,.;t.,.

I t •4t

ra

‘0;.,

I -

‘4.’ tt

- 78 -

Fig 19: P-45Oscc and vimentin expression in GLOM cells and rat lung fibroblasts

(R LF).

Cells at various times in culture (as noted below) were treated for 24 hr with lOnM

ACTH and then were fixed and double stained for vimentin and P-45Oscc. Colour

photographs were taken using a double exposure of both the vimentin (AMCA-blue) and

P-45Oscc (rhodamine-red) wavelengths.

GLOM cells at Day 3 are still epithelial and they are just beginning to spread at

edges of the colony. The cells are P-45Oscc positive (Red mitochondria) but vimentin

negative (blue).

Middle: GLOM cells at Day 9 have modulated to a fibroblastic form. They are now positive

for both P-45Oscc (red mitochondria) and vimentin (blue intermediate filaments).

Bottom: RLF in primary culture do not produce steroids (not shown). The cells are

vimentin positive (blue intermediate filaments) and P-45Oscc negative.

- 79 -

TABLE 3

Intermediate Filament Expression in FASC and GLOM Cells

CELLS KERATINa VIMENTINb. CK49 CK55 RAK

FASC Day 3-4 - - - -

FASC Day 9-10 - - - -

GLOM Day 3...4c - - - -

GLOM Day 910d - - -

ROSE 199k ++ - ÷Fibroblastsg ND - ND

FASC and GLOM cells in primary culture were assayed forintermediate filament expression by immunofluorescence.

a Keratin expression was assayed using 3 different antibodies that react with ratkeratins. CK49 and CK 55 are monoclonals raised against specific keratin species thatare found in glandular epithelia; RAK reacts with a number of different keratins.

b Vimentin expression was assayed using a monoclonal antibody raised against ratvimentin.

C At days 3-4 in culture both FASC and GLOM cells were epithelial in morphology.d At days 9-10 in culture FASC cells remained epithelial while GLOM cells had

modulated to a fibroblastic form.e Vimentin positive GLOM cells also expressed P-45Oscc.

ROSE 199 is a rat surface epithelial cell line that was used as a positive control forkeratin expression.

g Fibroblasts were explanted from the rat lung. They were used as a positive control forvimentin expression.

h Vimentin positive fibroblasts were P-45Oscc negative.

- 80 -

y) Catecholamine Activity

There was a concern that adrenal medullary cells might be a contaminant of the

adrenocortical parenchymal populations, particularly FASC. Therefore, primary

cultures at days 4-7 were probed histochemically for catecholamine expression

Adrenal medullary cells were isolated and cultured separately as they do not

survive the dissociation method used here for dissociating the adrenocortical cells (Bell

et al., 1978, Livett et al. 1984). Adrenomedullary cells would not attach to uncoated

plastic, but only to collagen-coated plates. Also, after separation on the percoll gradient

the overwhelming majority of the cells had a different density (Band 6, 1.061 g/ml)

than either FASC or GLOM cells. These cells were uniformly phase bright and they were

positive for catecholamine expression (Fig 20). In contrast, FASC cells, which are also

phase bright but mottled because of the presence of large amounts of cytoplasmic lipid,

were not catecholamine positive.

- 81

Fig 20: Comparison of morphology and catecholamine staining in adrenal medullary

cells (a.c.e) and FASC cells (b.d.f) in primary culture.

a.b phase microscopy: Adrenal medullary cells (a) are uniformly phase bright and are

smaller than FASC cells (b) which are heterogeneously phase bright because of an

abundance of cytoplasmic lipid vesicles. a,b bar=20 jim.

c-f catecholamine staining: Adrenal medullary cells (c,e) and FASC cells in primary

culture (day 2) were fixed and stained for catecholamine expression by histochemistry.

Medullary cells are positive, exhibiting intense fluorescence (the small negative cells

are red blood cells). FASC cells are negative. c,d fluorescence microscopy; e,f phase

microscopy; bar=20 jim.

81

r.% -‘I.

.

1’

IzL.

r .‘-

II

- 82 -

4. Angiotensin Il-mediated Siynal Transduction in GLOM and FASC Cells.

In the rat, cells of different adrenocortical zones have differing responses to the

trophic hormone angiotensin II (Ang II; Barrett et al., 1989). For example, Ang II

increases steroidogenesis in capsule derived zona glomerulosa cells but not in

fasciculata/reticularis cells derived from decapsulated glands (Haning et al., 1970;

Douglas et al., 1978; Braley et al., 1986). Utilizing the homogeneous GLOM (zona

glomerulosa) and FASC (zona fasciculata) cell populations, an attempt was made to

elucidate the basis for this developmentally associated phenotypic difference by closely

examining Ang Il-mediated signal transduction in the two cell types.

i) Any Il-mediated steroidogenesis

In suspension, immediately after density isolation, GLOM cells produced much

higher basal levels of aldosterone (Aldo) while FASC cells produced higher basal levels

of corticosterorie (CS) (Fig 21). Aldo and CS are the major endpoint mineralo- and

glucocorticoids respectively in the rat (Nussdorfer, 1986). GLOM cells in suspension

responded strongly to a 4 hour Ang II treatment with increased CS and ALDO production

(Fig 21). Ang II had little effect on steroidogenesis in FASC cell suspensions, except for a

small CS increase at high Ang II doses.

In primary culture (day 4), Ang II continued to increase steroidogenesis in GLOM

cells while FASC cells remained refractory to the effects of the trophic hormone (Fig

22). Therefore, the differential steroidogenic response of GLOM and FASC cells to Ang II

was maintained in culture. This allowed us to examine signal transduction in culture

where individual, morphologically identifiable, cells could be assayed. It should be noted

however, that the actual levels of steroid production were decreased in primary culture

compared to those seen in freshly isolated suspensions. This decreased response to acute

- 83 -

Ang II treatment (ie. 4 hr) is likely due to a partial de-differentiation that takes place

in the homogeneous GLOM and FASC populations in primary culture (Fig 9). Such de

differentiation can be partially overcome by chronic trophic hormone treatment.

ii) Inositol Phosphate Accumulation.

a lnsPl. lnsP2. lnsP3

Ang II treatment for 3 mm. increased the production of 3H inositol labelled

InsPi, lnsP2 and lnsP3 in both GLOM and FASC primary cultures (Fig 23a).

Qualitatively similar results were obtained after a 10 mm. stimulation although both

cell types showed greater increases in InsPi production at this time point (Fig 23b).

This indicates that Ang II induces inositol phosphate production in both cell types. It also

suggests that the metabolism of the inositol phosphates after their production is similar

in both cell types.

b) lnsP(1.4.5)P3

Utilizing an isomer specific radioligand binding assay we found that Ang Il

increased the total mass of lns(1 ,4,5)P3 to similar extents in both GLOM and FASC

primary cultures (Fig 24a). This increase occured rapidly, within 15 seconds of

exposure to the ligand. Ang II also induced lns(1,4,5)P3 increases in freshly isolated

suspensions of GLOM and FASC cells (Fig 24b). Therefore, the initiation of Ang II

mediated signal transduction in FASC cells was not a culture artifact.

FASC and GLOM cells in primary culture were also treated with the calcium

ionophore Br-A23187 in calcium containing HBSS (1.3 mM). This treatment resulted

in an increase in lns(1 ,4,5)P3 in both cell types (Fig 24c).

- 84 -

Fig 21: Any Il-mediated steroidoyenesis in freshly isolated suspensions of GLOM and

FASC cells.

Freshly isolated suspensions of GLOM and FASC cells were treated with or without the

indicated doses of Ang II for 4 hrs and the medium was assayed for steroid content by RIA.

A-aldosterone (ALDO); E-corticosterone (CS). All Treatments: 11 Basal, 11 1 nM

1OnM, •lOOnM Any II. Data represent steroid/b5 cells/4hrs., (n=3,

means ± SEM, *p<05 **p<o1 vs. basal).

-84a-

1600

0)0

800

0

20

0)C

10C-)

0

A

GLOM FASC

B

GLOM FASC

- 85 -

Fig 22: Any Il-mediated steroidogenesis of GLOM and FASC cells in primary culture.

Primary cultures of GLOM and FASC cells were treated without or with the indicated

doses of Ang II for 4 hrs and the medium was assayed for steroid content by RIA.

A-aldosterone (ALDO); .-corticosterone (CS). All Treatments: 111Basal, IJ 1 nM,

lOnM, • lOOnM Ang II. Data represent steroid/105 cells/4hrs. (n=3,

means ± SEM, *p<05 **p<o1 vs. basal).

CS

(ng)

ALD

O(p

g)

N)

C-

0)

00

I’)

0

C’)

0

OD

C,)

0

- 86 -

Fig 23: Ang II -mediated inositol phosphate production

& Primary cultures of GLOM and FASC cells were labelled with 3H myo-inositol and

treated with Ang II (lOOnM). InsPi, lnsP2, lnsP3 accumulations were assayed by ion

exchange chromatography. 11 Basal, • + Ang II, 3mm. (n=4, means ± SEM, *p<05

**p<O1 vs basal).

The % increase in inositol phosphates in GLOM and FASC cells after 100 nM Ang II

treatment for either 3 min.(1I1)or 10 mm (B) are shown. Unstimulated basal levels

represent 0% increase (n=4 for each, means).

-86a-

U)

U)>

ci)U)

ci)0

A

cv)

b

0

InsP 1 InsP2 InsP3 InsP 1 InsP2 InsP3

8.0

6.0

4.0

2.0

0.0

500

400

300

200

100

0

B

IP1 1P2 1P3 Ipi IF 1P3

- 87

Fig 24: Ang Il-mediated lns(1.4.5)Pp production.

& GLOM and FASC cells in primary culture were treated with Ang II (lOOnM) for the

indicated times, and Ins(1 ,4,5)P3 accumulations were assayed using an isomer specific

radioligand binding assay. D Osec, l5sec, • 3osec Ang II (Data represent pmoles

lns(1,4,5)P3/106cells, n=8, means ±SEM, * p<.05, **p.<.o1 vs. basal).

j GLOM and FASC cells in suspension prior to culture were treated with Ang 11(100

nM) and lns(1,4,5)P3 levels were determined (n=4, means ±SEM, * p<.05, vs.

basal).

j GLOM and FASC cells in suspension were treated with the calcium ionophore Br

A23187 (25 jim) and lns(1,4,5)P3 levels were determined (n=4, means ±SEM,

*p<05 vs. basal).

Ins(

1,4

,5)I

Ins(

1,4

,5)’

Ins(

1,4

,5)

-L

0N.

)4S

0)

0)

0

0

0F’

)4

G)

0’

r0

N)

- 88 -

iii) [Ca2]i in cell groups.

[Ca2]i was assayed in GLOM and FASC primary cultures (day 4-12) using

Fura-2 microfluorimetry. At rest, small groups of 10-30 GLOM and FASC cells had

mean [Ca2]i levels of 74 ± 7 nM and 80 ±20 nM (n=7) respectively. Transient Ang

II treatment (< 1 mm) resulted in rapid (<30 sec.) increases in [Ca2]i in GLOM

populations (Fig 25). The timing of the onset of the [Ca2]i response correlated with

the rapid rise in Ins(1 ,4,5)P3. The magnitude of the [Ca2]i response was dose

dependent in these GLOM cell populations. A representative graph for a single GLOM

population is shown in Fig 26a.

In FASC populations, transient Ang II treatment resulted in much smaller

[Ca2]i increases than those seen in GLOM populations (Fig. 25). At high Ang II doses,

the increase in FASC populations was approximately one fourth that of GLOM populations.

A representative graph of one FASC population is shown in Fig 26b.

Thapsigargin, which acts to release calcium from intracellular stores in an

lns(1,4,5)P3 independent manner (Thastrup et al., 1990), induced a stable [Ca2]i

increase in both GLOM and FASC populations. These [Ca2]i increases were stable in

calcium containing medium and transient in calcium-free medium (Fig 27).

- 89 -

Fig 25: Any Il-mediated changes in [Ca2]i in groups of FASC and GLOM cells in

primary culture.

GLOM and FASC cells were cultured on glass coverslips and groups of cells (10-30)

were assayed for [Ca2]i using Fura-2 microfluorimetry after transient (< 1 mm)

treatment with the indicated doses of Ang II.

El1nM 1OnM, •lOOnM Ang II. (n=7, means ±SEM, **p<o1 vs mM Ang II

treatment).

[C

a2]i

incr

ease

(nM

)L

C)

CC

C0

00

0

C,)

C)

cz cc 3

- 90 -

Fig 26: Examples of [Ca2ji changes in GLOM and FASC cell groups.

GLOM and FASC cells were cultured on glass coverslips and groups of cells (10-30)

were assayed for [Ca2ji using Fura-2 microfluorimetry after transient (< 1 mm.)

treatment with Ang II at the indicated dose (labelled arrows).

A GLOM: the population responds to Ang II treatment with an increase in [Ca2]i , the

magnitude of which is Ang II dose dependent (representative example taken from a

sample size, n=7).

B FASC: the population responds very little to Ang II at any dose (representative

example taken from a sample size, n=7).

-90a-

+c..J

c

A

+

c’Jctsq C

00

500

400

300

200

100

00

500

400

300

200

100’

0

2010

Time (mm)

B

q 0

0 10 20Time (mm)

- 91

Fig 27: Comparison of Any II and Thapsigargin induced [Ca 2+ changes in cell groups.

A GLOM: Ang II and thapsigargin both induce [Ca2]i increases in the population

(representative example taken from a sample size, n=4).

B FASC: Only thapsigargin induces a significant [Ca21i increase in the population

(representative example taken from a sample size, n=5).

Single Arrows: transient treatment with Ang II (lOOnM). Horizontal Bars: duration of

static treatment with thapsigargin (200nM).

-91a-

+c..J

ct

+c..J

A

00

1

5

Time (mm)

500-

400-

300

200

100

00

500

400-

300-

200-

100•

0

I • I -

10 15

B

001

0 10 20

Time (mm)30

- 92 -

iv) [Ca2]i in single cells.

As experiments with the small groups of cells suggested a differential regulation

of Ang Il-mediated [Ca2]i increases in GLOM and FASC cells, we assayed single cells to

determine whether similar differences could be observed at this level. In culture, all

GLOM cells assayed were characteristically flattened and contained only small amounts of

cytoplasmic lipid. All FASC cells assayed were laden with cytoplasmic lipid. None of the

cells examined had the morphologic characteristics of adrenal medullary cells.

At rest, individual GLOM and FASC cells had mean [Ca2ji levels of 109 ± 7 nM

(n=58) and 126 ± lOnM (n=39), respectively. Individual GLOM cells responded to

transient Ang II treatment with rapid increases in [Ca2]i over a dose range of 0.1 to

lOOnM. Once a threshold Ang II dose was reached in an individual GLOM cell the

magnitude of the [Ca2+li increase appeared to be near maximal, independent of

increasing dose (Fig. 28a). Ang Il-mediated [Ca2]i increases in single GLOM cells also

occured in Ca2+ free medium and in the presence of the calcium channel blocker

verapamil, indicating that the source of the calcium was an intracellular pool (Fig 29).

The majority of individual FASC cells did not respond to Ang II with [Ca2]i

increases, although they did respond to the non-fluorescent calcium ionophore Br A-

23187 (Fig 28b). The response to the calcium ionophore indicates that the lack of

response by FASC cells to Ang II was not due to a Fura-2 loading artifact.

In a comparison of individual cells, lOnM Ang II stimulated [Ca2ji increases of

>5OnM in 90% (53/58) of GLOM cells but in only 28% (11/39) of FASC cells. Only

cells that exhibited increases of >5OnM were considered responsive as this ensured that

the [Ca2+]i changes observed were not due to baseline variation. A frequency

distribution indicates that the magnitude of [Ca2]i increases exhibited by responsive

GLOM cells was highly variable (Fig 30). The mean [Ca2]i increases were

341 ±6OnM for responsive GLOM cells and 309 ±55nM for all GLOM cells tested. In

- 93 -

contrast, the mean [Ca2]i increases werel 84 ± 48nM in responsive FASC cells and

70 ± 1 7nM for all FASC cells tested. Therefore, much fewer individual FASC cells

responded to transient Ang II treatment, and those that did exhibited a smaller increase

in [Ca2]i compared to responsive GLOM cells.

All the above experiments were carried out using transient (<1 mm) Ang Il

treatment. Under these conditions there was little or no evidence of sustained [Ca2+]i

oscillations. However, if GLOM cells were perfused with Ang II an initial peak followed

by lesser oscillatory peaks occurred (Fig 31a).

As is the case in rat FASC cells, Ang II also increases inositol phosphate

production in Yl mouse adrenocortical tumour cells without a resultant increase in

steroidogenesis (Begeot et al., 1987). Therefore, we assayed [Ca2ii levels in these

cells in conjunction with Ang II treatment. We found that transient Ang II treatment, at

doses up to 1 aiM, did not increase [Ca2]i in 4 individual Yl cells assayed. A

respresentative example is shown in Fig 31b.

v) Calcium lonophore-Mediated Steroidoyenesis

The data presented above suggest a disconnection between Ang Il-mediated

increases in lns(1 ,4,5)P3 production and intracellular calcium release in FASC cells.

To determine whether there was a direct link between this observation and the

differences in steroidogenesis between the zones, we compared steroidogenesis in GLOM

and FASC cultures after [Ca2]i was raised by treatment with calcium ionophore. In

calcium containing medium, the ionophore induced dose dependent increases in

steroidogenesis in both GLOM and FASC cells (Fig 32). Therefore, like GLOM cells, FASC

cells have retained the intrinsic capacity to respond to elevated [Ca2ji levels by

increasing steroidogenesis.

- 94 -

Fig 28: Examples of [Ca2ji changes in individual GLOM and FASC cells.

GLOM and FASC cells were cultured on glass coverslips and individual cells were assayed

for [Ca2]i using Fura-2 microfluorimetry after transient (< 1 mm.) treatment with

Ang II or calcium ionophore at the indicated dose (labelled arrows).

A GLOM: the cell responds to Ang II treatment with an increase in [Ca2]i that is near

maximal once a threshold of 1.0 nM Ang II is reached (representative example taken

from a sample size, n=58 from 11 cultures).

B FASC: the cell responds very little to Ang II but does respond to calcium ionophore

treatment, indicating proper Fura-2 loading (representative example taken from a

sample size, n=39 from 8 cultures).

Single arrows: transient Ang II treatment with indicated dose. Double arrow: transient

non-fluorescent calcium ionophore Br-A23187 (IP) treatment (25 iiM).

-94a-

A

‘-: q 00 ‘-

I I

0 10 20

500-

400

300

200

100•

0

600

500

400

300

200

100-

0

+

c’Jct$

+c’Jc

Time (mm)

B

‘— c 00 0d

11

I0 10 20 30

Time (mm)

- 95 -

Fig 29: Ana Il-mediated [Ca2]i chanaes in individual GLOM cells in the absence of

extracellular calcium (A) and in the presence of verapamil (B).

& An individual GLOM cell was transiently treated with 100 nM Ang II (arrows) in the

presence of extracellular calcium (1 .8mM) and in the absence of extracellular calcium

(under horizontal bar). Notice that the first Ang II treatment in the absence of calcium

induced an increase in [Ca2]i comparable to that seen before the removal of calcium.

However, the second Ang II treatment did not increase [Ca2+]i presumably due to an

exhaustion of the intracellular calcium pool (Kojima et al., 1987). This pool refilled

after restoration of extracellular calcium as a further Ang II treatment resulted in

increased [Ca2+]i (a representative example taken from a sample size, n=8)

An individual cell in calcium containing medium was transiently treated with 100 nM

Ang II (arrows) in the absence or presence of the calcium channel blocker verapamil

(100 nM, under the horizontal bar). The [Ca2+]i increases were similar in the absence

or presence of verapamil (a representative example taken from a sample size, n=3).

-95a-

+c’J

as

A

C

+c..J

as

0 20 40 60

600

500

400

300

200

100

0

600

500

400

300

200

100-

0

Time (mm)80

B

I— I

0 10 20 30 40Time (mm)

- 96 -

Fig 30: Frequency distribution of increases in [Ca2]i in single GLOM and FASC cells

after lOnM Any II treatment.

Each [Ca2+]i interval represents 5OnM increases and the % frequency represents the

percentage of the total number of FASC or GLOM cells assayed which responded within

that interval. Cells which had increases in [Ca2]i of 50 nM were considered non

responsive. 10% of GLOM cells and 72% of FASC cells were non-responsive. Non

responsive cells are not included in the figure (ie.10% GLOM cells, 72% FASC cells).

-96a-

FASC

a)U)C”a)0

+C’.’

C”0

>750

700-749

650-699

600-649

550-599

500-549

450-499

400-449

350-399

300-349

250-299

200-249

150-199

100-149

50-99

C

C)U)C”a)C.)C

+C’.’

C”0

>750

700-749

650-699

600-649

550-599

500-549

450-499

400-449

350-399

300-349

250-299

200-249

150-1 99

100-1 49

50-99

0 10 20 0 10 20

% frequency % frequency

- 97 -

Fig 31: Any Il-mediated [Ca2]i responses in an individual GLOM cell (A) and a Yl

mouse adrenal cortical tumour cell (B).

An individual GLOM cell was treated either trasiently (arrow) or perfused (under the

horizontal bar) with 100 nM Ang II. Notice that the Ang II perfusion caused an

oscillatory pattern after the initial [Ca2ji peak increase. Oscillatory patterns were

seen with dual wavelength fluoresence as shown here (representative example taken

from a sample size, n=3), or by direct chart recording of fluctuations in single

(350nm) wavelength fluorescence (data not shown, n=4).

An individual Yl mouse adrenocortical tumour cell was treated with the indicated

doses of Ang Il (single arrows) or 25 tm of calcium ionophore Br-A23187 (IP, double

arrow). Ang II did not increase [Ca2]i significantly while the ionophore did

(representative example taken from a sample size, n=4).

-97a-

+c..J

c

A

400

300C

200

5 7 9 11 13

Time (mm)

15

B

100

0

700

600

500

400

300

200

100

0

C

C

‘if

C

C0

‘if

:i

‘if

2 4 6 8 10 12 14 16

Time (mm)

- 98 -

Fig 32: Calcium ionophore-mediated steroidogenesis of GLOM and FASC cells in primary

culture.

Primary cultures of GLOM and FASC cells were treated without or with the indicated

doses of the calcium ionophore A-Br 23187 for 4 hrs and the medium was assayed for

steroid content by RIA.

A-aIdosterone (ALDO); E.-corticosterone (CS). All treatments: C Basal, 1 0.1 i.LM,

1iiM, •1OItM Br A-23187. Data represent steroid/b5 cells/4hrs (n=3,

means ± SEM, **p<ol vs basal).

0

CS

(ng)

-

ALD

O(p

g)

001 0

0 001 0

>1

(I) C)

(I)

C)

- 99 -

5. ras Oncoprotein Induced Transformation and Differentiation of FASC and GLOM Cells.

FASC and GLOM cells were infected with high titres of Kirsten murine sarcoma

virus (KiMSV) in primary culture. This retrovirus contains the v-Ki-ras oncogene

(Shih et al., 1982). Morphological alterations, transformation parameters and

steroidogenic differentiation were then monitored in the KiMSV infected cells.

i) KiMSV Infection and Selection of Morphologically Altered Cells

In 3 separate experiments involving 3 or more cultures in each, KiMSV infection

had no discernable effects on FASC cells. The cells remained epithelial, monolayered and

stationary. In addition, very few FASC cells survived passaging with or without viral

infection. Therefore, FASC cells will not be considered further here.

After infection of GLOM cells in primary culture with KiMSV, discrete foci did

not appear. However, heterogeneity within the culture dish did arise. About one week

after viral infection, diffuse areas of multilayering developed among the flat fibroblastic

GLOM monolayer. Within these multilayers rounded cells emerged (Fig 33). In infected

GLOM cultures, the multilayered areas were often transient, reverting back to a

monolayer form after a further 1-2 weeks in primary culture or after enzymatic

passaging. The rounded cells that sat atop the infected GLOM multilayers were loosely

adherent and could be easily detached by gentle agitation. Once detached, these cells were

collected, washed by centrifugation and replated into secondary culture. Thus, we were

able to select cells which exhibited the strongest morphological response to KiMSV

infection. We have designated these cells KGLOM.

100 -

Fig 33: Morphology of KiMSV infected GLOM cells.

a) Uninfected GLOM cells Day 9. primary culture: The cells are flat, fibroblastic and

monolayered.

b) KiMSV infected GLOM cells Day 10. primary culture: An area of diffuse cellular

multilayering is shown.

c) KiMSV infected GLOM cells at day 16 in primary culture: An area of intense cellular

multilayering with detaching and loosely adherent rounding cells is shown.

d) K1MSV infected GLOM cells in secondary culture: Rounded cells were removed from

primary culture multilayers by gentle agitation and replated into secondary culture

were they exhibit a fusiform, transformed morphology.

Phase microscopy; bar=5Ojim.

/00&

- 101

ii) Transformation Associated Parameters of KGLOM (KiMSV infected ylomerulosa) Cells

a Transformation in vitro

In secondary culture, KGLOM cells took on a retractile, spindle shaped

‘transformed’ morphology (Fig 33d). There were very few (less than 1%) flat,

fibroblastic cells in these post-selection KGLOM secondary cultures. Thus, the selection

process efficiently separated the morphologically altered KGLOM cells from surrounding

morphologically normal glomerulosa cells. Immunotluorescence staining indicated that

the concentration of p21 ras was greater in KGLOM cells than in uninfected GLOM cells

(Fig 34,35). The ras p21 protein observed by immunotluorescence was at least in part

membrane associated as it could be extracted from membrane ruffles as well as cell

bodies by treatment with the non-ionic detergent triton X-100 (Fig 36).

Immunoprecipitation indicated that p21 ras was present in KGLOM cells at levels

similar to that of the original KNRK producer line (Fig 37a). In addition, the viral form

of the protein was expressed in KGLOM but not in unirifected GLOM cells. This was

illustrated by the second slightly more slowly migrating p21 species in KGLOM cells

(Fig 37b). This species arises due to phospholylation of the mutant protein at amino acid

position 59 (Shih et al., 1982). Interestingly, in unifected GLOM cells an additional 27

kd protein (p27) that was specifically immunoprecipitated by the anti-ras antibody

(Fig 37b). Tryptic mapping of p27 isolated from uninfected explant derived adrenal

fibroblasts suggests that p27 may be a cellular ras homologue that is not expressed in

transformed cells (MacAuley, 1987).

KGLOM cells grew rapidly and continued to divide after reaching confluence. In

passage 5, KGLOM cells grew to a saturation density of approximately 3 X cells/cm2

which was approximately lOX greater than that of uninfected glomerulosa cells in the

same passage. Also in passage 5, KGLOM cells grew rapidly in both 10% andl % serum

- 102 -

(FBS), indicating that they were not dependent upon high concentrations of serum

derived factors for growth. (Fig 38). In contrast, in the same passage, uninfected GLOM

cells remained strictly serum dependent.

b) Tumorigenicity in vivo

Passage 10 KGLOM cells were injected into various sites in immunosuppressed

male rats. Palpable tumours formed rapidly, within 9-12 days (Table 4). These

tumours were all >1 cm3 in size, contained areas of neo-vascularization, were often

centrally necrotic, and were usually locally invasive. Most tumours were pleomorphic.

Those that arose in connective tissue (le. sub-cutaneous, suprarenal fatpad) were

predominantly sarcomatous, consisting mostly of whorls of bipolar, attenuated cells

(Fig 39a). In contrast, when KGLOM cells were injected into the adrenal gland or invaded

the kidney, the tumours formed were usually more carcinomatous with cells in clumps

and chords. In one instance in the adrenal, the tumour was almost almost entirely

carcinomatous and undifferentiated, consisting of disorganized cell clumps and chords

(Fig 39b).

- 103

Fig 34: p21 ras expression in KGLOM cells (p2).

Immunofluorescence staining for p21 ras. The cell bodies and processes are

positive.

No first antibody control.

a,c) Fluorescence microscopy; b,d) phase microscopy; bar=20 j.tm.

103

- 104

Fig 35: p21 ras expression in uninfected GLOM cells (p2).

abi Immunofluorescence staining for p21 ras. The cells are slightly positive.

çJj. No first antibody control.

a,c) Fluorescence microscopy; b,d) phase microscopy; bar=20 tim.

104L

-a’-- ..

a

‘f;:.4..

9---

- 105

Fig 36: p1 ras extraction in KGLOM cells (p5).

j)j Immunofluorescence staining for p21 ras. The cell bodies, processes and

membrane ruffles are positive.

.d)j Immunofluorescence staining for p21 ras after a 2 minute treatment with 1%

triton X-1 00. Most of the positive staining has been extracted.

.J)j No first antibody control in the absence of triton extraction.

a,c)Fluorescence microscopy; b,d) phase microscopy; bar=20 jim.

- 106 -

Fig 37: p21 ras immunoprecipitation in GLOM and KGLOM cells (p5).

Cells were labelled with 355 methionine, lysed and p21 ras was immunoprecipitated

using the monoclonal antibody Y13-259. Equal amounts of TCA precipitable c.p.m per

sample were then loaded onto 12/5% SDS gels and separated by electrophoresis.

Immunoprecipitated 35S labelled proteins were then visualized by autoradiography.

.. p21 ras expression in GLOM and KGLOM cells was compared to NRK (v-Ki ras

negative control) and KNRK cells (v-Ki ras positive control). Lane 1, NRK; Lane 2,

KNRK; Lane 3, KGLOM; Lane 4, GLOM. Notice the great increase in p21 in the KNRK and

KGLOM cells as compared to their normal counterparts.

B. Specificity of the immunoprecipitation. Lane 1, GLOM; Lane 2 KGLOM (no 1st

antibody); Lane 3 KGLOM. The 21 kd protein is indeed p21 ras as it is absent in the no

first antibody condition (Lane 2). This autoradiogram was underexposed to show the

viral p21 doublet in KGLOM cells (lane 3). The slightly more slowly migrating p21 ras

species is formed by phosphorylation of the viral protein (Shih et aL, 1982). In GLOM

cells (lane 1) there was a second immunoprecipitated peptide p27 (kD) that was not

present in KGLOM cells (lane 3).

Ib Oh.

np

lAP50—

33—

2 8—

1 8—

aC.

IBI

ap2l ras

IthI27

j4P21 ras

a

4,18—

107 -

Fig 38: Proliferation of GLOM and KGLOM cells (p5).

GLOM and KGLOM cells were plated at 2 x io per 16 mm well and cultured with 1%

C) or 10% () fetal bovine serum. At the indicated times, the cells were trypsin

dissociated and the cell numbers were estimated by DNA assay. Data represent means

+1- SEM; each in quadrupilcate.

II

—I

I•

I•

oo

00

00

(0L()

cJC’)

C’J

c’J0

-co

C

>%

,c’J0cx

>

0I

•I

•I

•

(0(0

C)

CJ

(oix

)#iieo

‘cJ

00

#iieo

- 108 -

TABLE 4

KGLOM Tumour Histopathology

Injection Site Tumour Sizea Histopathologyb

Sarcomatous/Carcinomatous

Subcutaneous +

Suprarenal Fatpad ++ +++/+

Intraperitoneal ++•

Intradrenal/kidneyc + +/++

In an effort to determine if KGLOM tumour histopathology wasaffected by the site of origin, cells (passage 8) were injected into anumber of different anatomical sites. Each site was injectedseparately in 3 different animals. Palpable tumours, some verylarge, formed within 9-12 days at all sites injected.

a Tumour volume was estimated by measurement after excision. This was very difficultin the intraperitoneal and fatpad tumours as they were usually multifocal and locallyinvasive (+) 1-2cm3; (++) >2cm3.

b Usually tumours were pleiomorphic except for one intradrenal tumour that wasentirely carcinomatous. Areas consisting of whorls of attenuated, bipolar cells werecharacterized as sarcomatous. Areas consisting of clumps and chords of cells werecharacterized as carcinomatous. The ratio of the number of symbols in each catagoryrepresents an estimation of the relative proportion of sarcomatous vs. carcinomatousareas per tumour).

C Cells were injected into the adrenal in 3 animals and into the kidney once. Thesetumours were always more restricted in size than those produced in the connectivetissue sites. In addition, overall they were more carcinomatous than tumours thatarose in the connective tissue sites.

- 109 -

FiQ 39: KGLOM tumour histopathology.

1 X 106 KGLOM cells (plo) were injected into either the suprarenal fat pad or directly

into the adrenal gland of immunosuppressed rats. Two weeks later the animals were

sacrificed and the tumours removed and processed.

a) Suprarenal fatpad tumour: Cells are attenuated and align themselves in whorls and

parallel arrays, suggesting that the tumour is sarcomatous. At the bottom of the figure is

the outer region of the uninjected normal adrenal cortex. This was the predominating

histopathology of the tumours that formed in connective tissue (see Table 4).

b) Intradrenal tumour: The normal organization of the adrenal cortex is disrupted but

the cells are arranged in clumps and plates suggesting that the tumour is an

undifferentiated carcinoma. The normal suprarenal fat pad is seen at the top of the

figure. It should be noted that a mixture of sarcomatous and carcinomatous areas were

seen in the other intradrenal tumours (see Table 4).

Hematoxylin and eosin staining; light microscopy; bar=100 urn.

e

- 110 -

iii) Steroidogenic Differentiation of KGLOM cells

KGLOM cells with a transformed morphology expressed P-45Oscc and retracted

from the substratum after treatment with cAMP (Fig 40), Both of these traits are

markers of steroidogenic differentiation. Thus, the cells selected from KiMSV infected

GLOM cultures were not non-steroidogenic stromal fibroblasts.

More than 90% of KGLOM cells expressed P-45Oscc and also overexpressed p21

ras. We were unable to show definitively that this combination occurred within the same

KGLOM cells as a double label immunofluorescence could not be carried out. This was the

case because treatment with a detergent such as triton X-100, which is necessary to

expose the P-45Oscc protein in the inner mitochondrial membrane, extracts p21 ras

from the cells (Fig 36). Clearly however, morphologically transformed KGLOM cells,

which overexpressed p21 ras , also expressed P-45Oscc (compare Fig 34 and Fig 40).

KGLOM cells in passage 2 produced corticosterone basally and responded to both

ACTH and cAMP with increased corticosterone production (Fig 41). It should be noted

that the amount of corticosterone produced by KGLOM cells in passage 2 was

considerably less than that produced in primary cultures of uninfected GLOM cells (see

Fig 9). Interestingly however, uninfected GLOM cells in passage 2 had completely de

differentiated and no longer produced corticosterone under any conditions. Importantly,

this de-differentiation indicated that the corticosterone produced by KGLOM cells was not

derived from normal GLOM cells that may have been carried over during the original

selection/passaging process.

— 111

Fig 40: P-45Oscc expression in KGLOM cells.

KGLOM cells (p2) were treated for 24 hrs with cAMP and then stained by

immunofluorescence for P-45Oscc.

P-45Oscc immunofluorescence. The cell bodies and cell extensions are positive

with the staining confined to the mitochondria.

ç)j No first antibody control. There is no mitochondrial staining.

a,c) fluorescence microscopy; b,d) phase microscopy; bar=20 jim.

III o

rT

- 112 -

Fig 41: Corticosterone production by GLOM and KGLOM cells (p2).

Subconfluent passage 2 cultures in 16 mm wells were either not treated (D), treated

with 10 nM ACTH () or treated with 0.5 mM cAMP () for 72 hours.

Corticosterone in the media was determined by RIA. Data represent means +1- SEM each

in triplicate; corticosterone/105cells/24 hrs; ** p<.01 vs untreated.

CS

(ng)

-1 I’3

0F\

).

o•)0

II

I

0 6

- 113 -

iv) Inhibition of p21 ras Function in KGLOM cells

In order to determine whether v-Ki-ras p21 expression was directly

responsible for the maintainence of steroidogenic differentiation in ras transformed

KGLOM cells we treated the cells with lovastatin, a pharmacologic inhibitor of p21

function.

Lovastatin acts as a competitive inhibitor of HMG CoA reductase activity (Alberts

et al., 1980). Therefore, it inhibits p21 ras function by preventing the addition of a

prenyl group (C15-farnesyl) to the carboxy terminus. This covalent modification of

p21 is critical for the sub-plasmalemmal localization and biological function of the

protein (Schafer et al., 1989). As well as interrupting p21 ras function, lovastatin also

interferes with cholesterol biosynthesis. Therefore, pregnenolone (100 ng/ml) was

added to the culture medium in these experiments in an attempt to prevent the

interruption of steroidogenesis due to an inadequate production of cholesterol. This dose

was chosen as it was low enough to avoid cross-reactivity with corticosterone during the

radioimmunoassay.

GLOM cells early in primary culture (Day 4) were treated with increasing doses

of lovastatin for up to 26 hrs in the presence or absence of ACTH/cAMP. Lovastatin did

not alter basal CS production but it did slightly reduce the CS increase induced by either

acute (8hr) or chronic (26 hr) ACTH/cAMP treatment (Fig 42) . As has already been

shown, GLOM cells de-differentiate with increasing time in primary culture. By day 28,

both basal and ACTH/cAMP induced CS production were considerably less than that seen

at day 4 (compare Figs 42 and 43). In addition, at day 28, lovastatin did not decrease

either basal or ACTH/cAMP induced steroidogenesis (Fig 43). These data suggest that

lovastatin did not inhibit steroidogenesis per Se. However, when large amounts of steroid

are being produced, as occurred at day 4, lovastatin treatment may cause a cholesterol

deficit that cannot be completely overcome by the addition of 100 ng/ml pregnenelone.

- 114 -

Lovastatin did not decrease basal steroidogenesis in KGLOM cells. However,

lovastatin did inhibit ACTH/cAMP induced corticosterone production in a dose dependent

manner (Fig 44). This inhibition was more readily apparent in chronically treated cells

(26 hrs), as acute trophic hormone treatment (8 hrs) increased KGLOM steroidogenesis

to only a small degree. Also, this inhibition was considerably greater than that observed

in day 4 GLOM cells (compare Figs 42 and 44).

During the first 12 hours of lovastatin treatment KGLOM cells retracted from the

substratum and rounded up. However, by 24 hours many of the cells began to flatten and

revert somewhat to an untransformed morphology (Fig 45). In addition, in lovastatin

treated cells p21 ras staining was diffuse throughout the cytoplasm and there was no

longer any localized staining along the plasma membrane (Fig 45).

- 115 -

Fig 42: Corticosterone production in lovastatin treated early primary GLOM cultures.

GLOM cells at day 4 early in primary culture were treated with the indicated doses of

lovastatin for 8 or 26 hrs in the presence of 100 ng/ml pregnenolone. Concurrently,

either no further treatment was added (D), 10 nM ACTH was added (u), or 0.5 mM

8Br-cAMP () was added. Culture medium was then assayed for corticosterone by

RIA. Data represent means +1- SEM each in triplicate; corticosterone/105cells/time

period specified, *p<05 **p<01 vs same treatment (no addition, ACTH or cAMP) when

no lovastatin added.

-115a-

0)C

Cl)C-)

0)C

Cl)C)

1200

800

400

400

GLOM (Day 4) 8 hr

0.0 0.5 5.0 50.00• -

1200

800

LOVASTATIN iM

GLOM (Day 4) 26 hr

n0

0.0 0.5 5.0 50.0

LOVASTATIN .tM

- 116

Fig 43: Corticosterone production in lovastatin treated late primary GLOM cultures.

GLOM cells at day 28 late in primary culture were treated with the indicated doses of

lovastatin for 8 or 26 hrs in the presence of 100 ng/ml pregnenolone. Concurrently,

either no further treatment was added (III), 10 nM ACTH was added (), or 0.5 mM

8Br-cAMP (•) was added. Culture medium was then assayed for corticosterone by

RIA. Data represent means +1- SEM each in triplicate; corticosterone/105cells/time

period specified.

-116a-

0)

Cl)0

0)

Cl)0

GLOM (Day 28) 8 hr

rJ

0.0 0.5

100

50

0

100

50

0

I—

50.0

LOVASTATIN jtM

GLOM (Day 28) 26 hr

0.0 0.5 50.0

LOVASTATIN iiM

- 117 -

Fig 44: Corticosterone production in lovastatin treated KGLOM cultures.

KGLOM cells (p3) were treated with the indicated doses of lovastatin for 8 or 26 hrs in

the presence of 100 ng/ml pregnenolone. Concurrently, either no further treatment was

added ( III), 10 nM ACTH was added (), or 0.5 mM 8Br-cAMP ( ) was added. The

culture medium was then assayed for corticosterone by RIA. Data represent means +1-

SEM each in triplicate; corticosterone/1 cells/time period specified, *p<05

**p<ol vs same treatment (no addition, ACTH or cAMP) when no lovastatin added.

-117a-

0)C

C.’)C-)

20

10

0

KGLOM 8 hr

r

10

0

20

0.0 0.5 5.0 50.0

LOVASTATIN p.M

KGLOM 26 hr

0)C

C/)C-)

0.0 0.5 5.0 50.0

LOVASTATIN p.M

- 118 -

Fig 45: p21 localization in lovastatin treated KGLOM cells.

KGLOM cells (p2) were treated for 26 hrs with 5 im lovastatin and then fixed and

stained for p21 ras by immunofluoresecence.

a,b) Lovastatin treated: The cells are somewhat flattened and p21 ras is found diffusely

throughout the cytoplasm.

c,d) Untreated controls (no lovastatin): The cells are spindle shaped and there are

concentrations of p21 ras along the plasma membrane.

a,c flourescence microscopy; b,d phase microscopy; bar=20 jim.

118 o.

- 119 -

6. Parenchymal Differentiation of a Mixed Parenchymal-Stromal (MIX) Cell Population

in Culture

As already mentioned in Sec. 3. II. c., (FASC vs GLOM), homogeneous FASC and

GLOM parenchymal cell populations de-differentiated with increasing time in culture.

This was reflected by the decreased steroidogenic response to ACTH treatment late in

primary culture (see Fig 9, 42 vs. 43), and by the almost total non-responsiveness

after passaging (see Fig 10). This de-differentiaton was associated with a decrease in the

activity of the steroidogenic pathway enzyme A5,313-HSD in FASC and GLOM cells with

increasing time in culture. However, in a separate density-isolated adrenocortical

fraction which contained a mixture of stromal, endothelial and parenchymal cells

(designated MIX), i5,313-HSD activity was not reduced but became enhanced with time.

In addition, steroidogenic differentiation was maintained in long term primary culture

and after passaging. This phenomenom in MIX cultures will be described in some detail

here.

i) Characterization

MIX populations originated from Band 5 (1.057 g/ml) on the density gradient

(Table 2). These populations contained only 60% A5,313-HSD positive cells at the time

of isolation. In addition, the cells in this population were heterogeneous in both size and

lipid content and they tended to form cellular aggregates while they were within the

density gradient (Fig 6c).

- 120 -

ii Primary Culture MorpholoQy

As described previously, FASC cells formed homogeneous epithelial monolayers,

retained their original epithelial morphology, and underwent little proliferation in

primary culture. GLOM cells were initially epithelial but after the first week in culture

they formed monolayers of flat, fibroblastic cells that began to proliferate (Fig 11).

Unlike the two parenchymal populations, the MIX populations immediately

exhibited heterogeneity in culture. After 4 days, fibroblasts and endothellal cell colonies

were distinguishable from the lipid-containing parenchymal cells (Fig 46a). A few days

later MIX cultures contained epithelial islands surrounded by areas of bipolar

fibroblastic cells. After 1 week, cellular multilayers began to form as proliferation

continued (Fig 47). Initially these multilayers were predominantly fibroblastic, but

during the second week in culture refractile and rounded cells appeared (Fig 46b). This

multilayering morphology was reminiscent of that seen in KiMSV infected GLOM cultures

(see Fig 33). Monitoring by time lapse indicated that in the MIX multilayers flattened

cells gradually rounded, acquired cytoplasmic lipid and proliferated (Fig 48).

Occasionally large colonies of these rounded, lipid filled cells would detatch from the

multilayer and float in the medium (Fig 46c).

121

Fig 46: Morphology of MIX adrencocortical cell population in primary culture.

a) Day 4: A variety of cell types are present including epithelial adrenocortical

parenchymal cells, fibroblastic cells and endothelial cells.

b) Day 9: A multilayered culture is shown. Rounded, lipid containing containing cells are

located apically on an underlying bed of fibroblastic cells.

C) Day 16: A large colony of rounded cells that have emerged from a multilayer similar

to that shown in (b) above.

Phase microscopy; bar=50 jim.

- 122 -

Fig 47: Growth of FASC. GLOM and MIX cells in primary culture.

1 X MIX, FASC or GLOM cells were seeded in 16mm wells. At the times indicated,

triplicate cultures were trypsinized and counted. Data represent means, all SEM < 10%,

each time point is in triplicate. The results are representative of 3 experiments.

-122a-

C

ci)C-)

12

8

4

00 10 20 30

Days in Culture

- 123 -

Fig 48: Time lapse microscopy of proliferatino MIX cells in primary culture.

Frames from a time lapse microscopic analysis of a 10 day old multilayered culture of

MIX cells. This shows rounded cells in the same field that emerge from the multilayer

and undergo cell division over a 72 hour period, a) 0 hr; b) 20 hr; C) 28 hr; d) 45 hr;

e) 48 hr; f) 72 hr.

The small arrow designates a rounded cell which begins to divide in c); completes

division in d); and shows the daughter cells moving apart in d).

The large arrow shows a rounded cell which begins to divide in c); completes division in

e); and shows the daughter cells moving apart in f).

Phase microscopy; bar= 50 jim.

1i3&

- 124 -

iii) Steroidogenic Differentiation in Primary Culture.

When assayed after 4 days in primary culture, all 3 density-isolated populations

(FASC, GLOM, MIX), were steroidogenic and produced corticosterone in the absence of

trophic hormone treatment. Interestingly, on a per cell basis, MIX cultures produced the

most corticosterone (Fig 49) even though a significant proportion of this population was

not parenchymal. ACTH treatment (24 hr) resulted in dramatic increases in

corticosterone production in all 3 populations. FASC cultures produced the most

corticosterone in response to ACTH, which was to be expected given that the zona

fasciculata is the major site of glucocorticoid production in 3jQ.

When assayed after 16 days in primary culture, all 3 populations produced

fluorogenic steroid and responded to ACTH treatment with increased steroidogenesis (Fig

50a). At this time point, FASC cultures produced the most fluorogenic steroid in

response to ACTH. However, after 56 days in primary culture, the ACTH responsiveness

of FASC and GLOM cultures was greatly reduced (Fig 50b). On the other hand, the

steroidogenic response to ACTH was greatly increased in 56 day old MIX cultures

compared to day 16.

The increased responsiveness to ACTH treatment suggested that a greater degree

of cyto-differentiation took place in MIX cultures compared to that which occurred in the

FASC and GLOM cultures. A second possibility is that there is a proliferating,

differentiated cell population in the MIX cultures. To provide a quantitative measure of

cyto-differentiation in the absence of trophic hormone treatment, we estimated the

percentage of A5,313-HSD positive cells at various time points in primary culture. As

was expected (Ryback and Ramachandran, 1981b), the percentage of z5,3I3-HSD

positive FASC and GLOM cells decreased gradually during the first 21 days in culture

(Fig 51 a). In contrast, the percentage of 5,313-HSD positive cells decreased very little

in MIX cultures. This finding, coupled with the high rate of proliferation, indicated a

- 125

greater than 10 fold increase in the number of A5,313-HSD positive cells in the MIX

cultures after 21 days in culture (Fig 51b). When MIX multilayers were stained in

i1LL, it was the rounded cells that contained lipid and A5,36-HSD activity (Fig 52a,b).

These cells also expressed P-45Oscc, in the absence of ACTH treatment (Fig 52c-f).

Interestingly, under these conditions the underlying fibroblastic cells in the multilayer

were P-45Oscc negative.

- 126 -

Fig 49: Corticosterone production by FASC. GLOM and MIX cells in primary culture.

FASC ( III), GLOM (E1) and MIX () cells were seeded at approximately 1 X iO per

16 mm well. At Day 6 the cells were left untreated (A) or were treated with lOnM ACTH

(B) for 24 hrs. Medium was then collected and assayed by RIA for corticosterone. Data

represent means +1- SEM each in triplicate; corticosterone/1 cells/24hrs.

-126a-

200

C)

a)

0100

0

6000

a)

04000

2000

8000

0

- 127

Fig 50: Fluoroyenic steroid production by FASC. GLOM and MIX cells in primary

culture.

Cells were seeded at approximately 1 X 1 per 35 mm dish. Fluorogenic steroid

production was determined at either day 16 or day 56 in primary culture in the absence

( EJ) or presence of (•) 10 nM ACTH for 4 hours. Data represent means +1- SEM

each in triplicate; fluorogenic steroid production/dish/4 hrs.

Flu

orog

enic

Ste

roid

(ng)

010

010

010

010

00

00

00

00

00

00

00

00

00

Flu

orog

enic

Ste

roid

(ng)

p’.I (Ji

0)

Cl)

C)

><

-l

I\3 —a

><

- 128 -

Fig 51: A5.3f3-Hydroxysteroid dehydroQenase (5.3B-HSD activity of FASC. GLOM

and MIX cells in primary culture.

A) % A5.313-HSD positive cells. Cells were seeded at 1 X cells per 16 mm well. At

the indicated time in culture the cells were dissociated, plated on coverslips and stained

for A5,313-HSD activity. The percentage of positive cells was then determined by

microscopic examination. Data represent means of at least 10 fields of> 50 cells each.

All SEM < 15%.

B) Number of A5.313-HSD positive cells. Values were determined by multiplying

percent 5,3B-HSD positive in (A) with the total number of cells at indicated time

points shown in Fig 47.

-128a-

0

U)

ci0

>4-,

U)00

100

80

60

40

20

U)

ci0G)>4-,

U)00

00 7 14 21

Day

6

4

2

00 10 20

Days in Culture

- 129 -

Fig 52: Differentiation marker expression of multilayered MIX primary cultures in

1L

These cultures were not treated with ACTH or cAMP.

a) Oil Red 0: Day 12 in culture. Notice that the rounded cells emerging from the

multilayer are positive for lipid. Underlying fibroblastic cells (out of focus) are for the

most part negative.

b) z\5.313-HSD: Day 11 in culture. Notice that rounded cells are most A5,36-HSD

positive.

a,b) light microscopy; bar=20 jim.

c.d) P-45Oscc Day 7: (same field) Rounded cells are just beginning to emerge from the

multilayer, some of which are P-45Oscc positive. The underlying fibroblastic cells are

negative.

e.f) P-45Oscc Day 14: (same field) The rounded cells are now all P-45Oscc positive.

c,e) fluorescence microscopy; d,f) phase microscopy; bar=20 jim.

lzqL

LI

t2

S.

‘1 •

• -.

-9-9.•.

90• I. •

S..

•v-f?

4 -

Vt—

S

-S.

•

S

5 4-•

- •C

.4

C

- 130 -

iv) Secondary Culture Morphology

Rounded MIX cells, either detached or slightly adherent to the underlying

fibroblastic cells, were selectively passaged by gentle agitation using a pasteur pipette

in a fashion similar to that used for passaging KiMSV infected GLOM cells. Secondary

cultures of these cells were initially homogeneous (Fig 53a). The cells were elongated,

branched, refractile and contained cytoplasmic lipid. After a few days, a second,

morphologically distinct cell type appeared in these cultures (Fig 53b). Initially these

cells were rare, and they were large, flat, and fibroblastic. These fibroblastic cells

served as a substratum for the predominating retractile cells which selectively adhered

to the fibroblastic cells.

v) Steroidogenic Differentiation in Secondary Culture

In secondary culture, the initially homogeneous MIX cells were P-45Oscc

positive (Fig 54). When heterogeneous secondary MIX cultures were treated with ACTH,

or its second messenger cAMP, the retractile cells rounded up, while the underlying

fibroblastic cells did not (Fig 55a,b). As cAMP-induced rounding is considered to be a

characteristic of many steroidogenic cell types, this suggested that the fibroblastic cells

are stromally derived. However, after CAMP treatment both the refractile cells and the

underlying fibroblasic cells expressed P-45Oscc (Fig 55c,d), indicating a common

parenchymal origin for both cell types.

Using time lapse videomicroscopy we were unable to determine if the fibroblastic

cells arose by modulation from the refractile cells or if they represented a separate cell

type carried over from primary cultures. The retractile cells remained adherent and

continued to replicate in secondary culture only if they were in contact with the flattened

fibroblastic cells, suggesting a requirement for heterologous cell interactions.

- 131

Upon reaching confluence these heterogeneous secondary cultures once again

formed predominantly fibroblastic multilayers from which refractile and rounded cells

once again emerged in the absence of trophic hormone treatment (Fig 53c). After

another round of selective passaging, MIX cells in passage 2 continued to produce

fluorogenic steroid and responded to ACTH treatment with increased steroidogenesis (Fig

56a). After non-enzymatic, dissociation GLOM cells in passage 2 produced negligible

amounts of fluorogenic steroid either basally or after ACTH treatment. FASC cells were

not assayed in these experiments as they did not survive mechanical passaging. MIX cells

in passage 2 also produced corticosterone basally and responded to ACTH and cAMP,

although at reduced levels compared to primary cultures (Fig 56b). GLOM cells in

passage 2 produced negligible amounts of corticosterone either basally or after ACTH or

cAMP treatment.

- 132 -

Fig 53: Morphology of MIX adrencocortical cell population in secondary culture.

Loosely adherent and detaching rounded cells from multilayering primary MIX cultures

were removed by gentle agitation and passaged into secondary culture (p1).

a) Day 2: Cultures were relatively homogeneous. Cells contained lipid and often had

branching processes. Some cells did not spread and remained rounded (not shown here,

but see Fig 54).

b) Day 10: Cultures became heterogeneous. Small, refractile cells utilized large, flat

fibrobastic cells as an underlying substratum.

c) Day 21: Multilayers once again formed with emerging rounded cells that contained

lipid.

Phase microscopy; bar=50 lim.

‘sq

4

- 133 -

Fig 54: P-45Oscc expression in MIX (p1) cells.

Two days after selective passaging MIX cells were stained for P-45Oscc expression in

the absence of ACTH or cAMP treatment. The cells are P-45Oscc positive and individual

mitochondria are visible in flattened cells.

a) phase microcopy; b) fluorescence microscopy; bar=20 urn.

133 L

Fe

- 134 -

Fig 55: Response to cAMP and P-45Oscc expression in heterogeneous MIX (p1)

cultures.

Heterogeneous MIX (p1) cultures were treated with 0.5 mM 8Br-cAMP for 24 hours.

a) no cAMP: Refractile lipid containing cells adhering to underlying fibroblastic cell.

b) cAMP treated (morphology): The majority of the retractile cells responded to cAMP

by rounding up. Some cells detached from the underlying fibroblastic cell. The latter

remained flat. a,b) phase microscopy; bar=50 jim.

c.d) cAMP treated (P-45Oscc expresion): Both the rounded and fibroblastic cells are P

45Oscc positive. C) fluorescence microscopy; d) phase microscopy; bar=20 jim.

I3LIOL.

- 135 -

Fig 56: Steroidogenesis of passage 2 GLOM and MIX cultures.

GLOM cultures were passaged mechanically by scraping to avoid enzymatic dissociation.

The latter method has already been shown to result in loss of steroidogenic

differentiation (see Fig 10). MIX multilayered cultures were selectively passaged by

removing detached and loosely adherent rounded cells.

Fluorogenic steroid production: Subconfluent GLOM and MIX cultures in 35 mm dishes

were treated without () or with () 10 nM ACTH for 24 hours. Data represent

means ÷1- SEM; each in triplicate; fluorogenic steroid/dish/24 hours; **

p <.01 vs

untreated.

Corticosterone production: Subconfluent GLOM and MIX cultures in 16 mm Wells

were not treated (LII), treated with 10 nM ACTH () or treated with 0.5 mM cAMP

( •) for 72 hours. Corticosterone in the media was determined by RIA. Data represent

means +1- SEM each in triplicate; corticosterone/105cells/24 hrs; ** p<.O1 vs.

untreated.

00

1

CS

(ng)

Flu

orom

etri

cS

tero

id(n

g)

-I

C)

C.;,

001

01 00

010

0

- 136 -

IV. DISCUSSION

The initial attempts at adrenocortical parenchymal cell purification were not

wholly successful. However, density gradient centrifugation produced 2 homogeneous

populations, one derived from the zona glomerulosa (GLOM) and the other derived from

the zona fasciculata (FASC). In primary culture, these cells exhibited phenotypic

differences associated with their stage of adrenocortical development. For example,

GLOM cells responded to Ang II while FASC cells did not. An interruption of Ang II-

mediated signal transduction at the level of intracellular calcium release appeared to

contribute to this phenotype in FASC cells. Infection with KiMSV, which contains the ras

oncogene, was associated with oncogenic transformation and steroidogenic differentiation

in GLOM but not FASC cells. This difference may be representative of the state of the

signal transduction pathways in the two cell types. Both GLOM and FASC cells de

differentiated with time and/or passaging in culture. In contrast, in a mixed

parenchymal/stromal cell population (MIX) steroidogen ic differentiation occurred

spontaneously over the longterm. This phenomenom was associated with a cellular

multilayering that may mimic some aspects of the histological organization present

during adrenocortical development In yjyn.

1. Preliminary Attempts at Parenchymal Cell Separation

A major objective of this study was to produce homogeneous populations of

adrenocortical parenchymal cells. A number of different separation techniques were used

in an attempt to isolate such populations from crude, whole gland preparations. Long

term treatment of whole gland cultures with cAMP caused the majority of the

parenchymal cells to retract from the substratum and round up. This morphological

response is typical of many steroidogenic cell types in culture (Gill et al., 1980;

- 137

Amsterdam et al., 1989; Hornsby et al., 1989). The rounded cells were selectively

removed from the culture dish, thereby leaving behind a bed of non-responsive

fibroblastic cells. However, the detached cells did not reattach to the substratum when

passaged to new dishes. Differential substratum adhesion removed large numbers of

fibroblasts and factor VIII positive endothelial cells from whole gland suspensions prior

to culture. However, a small percentage of contaminating cells were also carried over

with the lipid containing parenchymal cells during serial transferring. Therefore, this

method enriched the parenchymal cell component but it did not purify it. Chemical anti

stromal cells agents such as d-valine (Ryback and Ramachandran, 1981 a) were not used

to try and eliminate contaminants from these populations as there was a concern that

they would also be detrimental to the growth and/or function of the mesodermally

derived parenchymal cells. Also, we did not attempt to clone the lipid containing

epithelial cells because rat adrenocortical cells have a short pre-senescent lifespan and

rapidly de-differentiate in monolayer culture (O’Hare and Neville, 1973b; Hornsby et

al., 1974, Ryback and Ramachandran, 1981b).

Removal of the capsule by dissection is often used to separate zones of the rat

adrenal cortex (Haning et al., 1970, Hornsby et al., 1974; Ryback and Ramachandran,

1981a,b; Kojima et al., 1984,1985; Payet et al., 1984; Woodcock 1990). This process

produces two fractions, a capsular adherent zona glomerulosa and the de-capsulated

zonae fasciculata/reticularis. While this method is efficient in producing populations

that have the overall characteristics of a particular zona, it does not remove the stromal

cells associated with the connective tissue framework of the gland or the endothelial cells

that are derived from the extensive the capillary network that courses throughout the

cortex. In explant culture, adrenocortical capsules, but not de-capsulated glands

produced large numbers of migrating fibroblastic cells. While these explant derived

fibroblasts resemble myofibroblasts ultrastucturally and produce large amounts of an

extracellular matrix that contains collagen, they also exhibit limited parenchymal

- 138 -

characteristics under some culture conditions, or after ras-mediated transformation

(Slavinski et al., 1974, 1976; Slavinski-Turley and Auersperg, 1978; Auersperg et

al., 1981,1990). The connective tissue capsule can be dissected away from the zona

glomerulosa prior to collagenase dissociation (Hornsby et al., 1974). This procedure

removes most myofibroblastic cells from the capsular fraction but it does not address

the presence of stromal and/or endothelial cells within the zona glomerulosa itself.

When de-capsulated glands were collagenase dissociated and seeded into culture,

the great majority of the cells contained large cytoplasmic lipid inclusions. During the

first week in culture, these cells formed epithelial monolayers except for small areas

containing fibroblastic cells. Over a period of weeks the latter have been observed to

overgrow the epithelial monolayer (Neville and O’Hare, 1973a; Ryback and

Ramachandran, 1981b). Thus, decapsulation provided a population that was enriched

with, but not homogeneous for, parenchymal epithelial cells.

2. Percoll Density Gradient Parenchymal Cell Separation

The parenchymal cells of the zona fasciculata and glomerulosa have a number of

different physical properties. There are large differences in cell size, nuclear to

cytoplasmic ratio, lipid content and cell density (Idleman, 1970; Tait et al., 1974;

Nickerson, 1976; Nussdorfer 1986). We attempted to obtain homogeneous parenchymal

cell populations by exploiting the extreme differences in cell density in these two cell

types.

Serum albumin gradients, used previously to estimate the density of rat zona

fasciculata and zona glomerulosa cells, do not separate the cells efficiently because they

are composed of both density and viscosity components (Tait et al., 1974). This dual

gradient produces a sedimentation rate that is dependent on both cell density and size.

Thus, the larger the cell and the greater its density the faster the sedimentation rate. As

- 139 -

zona fasciculata cells from decapsulated glands are large (18 j.tm diameter) and of low

density (1.040 g/ml), they tended to travel through.the albumin gradients at a rate

similar to capsular zona glomerulosa cells which were small (12 jim diameter) and of

high density (1.072 g/ml). Due to the large difference in density between the 2 cell

types, separation on the basis of density alone was predicted to be much more efficient.

Percoll, whose density differs with its concentration, is useful in this regard because its

viscosity is similar to that of tissue culture media. Thus the viscosity of percoll does not

vary with changes in concentration/density. Contaminating zona fasciculata cells have

been. removed from capsular glomerulosa cell preparations by sedimenting the cells in

continuous percoll gradients (Chu and Hyatt, 1986). We went one step further and

constructed discontinuous density gradients. This has two advantages. Firstly, the density

interfaces could be set at our choosing. Secondly, the cells settle out at the density

interface closest to their own density (isopycnic point). Therefore, sedimentation rates

are not a variable that has to be controlled for between experiments. In its final form, an

8 step discontinuous gradient was utilized. Density interfaces that were similar to those

determined previously for fasciculata and glomerulosa cells on albumin gradients were

positioned at opposite ends of the gradient. These were separated by numerous

intervening interfaces in an effort to achieve maximal parenchymal cell separation.

To initially characterize the constituents of each cell band obtained from the

density gradient, we used the presence of cytoplasmic lipid inclusions and A5,313-HSD

activity as markers of steroidogenic differentiation. The presence of the microsomal

enzyme A5,38-HSD is not an exclusive marker for endocrine derived steroidogenic

cells. For example, it is found in low amounts in some some peripheral tissues including

skin (Labrie, 1990). However, all adrenocortical parenchymal cells contain large

amounts of this enzyme when they are producing endpoint steroids (Simpson et al.,

1990).

- 140 -

The constituents of the lowest density cell band (#1) were morphologically

homogeneous. The cells were large and they contained large cytoplasmic lipid inclusions

that obscured the nucleus. However, only about half of these cells were steroidogenic.

The rest were probably capsular-adherent adipocytes, as the great majority of cells

from the adrenal fat pad settled at this density interface.

Bands obtained from the middle of the gradient had a heterogenous make-up, both

in cell size and lipid content. In addition, in these bands (#4-6) the cells tended to form

large (<50 cells) aggregates during the percoll separation. These cells were also

heterogeneous for steroidogenic differentiation. For example, Band 5 (1 .057 g/ml)

contained 60% A5,3B-HSD positive cells. On the basis of the absence of lipid and their

morphology in primary culture it is believed that the majority of the i5,3B-HSD

negative cells in band 5 consisted of both stromal and endothelial cells. Therefore, this

band was designated MIX (see below).

In bands #2 and #7 the great majority of the cells were A5,3B-HSD positive.

Within each of these bands the cells were morphologically homogeneous. However,

comparatively, the cells of each band were very different from each other. Band #2 cells

were large and filled with cytoplasmic lipid inclusions that obscured the nucleus. Band

#7 cells were small and contained only scanty amounts of cytoplasmic lipid. in yi,

the zona fasciculata contains columns of large, lipid laden cells, while the zona

glomerulosa contains clustered nests of small relatively lipid poor cells (Nussdorfer

1986). Therefore, the physical characteristics of the cells suggested that Band #2

arose predominantly from the zona fasciculata and Band #7 from the zona glomerulosa.

In addition, the densities of Band #2 (1 .034 g/mI) and Band #7 (1 .069 g/mI) were

similar to those determined for zona fasciculata and zona glomerulosa cells on serum

albumin gradients (Tait et al., 1974; Bell et al., 1978). Therefore Band #2 was

designated FASC (fasciculata) and Band #7 was designated as GLOM (glomerulosa).

Fractionation of adrenal capsules (glomerulosa) from inner glands

- 141 -

(fasciculata/reticularis), followed by density separation of the cells from each fraction

supported these conclusions. It should be noted at this point that neither of these bands

contained ]! the cells of a particular zona. Thus, other density bands likely contained

cells from these zonae that had slightly different physical characteristics.

3. in vitro Characterization of FASC and GLOM Cells

Due to their intra-population homogeneity and different parenchymal origins,

FASC and GLOM cells were characterized in depth. In suspension FASC and GLOM cells

exhibited the steroidogenic characteristics of fasciculata and glomerulosa cells

respectively (Haning at al., 1970; Tait et aI., 1974; Douglas et al., 1978, Braley et al.,

1986). In primary culture ACTH, but not Ang II, dramatically increased CS production

in FASC cells. This indicated that a fasciculata phenotype was maintained. Both ACTH and

Ang II increased Aldo production in GLOM cultures, which indicated that a glomerulosa

cell phenotype was maintained.

After one week in primary culture, FASC cells remained homogeneous, epithelial

and responded to a 48 hr treatment with cAMP by retracting from the substratum.

During the same period, GLOM cells rapidly lost lipid, spread extensively, and assumed

fibroblastic shapes that changed little after a 48 hour cAMP treatment. cAMP induced

retraction by adrenocortical cells in culture has long been used as an indicator of

parenchymal origin (Gill et al., 1980), although there are culture conditions that can

reduce this morphologic response (Hornsby et al., 1989). Regardless, the expression of

the steroidogenic enzyme P-45Oscc in the fibroblastic GLOM cells indicated that they

were of parenchymal rather than stromal origin. Therefore, cell morphology and

cAMP-induced rounding are not sufficient in delineating certain adrenocortical

parenchymal cells from stromal fibroblasts. Also, cAMP-mediated increases in steroid

production without GLOM cell rounding/retraction indicated that the induction of

- 142 -

steroidogenesis is not dependent on this morphological response as has been suggested by

others (Zor, 1983; Hall, 1984; Almahabohi and Hall, 1991).

Ultrastructurally, FASC cells maintained many of the characteristics of fasciculata

cells in JyQ. These included large cytoplasmic lipid inclusions and mitochoridria with

tubulovescicular cristae. GLOM cells that had modulated to a fibroblastic morphology

continued to also showed exhibit ultrastructural characteristics steroidogenic

differentiation. These included a well developed smooth endoplasmic reticulum and

mitochondria with tubular cristae. In addition, while fibroblastic GLOM cells did not

contain large lipid inclusions they did not exhibit many of the myofibroblastic

ultrastructural characteristics observed in explant derived adrenocortical fibroblasts

(Slavinski-Turley and Auersperg, 1978).

Parenchymal cells of glandular epithelia usually express cytokeratin intermediate

filaments (Steinert and Liem, 1990). However, there was no evidence of cytokeratin

expression in FASC or GLOM cells. The steroidogenic parenchymal cells of the rat

ovarian follicle (ie. granulosa cells) also do not express cytokeratiris (Amsterdam et aL,

1989). The absence of cytokeratins in steroidogenic rat cells may be species specific.

For example, keratins are expressed in human adrenocortical cells (Henzen-Logmans et

al., 1988).

The culture characteristics of FASC and GLOM cells may reflect the developmental

mechansims and the mesodermal origin of the rat adrenal cortex. Adrenocortical

cytogenesis appears to take place along a continuum as as an adrenocyte migrates inward

from the fibroblastic capsule through the zones of the gland before degenerating in the

zona reticularis (Gottschau, 1883; Ingle and Higgins, 1938; Wright and Voncina, 1977;

Taki and Nickerson 1985; lannacone and Weinberg, 1987). The rapid culture

modulation of GLOM cells to a fibroblastic morphology suggests that while they are

parenchymal in origin they are not far removed from a stromal cell phenotype. In

support of this suggestion, GLOM cells expressed the intermediate filament protein

- 143

vimentin after fibroblastic modulation. Vimentin is often expressed in connective

tissue/stromal cells (Steinert and Liem, 1990). It should be noted that the fibroblastic

modulation in GLOM cells is not an example of complete epithelial/mesenchymal

transformation (Greenberg and Hay, 1988). For example, fibroblastic GLOM cells

express both P-45Oscc and vimentin. Thus, GLOM cells are able to simultaneously

express parenchymal and fibroblastic characteristics. This is in contrast to explant

derived adrenocortical fibroblasts which are bipotential, but take on an epithelial

morphology and reduce their production of ECM when they express steroidogenic

characteristics (Turley 1980).

The fibroblastic modulation of GLOM cells is a phenomenon which is likely

enhanced in monolayer culture, but it is not trivial as FASC cells exhibit a more

restricted parenchymal phenotype under the same culture conditions. Therefore, the

ability of GLOM cells to express some characteristics of fibroblasts while remaining

steroidogenic suggests that these cells may be man intermediate position in the

adrenocortical developmental pathway, located somewhere between a bipotential

stromal/parenchymal capsular cell (explant derived fibroblast) and a fully committed

parenchymal cell (FASC). These conclusions are in agreement with the centripital

migration hypothesis (Gottschau, 1883; lannacone and Weinberg, 1 987) which suggests

that ‘adrenocyte’ development takes place along a single clonal continuum from the

capsule to the medulla.

4. Any ii- Mediated Signal Transduction in FASC and GLOM Cells

The centripital migration hypothesis states that zona glomerulosa cells

differentiate into zona fasciculata cells as they move inwards during normal

adrenocortical development. There is ample experimental evidence to support this

theory. Presumably this process is gradual, both temporally and spatially. The temporal

- 144 -

component can be followed by examining labelled cells as they move through the cortex.

The spatial component may be delineated by a non-descript “intermediate zone” that is

found between the two zonae (Nickerson, 1976). Regardless, as the migrating cells take

on a fasciculata phenotype they lose their ability to respond steroidogenically to Ang II

(Haning et al., 1970; Tait et al., 1974; Douglas et al., 1978; Braley et al., 1986). This

trophic hormone acts primarily by altering intracellular calcium [Ca2]i metabolism

and activating protein kinase C (Barrett et al., 1989). It does this by intially activating

phosphatidyl inositol specific phospholipase C which produces the second messengers

diacylgicerol (DAG) and lns(1 ,4,5)P3. We have demonstrated a loss in the capacity of

Ang II to trigger an elevation in [Ca2]i in rat fasciculata (FASC) cells despite the fact

that lns(1 ,4,5)P3 levels were initially increased. Interestingly, when [Ca2]i was

elevated independently in these cells, steroidogenesis was stimulated. Therefore, an

interruption of signal transduction at the level of increased [Ca2+]i may be one of the

factors that prevents Ang II from eliciting a steroidogenic response in FASC cells. In

contrast, the transduction pathway was intact in GLOM cells.

Rat fasciculata cells have much fewer Ang II receptors than their glomerulosa

cell counterparts (Douglas et al.,1978; Healy et al., 1984; Maurer and Reubi, 1986;

Husain et al., 1987). This difference between the zones has led to the speculation that

the lack of a steroidogenic response by rat fasciculata cells may be accounted for entirely

by the inability of the hormone to initiate signal transduction in these cells.

Nevertheless, in rat fasciculatalreticularis cell preparations isolated by dissection

prior to cell dissociation, Ang II increased phospholipid turnover and overall inositol

phosphate production (Whitley et al., 1984,1987). However, because of the possible

presence of contaminating cells in such preparations it has been difficult to precisely

identify the cell type which responded to Ang II in these studies. In addition it has not

been determined if production of the lns(1 ,4,5)P3 isomer is increased. Consequently, in

this study, the homogeneous sub-populations of rat glomerulosa (GLOM) and fasciculata

- 145 -

(FASC) cells were utilized to carry out a detailed comparison of Ang II mediated signal

transduction. As has been disscussed above, these sub-populations did not appear to be

contaminated by stromal, endothelial or adrenal medullary cells. The absence of

medullary cells which was determined by cell density, morphology, and the lack of

catecholamine staining is an important consideration as these cells have also been shown

to contain Ang II receptors in the rat (Healy et al., 1984; Maurer and Reubi, 1986).

We have confirmed that Ang II treatment increases the production of inositol

phosphates in FASC cells. We have also shown that Ang II specifically triggers the

increased production of lns(1,4,5)P3 in FASC as well as GLOM cells. Therefore, in spite

of the drastically reduced receptor number (Douglas et al., 1978; Healy et al., 1984;

Maurer and Reubi, 1986; Husain et al., 1987), Ang II initiates signal transduction in

rat fasciculata cells. Such inconsistency between receptor number and a functional

response is not unknown. For example, Ang II induces hypertrophy of rat fasciculata

cells jfly.j (Nussdorfer et al., 1981; Mazzocchi and Nussdorfer, 1986), increases

lnsP3 prQduction in mouse Y-1 adrenocortical tumour cells (Langlois et al., 1990) and

initiates signal transduction in bovine adrenal medullary cells (Zimlichman et al.,

1987; Stauderman and Pruss, 1989) even though few hormone receptors are detected in

any of these cell types (Healy et al., 1984; Maurer and Reubi, 1986; Husain et al.,

1987; Begeot et al., 1987).

In zona glomerulosa cells, Ang Il-mediated lns(1,4,5)P3 production elicits a

release of calcium from intracellular stores (Barrett et al., 1989). This produces an

intial elevation in [Ca2]i that falls within minutes. This [Ca2]i peak is essential for

the initiation of a steroidogenic response. Over a longer period, extending up to hours,

Ang II elicits a secondary increase in [Ca2]i which is associated with an influx of

extracellular calcium (Kojima et al., 1985). This secondary increase in [Ca 2]i is

essential for the maintenance of the steroidogenic response to Ang II and is strictly

dependent on the initial intracellular calcium release (Alkon and Rasmussen, 1988). At

- 146 -

the cell population level, this secondary response results in a raised plateau of [Ca2’ii

which is lower in magnitude than the initial peak. At the single cell level, the effect of

this calcium influx is unclear, although it may play a role in producing [Ca2]i

oscillations (Quinn et al., 1988; Johnson et al., 1989). Recently, it has been shown

that in addition to initiating the opening of calcium channels at the plasma membrane,

continued occupancy of Ang II receptors also helps trigger the secondary influx of

calcium into the cell via receptor-mediated endocytosis (Hunjady et al., 1991). Thus,

rather than acting to inhibit the generation of lnsP(1 ,4,5)P3, the reduced number of

Ang II receptors in rat fasciculata cells may inhibit the secondary influx of extracellular

calcium. If this is indeed the case, an interruption in signal transduction distal to

receptor-mediated lns(1 ,4,5)P3 generation must occur in order to prevent the

absoulute increase in steroidogenesis observed in these cells.

In GLOM cells, transient Ang II treatment (< 1 mm) produced sharp [Ca2]i

peaks that returned to baseline within minutes. These responses continued to occur in

the presence of the calcium channel blocker verapamil and in the absence of

extracellular calcium. A second Ang II treatment in calcium free medium did not increase

[Ca2]i suggesting that the intracellular calcium pool was exhausted after an initial

release (Kojima et al., 1987). There was no evidence of maintained [Ca2+]i plateaus or

oscillations in the GLOM cells unless Ang II treatment was continued for longer periods

(ie. 10 mm). Taken together, these results indicated that transient Ang II treatment

initiated an intracellular calcium release only.

Ninety percent of individual GLOM cells assayed responded to Ang II treatment

with increased [Ca2]i. This further attests to the parenchymal purity of the GLOM

population. The magnitude of the [Ca2hii increase was dependent on the dose of Ang II

added. In single GLOM cells however, the peaks were near maximal once a threshold dose

was reached. This direct comparison suggests that the dose-dependent increases in

[Ca2]i peaks observed in GLOM populations reflected an increased recruitment of

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responding single cells. This observation has been noted in other cell types where it has

been suggested that each individual cell has its own constant threshold for intracellular

calcium release under a particular set of conditions (Berridge and Irvine 1989).

Transient Ang II treatment of FASC populations resulted in [Ca2]i increases

that were much lower in magnitude than those seen in GLOM populations. Further, most

of the individual FASC cells assayed (72%) did not respond to Ang II at all, and those that

did exhibited mean [Ca2+]i increases that were only about one half of those observed in

responding GLOM cells. Therefore, it appeared that the initial Ang Il-mediated

production of lns(1 ,4,5)P3 was not efficiently translated into an intracellular calcium

release in rat fasciculata cells.

Yl mouse adrenocortical tumour cells respond steroidogenically to ACTH

treatment but not to Ang II, although the latter does increase the generation of

lns(1,4,5)P3 (Begeot et al., 1987; Langlois et al., 1990). However, in a small sample

assayed in this study, Ang II treatment did not induce an intracellular calcium release.

Thus, as was seen in FASC cells, Ang Il-mediated signal transduction may be initiated but

not completed in steroidogenically refractory Yl cells. Interestingly, rat fasciculata and

Yl cells have very similar Ang II receptor numbers, approximately 10,000 per cell

(Douglas et al., 1978; Begeot et al., 1987).

If the reduced ability of Ang II to increase [Ca2]i is an important negative

regulatory control mechanism in the rat zona fasciculata, then an artificial [Ca2ji

increase should overcome this control and stimulate steroidogenesis in FASC cells. This

was indeed the case. Calcium ionophore treatment, which increased [Ca2]i, also

elicited a dose dependent increase in steroidogenesis in both GLOM and FASC cells.

Calcium ionophore treatment alone, without an additional stimulation of PKC, has been

reported to be a weak stimulator of steroidogenesis in glomerulosa cells (Kojima et al.,

1984, Capponi et al., 1988). The observation that such a weak stimulator is sufficient

to increase steroidogenesis in FASC cells suggests that the portion of the Ang Il-mediated

- 148 -

signal transduction pathway distal to intracellular calcium release is maintained in

these cells. This is in contrast to ovine fasciculata cells where Ang II treatment elicits a

[Ca2+]i increase but is unable to increase steroidogenesis, presumably because of a

pathway interruption further downstream (Viard et al., 1990).

Interestingly, calcium ionophore treatment also increased lns(1 ,4,5)P3

production in GLOM and FASC cells. This suggests that extracellular calcium influx may

be important in maintaining protein kinase C activation (via DAG production) as well as

re-initiating a secondary lns(1 ,4,5)P3-mediated intracellular calcium release after an

initial exhaustion and re-filling of the intracellular calcium pool. As has been discussed

above, refilling of the intracellular calcium pool is also dependent upon an extracellular

calcium influx. Thus, such a cycling of intracellular release and extracellular calcium

influx may be self-perpetuating, thereby leading to the [Ca2]i oscillations observed

during long term Ang II treatment. In support of this suggestion, it has recently been

determined that extracellular calcium is required for the maintained stimulation of

PIP2-specific PLC during Iongterm Ang II treatment of rat glomerulosa cells (Foster et

al., 1991). Also, growth factor induced [Ca2]i oscillations in fibroblast lines are

dependent upon the continued, intermittant generation of InsP3 (Harootunian et al.,

1991).

There are a number of possible explanations for the interruption of

lns(1 ,4,5)P3-mediated intracellular calcium release in FASC cells. One could involve

qualitative differences among the Ang II receptors present in GLOM and FASC cells as 2

different receptor sub-types have been identified, each with different signal

transduction capabilities (Johnson and Aguilera, 1991). Another explanation could

involve the absence of an intracellular calcium pool in FASC cells. This does not appear

to be the case as thapsigargin, which releases intracellular calcium in an lns(1 ,4,5)P3

independent manner (Takemura et al., 1989; Thastrup et al., 1990), increased [Ca2]i

in both GLOM and FASC cells. However, this increase was smaller in magnitude and

- 149 -

slower in onset in FASC cells. Thus, it is possible that the intracellular calcium pool is

reduced or restricted in FASC cells.

Differential InsP3 metabolism could also inhibit [Ca2]i increases in FASC

cells. For example, a rapid conversion of lnS(1,4,5)P3 into InS(1,3,4)P3 could

impede intracellular calcium release (Berridge and Irvine, 1989). In fact, this type of

differential lnsP3 metabolism appears to be responsible for the inability of the peptide

hormone endothelin to increase intracellular calcium or steroidogenesis in capsule

derived rat glomerulosa cells (Woodcock et al., 1990). If it occured in Ang II treated

FASC cells, this lnsP3 conversion would also produce inositol 1,3,4,5-

tetrakisphosphate (lnsP4) as an intermediate (Berridge and Irvine, 1989). lnsP4 has

been shown to further prevent [Ca2+]i increases in other cell types by causing a

sequestration of intracellular calcium (Hill et al., 1988). However, in a crude

comparison of InsPi, lnsP2 and lnsP3 production after 3 and 10 minute Ang II

treatments no large differences in inositol phosphate metabolism were evident in GLOM

and FASC cells. For example, in both cell types lnsPi production increased similarly

after 10 minutes due to inositol phosphate phosphatase activity.

The status of intracellular Ins(1 ,4,5)P3 receptors within the cell could also be

a factor in the decreased [Ca2]i release in FASC cells. There is some evidence of

heterogeneity among these receptors in the adrenal cortex (Enyedi and Williams, 1988;

Challiss et al., 1990). Also, signal pathway cross-talk can modify the function of these

receptors. For example, cAMP-mediated activation of protein kinase A induces the

phosphorylation of lns(1 ,4,5)P3 receptors in the rat cerebellum, thereby decreasing

their ability to release intracellular calcium by up to 90% (Supattapone et al., 1988).

in yitrQ, chronic ACTH treatment of rat glomerulosa cells results in the appearance of a

fasciculata-like phenotype (Hornsby et al., 1.974; Roskelley and Auersperg, 1990), and

ACTH plays a major role in the emergence of the fasciculata phenotype inyJ2

(Nussdorfer, 1986). As the major second messenger of ACTH action is cAMP, it is

- 150 -

possible that the ability of the Ins(1 ,4,5)P3 receptor to induce calcium fluxes across

intracellular membranes is decreased in rat fasciculata cells. Therefore, heterogeneity

among lns(1 ,4,5)P3 receptors or alterations to receptor phosphorylation states could

contribute to the differences observed in Ang Il-mediated [Ca2]i responses in GLOM

and FASC cells.

Locally acting paracrine or autocrine factors also modulate steroidogenic

function. For example, TGF-13, which is expressed in the bovine adrenal cortex, strongly

inhibits Ang II mediated steroidogenesis in bovine adrenocortical cells in yJljQ. (Hotta

and Baird, 1986; Feige et al., 1991). Thus, a zone specific expression of TGF-13 in the

rat could help to facilitate the differential Ang II responses of FASC and GLOM cells. To

our knowledge however, levels of TGF-13 expression in the different zones of the rat

adrenal cortex have as yet not been determined.

5. Expression of the ras Oncopene in GLOM Cells

After infection with KiMSV, primary GLOM cultures produced morphologically

altered cells that could be selectively passaged into secondary culture. These cells

(designated KGLOM) contained viral p21 ras and rapidly became oncogenically

transformed. Therefore, expression of the v-Ki ras oncogene and its effect on signal

transduction is compatible with both steroidogenic differentiation and oncogenic

transformation in pre-senescent rat zona glomerulosa cells.

Both clinical and experimental evidence strongly suggest that

carcinogenesis is a multistep process (Bishop, 1987). Initial assays utilizing DNA

isolated from tumour tissues identified activated ras genes on the basis of their ability to

transform immortal fibroblast lines (Barbacid, 1987). However, it has become

apparent that ras alone is inefficient in fully transforming numerous pre-senescent cell

types in i1r. (Weinberg, 1985). Therefore, it has been hypothesized that along with

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the expression of activated ras, further genetic or epigenetic changes are required to

fully transform pre-senescent cells. In certain experimental situations activation of

secondary co-operating oncogenes, or a loss of a tumour suppressor can act as the

additional genetic component (Land et al., 1983; Hunter, 1991). In others, tumour

promoters or escape from the control of surrounding normal cells can act as the

additional epigenetic factors (Dotto et al., 1988; Missero et al., 1990). In pre

senescent rat adrenocortical fibroblasts derived from tissue explants, v-Ki ras

expression eventually produces cells that are tumorigenic (Auersperg et al., 1977,

1981; Auersperg, 1978). However, in mass cultures complete oncogenic

transformation occurs very slowly over multiple passages and tends to be completed at

the time when surrounding untransformed cells senesce (Auersperg and Calderwood,

1984; Auersperg et aI., 1986). This process takes place independently of the levels of

ras expression and it can be accelerated if the ras and src oncogenes are co-expressed

(MacAuley et al., 1986; MacAuley and Pawson, 1988). These results suggest that

expression of the ras oncogene alone was not sufficient to fully transform adrenocortical

fibroblasts in mass culture. Thus, at first glance it was surprising that GLOM cells were

so rapidly transformed by v-Ki ras in this study. However, it should be noted that we

selected morphologically altered GLOM cells after K1MSV infection in primary culture.

In this way we isolated those cells that were most susceptible to ras-mediated

morphological changes and we removed them from the influence of surrounding normal

cells. Previously, simple genetic selection of pre-senescent rat embryo fibroblasts that

express activated ras p21 has been shown to abrogate the need for a second co-operating

oncogene to induce transformation (Land et al., 1 986). Therefore, it was not

unreasonable to find that a more phenotypically rigorous method of selecting v-ras

expressing cells (ie. morphologic alteration) resulted in the rapid transformation of

pre-senescent GLOM cells.

- 152 -

Interestingly, rapidly transforming KGLOM celisdid not express a 27 kD protein

(p27) that was immunoprecipitated from normal GLOM cells using the anti-ras

antibody. p27 is also absent from slowly transforming adrenocortical fibroblasts, but

not from their normal counterparts. Tryptic mapping of p27 suggests that it is a ras

homologue (MacAuley, 1987). Thus p27 may be an example of a ras related protein that

acts in the manner of a tumour suppressor, as it is no longer expressed in transformed

cells.

KGLOM cells formed tumours in immunosuppressed rats. Palpable (>1 cm3)

tumours formed rapidly, in less than two weeks, regardless the site of injection. In

general, the tumours were sarcomatous, although there were occasional carcinomatous

areas and rare carcinomas. The latter were composed of undifferentiated epithelial

clumps and cords and they were found where the tumours had formed within, or had

invaded into, the kidney or the adrenal gland. The ability of KGLOM cells to form both

sarcomas and carcinomas is consistant with the dual parenchymal/stromal nature of

these cells as discussed above. This same duality has been observed in ras transformed

explant-derived fibroblasts (Auersperg et al., 1981).

Due to its biochemical function, sub-plasmalemmal location, and transforming

potential, it has long been assumed that ras p21 is involved in the transduction of

proliferative signals (Macara, 1991). However, jnyjy, cellular ras p21 is expressed

in a number of non-proliferating, differentiating tissues (Chesa et al., 1987; Furth et

al., 1987). While the oncogenic transformation elicited by the expression of activated

ras p21 is usually associated with increased proliferation and a loss of differentiation,

there are cell types in which the opposite occurs. For example neuronal differentiation

is induced in adrenomedullary PC-12 tumour cells (Noda et al., 1985; Kremer et al.,

1991) and adipocyte differentiation occurs in 3T3-L1 pre-adipocytes (Benito et al.,

1991). Neuronal cell differentiation in PC-12 cells is ras specific as it can be blocked

- 153 -

by lovastatin, an inhibitor of ras function (Mendola and Barker, 1990). In PC-12 cells

p21 ras appears to impinge upon a pathway that stimulates protein kinase C, while in

3T3-L1 cells it may transduce signals normally initiated activation of the insulin

receptor which is a tyrosine kinase. Therefore, the effect of activated p21 ras

expression on signal transduction, proliferation or differentiation appears to be cell

type specific.

v-Ki ras mediated transformation of GLOM cells was associated with both an

increased proliferative potential and the maintenance of steroidogenic differentiation.

This suggested that v-Ki ras expression was associated with greater degree of

proliferative and steroidogenic autonomy. Such autonomy is a hallmark of malignant

cells. KGLOM cells produced corticosterone constitufively and responded to ACTH and

cAMP treatment with increased steroidogenesis. However, this steroidogenic increase

was the greatest after after a chronic (ie. >24 hr) trophic hormone treatment. Acute (ie.

8 hr) trophic hormone treatment resulted in only a small increase in corticosterone

production.

Acute trophic hormone treatment of adrenocortical cells activates signal

transduction pathways leading to substrate delivery to steroidogenic enzyme systems.

For example, acute ACTH treatment induces steroidogenesis in a cAMP dependent manner

by increasing the association of cholesterol with P-45Oscc (Jefcoate et al., 1987). In

contrast, chronic ACTH treatment acts by inducing steroidogenic enzyme gene expression

(Simpson et al., 1990). Thus, it is probable that steroidogenic enzyme expression is

required prior to the maximal ACTH or cAMP-mediated steroidogenic increases in

KGLOM cells. This also occurs in ras transformed ovarian granulosa cells which respond

to chronic cAMP treatment with increased progesterone production (Amsterdam et al.,

1988; Pan et al., 1991). Importantly, after passaging, uninfected GLOM cells were no

longer steroidogenic under any conditions. Therefore, the maintenance of steroidogenesis

- 154 -

and ACTH/cAMP responsivenss observed in KGLOM cells was not due to the carry over of

normal GLOM cells during the selection process.

Lovastatin, which acts as a pharmacological inhibitor of p21 ras function by

preventing membrane localization of the protein (Schafer et al., 1989; Chen et al.,

1991), caused some reversion to a flattened fibroblastic phenotype in KGLOM cells after

24 hrs. This occured after an initial rounding of the cells during the first 12 hrs after

treatment. In addition, lovastatin inhibited steroidogenesis in KGLOM cells to a much

greater degree than it did in primary cultures of GLOM cells. Therefore, the maintenance

of differentiation appeared to depend on v-ras expression rather than on other aspects of

the transformed phenotype.

Expression of activated ras genes has been associated with both the

transformation and steroidogenic differentiation of other cell types as well. For example,

v-Ki ras transformation enhances the limited steroidogenic potential of adrenocortical

fibroblasts (Auersperg et al., 1990) and it is associated with the maintenance of

progesterone production in ovarian granulosa cells (Pan et al., 1991). Specifically,

granulosa cells immortalized by transfection of a complete SV-40 genome are not

steroidogenic but they can be induced to produce progesterone in response to cAMP after

expression of an activated Ha-ras oncogene (Amsterdam et al., 1988). Activated Ki-ras

p21 expression also induces non-steroidogenic rat ovarian surface epithelial cells to

produce progesterone (Pan et al., 1991). Therefore, it appears that in general,

steroidogenic differentiation is positively influenced by ras p21 expression. The

mechanism responsible for this differentiative effect is unknown, but presumably it

involves the stimulation of steroidogenic, “template” specific signal transduction

pathways which are impinged upon by the ras oncogene. It must be noted that

steroidogenic differentiation can be associated with the expression of oncogenes other

than ras. For example, it has recently been reported that early gene regions of the SV

- 155 -

40 virus alone are able to produce immortalized ovarian granulosa cells that respond to

cAMP by producing progesterone (Rao et al., 1991).

Steroidogenic cells respond to the presence of numerous trophic hormones. While

they always stimulate steroidogenesis, these trophic hormones can have opposing effects

on cell proliferation depending on the signal transduction pathway that is activated. For

example, the activation of adenylate cyclase by ACTH increases steroidogenesis and

inhibits ceilular proliferation in adrenocortical cells (Ramachandran and Suyama,

1975). Adrenocortical cells also respond to angiotensin II, which activates the

phosphatidyl inositol specific PLC pathway (Barrett et al., 1989). The resulting

production of DAG and lns(1 ,4,5)P3 increases both steroidogenesis and cell

proliferation, presumably by stimulation of PKC (Gill et al., 1980). As we have shown,

expression of viral p21 ras is also associated with a steroidogenic and proliferative

phenotype in zona glomerulosa (GLOM) cells. Thus, p21 ras could act by impinging upon

the PLC mediated pathway in these cells. If this were the case it could explain the

inability of K1MSV to transform zona fasciculata (FASC) cells. As discussed above, there

is an interruption in the Ang Il-mediated pathway at a point distal to PLC stimulation but

proximal to PKC stimulation in these cells. Therefore, the differentiation specific

alteration in the signal transduction template of FASC cells may make them non-

responsive to the transforming effect of the ras oncogene. This is further supported by

the finding that stimulation of PKC directly with phorbol esters increases proliferation

of fasciculata/reticularis parenchymal cells (McAllister and Hornsby, 1987). It should

be noted however, that in our experiments other possible explanations for the non

responsiveness of FASC cells to viral ras transformation have not been ruled out. These

include possible differences in KiMSV infectivity, and the low rate of proliferation of

FASC cells in primary culture which may suppress pro-virus integration and resulting

viral gene expression (Shih et al., 1982). Oncogenic ras p21 could also act to enhance

steroidogenic differentiation by autonomously activating tyrosine kinase-mediated

- 156 -

pathways. For example, IGF-1, whose receptor is a transmembrane tyrosine kinase,

enhances the cAMP-induced expression of steroid hydroxylase genes (Naseerudin and

Hornsby 1990).

6. Parenchymal Cell Proliferation and Differentiation in MIX Cultures

Homogeneous FASC and GLOM cells did not retain their steroidogenic

differentiation in long term primary culture or after passaging. This decline has been

noted previously in rat fasciculata cells obtained by de-capsulation (Ryback and

Ramachandran, 1981b). Obviously, the rather primitive culture conditions used in

these studies may have played a part in this loss of differentiation. For example,

culturing adrenocortical cells on extracellular matrices or in atmospheres with a low

02 tension enhances long term differentiation by maintaining the expression and

preventing the oxidative degradation of steroidogenic enzymes (Gill et al., 1980;

Hornsby, 1980; Georgiu et at., 1987). However, in the present study, an adrenocortical

population which consists of a mixture of parenchymal, endothelial and stromal cells

(designated MIX) has been identified that both maintains and produces large numbers of

differentiated steroidogenic cells in long term primary culture. This differentiation

occured in the absence of trophic hormone treatment, and it was associated with a high

rate of proliferation and a cellular multilayering that did not occur in the homogeneous

FASC and GLOM cultures.

During embryogeriesis, the adrenal cortex arises from a condensation of

mesodermal cells that proliferate rapidly and initiate steroidogenic differentiation prior

to the production of ACTH by the pituitary (Idleman, 1970; Jost, 1975; Nussdorfer,

1986). With the appearance of ACTH, adrenocortical growth and differentiation are

greatly accelerated and brought to completion. In the adult, the adrenal cortex retains a

regenerative potential as evidenced by the the re-appearance of a complete gland after

- 157 -

enucleation removes all but a sub-capsular layer of parenchymal cells (lngle and

Higgins, 1938; Taki and Nickerson 1985). Therefore, the adult adrenal cortex is also

capable of both proliferation and steroidogenic differentiation. It appears that MIX

cultures contain the cells and the conditions that allow for similar, comcomitant

proliferation and differentiation.

When adrenocortical parenchymal cells are treated with ACTH or cAMP,

steroidogenesis is increased and cells often morphologically round up after retracting

from the underlying substratum (Hall, 1984; Hornsby et al., 1989). Long term MIX

cultures produced increasing numbers of rounded cells that emerged from an underlying

bed of fibroblastic cells. This process occurred in the absence of ACTH or cAMP

treatment. The rounded MIX cells were positive for markers of steroidogenic

differentiation including: cytoplasmic lipid; t5,313-HSD activity; P-45Oscc

expression; and the production of fluorogenic steroid and corticosterone. Therefore, in

MIX cultures parenchymal cell differentiation occurred independently of trophic

hormone treatment.

In multilayered MIX cultures, the underlying fibroblastic cells may act as a

feeder layer that is conducive to the differentiation of an associated population of pre

existing parenchymal stem cells. In a number of jnyJiio systems, feeder layers act to

support proliferation rather than differentiation (Watt, 1991). However, the formation

of MIX multilayers was associated with sustained proliferation after confluence. This led

to high saturation densities and extreme cell crowding that, in turn, may have been

conducive to the induction of proliferation and differentiation. The heterogeneous

cellular multilayering in MIX cultures (ie. fibroblastic cells on one side of the

multilayer and spherical, parenchymal cells on the other) may represent an iny.Ltrn

recapitulation of the normal in yJy.Q adrenocortical histoarchitecture where

proliferating parenchymal cells associate with subcapsular fibroblast-like cells prior

to differentiating and moving inwards within the cortex. In support of this suggestion, a

- 158 -

similar cellular multilayering occurred after KiMSV (ras) infection of GLOM cultures.

This also led to the production of proliferating, steroidogenic cells.

As discussed above, it is likely that parenchymal cells arise from multipotent

sub-capsular fibroblasts that differentiate into steroidogenic cells as they move into the

zonae of the adrenal cortex (lannacone and Weinberg, 1987). Thus, it is reasonable to

speculate that the fibroblastic cells themselves could have given rise to the steroidogenic

cells that emerged from the MIX culture multilayers. However, the experimental

evidence does not exclude the possibility of another alternative, that the flattened cells

which undergo differentiation in MIX cultures represent parenchymal cells that have

initially modulated from an epithelial to a fibroblastic morphology, as was seen in

normal GLOM cultures.

The production of soluble factors which act in a paracrine fashion may also play a

role in the differentiative process that takes place in MIX cultures. For example, long

term bone marrow cultures, which contain a mixture of adherent stromal cells,

hematopoietic stem cells and detaching colonies of differentiating hematopoietic cells,

have been utilized to identify a number of the critical cytokines that act in a paracrine

fashion during stem cell renewal and differentiation (Dexter et al., 1979; Humphries et

al., 1981; Metcalf, 1989; Eaves et al., 1991). As MIX cultures also produce

differentiated cells that can be removed from an underlying stroma, they could prove

useful in identifying those soluble factors that are important in the renewal and

differentiation of cells derived from steroidogenic tissues. One factor that could be

involved is IGF-1 which is produced in the adrenal cortex kuthi2. (Han et al., 1987) and

enhances both the proliferation (Horiba et al., 1987; van Dijk et al., 1988) and

steroidogenesis (Nasserrudin and Hornsby, 1990) of adrenocortical cells in yJfl.

Extracellular matrix (ECM) production in MIX cultures may also effect

steroidogenic differentiation. It has been shown that fibronectin and Matrigel, a

basement membrane derived ECM, will enhance steroidogenic enzyme expression in

- 159 -

bovine adrenocortical cells (Flasch et al., 1990; Hornsby, 1991). In addition,

adrenocortical fibroblasts derived from explant cultures have been found to produce

large amounts of an heterogeneous ECM, the production of which varies depending on the

differentiation state of the cells (Turley, 1980; Auersperg et al., 1990). Therefore, the

fibroblastic cells in MIX cultures may produce an ECM that is conducive to steroidogenic

differentiation of a parenchymal population. This could be tested by treating MIX

cultures with ECM production inhibitors such as cis-hydroxy-L-proline and

subsequently determining the number of differentiating A5,313-HSD positive cells.

Treatment of bone marrow cultures with this inhibitor prevents the stromal cell-

dependent growth and differentiation of hematopoietic stem cells (Zuckerman et al.,

1985).

After selective passaging, rounded MIX cells re-formed multilayers wherein

refractile, lipid containing cells adhered to large, flattened fibroblasts. Although only

the refractile cells responded morphologically to cAMP treatment, both cell types

expressed P-45Oscc. At this point it cannot be stated with certainty that the appearance

of differentiated steroidogenic cells in MIX cultures was not due to the presence of an

autonomously differentiating cell population that was not present in FASC or GLOM

cultures. However, the progressive production of these steroidogenic cells in longterm

primary culture, and the intimate relationship between refractile and fibroblastic cells

in secondary culture suggests that heterologous cell-cell interactions are involved.

7. Conclusions

Utilizing discontinuous density gradients we have separated adrenocortical

parenchymal cells into homogeneous populations which have the characteristics of zona

fasciculata (FASC) and zona glomerulosa (GLOM) cells respectively. This method

provides a quick and easy alternative to decapsulation for separating adrenocortical

- 160 -

parenchymal cell types, and results in preparations of superior purity with minimal

stromal or endothelial cell contamination.

Both FASC and GLOM cells were steroidogenic in primary culture. However, they

exhibited very different morphologies. FASC cells formed epithelial monolayers while

GLOM cells modulated to a fibroblastic form and expressed vimentin. This suggests that

GLOM cells may have both parenchymal and stromal characteristics, while FASC cells

have a more restricted parenchymal phenotype.

In the course of differentiation, cells arising from a common precursor often

acquire similar but subtly different phenotypes. The cells of the different adrenocortical

zones have a common origin and appear to differentiate along a spatial and developmental

continuum. It appears that a mesodermally derived sub-capsular stem cell gives rise to

zona glomerulosa cells which in turn give rise to zona fasciculata cells (Wright and

Voncina, 1977; Ueberberg et al., 1982; Farcnik and Auersperg, 1984; Taki and

Nickerson, 1985; Zajicek et al., 1986; lannaccone and Weinberg, 1987). The dual

stromal and parenchymal charateristics exhibited by GLOM cells in primary culture are

consistant with them being in an intermediate developmental position, located between

the mesodermal stem cells and the parenchymal zona fasciculata (FASC) cells.

In the rat adrenal cortex, a specific phenotypic consequence of the zonal

interconversion from a glomerulosa to a fasciculata cell type is the loss of steroidogenic

responsiveness to the trophic hormone Ang II (Haning et al., 1970; Tait et al., 1974;

Douglas et al., 1978; Braley et al., 1986; Whitley et al., 1987). Our findings suggest

that this lost responsiveness arises, at least in part, due to a modification within the Ang

Il-mediated signal transduction pathway. That this modification takes place precisely at

the level of increased intracellular calcium indicates that developmental decisions can be

enacted via alterations within signal transduction pathways distal to initial receptor

mediated events and proximal to downstream effector events.

- 161

The phenotypic effects of activated p21 ras expression are often associated with

alterations to signal transduction pathways (Macara, 1991). It appears that these

alterations are cell-type, and thus transduction-template, specific (Auersperg and

Roskelley, 1991). In GLOM cells, activated ras expression was associated with both

oncogenic transformation and the enhanced maintenance of steroidogenic differentiation.

Therefore, ras may lead to the autonomous activation of PLC, or tyrosine kinase

mediated, signal transduction pathways in GLOM cells as these pathways are normally

associated with differentiation and proliferation in steroidogenic cells (Gill et al., 1980;

Barrett et al; 1989; van Dijk et al., 1988, Nasseerudin and Hornsby 1990).

While FASC and GLOM cells de-differentiated in long term primary culture, a

mixed parenchymal/stromal (MIX) population did not. Instead, MIX cultures produced

large numbers of differentiated cells that could be selectively passaged. These

differentiated cells emerged from highly proliferative cellular multilayers that were

morphologically similar to ACTH/cAMP treated cells or to those cells that appeared in

KiMSV (ras) infected GLOM cultures. Thus, cellular multilayers may produce a

microenvironment that is conducive to the steroidogeriic differentiation of adrenocortical

stem-like cells. The specific characteristics associated with this microenvironment that

are crucial for its differentiative effect are as yet unknown. Some possibilites include:

the establishment of heterologous cell-cell interactions; production of extracellular

matrices; and production of soluble growth factors that act in a paracrine fashion.

Further examination and manipulation of this novel adrenocortical culture system

should provide clues as to the specific factors which are crucial to the modulation of the

steroidogenic phenotype in a differentiative setting.

- 162 -

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177

Appendix A: Percoll Gradient Preparation

Stock Isosmotic Percoll

Percoll consists of a colloidal suspension of microscopic polymer coated silica

particles in water. While it has a high density (- 1.130 g/rnl), the osmalality is low

(5-10 mOsM). Thus, mixing 9 parts percoll with 1 part lox concentrated tissue cultue

medium produces a solution that is isosmotic with 1 X culture medium. This is known as

stock isosmotic percoll (SIP). Typically this was prepared by mixing 90m1 percoll with

10 ml iox DME/F12 medium.

Percentage SIP Steps

Percoll also has a low viscosity. Therefore, separation in percoll gradients is

dependent solely on particle (ie. cell) density. In a discontinuous, pre-made gradient the

cells settle at interfaces that have a density nearest their own. Discontinuous gradients

are advantageous because they can be custom made and they are highly reproducible. The

gradient used here consited of 8 steps with a total of7 density interfaces between the

steps. Each step consisted of a particular percentage of SIP diluted with 1X DME/F12.

Step # %SIP SIP (mfl ix DME/F12 (ml)

1 15% 1.5 8.5

2 25% 2.5 7.5

3 30% 3.0 7.0

4 35% 3.5 6.5

5 40% 4.0 6.0

6 45% 4.5 5.5

7 50% 5.0 5.0

8 65% 6.5 3.5

Gradient Preparation

The gradients were prepared in clear plastic, round bottom centrifuge tubes

(15m1; 13 x 100 mm; Nalgene). Before using, each % SIP step was thoroughly mixed

by pipetting. One ml of each step was then layered carefully into the tube from heaviest

178 -

to lightest (ie. step #8 first, step #1 last). This was done with an automatic pipettor to

reduce turbulence that can occur when using manual bulbs. Each step was slowly

released onto the step below it ( ie. #7 on top of #8) at a rate of approximately 30

sec/mi. As this was done, the pipette tip was kept just above the meniscus and fluid was

released along the wall of the tube to minimize turbulence or mixing with the step below.

Upon completion, the interfaces between % SIP steps could be seen as sharp refractory,

horizontal planes within the gradient. The gradients were stable for several hours.

Usually, they were prepared the morning of the experiments, prior to cell dissociation.

They were not constructed the night before, as the interfaces became slightly fuzzy

which reduced the efficiency of the cell separation.

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Appendix B: lns(1 .4.5)PlDetermination

The mass of lns(1 ,4,5)P3 in Ang II or calcium ionophore treated cells was determined

using a commercially available, competitive, isomer-specific radioligand binding assay

(Amersham). The assay was carried out essentially as to manufacturers specifications

with slight modifications (see Palmer et al., 1989).

lns(1 .4.5)Pp Extraction

Cells in suspension or in culture were incubated with Ang II or calcium ionophore ABr

231 87 in 125 tl of HBSS. At timed inteivals the incubations were terminated by the

addition of 25 il of cold 10% (v:v) percioric acid. Inositol phosphates were then

extracted into the medium during a 20 mm period on ice. The acidified samples werethen centrifuged 12,000 Xg for 2 mm (4°C) to remove cellular debris. Supernatants

were neutralized to pH 9.0 by the addition of 12.5 tl of 1.5M KOH with 60mM HEPES.

100 j.tl aliquots of the neutralized samples were then used for assay.

Assay Procedure

Reagents

-Assay buffer: 0.1 M Trix, 4mM EDTA, 4mg/mi BSA, pH 9.03H lns(1 ,4,5)P3 tracer: Enough tracer was used to give 4-6000 cpm per lOOjil in

water.

-Ligand binding protein: Isolated from bovine adrenocortical microsomes a slurry was

diluted in 10Oil assay buffer to give 25-40% binding of the

labelled tracer.

-lns(1 ,4,5)P3 standards: From a stock solution of 4iiM, standards were diluted to give

0.19, 0.38, 0.76, 1.5, 3.1, 12.5 and 25 pmol per 100111 ifl

water. For non-specific binding, 400pmol of standard was

used to fully compete out the tracer.

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Protocol

Reagents were added to 10 X 55 mm polypropylene tubes for each of the following

categories as summarized below (all volumes in ill):

Total Counts Non-Specific Blank Standards Samples

(TC) Binding (NSB) (Bo) (lñP’). (Extract)

Buffer 100 100 100 100 100ddH2O 200 - - -

Extracted medium - - 1 00

Standard - 1 00 1 00 -

Sample - - - 100

Tracer 100 100 100 100 100

Binding Protein - 1 00 1 00 1 00 1 00

The reagents were added in the order listed from top to bottom in the above summary. All

operations were carried out in a walk in fridge (4°C). Tubes were thoroughly vortexed

and left to stand for 15 minutes on ice. To separate free and bound tracer the tubes were

centfrifuged at 2000 Xg for 10 mm followed by decanting of the supernatant. The bindingprotein pellet, with bound tracer, was then resuspended in 200 jil dH2O by thorough

vortexing. The suspension was then placed into scintillation vials with 3 ml scintillation

fluid (ACS, Amersham) and counted for 5 mm.

Calculation of Results

1) Percent of tracer bound in the absence of standard/sample (% Bo) was calculated

using the following equation (25-40% was considered to be acceptable):

%Bo = (Bo cpm-NSB cpm) X100

(TC cpm)

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C)a-C)

0E00)0

2) Normalized percent bound (%B/Bo) for each standard/sample was calculated using

the following equation:

%B/Bo = (Standard or Sample cpm-NSB cpm) X 100(Bo cpm-NSB cpm)

A standard curve was generated by plotting %B/Bo as a function of the logo lnsP3 per

tube. Amounts of lnsP3 per sample were then extrapolated from this curve. Medium

blanks were then subtracted from all samples (these were negligable).

What follows is a representative standard curve from one experiment.

2

0

log pmole lnsP3 = 1.65 - 0.0243 %B/Bo R = 1.00

(each standard in duplicate)

0 20 4Ô 60 80 100

%B/Bo

- 182 -

Appendix C: p21 ras lmmunoprecipitation

GLOM or KGLOM cells in passage 5 were grown to subconfluence in 60mm dishes

and then labelled with 35S methionine prior to lysis and immunoprecipitation.

Protocol

1)CelI Labelling

-Rinse 2 times with HBSS.

-Pre-incubate 1 hour with methionine free DMEM medium (Sigma) supplemented with

5% dialyzed FBS.

-Incubate in 2m1 of the methionine free medium with 200 j.tCi 35S methionine (35S

Trans label, ICN) for 24 hours.

-Chase for 1 hour with unlabelled methionine containing medium and 10% FBS.

2) Cell Lysis

-Rinse cells with cold HBSS and stand on ice.

-Add 500 p.1 radiolabelled immunoprecipitation assay (RIPA) buffer with protease

inhibitors. -Swirl RIPA buffer over surface of cells evenly for 2 minutes being

careful to keep on ice to minimize proteolysis.

-Scrape lysed cells from dish and wash and scrape again with 500 jil RIPA buffer.

-Clear lysates by centrifuging 25,000 Xg for 90 mm at 4°C.

-Calculate volume of cleared lysate required to yield 1 X 108 TCA precipitable c.p.m. by

scintillation counting.

3) Immunoprecipitation

-On ice, dilute 108 c.p.m. aliquots of cell lysate to 250 p.1 with RIPA buffer.

-Add 70 p.1 of protein A sepharose (PAS) linked to 2nd antibody with (experimental) or

without. (control) 1 St antibody.

-Incubate and rotate (—20 r.p.m.) for 4 hours at 4°C.

-Wash immunoprecipitates by centrifuging 16,000 Xg 5 mm. and resuspending in 500

jii fresh RIPA buffer 4X.

-Release PAS bound proteins by boiling in 50 p.1 sample buffer for 5 mm.

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-Separate immunoprecipitated proteins by SDS-PAGE on 12.5% acrylamide gels.

-Visualize immunoprecipitated proteins by autoradiography by placing the dried gel

against X-ray film for 3-6 days (XAR-5 film, Kodak).

Reagents

1) Radiolabelled Immunoprecipitation Assay (RIPA) Buffer

Compound [Stock] Volume [Final]

Sodium phosphate buffer 1 M 1.0 ml 10 mMNa2HPO4 adusted to pH7.2

NaCI 5M 3.1 ml 154mM

Triton X-100 (Sigma) 100% 1.0 ml 1% v:v

Sodium deoxycholate 5% w:v 10 ml 0.5%w:v(Fischer)

Sodium dodecylsulfate 10% w:v 1 .0 ml 0.1 %w :v(Sigma)

ddH2O N/A 84 ml N/ATOTAL 100 ml

- 184 -

2) Protease Inhibitors

Just before lysis the following protease inhibitors are added to 4.85 ml RIPA buffer

Inhibitor [Stock] Volume [Final]

Aprotinin (Sigma) 10 mg/mI 50 jil 100.tg/ml

Leupeptin (Sigma) 100 mM 50 tl 1 mM

Phenylmeghylsulfonyl 100 mM 50 jil 1 mMflouride (PMSF, Sigma)

3) Protein A Sepharose (PAS)

-Suspend 100 mg of PAS in 1 ml of 0.1 M glacin (pH 2.5) and rotate 20 mm at 4°C.

-Wash with by centrifuging 10,000 Xg and resuspending in PBS (pH 7.2) 5 times;resuspend final time in 250 jtl PBS with 1 mg/mI BSA.

- Add 30 ml of 2nd antibody (Rabbit anti rat lgG, Cappel, manufacturers optimaldilution); rotate 4°C overnight.

- Wash 2X with 280 jil PBS containing 1 mM PMSF.

-Add 8 jil anti ras 1st antibody (monoclonal Y13-259, 1.2 mg/mI); rotate 4°C for 2hours.

-Wash 2 times with 280 .tl PBS/PMSF.

-Add 70 j.tl to each aliquot of 250 tl cleared lysate.

- 185 -

41 Sample Buffer

Compound [Stock] Volume [Final]

glycerol (Sigma) 1 00% 0.8 ml 8%w :v

2-mercaptoethanol 1 00% 0.5 ml 5% v:v(Sigma)

Sodium dodecylsulfate 1 0% 2.3 ml 2.3%

Tris (Sigma) 1.0 M 625 LtI .0625M

ddH2O N/A 5.775 ml N/ATOTAL 10.0 ml