Intracellular angiotensin II inhibits heterologous receptor stimulated Ca2+ entry

I do not defeat the world's crown of miracles

Nor does my path wreck the mysteries I meet

in flowers, eyes, lips or graves.

While light of others

Scatters the magic of the sealed unseen,

In darkness depths,

But me, with my opaque spark,

I widen the world's own mysteries -

and, just as moonshine deepens

rather than diminishes the night's obscurity,

so I enrich the dark horizon with ample thrills of sacred

mystery

and all incomprehensible transforms

into even greater unknowns

right before my eyes -

because I love

flowers and eyes and lips and graves.

Lucian Blaga (1895-1961)

Translation by Cătălin Filipeanu and Robert Henning

Rijksuniversiteit Groningen

Intracellular angiotensin II: from myth to reality

ProefschriftProefschriftProefschriftProefschrift

ter verkrijging van het doctoraat in de

Medische Wetenschappen

aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magnificus, dr. D.F.J. Bosscher,

in het openbaar te verdedigen op

woensdag 20 juni 2001

om 10.30 uur

door

Cătălin Filipeanu

geboren op 11 oktober 1968

te Vaslui, Roemenië

Promotor: Prof. dr. D. de Zeeuw

Referenten: dr. S.A. Nelemans

dr. R.H. Henning

ISBN 90-367-1414-1

NUGI 743

Reading Committee: Prof. dr. H. Haller

Prof. dr. J.G. De Mey

Prof. dr. J. Zaagsma

Paranimfen: dr. H. Bos

dr. L.E. Deelman

Financial support by the Netherlands Heart Fundation and The Groningen

University Institute for Drug Exploration (GUIDE) for the publication of

this thesis is gratefully acknowledged.

Copyright © - 2001 by Cătălin Filipeanu. No part of this publication may bereproduced or transmitted in any form or by any means, without writtenpermission from the author.

Typesetting and layout by Cătălin FilipeanuCover design: Cătălin Filipeanu and Leo Deelman

Printed by van Denderen B.V. Groningen

Financial support by AstraZeneca BV, Bristol-Myers Squibb BV and

Medtronic Bakken Research Center BV for publication of this thesis is

greatly acknowledged.

7

CONTENTS

Chapter 1: General introduction and aim of this thesis. 9

Chapter 2: Contractile effects by intracellular angiotensin II via receptorswith a distinct pharmacological profile in rat aorta. 21

Chapter 3: Intracellular angiotensin II elicits Ca2+ increases in A7r5vascular smooth muscle cells. 39

Chapter 4: Intracellular angiotensin II inhibits heterologous receptorstimulated Ca2+ entry. 63

Chapter 5: Intracellular angiotensin II and cell growth of vascularsmooth muscle cells. 77

Chapter 6: Contribution of internalization and recycling to angiotensinAT1 receptor desensitization to rat aorta contractility. 97

Chapter 7: Intracellular angiotensin II: from myth to reality. 113

English summary. 137

Nederlandse samenvatting. 141

Sumarul in limba român!. 145

Curriculum vitae. 149

Publication List. 151

Acknowledgements. 155

Chapter 1

General introduction and aim of this thesis.

Chapter I

10

Receptors and messengers

The cell is the ultimate target for all physiological or pharmacological stimuli.

At this level, the whole array of input signals is integrated, ensuring the

adaptation of the cells to environmental stimuli. Most of these stimuli consist of

chemical compounds transporting a biologic information, and represent the so

called ‘first messengers’. They deliver their message by binding to specialized

proteins, localized at the level of plasma membrane. These proteins have a

specific structure enabling them to recognize specific first messengers and

related molecules and have therefore been designated the name ‘receptors’.

After binding the specific signaling compound, the receptor is transmitting the

information to the cell, generating a specific response. This is accomplished by

an intricate system that may be different from receptor to receptor. In the case

of the seven transmembrane spanning receptors it involves the binding of the

activated receptor to G proteins. In turn, activated subunits of G-proteins have

multiple cellular targets, thus activating further signaling. Although the

receptors are specific, multiple first messengers can use the same G protein.

Stimulation of the receptor can induce rapid or long-term cellular responses.

The rapid cellular responses (e.g. smooth muscle contraction) are triggered by

production of intracellular ‘second messengers’. The definition of second

messengers is yet a matter of debate, but amongst the best characterized are

cyclicAMP (cAMP) and cyclicGMP (cGMP), inositol (1,4,5)trisphosphate

(InsP3), diacylglycerol (DAG) and calcium (Ca2+). Long-term cellular effects of

the G protein coupled receptor activation (i.e. cellular growth) are driven by

‘third messengers’ represented by kinases and phosphatases, which are

activated by second messengers (fig 1).

Angiotensin II receptors

Angiotensin II is one of the many hormones modulating biologic events in life

forms ranging from insects to humans. It is formed from the decapeptide

angiotensin I under the action of various enzymes like angiotensin converting

enzyme and chymase (fig 2). Angiotensin II is particularly important because

General introduction

11

of its involvement in many kidney and cardiovascular diseases (table 1). It has

also been shown that angiotensin II modulates neuronal cell differentiation

during fetal development.

first messenger

Receptor extracellular

intracellular

G protein

second messengers

third messengers

Angiotensin II exerts its effects by interaction with specific receptors localized

in cell plasma membrane. The existence of angiotensin II receptors was first

implied in the 70’s when it was demonstrated that radioactive angiotensin II

binds to the crude membranes from adrenal gland (Lin & Godriend, 1970; Catt

et al., 1974). Further characterization of the receptor types in rat liver (Gunther,

1984) and renal tubular membranes (Brown and Douglas, 1983) indicated the

existence of the multiple receptor subtypes. The synthesis of selective receptor

antagonists such as losartan and PD123319 enabled a further pharmacological

characterization of two distinct angiotensin II receptors, called AT1 and AT2

(Bumpus et al., 1991).

plasma membrane

Figure 1. Schematic representation of the steps involved in cellular signal

Chapter I

12

Advances in molecular biology made it possible to clone the type 1 angiotensin

receptor from bovine adrenal (Sasaki et al., 1991) and of the type 2 from a PC12

rat pheochromocytoma cell line (Kambayashi et al., 1993). The human AT1

receptor gene is located on the q22 band of chromosome 3 (Curnow et al.,

1992) and produces a receptor with 359 amino acids having a predicted

molecular weight of 40.9 kD. In fact, its molecular weight is increased by

posttranscriptional glycosilation (Murphy et al., 1991). In rodents, this receptor

is found in two isoforms: AT1A and AT1B, which share a high degree of

homology (Sasanura et al., 1992). In contrast, only one isoform exists in

humans, with an amino acid sequence 95 % similar to that of bovine or rat AT1

receptor (Bergsma et al, 1992).

Surprisingly, the AT2 subtype angiotensin receptor has only 34 % homology

with the AT1 receptor but is highly conserved among different species

(Mukoyama et al., 1993). It has 363 amino acids and its gene is localized on

q22 region of long arm of chromosome X in humans. AT2 molecular weight is

estimated between 68 kDa (human myometrium) to 113kDa in PC12 cells.

However, this is reduced to 31 kDa after proteolytic digestion suggesting a

substantial glycosilation of the receptor (Servant et al., 1994).

Both types of AT receptors show different patterns of localization. AT1 is the

main subtype found in heart and adult vasculature, kidney and some parts of

the brain. In contrast, AT2 is the prominent angiotensin receptor subtype in

fetal tissues, in the uterus and skin, and some parts of the brain.

O

OH

2HN

NHNH

NH2

2HN

NH

2HN

NH

ASP ARG VAL TYR ILE HIS PRO PHE HIS LEUH OH

OH

+

+

Angiotensin II

Angiotensin I

+

R:

Figure 2. Amino acid composition of human angiotensin I and angiotensin II.


13

Table 1. Selected diseases demonstrated to be induced by alteredangiotensin II levels

organ Disease Mechanism Referenceblood

vesselsHypertension Smooth muscle

contraction, cellproliferation

Menard et al., 1997Janssen et al., 1996

bloodvessels

Atherosclerosis cell migration,inflammatory

response

Arakawa et al., 2000

heart Hypertrophy cell proliferation,matrix protein

production

Dostal and Baker,1992

kidney Glomerulosclerosis TGF!, matrix proteinproduction

Miller et al., 1991Wolthuis et al., 1991

The signal transduction pathways mobilized by these two subtypes are partially

characterized (table 2). Although it has been shown that AT1 receptor interacts

with several G protein subtypes, its major actions occur through Gq interaction.

Via this G protein subtype, it stimulates phospholipase C, which is

accompanied by a rapid generation of InsP3 and diacylglycerol. These events,

accompanied by activation of different Ca2+ channels, induces intracellular

calcium ([Ca2+]i) increase.

Table 2. Effects of AT1 and AT2 subtype receptors on various signal

transduction pathways.

AT1 AT2

Phospholipase C +++ No effect/-

Phospholipase D + No effect

Phospholipase A2 ++ +

T-type Ca2+ channels ++ --

MAP kinase +++ ---

Ceramide production No effect ++

Further, AT1 activity stimulates phospholipase D and phospholipase A2,

generating diacylglycerol and arachidonic acid. Phosholipase A2 may be also

stimulated by the AT2 subtype receptor, which has also been found to mediate

Chapter I

14

ceramide production (Gallinat et al., 1999). Earlier studies proposed that AT2

receptor is not interacting with G proteins (Bottari et al., 1991), but more

recently it has been shown that AT2 binds to Gi"2 and Gi"3 subunits (Zhang and

Pratt, 1996). Also, it has been suggested that AT2 receptor can inhibit

phospholipase C, but this evidence should be used with caution as it has been

obtained at very high concentrations of angiotensin II and AT1 or AT2

antagonists (#10-4 M, Gyurko et al., 1992). The two AT receptor subtypes have

opposite effects at least on T-type voltage dependent Ca2+ channels: AT1

stimulate the channel, whereas AT2 has an inhibitory effect (Buisson et al.,

1994).

At the functional level, the opposite actions of the two subtypes are clearer.

AT1 stimulation mirrors most of the hypertensive effects of angiotensin II,

inducing vascular smooth muscle contraction, cell growth, inhibition of

apoptosis, extracellular matrix deposition and cell migration (Unger et al.,

1996). In contrast, AT2 is promoting apoptosis and inhibition of cell growth,

cell differentiation, collagen production and fibrosis (Horiuchi et al., 1999).

Specific pharmacological tools have been developed for these two angiotensin

receptor subtypes. Several AT1 receptor antagonists have become available

(Vanderheyden et al., 1999), of which the non-peptide ligand losartan with a Ki

of 30 nM is most widely employed (Whitebread et al., 1989). No AT1 specific

agonists have been developed to date, whereas (p-amino-Phe6)-angiotensin II

and CGP42112A are preferential agonists of AT2 subtype. PD123177 with a Ki

of 50 nM and the related compound PD123319 are selective non-peptide AT2

receptor antagonists.

In addition to the AT1 and AT2 receptor, other angiotensin receptor subtypes

have been reported. The first additional subtype has been found in a

neuroblastoma that does not bind either losartan or PD123319 (Chaki and

Ingami, 1992). It was designated AT3 receptor and to date have been found

only in cell lines. Another subtype, called the AT4 receptor recognizes

preferentially angiotensin IV and has a widespread localization (Harding et al.,

1994). Further, other nonAT1/nonAT2 receptors were found in chicken vascular


15

smooth muscle (le Noble et al., 1996), human placenta (Li et al., 1998),

intestinal epithelium (Smith, 1995) and human heart (Regitz-Zagrosek et al.,

1996). Thus, although AT1 and AT2 mediate many angiotensin II effects, other

additional angiotensin receptor subtypes may explain patho-physiological

effects of angiotensin II as well.

An intracellular RAAS system?

Endogenous angiotensin II activating these multiple receptor subtypes may

originate from circulating or locally formed angiotensin II. For a long time the

renin-aldosteron-angiotensin was regarded as an endocrine circulatory system

(Dzau 1988). However, in the past two decades the existence of a local renin

angiotensin systems has been demonstrated (Lee et al., 1993; Pinto et al., 1996;

Danser et al., 1999). Based on different observations, it has been suggested that

the demonstrated local renin-angiotensin systems might be extended to the

intracellular level. In addition, circulating angiotensin II accumulates in tissues

and this process extends its half-life (van Kats et al., 1997). Physiologically

significant amounts of angiotensin II were found stored in kidney cortex and

proximal tubular cells (Imig et al., 1999) and in lymphocytes (Herman and

Ring, 1995). These levels are influenced by various pathological situations

suggesting an active role for stored angiotensin II (Imig et al., 1999; Herman

and Ring, 1995). It has been suggested that the stored angiotensin II can be

released in pathological situations (Sadoshima et al., 1993), but this theory was

not supported by others (De Mello and Danser, 2000). However, it has been

shown that intracellular angiotensin II inhibited junctional conductance in heart

muscle (De Mello et al., 1994). Moreover, Haller and co-workers showed that

intracellular injected angiotensin II raise [Ca2+]i in cultured vascular smooth

muscle cells (Haller et al., 1996).

AIM OF THIS THESIS

When the work on this thesis started in 1997, only the above mentioned two

papers were published concerning the effects of intracellular angiotensin II (de

Chapter I

16

Mello, 1994; Haller et al., 1996). Both studies characterized these effects using

a single method for intracellular delivery (cell injection) and measured one

parameter. The existence of such intracellular angiotensin receptors may

contribute to further understanding of the patho-physiological effects of the

renin-angiotensin system. Thus, we decided to investigate the importance of

such receptors in vascular smooth muscle, one of the main sites of action of

angiotensin II. Because cell injection limits the investigation of the single cell

we developed two additional techniques for intracellular delivery of

angiotensin II, cell permeabilization and liposomes. We used these techniques

in order to:

- investigate the cell functions modulated be intracellular angiotensin II

- characterize the receptor(s) pharmacologically

- identify the associated signal transductions mechanisms

As opposed to cell culture, in chapter 2 the effects of intracellular angiotensin

II, delivered by liposomes, on rat aortic smooth muscle contraction were

investigated.

In order to characterize the Ca2+ pathways mobilized by intracellular

angiotensin II we used in chapter 3 A7r5 vascular smooth muscle cells, a cell

line derived from fetal rat aorta. This cell line has been choosen because

extracellular angiotensin II has no functional effects.

To integrate the effects of intracellular angiotensin II in the cell patho-

physiology, in chapter 4 we studied the interactions of intracellular angiotensin

II with heterologous plasma membrane receptors.

In chapter 5 we investigated the effects of intracellular angiotensin II on

another major target of cardio-vascular diseases, cellular growth.

In chapter 6 we investigated the pathways involved in plasma membrane AT1

internalization, as possible source of intracellular angiotensin receptors.

In chapter 7 the present knowledge on intracellular angiotensin II is reviewed

and possible future directions are indicated.


17

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Bergsma DJ, Ellis C, Kumar C, Nuthulaganti P, Kersten H, Elshourbagy N, Griffin E,Stadel JM, Aiyar N. Cloning and characterization of a human angiotensin II type 1receptor. Biochem Biophys Res Commun 1992; 183: 989-95.

Bottari SP, Taylor V, King IN, Bogdal Y, Whitebread S, de Gasparo M. AngiotensinII AT2 receptors do not interact with guanine nucleotide binding proteins. Eur JPharmacol 1991; 207(2):157-63.

Brown GP, Douglas JG. Angiotensin II-binding sites in rat and primate isolated renaltubular basolateral membranes. Endocrinology 1983; 112(6): 2007-14.

Buisson B, Laflamme L, Bottari SP, de Gasparo M, Gallo-Payet N, Payet MD. A Gprotein is involved in the angiotensin AT2 receptor inhibition of the T-type calciumcurrent in non-differentiated NG108-15 cells. J Biol Chem 1995; 270(4): 1670-4.

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Catt K, Baukal A, Ketelslegers JM, Douglas J, Saltman S, Fredlund P, Glossmann H.Angiotensin II receptors of the adrenal gland: location and modulation by cations andguanyl nucleotides. Acta Physiol Lat Am 1974; 24(5): 515-9.

Chaki S, Inagami T. Identification and characterization of a new binding site forangiotensin II in mouse neuroblastoma neuro-2A cells. Biochem Biophys ResCommun 1992; 182(1): 388-94.

Curnow KM, Pascoe L, White PC. Genetic analysis of the human type-1 angiotensinII receptor. Mol Endocrinol 1992; 6(7): 1113-8.

Danser AH, Saris JJ, Schuijt MP, van Kats JP. Is there a local renin-angiotensinsystem in the heart? Cardiovasc Res 1999; 44(2): 252-65.

De Mello WC. Influence of intracellular renin on heart cell communication.Hypertension. 1994; 25(6): 1172-7.

De Mello WC, Danser AH. Angiotensin II and the heart: on the intracrine renin-angiotensin system. Hypertension. 2000; 35(6): 1183-8.

Dostal DE and Baker KM (1992) Angiotensin II stimulation of left ventricularhypertrophy in adult rat heart: Mediation by the AT1 receptor. Am J Hypertens 5:276-280.

Chapter I

18

Dzau VJ. Circulating versus local renin-angiotensin system in cardiovascularhomeostasis. Circulation 1988; 77: I4-13.

Gallinat S, Busche S, Schutze S, Kronke M, Unger T. AT2 receptor stimulationinduces generation of ceramides in PC12W cells. FEBS Lett. 1999; 443(1): 75-9.

Gunther S. Characterization of angiotensin II receptor subtypes in rat liver. J BiolChem 1984; 259(12): 7622-9.

Gyurko R, Kimura B, Kurian P, Crews FT, Phillips MI. Angiotensin II receptorsubtypes play opposite roles in regulating phosphatidylinositol hydrolysis in rat skinslices. Biochem Biophys Res Commun 1992; 186(1): 285-92.

Haller H, Lindschau C, Erdmann B, Quass P, Luft FC. Effects of intracellularangiotensin II in vascular smooth muscle cells. Circ Res. 1996; 79(4): 765-72.

Harding JW, Wright JW, Swanson GN, Hanesworth JM, Krebs LT. AT4 receptors:specificity and distribution. Kidney Int 1994; 46(6): 1510-2.

Hermann K, Ring J. Association between the renin angiotensin system andanaphylaxis. Adv Exp Med Biol 1995; 377: 299-309

Horiuchi, M., Akishita, M., Dzau, V.J. Recent progress in angiotensin II type 2 receptorresearch in the cardiovascular system. Hypertension 1999; 33: 613-21.

Imig JD, Navar GL, Zou LX, O'Reilly KC, Allen PL, Kaysen JH, Hammond TG,Navar LG Renal endosomes contain angiotensin peptides, converting enzyme, andAT(1A) receptors. Am J Physiol 1999; 277: F303-11.

Janssen WM, de Jong PE, de Zeeuw D. Hypertension and renal disease: role ofmicroalbuminuria. J Hypertens Suppl 1996; 14(5): S173-7.

Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, Inagami TMolecular cloning of a novel angiotensin II receptor isoform involved inphosphotyrosine phosphatase inhibition. J Biol Chem 1993; 268(33): 24543-6.

van Kats JP, de Lannoy LM, Jan Danser AH, van Meegen JR, Verdouw PD,Schalekamp MA. Angiotensin II type 1 (AT1) receptor-mediated accumulation ofangiotensin II in tissues and its intracellular half-life in vivo. Hypertension 1997; 30:42-9.

van Kats JP, Duncker DJ, Haitsma DB, Schuijt MP, Niebuur R, Stubenitsky R,Boomsma F, Schalekamp MA, Verdouw PD, Danser AH. Angiotensin-convertingenzyme inhibition and angiotensin II type 1 receptor blockade prevent cardiacremodeling in pigs after myocardial infarction: role of tissue angiotensin II.Circulation 2000; 102(13): 1556-63.

Lee MA, Bohm M, Paul M, Ganten D. Tissue renin-angiotensin systems. Their role incardiovascular disease. Circulation 1993; 87: IV7-13.


19

Li X, Shams M, Zhu J, Khalig A, Wilkes M, Whittle M, Barnes N, Ahmed A. Cellularlocalization of AT1 receptor mRNA and protein in normal placenta and its reducedexpression in intrauterine growth restriction. Angiotensin II stimulates the release ofvasorelaxants. J Clin Invest. 1998; 101(2): 442-54.

Lin SY, Goodfriend TL. Angiotensin receptors. Am J Physiol 1970; 218(5): 1319-28.

Menard J, Campbell DJ, Azizi M, Gonzales MF. Synergistic effects of ACE inhibitionand Ang II antagonism on blood pressure, cardiac weight, and renin in spontaneouslyhypertensive rats. Circulation 1997; 96(9): 3072-8.

Miller PL, Rennke HG, Meyer TW. Glomerular hypertrophy accelerates hypertensiveglomerular injury in rats. Am J Physiol 1991; 261: F459-F465.

Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau VJ. Expressioncloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors. J Biol Chem 1993; 268(33): 24539-42.

Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE.. Isolation of acDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991; 351(6323):233-6.

Pinto YM, Buikema H, van Gilst WH, Lie KI Activated tissue renin-angiotensinsystems add to the progression of heart failure. Basic Res Cardiol 1996; 91: 85-90.

Regitz-Zagrosek V, Neuss M, Warnecke C, Holzmeister J, Hildebrandt AG, Fleck E.Subtype 2 and atypical angiotensin receptors in the human heart. Basic Res Cardiol1996; 91: 73-7.

Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediatesstretch-induced hypertrophy of cardiac myocytes in vitro. Cell. 1993; 75(5): 977-84.

Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ, Hasegawa M, Matsuda Y,Inagami T Cloning and expression of a complementary DNA encoding a bovineadrenal angiotensin II type-1 receptor. Nature 1991; 351(6323): 230-3.

Sasamura H, Hein L, Krieger JE, Pratt RE, Kobilka BK, Dzau VJ Cloning,characterization, and expression of two angiotensin receptor (AT-1) isoforms from themouse genome. Biochem Biophys Res Commun 1992; 185: 253-9.

Servant G, Dudley DT, Escher E, Guillemette G The marked disparity between thesizes of angiotensin type 2 receptors from different tissues is related to differentdegrees of N-glycosylation. Mol Pharmacol 1994; 45(6): 1112-8.

Smith RD. Identification of atypical (non-AT1, non-AT2) angiotensin binding siteswith high affinity for angiotensin I on IEC-18 rat intestinal epithelial cells. FEBS Lett1995; 373(3): 199-202.

Chapter I

20

Wolthuis A, Boes A, Grond J. Cell density modulates growth, extracellular matrix,and protein synthesis of cultured rat mesangial cells. Am. J Pathol. 1993; 143(4):1209-19.

Unger, T., Chung, O., Csikos, T., Culman, J., Gallinat, S., Gohke, P., Hohle, S.,Meffert, S., Stoll, M., Stroth, U., Zhu YZ. Angiotensin receptors. J. Hypertens. 1996;14(5): S95-S103.

Vanderheyden PM, Fierens FL, De Backer JP, Fraeyman N, Vauquelin G. Distinctionbetween surmountable and insurmountable selective AT1 receptor antagonists by useof CHO-K1 cells expressing human angiotensin II AT1 receptors. Br J Pharmacol1999; 126(4): 1057-65.

Whitebread S, Mele M, Kamber B, de Gasparo M. Preliminary biochemicalcharacterization of two angiotensin II receptor subtypes. Biochem Biophys ResCommun 1989; 163(1): 284-91.

Zhang J, Pratt RE. The AT2 receptor selectively associates with Gialpha2 and Gialpha3 inthe rat fetus. J Biol Chem 1996; 271(25): 15026-33.

Chapter 2

Contractile effects by intracellular angiotensin II via

receptors with a distinct pharmacological profile in rat

aorta.

Eugen Brailoiu, Catalin M. Filipeanu, Andrei Tica, Catalin P. Toma, Dick de

Zeeuw and S. Adriaan Nelemans.

British Journal of Pharmacology 126, 1133-1138, 1999.

Chapter II

22

SUMMARY

1 We studied the effect of intracellular angiotensin II (Ang II) and related

peptides on rat aortic contraction, whether this effect is pharmacologically

distinguishable from that induced by extracellular stimulation, and determined

the Ca2+ source involved.

2 Compounds were delivered into the cytoplasm of de-endothelised aorta rings

using multilamellar liposomes. Contractions were normalised to the maximum

obtained with phenylephrine (10-5 M).

3 Intracellular administration of angiotensin II (incorporation range: 0.01-300

nmol mg-1) resulted in a dose-dependent contraction, insensitive to extracellular

administration (10-6 M) of the AT1 receptor antagonist CV11947, the AT2

receptor antagonist PD 123319, or the non-selective AT receptor antagonist and

partial agonist saralasin ([Sar1,Val5,Ala8]-angiotensin II (P<0.05).

4 Intracellular administration of CV11947 or PD 123319 right shifted the dose-

response curve about 1000-fold or 20-fold, respectively. PD 123319 was only

effective if less than 30 nmol mg –1 Ang II was incorporated.

5 Contraction was partially desensitised to a second intracellular Ang II addition

after 45 min (P<0.05).

6 Intracellular administration of Ang I and saralasin also induced contraction

(P<0.05). Both responses were sensitive to intracellular CV11947 (P<0.05), but

insensitive to PD 123319. The response to Ang I was independent of intracellular

captopril.

7 Contraction induced by extracellular application of Ang II and of Ang I was

abolished by extracellular pre-treatment with saralasin or CV11947 (P<0.05), but

not with PD 123319. Extracellular saralasin induced no contraction.

8 Intracellular Ang II induced contraction was not affected by pre-treatment with

heparin filled liposomes, but completely abolished in Ca2+ -free external medium.

9 These results support the existence of an intracellular binding site for Ang II in

rat aorta. Intracellular stimulation induces contraction dependent on Ca2+-influx

but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+-stores.

Intracellular angiotensin II induced contraction

23

Intracellular Ang I and saralasin induce contraction, possibly via the same

binding site. Pharmacological properties of this putative intracellular receptor are

clearly different from extracellular stimulated AT1 receptors or intracellular

angiotensin receptors postulated in other tissue.

Key Words: Intracellular angiotensin II, Angiotensin I, Saralasin, Liposomes, Vascular

Smooth Muscle.

Abbreviations: Ang II, angiotensin II; AT1 receptor, angiotensin II type 1 receptor;

AT2 receptor, angiotensin II type 2 receptor; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate;

saralasin, ([Sar1,Val5,Ala8]-angiotensin II, EGTA, [ethylenebis (oxyethylenenitrilo)

]tetra-acetic acid;

1. Introduction

Since the identification of angiotensin I and angiotensin II (Ang I and Ang II;

Skeggs et al., 1956), the fundamental role of the renin-angiotensin system in the

regulation of cardiovascular function is widely accepted. The cellular effects of

angiotensin are a consequence of its coupling to specific receptors in the plasma

membrane, namely angiotensin (AT) receptors (Timmermans et al., 1993). Until

now, cDNA was cloned for two subtypes, AT1 and AT2, of these receptors, but

the existence of possible others subtypes is under investigation (Griendling et al.,

1997). Most of the vascular effects of Ang II are mediated via the AT1 receptor

subtype by activating phospholipase C, and Ins(1,4,5)P3 and diacylglycerol

formation (Griendling et al., 1986; 1997; Smith, 1986; Danthluri and Deth, 1986;

Murphy et al., 1991). However, recent studies suggest that Ang II might

influence cellular function also from the intracellular side of the plasma

membrane (De Mello et al., 1994). The presence of intracellular Ang II binding

sites was described recently in rat cerebellar cortex (Erdmann et al., 1996), and in

human placenta, the latter identified as a non-AT1/non-AT2 recognition site (Li et

al., 1998). Intracellular injection of Ang II elicited increases in [Ca2+]i in vascular

smooth muscle cells (Haller et al., 1996) and recently we reported a role for

Chapter II

24

intracellular Ang II in signal transduction of an aortic smooth muscle cell line

(Filipeanu et al., 1998b).

Multilamellar liposomes can be directed to and captured in cells, and are

subsequently able to transfer their content into the cell. This was shown for

smooth muscle cells from rat aorta (Brailoiu et al., 1993; 1995; Filipeanu et al.,

1998a), trachea (Costuleanu et al., 1995) or for the neuromuscular junction

(Brailoiu and Van der Kloot 1996). Besides aqueous signal transduction

intermediates, like inositol polyphosphates (Brailoiu et al., 1993; 1995), this type

of vesicles is also suitable to deliver other compounds like, peptides and proteins

into target cells (Costuleanu et al., 1996; Crommelin et al., 1997). Therefore, in

this paper we use this technique to deliver Ang II or related peptides and AT

receptor antagonists into the cytoplasm of rat aortae. The aim of the study was to

investigate 1) if these peptides elicit an effect on aortic contraction, and 2) if this

effect can be pharmacologically distinguished from that induced by extracellular

Ang II or related peptides, and 3) the Ca2+ source involved in this intracellular

induced effect.

2. Methods

2.1 Liposomes preparation

The liposomes used in these physiological studies were prepared from egg

phosphatidylcholine, 60 mg lipid per ml of solution to be incorporated, according

to the method described by Bangham et al., (1965) as modified by us (Brailoiu et

al., 1993). Control liposomes contained only KCl (140 mM, pH adjusted to 6.9).

The same solution was used to prepare liposomes containing the desired

compound. To maximise contractile effects liposomes batches (0.5 ml) were

added to the 2-ml organ bath containing 1.5 ml of Krebs-Henseleit buffered

solution (Brailoiu et al., 1995), with the following composition (mM): NaCl,

118; KCl, 4.8; CaCl2, 2.5; MgSO4, 1.6; KH2PO4, 1.2; NaHCO3, 25; glucose, 5.5;

pH 7.4. In order to remove non-incorporated solutes, liposomes batches were

subjected to dialysis (Sigma dialysis tubing, molecular weight cut-off: 12400


25

dalton) in Krebs-Henseleit buffered solution (150 min, 1 to 600 volume ratio, the

buffer being exchanged every 30 min). To control if not incorporated substances

were still present in the dialysis buffer, and thereby contributing to the liposomal

effects, a similar amount of dialysis buffer was added to control rings in each

experiment in a 1 to 4 volume ratio similar to the procedure as used in the

liposome experiments. Effects on contraction were never observed.

2.2 Determination of the Ang II concentration delivered into rat aortic smooth

muscle cells.

The liposomes were prepared exactly as described in section 2.1, but

[125I]angiotensin II (specific activity 1-5 Ci/mmol) was dissolved in aqueous

phase (final Ang II concentration of 35 nM). After dialysis, 6.2 ± 0.4 % (n=5) of

the initial amount dissolved in the aqueous phase was entrapped into liposomes

(n=5). The liposomes were incubated with rat aortic rings in Krebs Henseleit

solution at 37o C for 10 min. At the end of this period the rings were washed five

times with Krebs Henseleit buffer at room temperature in order to remove all

possible extracellular radioactivity. In the last three washings radioactivity was

below the detection limit, excluding extracellular contamination of our samples.

The amount of radioactivity captured by rat aortic rings was 0.3 ± 0.1 % (n=5) of

the initial amount in aqueous solution. Radioactivity of the rings was measured

by $ counting (RiaStar System, Packard). Based on these values the contractile

data are expressed as the amount of angiotensin peptides delivered into the aorta

ring (nmol mg-1 wet weight). The average weight of the rings was 1.6 ± 0.1 mg

(n=6).

2.3 Tissue preparation

The investigation conforms to the Guide for the Care and Use of Laboratory

Animals published by the US National Institutes of Health (NIH Publication No.

85-23, revised 1985). Male Wistar rats (150-200 g body weight) were

decapitated and exsanguinated. The thoracic aorta was rapidly removed and cut

Chapter II

26

into rings, 2 mm wide. Endothelium was rubbed off gently with a smooth

softwood stick. The rings were then mounted between hooks and their

mechanical activity was monitored using an isometric force transducer and a

potentiometric pen recorder (Linseis L650). The volume of Krebs-Henseleit

buffered solution present in the organ-bath was 2.0 ml. This solution was kept at

37oC and aerated continuously with 95% O2 + 5% CO2. In order to obtain Ca2+-

free Krebs-Henseleit solution, NaCl (equimolar) replaced CaCl2 and EGTA was

added (2 mM).

2.4 Experimental protocols

A pre-tension of 20 mN (2 g) was imposed on each preparation; this ensured

maximal contractile responses on pharmaco-mechanical stimulation. After an

equilibration period of 60-90 min, the arteries were stimulated 2-3 times with 10-

5 M phenylephrine until at least two successive contractions differing by less than

5 % were obtained. The amplitude of this contractile response can be reproduced

for at least 3 hours. At the plateau of one of these contractions the efficient

removal of endothelium was tested by the absence of relaxation in response to

10-5 M carbachol. The amplitude of the last phenylephrine 10-5 M-induced

contraction was considered as control (100 %) for further comparisons. Rings

that did not develop 0.8 g active force in response to phenylephrine were

discarded. Thereafter the experimental protocols were performed. For

extracellular experiments the desired compounds were added from 100 times

more concentrated stock solutions in a maximal volume of 20 µl. The antagonists

were added 15 min before addition of agonists. Antagonist containing liposomes

were added 15 min before addition of agonist filled liposomes. Tachyphylaxis of

the initial contraction to Ang II filled liposomes was studied after washing and

re-equilibrating the preparations for 45 min (bath solution changed every 10 min)

and monitoring in the same ring the second contraction induced by liposomes

filled with the original concentration of Ang II. In experiments using Ca2+-free

Krebs-Henseleit buffered solution (containing 2 mM EGTA) the normal


27

extracellular Ca2+ concentration was restored by addition of 5 mM CaCl2.

2.5 Chemicals.

Angiotensin II, angiotensin I and saralasin ([Sar1,Val5,Ala8]-angiotensin II) were

obtained from Serva; [125I]- angiotensin II was obtained from Nichols Institute

Diagnostics; phenylephrine, captopril, heparin (MW 4000-6000 dalton),

carbachol and phosphatidylcholine (type X-E) were obtained from Sigma,

CV11947 and PD 123319 were a kind gift of Dr. O. Balatu (Hypertension

Research, Max-Delbrueck Center for Molecular Medicine, Berlin, Germany). All

other compounds used were of analytical grade.

2.6 Statistics

The results are expressed as % of the control contraction to 10-5 M phenylephrine

(mean ± SD) or other proper controls when stated. Rings from different animals

were used to obtain the number of experiments (n). From one animal a ring was

used only once at the desired concentration of (ant)agonist. Statistical

significance was tested with unpaired Student t-test or by ANOVA followed by

Bonferonni test, P<0.05 being considered significantly different.

3. RESULTS

3.1 Intracellular effects of angiotensin related compounds

The basal tone of the aortic ring was neither modified by control liposomes,

containing 140 mM KCl, nor by control dialysis buffer. Complete de-

endothelisation of the ring was verified by addition of carbachol (10-5 M) on top

of the maximum phenylephrine (10-5 M) contraction. As shown in the typical

tracings (Fig 1), no relaxation occurred until the washout procedure was started.

In contrast to control liposomes, liposomes filled with 10-6 M Ang II induced

contraction (Fig 1A). Extracellular addition of the selective AT1 receptor

antagonist CV11947, the AT2 receptor antagonist PD 123319 or the non-selective

AT receptor antagonist saralasin (all at 10-6 M) did not influence these

Chapter II

28

contractions (Fig 1B).

The contractile effect of Ang II filled liposomes was dose dependent (Fig 2). An

increase in contraction was observed after varying the amount of Ang II

delivered into the aortic ring over a range of three orders of magnitude. In

contrast to the lack of effect of extracellular addition of antagonists, contractions

are sensitive to pre-treatment with liposomes containing the AT1 receptor

antagonist CV11947 over the entire range of Ang II delivered into the tissue. The

dose response curve was right shifted by about 1000-fold. Pre-treatment with

liposomes filled with the AT2 receptor antagonist PD 123319 also inhibited

angiotensin II induced contractions, although less effective than observed for

CV11947. This inhibition was only observed at contractions induced by amounts

Figure 1 Contraction of rataorta in response toadministration of Ang II filledliposomes. Representativetracings out of 12 experimentsshow the control phenylephrine(Phe, 10-5 M) response and thelack of response to carbachol(Carb, 10-5 M), indicatingeffective removal of theendothelium. After washout(w) liposomes filled with AngII (10-6 M, LAngII) inducedcontraction (A), also in thepresence of extracellularsaralasin (10-6 M, SAR) (B).


29

of Ang II incorporated into the aorta of less than 30 nmol/mg. A right shift of

about 20-fold was obtained for this part of the dose response curve.

The intracellular Ang II induced contractile response is transient (Fig 1). This

might reflect a desensitization process of the receptor comparable to the

mechanisms described for extracellular Ang II stimulation (Danthuluri & Deth,

1986; Boulay et al., 1994; Griendling et al., 1997). Indeed, a washing period of

45 min was not long enough to restore the initial response to a similar dose of

intracellular Ang II (Fig 3A).

Desensitization became already apparent for the lowest amounts of Ang II

incorporated intracellularly (P<0.05), but was more prominent if higher amounts

were incorporated, reaching a level of about 50 % of the initial contraction for the

second response elicited after 45 min (Fig 3B). Under these circumstances the

Figure 2 Doses-effect curveof the LAngII inducedcontraction and the effect ofAT receptor antagonists. Theamount of Ang IIincorporated in the aorta wasvaried using liposomes filledwith different concentrationsof Ang II (range 10-9 - 3x10-5

M). Antagonists wereadministered intracellularlyby liposomes filled withCV11947 (10-6 M) or PD123319 (10-6 M). The dataare presented as % of Phecontraction and given asmean + SD (n=6).Significance level:* P<0.05 vs LAngII, unpairedStudent’s t-test.

Chapter II

30

rings were fully responsive to phenylephrine (data not shown).

The particular properties of the postulated intracellular Ang II receptor become

evident from experiments showing that contraction can be induced also by

intracellular administered Ang I and saralasin (Fig 4). In comparison with

intracellular Ang II, similar and even stronger contractions were obtained for

saralasin (10-6 M filled liposomes) and Ang I (10-6 M filled liposomes),

respectively. As observed for the Ang II induced contraction, those of Ang I and

saralasin were also not affected by pre-treatment with liposomes filled with PD

Figure 3 Desensitization ofthe LAngII-induced contraction.A representative tracing of theresponse to second addition ofLAngII (10-6 M filled liposomes)after a 45 min washout period(A). Doses-response curve ofthe desensitization effect (B).The data are presented as % ofthe initial contraction to LAngII

and given as mean + SD (n=6).All values are different fromcontrol (P<0.05, one wayANOVA followed byBonferonni test).


31

123319 (10-6 M), but could be inhibited if filled with CV11947 (10-6 M). If

possibly intracellular Ang I-converting enzyme activity is present in the aortic

smooth muscle cells, this activity is not involved in the contractile effects

observed, since the Ang I contraction was unaffected by liposomes filled with

captopril (10-6 M).

3.2 Extracellular effects of Ang II, Ang I, and saralasin

The following experiments were done to establish whether the effects observed

so far are pharmacologically distinguishable from those induced after stimulation

of plasma membrane receptors. Extracellular administration of Ang II (10-6 M)

induced a contraction of 39 ± 2 % (n=12, P<0.05) as compared to control

Figure 4 Contractile effects ofintracellular delivered Ang I,saralasin and various inhibitors incomparison with LAngII inducedcontraction. Liposomes filled witheither 10-6 M of Ang II, Ang I(LAngI) or saralasin (LSar) inducedcontraction (significance level: *P<0.001 vs control liposomescontaining 140 mM KCl., one wayANOVA followed by Bonferonnitest). LAngI induced a morepronounced contraction(significance level: $ P<0.001 vsLAngII, one way ANOVA followedby Bonferonni test). LAngI and LSar

contraction was inhibited byliposomes filled with CV11947 (10-6

M), but not by liposomes filled withPD 123319 (10-6 M; significancelevel: # P<0.001 vs LAngI and vsLSar, respectively, one way ANOVAfollowed by Bonferonni test). LAngI

contraction was also not inhibited byliposomes filled with captopril (10-6

M). The data are presented as % ofPhe contraction and given as mean +SD (n=6).

Chapter II

32

contractions induced by phenylephrine (10-5 M). Ang I (10-6 M) contracted the

rat aorta rings to 38 + 2 % of the control contraction (n=12, P<0.05). Pre-

incubation (15 min) with the non-selective plasma membrane AT1 receptor

antagonist CV11947 (10-6 M) completely abolished the contractions induced by

Ang II (n=4, P<0.05). The AT1 and AT2 receptor antagonist saralasin (10-6 M, 15

min) completely inhibited contraction triggered by both Ang II and Ang I (n=4,

P<0.05). However, pre-treatment with the AT2 antagonist PD 123319 (10-6 M, 15

min) did not affect the contractions (38 ± 4 %, (n=4) and 35 ± 3 %, (n=4) for

Ang II and Ang I, respectively). In view of the lack of antagonistic properties of

extracellular addition of these compounds on the intracellular effects induced by

the angiotensin peptides, it is not likely that the contractile effect of angiotensin

filled liposomes is mediated by activation of plasma membrane AT receptors.

3.3 Ca2+ source in contraction

To gain insight into possible subcellular mechanisms used by intracellular

angiotensin II to initiate contraction, we investigated the contribution of Ca2+

influx and intracellular Ca2+ mobilisation to the observed contractile responses.

Administration of intracellular heparin, a specific blocker of Ins(1,4,5)P3-

mediated Ca2+ release from intracellular Ca2+ stores, by pre-treatment with

heparin filled liposomes (20 mg/ml) did not affect the contractions induced by

intracellular Ang II (93 + 6%, n=8).

Figure 5 The effect ofextracellular Ca2+ on the LAngII

induced contraction. LAngII (10-6

M filled liposomes) wasadministered in buffer solutionwithout Ca2+ (Ca2+- free). Acontraction could only beobtained after restoring theoriginal extracellular Ca2+

concentration. A representativetracing is shown out of 6experiments.


33

In contrast omission of Ca2+ from the external medium completely abolished

these contractions. Restoring the normal external Ca2+ concentration by addition

of 5 mM CaCl2 in the continuous presence of Ang II filled liposomes re-

established contraction (Fig 5).

4. Discussion

The main objective of this study was to investigate if intracellular Ang II or

related peptides affect vascular smooth muscle contraction, and subsequently to

characterise this response. We used liposomes to test the effects of intracellular

administration of compounds on contraction of de-endothelised rat aorta rings.

We previously demonstrated the effective use of liposomes to study intracellular

effects of various compounds in rat aorta (Brailoiu et al., 1993; 1995; Filipeanu

et al., 1998a). Integrity of angiotensin containing liposomes is maintained for

concentrations less than 10-5 M Ang I and 10-4 M Ang II, respectively (Brailoiu

et al., 1997).

Intracellular administration of Ang II induces a dose-dependent contraction. This

effect is not due to a non-specific effect of the liposomes, since control liposomes

were ineffective. Plasma membrane AT1 receptor activation is also not involved,

in view of the lack of effect of extracellular addition of saralasin, a non-specific

AT receptor antagonist and partial agonist of AT receptors (Gavras and Salerno,

1996), or of extracellular addition of the selective AT1 receptor antagonist

CV11947. Apparently, contraction is stimulated by activation of an intracellular

binding site for Ang II. Internalisation of the plasma membrane AT receptor

complex after receptor stimulation might contribute to the intracellular pool of

receptors and peptides (Anderson et al., 1993; Van Kats et al., 1996). The

binding site observed in our experiments resembles to certain extent the 'normal'

plasma membrane AT1 receptor (Griendling et al., 1997), but is different in

several aspects. Both types of receptors are insensitive to extracellular addition of

the AT2 receptor antagonist PD 123319. The intracellular receptor induced

contraction is subjected to a marked dose-dependent desensitization similar to the

Chapter II

34

tachyphylaxis observed after plasma membrane AT receptor stimulation in rat

aorta (Danthluri and Deth, 1986). Both types of receptors are inhibited by

CV11947, but the intracellular receptor is only inhibited if this compound is

applied from within the cell. The intracellular receptors are also different with

respect to the observed sensitivity to intracellular PD 123329.

The related peptides Ang I and saralasin also induce contraction if delivered via

liposomes into aortic rings, as tested in a single dose experiment. Both responses

are insensitive to intracellular PD 123319, but sensitive to intracellular CV11947.

The response to Ang I was independent of Ang I-converting enzyme activity,

since captopril did not affect this response. This might indicate that either 1)

intracellular chymase activity or 2) smooth muscle specific neutral

metalloendopeptidase activity is present in our preparation (Ferrario et al., 1997),

or 3) that these peptides also bind to the active side of the putative intracellular

Ang II receptor. However, cleavage of Ang I by chymase activity does not result

in Ang II formation in the rat (Yamamoto et al., 1998), and neutral

endopeptidase activity would result in angiotensin - (1-7) that exhibits opposite

characteristics than Ang II (Ferrario et al., 1997). Therefore, the third suggestion

is more likely.

Several intracellular binding proteins for angiotensin have been recognised. A

protein with high affinity binding for Ang II was purified from rabbit liver (Kiron

and Soffer, 1989). Saralasin bound even more tightly to this protein, while also

Ang I was competing for this binding site. More recently subcellular localisation

of Ang II immunoreactivity was observed in cerebellar cortex (Erdmann et al.,

1996) and a non-AT1/non-AT2 binding site was reported within the cytosolic

compartment of placenta (Li et al., 1998). This latter protein resembles the non-

AT1/non-AT2 receptor involved in the angiogenesis in chick embryo (Le Noble

et al., 1993). These receptors are insensitive to PD 123319 and the AT1 receptor

antagonist losartan, and at least in the chicken Ang I and [Sar1, Ile8]-angiotensin

II bind with high affinity to this receptor too. The intracellular receptor proposed

by us has a number of properties in common as described for these binding


35

proteins until now, but seems to be dissimilar from these binding proteins.

The different physiological responses reported on intracellular application of Ang

II might be ascribed to stimulation of (one of) these intracellular receptors.

Different aspects of Ca2+ homeostasis are known to be affected, like Ca2+ influx

in vascular smooth muscle cells (Haller et al., 1996), gap junction conduction of

heart muscle (De Mello, 1994) and on 45Ca2+ release from permeabilised A7r5

cells (Filipeanu et al., 1998b). In the present study, contraction was completely

abolished in Ca2+-free medium, and Ca2+ mobilisation from Ins(1,4,5)P3 sensitive

Ca2+ stores appears not to be involved because heparin-filled liposomes failed to

affect these contractions, a treatment shown to completely abolish contractions

induced by Ins(1,4,5)P3 in rat aorta (Brailoiu et al., 1993). Therefore, intracellular

Ang II induced contraction is entirely dependent on Ca2+ influx from the

extracellular medium. Ca2+ influx is also prominent for the intracellular Ang II

induced responses in cultured cells from vascular smooth muscle, but

Ins(1,4,5)P3 -mediated mechanisms are not excluded (Haller et al., 1996;

Filipeanu et al., 1998b). In contrast to the present results with intracellular

stimulation of Ang II, Ca2+ mobilisation from internal stores plays a major role in

extracellular stimulation of vascular smooth muscle with Ang II. Receptor

stimulation activates phospholipase C and formation of Ins(1,4,5)P3, which

subsequently discharges Ca2+ from internal stores (Alexander et al., 1985;

Smith, 1986), a mechanism directly related to rat aorta contraction

(Manolopoulos et al., 1991).

In summary, these results support the existence of an intracellular binding site for

Ang II in rat aorta. Intracellular administration via treatment with Ang II filled

liposomes results in muscle contraction. Also Ang I and saralasin respond in a

similar fashion, possibly via the same binding site. The pharmacological

properties of this putative intracellular receptor are clearly different from that of

the extracellular stimulated plasma membrane AT1 receptor or that of

intracellular angiotensin receptors postulated in other tissue. Contraction induced

by intracellular Ang II is solely dependent on Ca2+ -influx and not on Ins(1,4,5)P3

Chapter II

36

mediated release from intracellular Ca2+-stores.

Acknowledgements

The Romanian Ministry of Science and Education (CNCSU Grant) supported this work

through the Romanian Ministry of Research and Technology (C-D Grant). We thank Dr.

Mihai Todiras for his involvement in the preliminary experiments and Mr. Adrian Zosin

for his excellent technical assistance. C.M.F. is a recipient of an Ubbo Emmius

Fellowship from the Groningen Utrecht Institute for Drug Exploration (GUIDE).

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Anderson, K.M., Murahashi, T., Dostal, T.E. & Peach M.J. (1993). Morphological andbiochemical analysis of angiotensin II internalization in cultured rat aortic smoothmuscle cells. Am J Physiol, 46, C179-188.

Bangham, A.D., Standish, M.M. & Watkins J.C. (1965) Diffusion of univalent ionsacross the lamellae of swollen phospholipids. J Mol Biol, 13, 238-252.

Brailoiu, E., Baltatu, O., Costuleanu, M., Slatineanu, S., Filipeanu, C.M. & BranisteanuDD. (1995). Effects of "-trinositol administered extra- and intracellularly (usingliposomes) on rat aorta rings. Eur J Pharmacol, 281, 209-212.

Brailoiu, E., Serban, D.N., Popescu, L.M., Filipeanu, C.M., Slatineanu, S. & Branisteanu, D,D. (1993). Effects of liposome-entrapped Ins(1,4,5)P3 andIns(1,3,4,5)P4 in the isolated rat aorta. Eur J Pharmacol, 250, 493-495.

Brailoiu, E., Todiras, M., Margineanu, A., Costuleanu, M., Brailoiu, C., Filipeanu,C.M., Costuleanu, A., Rusu, V. & Petrescu G. (1997). TLC characterisation ofliposomes containing angiotensinogen, angiotensin I, angiotensin II and saralasin.Biomed Chromatogr, 11, 160-163.

Brailoiu, E. & Van der Kloot, W. (1996). Bromoacetylcholine and acetylcholinesteraseintroduced via liposomes into motor nerve endings block increases in quantal size.Pfluegers Arch/Eur J Physiol, 432, 413-418.

Boulay, G., Chretien, L., Richard, D.L. & Guillemette, G. (1994). Short-termdesensitization of the angiotensin II receptor of bovine adrenal glomerulosa cellscorresponds to a shift from a high to a low affinity state. Endocrinology, 135, 2130-2136.


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Costuleanu, M., Brailoiu, E., Filipeanu, C.M., Baltatu, O., Slatineanu, S., Nechifor, M.& Branisteanu, D.D. (1995). Effects of platelet-activating factor-entrapped liposomesupon rat trachea. Eur J Pharmacol, 281, 89-93.

Crommelin, D.J.A., Daemen, T., Scherphof, G.L., Vingerhoeds, M.H., Heeremans,J.L.M., Kluft, C. & Storm, G. (1997). Liposomes: vehicles for the targeted andcontrolled delivery of peptides and proteins. J Control Rel, 46, 165-175.

Danthuluri, N.R. & Deth, R.C. (1986). Acute desensitization to angiotensin II: evidencefor a requirement of agonist induced diacylglycerol production during tonic contractionof rat aorta. Eur J Pharmacol, 126, 135-139.

De Mello, W.C. (1994). Is an intracellular renin-angiotensin system involved in controlof cell communication in heart? J Cardiovasc Pharmacol, 23, 640-646.

Erdmann, B., Fuxe, K. & Ganten D. (1996). Subcellular localization of angiotensin IIimmunoreactivity in the rat cerebellar cortex. Hypertension, 28, 818-824.

Ferrario, C.M., Chappell, M.C., Tallant, E.A., Brosnihan, K.B. & Diz, D.I. (1997).Counterregulatory actions of angiotensin-(1-7). Hypertension, 30, 535-541.

Filipeanu, C.M., Brailoiu, E., Petrescu, G. & Nelemans, S.A. (1998a). Extracellular andintracellular arachidonic acid-induced contractions in rat aorta. Eur J Pharmacol, 349,67-73.

Filipeanu, C.M., Henning, R.H., de Zeeuw, D. & Nelemans, S.A. (1998b). Functionalevidence for a role of intracellular angiotensin II in A7r5 cells. Br J Pharmacol, 123,141 P.

Gavras, H.P. & Salerno, C.M. (1996). The angiotensin II type 1 receptor blockerlosartan in clinical practice: review. Clin Ther, 18, 1058-1067.

Griendling, K.K., Rittenhouse, S.E., Brock, T.A., Ekstein, L.S., Gimbrone, M.A. Jr. &Alexander, R.W. (1986). Sustained diacylglycerol formation from inositolphospholipids in angiotensin II-stimulated vascular smooth muscle cells. J Biol Chem,261,5901-5906.

Griendling, K.K., Ushio-Fukai, M., Lassegue, B. & Alexander RW (1997). AngiotensinII signalling in vascular smooth muscle. New concepts. Hypertension, 29, 366-373.

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Physiol, 264, R460-R465.

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Van Kats, J.P., de Lannoy, L.M., Danser, J.A.H., van Meegen, J.R., Verdouw, P.D. &Schalekamp, M.D. (1997). Angiotensin II type 1 (AT1) receptor mediated accumulationof angiotensin II in tissues and its intracellular half-life in vivo. Hypertension, 30, 42-49.

Yamamoto, D., Shiota, N., Takai, S., Ishida, T, Okunishi, H. & Miyazaki, M (1998).Three-dimensional molecular modeling explains why catalytic function for angiotensin-I is different between human and rat chymases. Biochem Biophys Res Commun, 242,158-163.

Chapter 3

Intracellular angiotensin II elicits Ca2+ increases in

A7r5 vascular smooth muscle cells

Catalin M. Filipeanu, Eugen Brailoiu, Jan Willem Kok, Robert H. Henning,

Dick de Zeeuw and S. Adriaan Nelemans.

European Journal of Pharmacology – in press

Chapter III

40

Abstract

Recent studies show that angiotensin II can act within the cell, possibly via

intracellular receptors pharmacologically different from typical plasma membrane

angiotensin II receptors. The signal transduction of intracellular angiotensin II is

unclear. Therefore, we investigated the effects of intracellular angiotensin II in

cells devoid of physiological responses to extracellular angiotensin II (A7r5

vascular smooth muscle cells). Intracellular delivery of angiotensin II was

obtained by using liposomes or cell permeabilisation. Intracellular angiotensin

II stimulated Ca2+-influx, as measured by 45Ca2+-uptake and single-cell

fluorimetry. This effect was insensitive to extracellular or intracellular addition of

losartan (angiotensin AT1 receptor antagonist) or PD123319 ((s)-1-(4-

[dimethylamino] - 3-methyl-phenyl) methyl -5- (diphenylacetyl) -4,5,6,7-

tetrahydro-1H-imidazo[4,5-c] pyridine-6-carboxylate) (angiotensin AT2 receptor

antagonist). Intracellular angiotensin II stimulated inositol-1,4,5-trisphosphate

(Ins(1,4,5,)P3) production and increased the size of the Ins(1,4,5,)P3 releasable45Ca2+ pool in permeabilised cells, independent of losartan and PD123319.

Small G-proteins did not participate in this process, as assessed by using

GDP!S. Intracellular delivery of angiotensin I was unable to elicit any of the

effects elicited by intracellular angiotensin II. We conclude from our

intracellular angiotensin application experiments that angiotensin II modulates

Ca2+ homeostasis even in the absence of extracellular actions. Pharmacological

properties suggest the involvement of putative angiotensin non- AT1-/non- AT2

receptors.

Key words: Angiotensin II, intracellular; Liposomes; Ca2+ influx; Ca2+ release;

Ins(1,4,5)P3; A7r5 cells

Intracellular angiotensin II signal transduction

41

1. Introduction

Angiotensin II is an important effector peptide involved in the regulation of

cardiovascular and renal function. Although classical physiology attributes its

effects to circulating angiotensin II, angiotensin II can act locally as an autocrine

hormone, producing patho-physiological effects at its production site (Dell’Italia

et al., 1997; Van Kats et al., 1997). The effects of angiotensin II occur through

interaction with specific plasma membrane receptors. To date, two such receptors

have been identified, namely angiotensin AT1 and AT2 (Chiu et al., 1989;

Griendling et al., 1997). Both of them belong to the family of seven

transmembrane G protein-coupled receptors but are coupled to different signal

transduction pathways. Stimulation of angiotensin AT1 receptors activates

phospholipase Cand the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5,)P3),

which subsequently discharges Ca2+ from internal stores, activates mitogen-

activated protein (MAP) kinase and stimulates cell growth, whereas angiotensin

AT2 receptors increase cGMP levels and inhibit cell growth (Unger et al., 1996;

Hunyady et al., 1996 Horiuchi et al., 1999). The existence of additional sub-

types of angiotensin II receptors is under investigation and studies suggest that

angiotensin non-AT1/non-AT2 binding sites are involved in angiogenesis (Le

Noble et al., 1996) and are present within the cytosolic fraction of placenta (Li et

al., 1998).

Several studies have drawn attention to the effects of angiotensin II in the cell.

Intracellular application of angiotensin II induces a [Ca2+]i increase in vascular

smooth muscle cells (Haller et al., 1996), whereas in heart muscle it inhibits the

functioning of gap-junctions (De Mello, 1996) and L-type Ca2+ currents (De

Mello, 1998). Furthermore, the presence of specific intracellular angiotensin II

binding proteins has been reported in other preparations, such as liver (Kiron

and Soffer, 1989), cardiovascular myocytes (Robertson and Khairallah, 1971;

Sadoshima et al., 1993) and mesangial cells (Mercure et al., 1998). However, it

is not clear whether these intracellular angiotensin II binding proteins represent

internalised plasma membrane receptors or a genuine new class of angiotensin

II receptors. We recently reported that intracellular angiotensin II induces

Chapter III

42

contraction of rat aortic muscle by a mechanism independent of extracellular

AT receptors (Brailoiu et al., 1999). The effects of intracellular angiotensin II

in smooth muscle have been attributed to an interaction with specific

intracellular AT receptors, because the effects were sensitive to specific AT1

(Brailoiu et al., 1999; Haller et al., 1996) or partly sensitive to AT2 receptor

antagonists (Brailoiu et al., 1999).

The aim of the present work was to demonstrate that intracellularly

administered angiotensin II induces cellular effects in a cell line that does not

respond to extracellular angiotensin II. To this end, we used A7r5 vascular

smooth muscle cells. Intracellular angiotensin II effects on [Ca2+]i homeostasis

were studied, since this parameter is of major importance in smooth muscle

physiology. A7r5 cells lack functional responses typical for extracellular

angiotensin II stimulation. However, after intracellular application, we found

that angiotensin II is able to modulate [Ca2+]i homeostasis at different levels.

Part of this work has been communicated in abstract form (Filipeanu et al.,

1998a).

2. Material and methods

2.1. Chemicals

All culture media were obtained from Gibco BRL (U.S.A.). Inositol 1,4,5-

trisphosphate sodium salt (Ins(1,4,5)P3) was obtained from Boehringer

(Germany). Losartan and PD123319 ((s)-1-(4-[dimethylamino]-3-

methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-

c]pyridine-6-carboxylate) were kindly provided by Merck Sharp and Dohme

(U.S.A.), and Park Davis Company (U.S.A.), respectively. Angiotensin II was

supplied by the Academic Hospital Pharmacy of the University of Groningen.

Fura-2 acetoxymethylester and angiotensin II-fluorescein were obtained from

Molecular Probes (U.S.A.). 45CaCl2 (specific activity: 19.3 Ci /g) and D-[inositol-

1-3H(N)]-inositol 1,4,5-trisphosphate (specific activity: 21.0 Ci /mmol) were

obtained from Dupont-NEN (U.S.A.). [3H] thymidine (specific activity: 24 Ci


43

/mmol) was from Amersham Nederland (the Netherlands). GDP!S and all other

compounds were obtained from Sigma (U.S.A.).

2.2. Cell culture

A7r5 cells (a stable cell line derived from foetal rat aorta) were kindly provided

by Dr. H. De Smedt (K. U. Leuven, Belgium) and were cultured in Dulbeco’s

modified Eagle’s medium (DMEM) including antibiotics and supplemented with

7 mM NaHCO3, 10 mM HEPES at pH 7.2 and 10 % foetal calf serum at 37 0C in

95% air, 5% CO2. Confluent monolayers in 75-cm2 flasks (Costar) were

subcultured by trypsinisation. The medium was changed twice a week.

2.3 Liposomes preparation and intracellular application

Liposomes were prepared as described previously (Brailoiu et al., 1993; 1999),

using 10 mg phosphatidylcholine per ml of solution containing the substance to

be incorporated. The number of lamellae was decreased by addition of diethyl

ether in a ratio of 1/10 (v/v). Angiotensin II was dissolved at a concentration of

10-6 M in 140 mM KCl solution (pH 7.0). Control liposomes contained only

140 mM KCl. In order to remove non-incorporated solutes, liposomes were

subjected to dialysis, 2 times during 120 min, against buffer solution in a ratio

of 1/600 v/v. (Sigma dialysis tubing, molecular weight cut-off: 12400 Da). The

buffer solution had the following composition (mM): 145 NaCl, 5 KCl, 0.5

MgSO4, 0.5 CaCl2, 10 glucose and 10 HEPES (pH adjusted to 7.4 with NaOH).

Angiotensin II delivery into the cells was monitored by fluorescence

microscopy as described previously (Kok et al., 1998). Cells were loaded with

fluorescein-angiotensin II-filled liposomes according to the protocol as

described below for 45Ca2+ uptake by intact cells. The cells were washed 3 times

with liposome-free buffer solution after loading for 5 min at room temperature

and kept on ice until the photomicrographs were taken. The amount of

angiotensin II delivered into the cells by this procedure was estimated by

loading the cells with 10-6 M fluorescein-angiotensin II-filled liposomes and

subsequent cell permeabilisation with saponin comparable to the method

Chapter III

44

described for intracellular adenosine delivery (Brailoiu et al., 1993).

Fluorescence was measured with an excitation wavelength 470 nm, an emission

wavelength 520 nm, and a bandpass filter of 4 nm (Aminco Bowman LS Series

2). Liposome-encapsulated fluorescein-angiotensin II amounted to 12.4 ± 0.9

% (n=4) of the initial amount in the aqueous phase. Angiotensin II incorporated

into the cells with an efficiency of 2.3 ± 0.4 % (n=4). The actual intracellular

[angiotensin II] can be calculated based on an estimation of the cell volume.

The volume of A7r5 cells was estimated by determination of the maximal

radius (r) of spherical cells and calculation of the volume (V) according to

V=4/3*%*r3. Osmotic swelling of cells attached to the dish was obtained by

sequential dilution of the culture medium by addition of water. The osmotic

swelling process was monitored under microscopy and the maximal diameter

reached was estimated with a standard micrometer. The maximal diameter was

13.4 & 0.2 'm (n=64), resulting in a volume of 1.26 & 0.02 pl /cell. Therefore,

with the protocol used to deliver liposomes filled with 10-6 M angiotensin II to

the cells, the estimated intracellular [angiotensin II] is 18 & 3 'M.

2.4. 45Ca2+ uptake by intact cells45Ca2+ uptake was measured essentially as described previously (Sipma et al.,

1996). A7r5 cells were plated in 6 well plates 24 h prior to the experiment at a

density of 105 /well. Culture medium was replaced 1 h before the start of the

experiment with a buffer solution of room temperature (22-24 oC) containing (in

mM): 145 NaCl, 5 KCl, 0.5 MgSO4, 0.5 CaCl2, 10 glucose and 10 HEPES (pH

adjusted to 7.4 with NaOH). Uptake of 45Ca2+ was measured at room temperature

and was started by removing the solution and replacing it with the same buffer (1

ml) supplemented with 10 'Ci 45Ca2+ (specific activity 19.3 Ci /g) and indicated

compounds. The liposomes were added at a ratio of 1/20 (v/v) in the buffer

solution. Aspiration of the solution and addition of 1ml ice-cold buffer in the

absence of CaCl2 stopped the uptake of 45Ca2+ after 5 min. After this procedure,

cells were washed 3 times with buffer without CaCl2 but containing 2 mM

EGTA. Cells were lysed in the presence of NaOH (1 ml, 1 M) and radioactivity


45

was measured by liquid scintillation counting. Data are corrected for non-specific

binding as determined by addition of buffer with 45Ca2+ and immediate

termination of uptake.

2.5. [Ca2+]i measurements

Cells were loaded with the fluorescent Ca2+ indicator fura-2 acetoxymethylester

(5 'M) for 45 min at 37 0C. Ca2+ measurements were performed using a S100

Axiovert inverted microscope (Zeiss). The 340/380 ratio was acquired at room

temperature at a frequency of 1 Hz using a cooled CCD camera (SensiCam) and

Workbench 2.2. Imaging software (Axon.Instruments). Liposomes were added at

a ratio of 1/20 v/v, 5 min before agonist addition. Ratio values were transformed

into [Ca2+]i at the end of the experiment (Grynkiewicz et al., 1985).

2.6. 45Ca2+ efflux measurements in permeabilised cells

The cells were plated 24-48 h before the experiment in 6 well plates (Costar) at a

density of 1-1.5x105 cells /well. The experiments were carried out at room

temperature (22-24 0C) exactly as described previously (Missiaen et al., 1990;

Van der Zee et al., 1995). In brief, the cells were equilibrated for 1 h with a

modified buffer solution of the following composition (in mM): 135 NaCl, 5.9

KCl, 2.5 CaCl2, 1.2 MgCl2, 11.6 HEPES and 11.5 glucose (pH adjusted to 7.4

with NaOH). Cells were permeabilised for 10 min using 40 'g /ml saponin in a

solution containing (in mM): 100 KCl, 30 imidazole, 2 MgCl2, 1 ATP and 1

EGTA (pH adjusted to 7.0 with KOH). Subsequently, the calcium stores were

loaded with 45Ca2+ by exposure for 5 min to 500 'l buffer solution containing

10.5 'Ci /ml 45CaCl2 (specific activity 19.3 Ci /g) with a final composition (in

mM) of 100 KCl, 5 MgCl2, 5 ATP, 5 NaN3, 0.44 EGTA and 0.12 CaCl2. The

final [Ca2+]free of this solution was calculated to be 150 nM. Efflux buffer solution

containing (in mM) 100 KCl, 30 imidazole, 2 MgCl2, 1 EGTA, and 5 NaN3 (pH

adjusted to 7.0 with KOH) was added (1 ml) and replaced every 2 min for 30 min.

The 45Ca2+ remaining in the cells at the end of the efflux procedure was extracted

with 1 ml of 1 M NaOH. 45Ca2+ release is expressed as the fractional loss per

Chapter III

46

minute, representing the amount of 45Ca2+ leaving the cell, normalised to the total

amount of 45Ca2+ in the cell.

2.7. Measurement of Ins(1,4,5)P3

Mass measurement of Ins(1,4,5)P3 was performed as described earlier (Sipma et

al., 1996), using a standard curve of Ins(1,4,5)P3 in ether-extracted trichloroacetic

acid solution. The samples were assayed in 25 mM Tris/HCl (pH=9), 1 mM

EDTA, 1 mg bovine serum albumin, [3H]Ins(1,4,5)P3 (3.3 Ci/mmol, 2000

cpm/assay) and about 1 mg binding protein (isolated from fresh cattle liver) for

15 min. Bound and free radioactivity were separated by centrifugation. The

radioactivity in the pellet was determined by scintillation counting.

2.8. Measurement angiotensin-converting enzyme activity

To determine angiotensin-converting enzyme activity cells were plated 48 h

before the experiment in 25-cm2 flasks (Costar) at a density of 105 cells /flask.

Cells were trypsinized at confluence, centrifuged at 2500 g and the pellet was

resuspended in 0.2 ml phosphate-buffered saline solution and homogenized by

sonification. Cell homogenates were assayed as described before (Roks et al.,

1999). The lower detection limit of the assay was 2 pmol/mg protein/min.

2.9. Statistics

All experiments were performed in series with n # 4 on different days using

different cell passages. The results are expressed as means ± S.D. Statistical

differences were tested either by analysis of variance (ANOVA) followed by

Bonferroni post-test or by unpaired Student’s t-test considering P ( 0.05

significantly different.


47

3. Results

We used liposomes to administer angiotensin II intracellularly. Various

compounds can be delivered by liposomes, while plasma membrane integrity is

maintained (Brailoiu et al., 1993; Brailoiu and Van der Kloot, 1996; Filipeanu

et al., 1998b). Cells are not metabolically compromised by liposome treatment,

since methylene blue is still excluded and incubation with control liposomes (24

h) did not affect cell growth (data not shown). Intracellular delivery of

Angiotensin II was also followed by fluorescence microscopy of the cells

incubated with liposomes containing fluorescein-angiotensin II.

These experiments confirmed the intracellular delivery of fluorescein-

angiotensin II. At a concentration of 10-6 M fluorescein-angiotensin II, the

fluorescence pattern showed a relatively uniform cytosolic distribution in

comparison to control liposomes filled with 140 mM KCl (Fig. 1, middle and

left panel). At much higher concentrations (3x10-5 M filled liposomes)

fluorescein-angiotensin II appeared also in more vesicular-like structures,

indicating the uptake of fluorescence by internal organelles (Fig.1, right panel).

No fluorescence was observed in the nucleus at any concentration. It should be

noted that the [angiotensin II] used to generate all functional data (10-6 M

Fig. 1. Intracellular delivery of fluorescein- angiotensin II in A7r5 cells. Cells wereincubated with: A, control liposomes containing KCl (140 mM); B, liposomes containingfluorescein-angiotensin II (10-6 M) or C, liposomes containing fluorescein-angiotensin II(3x10-5 M). Representative photomicrographs of 3-6 coverslips are shown.

Chapter III

48

angiotensin II filled liposomes) ensured a relatively uniform intracellular

distribution.

3.2. 45Ca2+ uptake in intact cells

To examine the effects of intracellular angiotensin II on Ca2+ homeostasis in

A7r5 cells we first used 45Ca2+ flux as a parameter. Addition of extracellular

angiotensin II did not modify the basal 45Ca2+ uptake (2.2 & 3.8 % vs. control). In

contrast, addition of 10-6 M angiotensin II-containing liposomes induced a

moderate increase in 45Ca2+ uptake by 32.2 & 11.2 % (n=16), which was not

sensitive to the intracellular administration of the angiotensin AT1 receptor

antagonist losartan (10-6 M) or to the angiotensin AT2 receptor antagonist

PD123319 (10-6 M; Fig. 2). Control liposomes did not change basal 45Ca2+

uptake. Extracellular addition of losartan or PD123319 was ineffective in

reversing the intracellular angiotensin II effect. Angiotensin I (10-6 M)-filled

liposomes did not modify basal 45Ca2+ uptake.

100

110

120

130

140

150

45C

a2+

upt

ake

(% o

f con

trol

)

Lcon LAngII+Llos +LPD

LAngII LAngII

*

**

Fig. 2. Effects of intracellularangiotensin II on 45Ca2+ uptakeby intact A7r5 cells. Uptake wasmeasured for control liposomesfilled with 140 mM KCl (Lcon ,n=18) or liposomes filled withangiotensin II alone (10-6 M,LAngII, n=18) or in the presence oflosartan (10-6 M, LangII+Llos,n=12) or PD123319 (10-6 M,LangII+LPD, n=12). Net uptakewas measured for 5 min. Data arepresented as mean ± S.D. Thebasal 100 % level corresponds to57 & 3 dpm (n=48). Significanceindications: * P<0.05 vs. Lcon.


49

3.3. [Ca2+]i measurements

Fig. 3. Effect of intracellular angiotensin II on [Ca2+]i in intact A7r5 cells. Panel A: typicaltrace showing the effects of angiotensin II-containing liposomes (10-6 M, LAngII) in normalCa2+ containing medium. Panel B: Concentration dependency of Ca2+ increases above controlvalues using different concentrations of angiotensin II filled liposomes. Data are presented asmean & S.D. (n=6-8). Panel C: typical trace showing the effect of angiotensin II-containingliposomes (10-6 M, LAngII) in Ca2+ free extracellular medium followed by restoration ofnormal Ca2+ concentration. Panel D: statistics showing the effects on [Ca2+]i of intracellularangiotensin II, intracellular angiotensin II antagonists and extracellular angiotensin II.Control liposomes were filled with KCl (140 mM, Lcon). Liposomes were filled either withangiotensin II (10-6 M, LAngII), angiotensin II together with losartan (10-6 M, LAngII +Llos),angiotensin II together with PD123319 (10-6 M, LAngII +LPD) or angiotensin II–containingliposomes were combined with extracellular angiotensin II (10-6 M, LAngII +AngII). Data arepresented as mean & S.D. (n=12 for each condition). Significance indications: * P<0.05 vs.Lcon.

tim e (s)

0 60 120 180 240

0

20

40

60

80

100

tim e (s)

0 60 120 180 240

0

20

40

60

80

100

0

10

20

30

40

50

60

Lc

on

L AngII

LAngII

Ca2+-free *

** *

log L[AngII] (M )

-10 -9 -8 -7 -6

0

10

20

30

40

50

LAngII

+L

los

+L

PD

+A

ng

II

A B

C D[C

a 2

+ ] i (

nM

)

[Ca

2+ ] i

(n

M)

[Ca

2+ ] i

(n

M)

inc

rea

se

in

[C

a

2

+ ] i (

nM

)

Chapter III

50

Similar results were obtained by measuring [Ca2+]i with fura-2 fluorescence.

Basal [Ca2+]i amounted to 57 & 4 nM (n=54). Addition of extracellular

angiotensin II (10-6 M) did not change this value (58 & 6 nM, n=12). In

contrast, angiotensin II-filled liposomes induced a slowly developing Ca2+

increase (Fig. 3A). A dose-dependent effect was observed, starting from 10-9 M

angiotensin II and reaching a maximal increase of 34 & 6 nM (n=8) at 10-6 M

angiotensin II (Fig. 3B). The EC50 value was obtained with liposomes filled

with 3 10-8 M angiotensin II. Taken into account the delivery efficiency and the

determined cell volume, this value can be extrapolated to an actual effective

intracellular [angiotensin] of approximately 200 nM. Angiotensin II-filled

liposomes failed to increase [Ca2+]i in Ca2+-free medium, but induced a normal

response after restoration of the normal extracellular Ca2+ concentration (Fig.

3C). The voltage-dependent Ca2+ channel blocker verapamil (10-5M) did not

modify the angiotensin II-induced Ca2+ influx (30 & 5 nM, n=4). Inclusion of

angiotensin AT1 and angiotensin AT2 receptor antagonists, losartan and

PD123319, into the liposomes did not alter the effects of angiotensin II-filled

liposomes (Fig. 3D). Extracellular angiotensin II administration prior to

intracellular angiotensin II application also did not modify the intracellular

angiotensin II effect (Fig. 3D).

3.4. Ins(1,4,5)P3 production

The involvement of phospholipase C activation after stimulation with

intracellular angiotensin II was examined by measuring Ins(1,4,5)P3 mass

production (Table 1). Extracellular serotonin (5-HT) stimulated Ins(1,4,5)P3

production substantially (2.2-fold increase), indicating the presence of a

functional G-protein/phospholipase C system in these cells. Stimulation with

angiotensin II-filled liposomes resulted in a 1.7-fold stimulation of Ins(1,4,5)P3

formation. As expected in view of the absence of functional plasma membrane

angiotensin AT1 receptors no net increase was observed after stimulation with

extracellular angiotensin II.


51

Table 1. Effects of intracellular angiotensin II application on Ins(1,4,5)P3

formation in intact A7r5 cells in comparison to extracellular stimulation

_________________________________________________________________

treatment Ins(1,4,5)P3 formation (pmol 105 cells-1)

_________________________________________________________________basal 0.23 ± 0.05 (12)

control liposomes (Lcon) 0.21 ± 0.07 (6)

angiotensin II filled liposomes 0.37 ± 0.05 (6) a, b

extracellular angiotensin II 0.22 ± 0.04 (6)

extracellular 5-HT 0.52 & 0.08 (6) a

__________________________________________________________________

Cells were stimulated with control liposomes filled with 140 mM KCl , with liposomesfilled with angiotensin II (10-6 M) or with extracellular angiotensin II (10-6 M).Ins(1,4,5)P3 formation was measured 1 min after stimulation. Serotonin (5-HT, 10-5 M, 1min) was used as positive control for G-protein coupled receptor stimulation. Data arepresented as mean ± S.D. (number of experiments). Significance indications: a P<0.05 vs.basal, b P<0.05 vs. control liposomes

3.5. 45Ca2+ fluxes in permeabilised cells

To further investigate signal transduction pathways influenced by intracellular

angiotensin II, we used permeabilised A7r5 cells after pre-loading the calcium

stores with 45Ca2+. The size of the Ins(1,4,5)P3-sensitive store is represented by

the fractional loss of 45Ca2+ in response to 10-5 M Ins(1,4,5)P3 from t=16 to

t=18 min after the start of the efflux measurement, amounted to 0.14 & 0.01

min-1 (n=36; Fig. 4A). The basal fractional loss was not changed by addition of

10-6 M angiotensin II at time t=12 min. However, this pre-treatment potentiated

the subsequent 45Ca2+ release induced by Ins(1,4,5)P3 by 21 & 3 % (P<0.05;

Fig. 4A). The stimulatory effect of angiotensin II on Ins(1,4,5)P3-induced45Ca2+ release was dependent on the Ins(1,4,5)P3 concentration, becoming

apparent above 3x10-6 M Ins(1,4,5)P3 (Fig. 4B).

Chapter III

52

Furthermore, this potentiation was dependent on the angiotensin II

concentration with an EC50 value of approximately 2 nM (Fig. 5A). The effect

of angiotensin II was not sensitive to the angiotensin AT1 receptor antagonist

losartan or to the angiotensin AT2 receptor antagonist PD123319 (Fig. 5B). As

in the case of 45Ca2+ flux in intact cells, the related peptide angiotensin I (10-6

M) modified neither Ins(1,4,5)P3-mediated 45Ca2+ release (n=4, data not shown)

nor basal 45Ca2+ release (99 & 2 % vs control, n=4). Cell permeabilisation with

saponin is expected to destroy any plasma membrane receptor signal

time (min)4 8 12 16 20 24

fra

cti

on

al

los

s (

min

-1

)

0.00

0.03

0.06

0.09

0.12

0.15

0.18

0.21

Ins(1,4,5)P3

*

log [Ins(1,4,5)P3] (M)-7 -6 -5

*

*

AngII

A B

Fig. 4. Effects of angiotensin II on Ins(1,4,5)P3 -induced 45Ca2+ release inpermeabilised A7r5 cells. A: Typical experiment in permeabilised cells withpreloaded non-mitochondrial 45Ca2+ stores. Control conditions ()) or (o) in thepresence of angiotensin II (10-6 M) added at t=12. Horizontal bars represents thepresence of Ins(1,4,5)P3 (10-5 M) and angiotensin II. Note that angiotensin II did notrelease 45Ca2+ by itself, but potentiated the Ins(1,4,5)P3 induced 45Ca2+ release. B:Stimulatory effect of angiotensin II (10-6 M) depends on [Ins(1,4,5)P3]. Data arepresented as mean & S.D. ([Ins(1,4,5)P3] below 10-5 M: n=4, at 10-5 M: n= 36 in theabsence and n=12 in the presence of angiotensin II). Significance indications: *P<0.05 vs. Ins(1,4,5)P3 alone.


53

transduction. Accordingly, 5-HT (10-5 M) was unable to induce 45Ca2+ release

in permeabilised cells (101 & 2 % vs control, n=4).

Small G proteins were previously reported to modulate Ca2+ mobilisation (Xu

et al., 1996a, 1996b; Loomis-Husselbee et al., 1998). To investigate whether

such proteins are involved in the observed angiotensin II effects, we tested the

responses in the presence of GDP!S. Although this compound greatly

attenuated the effects of Ins(1,4,5)P3, the potentiating effect of angiotensin II

was still present (Table 2).

log [AngII] (M)

-10 -9 -8 -7 -6

Ins

(1,4

,5) P

3 -

indu

ced

45C

a2+

rele

ase

(%

of c

ontro

l)

100

105

110

115

120

125

130

80

90

100

110

120

130

control AngII AngII +PD

* **

*

**

*

AngII +los

A

B

Fig. 5 Pharmacological propertiesof the angiotensin II effect onIns(1,4,5)P3-induced 45Ca2+

release in permeabilised cells. A:Concentration dependency ofangiotensin II on 10-5 MIns(1,4,5)P3 induced 45Ca2+

release. The experiment wasperformed as described in thelegend of Fig. 4. Data arepresented as mean & S.D., (n=12at 10-6 M and n=4 at lowerconcentrations). Significanceindications: * P<0.05 vs.Ins(1,4,5)P3 alone. B: Effects ofthe angiotensin AT1 receptorantagonist losartan (10-6M, n=4)and the angiotensin AT2 receptorantagonist PD123319 (10-6M,n=4) on angiotensin II (10-6M)stimulated 45Ca2+ release inducedby Ins(1,4,5)P3 (10-5 M). Numberof control experiments: n=36 inthe absence and n=12 in thepresence of angiotensin II Dataare presented as mean ± S.D.Significance indications: * P<0.05vs. Ins(1,4,5)P3 alone.

Chapter III

54

Table 2 Effect of GDP!!!!S on Ins(1,4,5)P3 induced 45Ca2+ release in

permeabilised A7r5 cells

_________________________________________________________________

treatment effect (%)

_________________________________________________________________

control 100

+ GDP!S 55 & 12 a

+ angiotensin II 122 ± 4 a

+ GDP!S + angiotensin II 78 & 18 b, c

__________________________________________________________________

45Ca2+ release was initiated by 10-5 M Ins(1,4,5)P3 as described in the legend of Fig. 4.Angiotensin II (10-6 M) and GDP!S (5 10-6 M) were added 4 min prior to this stimulus.Data are presented as mean ± S.D. (4 experiments). Significance indications: a P<0.05vs. control, b P<0.05 vs. GDP!S, c P<0.05 vs. angiotensin II.

3.6. Intracellular ACE activity

Intracellular ACE activity was determined in exponentially growing A7r5 cell

cultures. No activity above the lower detection limit was observed (n=3).

4. Discussion

Although intracellular effects of angiotensin II have been shown previously

(Brailoiu et al., 1999; Haller et al., 1996; De Mello 1996; 1998), this is the first

report demonstrating its effects in a cell line devoid of extracellular angiotensin

II effects. Because of the presence of plasma membrane angiotensin II

receptors in previous studies, one could argue that the effects observed were

possibly mediated by internalisation of plasma membrane receptors occupied

by angiotensin II. A7r5 vascular smooth muscle cells lack functional responses

to extracellular angiotensin II, such as increases in Ins(1,4,5)P3 formation and

[Ca2+]i, which are typical for extracellular angiotensin II stimulation

(Griendling et al., 1997; Horiuchi et al., 1999). To administer intracellular

angiotensin II we used either liposomes or delivered angiotensin II directly into

the cytosol of permeabilised cells. With the liposome technique the integrity of


55

the plasma membrane is maintained (Brailoiu et al., 1993; Brailoiu and Van der

Kloot, 1996) and fluorescence microscopy confirmed the intracellular delivery of

angiotensin II. The intracellular actions of angiotensin II were not modified by

extracellular application of angiotensin II and/or its antagonists. Together, these

data preclude that the intracellular actions of angiotensin II were mediated by

an internalised angiotensin AT receptor. Therefore, the first conclusion drawn

from our study is that intracellular angiotensin II acts independently of

extracellular angiotensin II receptor stimulation.

Our results obtained for both 45Ca2+ uptake and [Ca2+]i measurements

unequivocally demonstrate that intracellular angiotensin II stimulates Ca2+ influx

via non-voltage-dependent Ca2+ channels in A7r5 cells. A similar observation was

made in adult rat aorta, showing that the intracellular angiotensin II-induced

contraction was entirely dependent on Ca2+ influx from the extracellular space via

non-voltage-dependent Ca2+ channels (Brailoiu et al., 1999). The action of

intracellular angiotensin II in permeabilised cells has not been studied before.

Angiotensin II did not release Ca2+ from intracellular stores, but potentiated the

effects of Ins(1,4,5)P3 in a losartan- and PD123319-insensitive manner. The

observed increase in Ins(1,4,5)P3-induced Ca2+ release by intracellular

angiotensin II was of a similar magnitude to that observed for various other

compounds in permeabilised A7r5 cells (Missiaen et al., 1997) and other cell

types (Van der Zee et al., 1995; Loomis-Husselbee et al., 1998). The involvement

of G-proteins was noticed in Ins(1,4,5)P3 -induced Ca2+ channel activation in

pancreatic acinar cells (Xu et al., 1996a; Xu et al., 1996b) and in Ins(2,4,5)P3 -

activated Ca2+ mobilisation in L1210 cells (Loomis-Husselbee et al., 1998).

Although we observed a large inhibition of Ins(1,4,5)P3-activated Ca2+

mobilisation by GDP!S, as observed in acinar cells (Xu et al., 1996b), the

stimulation of Ca2+ release by angiotensin II was unaffected. This indicates that

G-proteins are not involved in the modulation of Ins(1,4,5)P3-activated Ca2+

mobilisation by intracellular angiotensin II. In saponin-permeabilised A7r5 cells,

Ca2+ was no longer released upon stimulation with 5-HT, suggesting the absence

of functional plasma membrane receptors under this condition. The effective

Chapter III

56

concentration range for the effects of intracellularly delivered angiotensin II

observed in the present study was almost similar to that obtained for intracellular

angiotensin II-induced rat aorta contraction (Brailoiu et al., 1999) and

intracellular angiotensin II induced-growth of A7r5 cells (Filipeanu et al., 2001).

From the observed EC50 values in the present study, one can extrapolate the

effective intracellular [angiotensin II] to be approximately between 2-200 nM,

depending on the data obtained for Ins(1,4,5)P3-induced Ca2+ release or the data

for liposomal angiotensin II-induced Ca2+ increases. Reported values of

intracellular [angiotensin II] are in the range of low pM to low nM in cardiac

tissue and cardiomyocytes (De Mello and Danser, 2000; Sadoshima et al.,

1993). Therefore, it is not unlikely that intracellularly effective concentrations

can accumulate in the cell. Whether physiological stimuli can induce these

particular cellular concentrations or whether specific intracellular compartments

are involved remains to be established.

We have shown that both stimulation of Ca2+ influx and potentiation of

Ins(1,4,5)P3-mediated 45Ca2+ release are not affected by the typical angiotensin

AT1 receptor and angiotensin AT2 receptor antagonists, losartan and PD123319.

Together, these results obtained with different techniques demonstrate that

modulation of [Ca2+]i by intracellular angiotensin II occurs at different subcellular

levels. Angiotensin II acts via angiotensin non-AT1-/non-AT2 type receptors to

promote basal Ca2+ influx and to increase the Ins(1,4,5)P3 releasable Ca2+ pool.

The involvement of angiotensin non-AT1/non-AT2 type receptors in the

intracellular effects of angiotensin II has been suggested before. Intracellular

angiotensin II inhibited the L-type Ca2+ current via a non-AT1/non-AT2

mechanism in rat cardiac myocytes, but stimulated this current in hamster cardiac

myocytes (De Mello, 1998). In some aspects, the intracellular effects of

angiotensin II described in this study are different from those seen in other cell

types. In adult vascular smooth muscle cells, intracellular angiotensin II induced

Ca2+ influx as well, but its effect was completely abolished by an angiotensin AT1

receptor antagonist (Haller et al., 1996). An angiotensin AT1 receptor-like

mechanism was also reported for the effect of intracellular angiotensin II on cell-


57

to-cell communication in cardiomyocytes (De Mello, 1996). In rat aorta rings,

both intracellular angiotensin II and angiotensin I mediated smooth muscle

contraction through mechanisms sensitive to both angiotensin AT1 receptor or

angiotensin AT2 receptor antagonists (Brailoiu et al., 1999). Angiotensin I was not

a substitute for intracellular angiotensin II in A7r5 cells. This was also observed

for the intracellular angiotensin-induced growth of these cells (Filipeanu et al.,

2001). These studies, including the present observations, indicate the

heterogeneity of intracellular angiotensin II receptors with respect to modulation

of Ca2+ homeostasis among different cell types.

The physiological function of intracellular angiotensin II receptors is still unclear.

A possible (patho-) physiological role for intracellular angiotensin II is supported

by the presence of specific intracellular angiotensin II binding proteins

(Robertson and Khairallah, 1971; Kiron and Soffer, 1989; Li et al., 1998). The

only data concerning the functionality of the angiotensin non-AT1/non-AT2 type

receptor is the involvement of this receptor in angiogenesis (Le Noble et al.,

1996). The presence of intracellular angiotensin II in cardiovascular tissue is

established (Van Kats et al., 1997), supporting the concept of angiotensin

synthesis at cardiac sites, possibly after internalisation of plasma-derived renin

into the cells (De Mello and Danser, 2000). In addition to the heart, in

cardiomyocytes (Sadoshima et al., 1993) and in various other cell types (Erdmann

et al., 1996, Hermann and Ring, 1994; Mercure et al., 1998) the subcellular

localisation or measurable levels of intracellular angiotensin II, angiotensin I, or

other angiotensin metabolites have been reported. Endogenous angiotensin II

might be a physiological substrate of putative intracellular angiotensin receptors.

Internalisation of angiotensin II together with the receptor complex after

stimulation of the plasma membrane angiotensin AT1 receptor might contribute to

intracellular angiotensin II levels (Anderson et al., 1993; Hein et al., 1997). The

absence of measurable functional responses to extracellularly applied angiotensin

II on [Ca2+]i and the persistence of an intracellular angiotensin II effect after cell

permeabilisation makes this supposition unlikely in A7r5 cells. The absence of

plasma membrane angiotensin II receptors, the presence of putative angiotensin II

Chapter III

58

intracellular receptors together with the absence of angiotensin-converting

enzyme activity in A7r5 cells make these cells a valuable model for studying the

intracrine angiotensin system.

In summary, we showed that angiotensin II is an important modulator of cell

function even in the absence of extracellular actions. Intracellular application of

angiotensin II stimulates Ca2+ influx and Ins(1,4,5)P3 formation and increases

Ins(1,4,5)P3-inducible Ca2+ release. The pharmacological properties suggest the

involvement of putative intracellular angiotensin non-AT1/non-AT2 receptors.

Acknowledgements

C.M. Filipeanu is recipient of an Ubbo Emmius fellowship from the Groningen

University Institute for Drug Exploration (GUIDE). We thank Alex Kluppel for

technical assistance.

References

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Brailoiu, E., Van der Kloot, W.V., 1996. Bromoacetylcholine and acetylcholinesteraseintroduced via liposomes into motor nerve endings block increases in quantal size.Pfluegers Arch.-Eur. J. Physiol. 432, 413-418.

Brailoiu, E., Serban, D.N., Slatineanu, S., Filipeanu, C.M., Petrescu, B.C., Branisteanu,D.D., 1993. Effects of liposome-entrapped adenosine in the isolated rat aorta. EuropeanJ. Pharmacol. 250, 489-492.

Brailoiu, E., Filipeanu, C.M., Tica, A., Toma, C.P., De Zeeuw, D., Nelemans, S.A.,1999. Contractile effects by intracellular angiotensin II via receptors with a distinctpharmacological profile in rat aorta. Br. J. Pharmacol. 126, 1133-1138.

Chiu AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ, Duncia JV, Pease LJ, WongPC, Wexler RR, Johnson AL, et al., 1989. Identification of angiotensin II receptorsubtypes. Biochem. Biophys. Res. Commun. 165, 196-203.

Dell'Italia, L.J., Meng, Q.C., Balcells, E., Wei, C.C., Palmer, R., Hageman, G.R.,Durand, J., Hankes, G.H., Oparil, S., 1997. Compartmentalisation of angiotensin IIgeneration in the dog heart. Evidence for independent mechanisms in intravascular andinterstitial spaces. J. Clin. Invest. 100, 253-258.


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De Mello, W.C., 1996. Renin-Angiotensin system and cell communication in the fallingheart. Hypertension 27, 1267-1272.

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De Mello, W.C., Danser, A.H.J., 2000. Angiotensin II and the heart. On the intracrinerenin-angiotensin system. Hypertension 35, 1183-1188.

Erdmann, B., Fuxe, K., Ganten, D., 1996. Subcellular localisation of angiotensin IIimmunoreactivity in the rat cerebellar cortex. Hypertension 28, 818-824.

Filipeanu, C.M., Henning, R.H., De Zeeuw, D., Nelemans, S.A., 1998a. Functionalevidence for a role of intracellular angiotensin II in A7r5 cells. Br. J. Pharmacol. 123,141P (abstract).

Filipeanu, C.M., Brailoiu, E., Petrescu, G., Nelemans S.A., 1998b. Extracellular andintracellular arachidonic acid-induced contractions in rat aorta. European J.Pharmacol. 349, 67-73.

Filipeanu, C.M., Henning, R.H., De Zeeuw, D., Nelemans.A., 2001. Intracellularangiotensin II and cell growth of vascular smooth muscle cells. Br. J. Pharmacol. 132,1590-1596.

Griendling, K.K., Ushio-Fukai, M, Lassegue, B., Alexander, R.W., 1997. Angiotensin IIsignaling in vascular smooth muscle. New concepts. Hypertension 29, 366-373.

Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca2+ indicatorswith greatly improved fluorescence properties. J. Biol. Chem. 260, 3440-3449.

Haller, H., Lindschau, C., Erdmann, B., Quass, P., Luft, F.C., 1996. Effects ofintracellular angiotensin II in vascular smooth muscle cells. Circ. Res. 79, 765-772.

Hein, L., Meinel, L., Pratt, R.E., Dzau, V.J., Kobilka, B.K., 1997. Intracellulartrafficking of angiotensin II and its AT1 and AT2 receptors: evidence for selectivesorting of receptor and ligand. Mol. Endocrinol. 11, 1266-1277.

Hermann, K., Ring J., 1994. Human leukocytes contain angiotensin I, angiotensin II andangiotensin metabolites. Int. Arch. Allergy Immunol. 103, 152-159.

Horiuchi, M., Akishita, M., Dzau, V.J., 1999. Recent progress in angiotensin II type 2receptor research in the cardiovascular system. Hypertension 33, 613-21.

Hunyady, L., Balla, T., Catt, K.J., 1996. The ligand binding site of the angiotensin AT1receptor. Trends Pharmacol. Sci. 17, 135-140.

Kiron, M.A., Soffer, R.L., 1989. Purification and properties of a soluble angiotensin II-binding protein from rabbit liver. J. Biol. Chem., 264, 4138-4142.

Chapter III

60

Kok, J.W., Babia, T., Filipeanu, C.M., Nelemans, A., Egea, G., Hoekstra, D., 1998.PDMP blocks brefeldin A-induced retrograde membrane transport from golgi to ER:evidence for involvement of calcium homeostasis and dissociation from sphingolipidmetabolism. J. Cell Biol. 142, 25-38.

Le Noble, F.A., Kessels-Van Wylick, L.C., Hacking, W.J., Slaaf, D.W., Oude Egbrink,M.G., Struijker-Boudier, H.A., 1996. The role of angiotensin II and prostaglandins inarcade formation in a developing microvascular network. J. Vasc. Res. 33, 480-488.

Li, X., Shams, M., Zhu, J., Khalig, A., Wilkes, M., Whittle, M., Barnes, N., Ahmed, A.,1998. Cellular localisation of AT1 receptor mRNA and protein in normal placenta andits reduced expression in intrauterine growth. angiotensin II stimulates the release ofvasorelaxants. J. Clin. Invest. 101, 442-454.

Loomis-Husselbee, J.W., Walker, C.D., Bottomly, J.R., Cullen, P.J., Irvine, R.F.,Dawson, A.P., 1998. Modulation of Ins(2,4,5)P3-stimulated Ca2+ mobilisation byIns(1,3,4,5)P4: enhancement by activated G-proteins, and evidence for the involvementof a GAP1 protein, a putative Ins(1,3,4,5)P4 receptor. Biochem. J. 331, 947-952.

Mercure, C., Ramla, D., Garcia, R., Thibault, G., Deschepper, C.F., Reudelhuber, T.L.,1998. Evidence for intracellular generation of angiotensin II in rat juxtaglomerular cells.FEBS Lett. 422, 395-399.

Missiaen, L., Declerck, I., Droogmans, G., Plessers, L., De Smedt, H., Raeymaekers, L.,Casteels, R., 1990. Agonist-dependent Ca2+ and Mn2+ entry dependent on state of fillingof Ca2+ stores in aortic smooth muscle cells of the rat. J. Physiol. (Lond), 427, 171-186.

Missiaen, L., Parys, J. B., De Smedt, H., Sienaert, I., Sipma, H., Vanlingen, S., Maes, K.,Casteels, R., 1997. Effect of adenine nucleotides on myo-inositol-1,4,5-trisphosphate-induced calcium release. Biochem. J. 325, 661-666.

Robertson, A.L.Jr., Khairallah, P.A., 1971. Angiotensin II: rapid localisation in nuclei ofsmooth and cardiac muscle. Science 172, 1138-1139.

Roks, A.J., Van Geel, P.P., Pinto, Y.M., Buikema, H., Henning, R.H., De Zeeuw, D.,Van Gilst, W.H., 1999. Angiotensin-(1-7) is a modulator of the human renin-angiotensinsystem. Hypertension 34, 296-301.

Sadoshima, J., Xu, Y., Slayter, H.S., Izumo, S., 1993. Autocrine release of angiotensin IImediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell 75, 977-984.

Sipma, H., Van der Zee, L., Van den Akker, J., Den Hertog, A., Nelemans A., 1996. Theeffect of the PKC inhibitor GF109203X on the release of Ca2+ from internal stores andCa2+ entry in DDT1 MF-2 cells. Br. J. Pharmacol. 119, 730-736

Unger, T., Chung, O., Csikos, T., Culman, J., Gallinat, S., Gohke, P., Hohle, S., Meffert,S., Stoll, M., Stroth, U.,, Zhu YZ. , 1996. Angiotensin receptors. J. Hypertens. Suppl. 14,S95-S103.


61

Van der Zee, L., Sipma, H., Nelemans, A., Den Hertog A., 1995. The role of inositol1,3,4,5-tetrakisphosphate in internal Ca2+ mobilisation following histamine H1 receptorstimulation in DDT1 MF-2 cells. European J. Pharmacol. 289, 463-469.

Van Kats J.P., De Lannoy, L.M., Danser, J.A.H., Van Meegen, J.R., Verdouw, P.D.,Schalekamp, M.A., 1997. Angiotensin II type 1 (AT1) receptor-mediated accumulationof angiotensin II in tissues and its intracellular half-life in vivo. Hypertension 30, 42-49.

Xu, X., Zeng, W., Muallem, S., 1996a. Regulation of the inositol 1,4,5-trisphosphate -activated Ca2+ channel by activation of G proteins. J. Biol. Chem. 271, 11737-11744.

Xu, X., Zeng, W., Diaz, J., Muallem, S., 1996b. Spatial compartmentalisation of Ca2+

signalling complexes in pancreatic acini. J. Biol. Chem. 271, 24684-24690.

Chapter 4

Intracellular angiotensin II inhibits heterologous

receptor stimulated Ca2+ entry.

Catalin M. Filipeanu, Eugen Brailoiu, Robert H. Henning, Leo E. Deelman,

Dick de Zeeuw, S. Adriaan Nelemans

Life Sciences, provisionally accepted

Chapter IV

64

Abstract

Recent studies show that angiotensin II (AngII) can act from within the cell,

possibly via intracellular receptors pharmacologically different from typical

plasma membrane AngII receptors. The role of this intracellular AngII (AngIIi) is

unclear. Besides direct effects of AngIIi on cellular processes one could

hypothesise a possible role of AngIIi in modulation of cellular responses induced

after heterologous receptor stimulation. We therefore examined if AngIIi

influences [Ca2+]i in A7r5 smooth muscle cells after serotonin (5HT) or UTP

receptor stimulation. Application of AngIIi using liposomes, markedly inhibited45Ca2+ influx after receptor stimulation with 5HT or UTP. This inhibition was

reversible by intracellular administration of the AT1-antagonist losartan and not

influenced by the AT2-antagonist PD123319. Similar results were obtained in

single cell [Ca2+]i measurements, showing that AngIIi predominantly influences

Ca2+ influx and not Ca2+ release via AT1-like receptors. It is concluded that AngIIi

modulates signal transduction activated by heterologous receptor stimulation.

Abbreviations used: AngIIi, intracellular Angiotensin II; 5HT, serotonin; Ins(1,4,5)P3,

inositol 1,4,5-trisphosphate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetra-acetic acid;

losartan, (2-n- butyl-4-chloro-5-hydroxymethyl-1-[(2’-(1H-tetrazol-5-yl)biphenyl-4-

yl)methyl]imidazole); PD123319, (s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-

5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate

Introduction

Cellular effects of angiotensin II (AngII) observed under physiological and patho-

physiological conditions are attributed to the interaction with specific AT1- and

AT2- type AngII receptors in the plasma membrane [1]. These receptors are

coupled to different signal transduction pathways. Stimulation of AT1-receptors

activates phospholipase C, formation of Ins(1,4,5)P3, which subsequently

discharges Ca2+ from internal stores and activates MAP kinase, whereas AT2-

receptors increase cGMP levels and inhibit cell growth [2, 3]. Intracellular AngII

Intracellular angiotensin II crosstalk with extracellular hormones

65

binding proteins have been reported in liver [4], storage granules of cardiac

myocytes [5], mesangial cells [6], and within the cytosolic fraction of placenta

[7]. Recent studies show that AngII can act from within the cell, possibly via

intracellular receptors, which differ in their pharmacology from typical plasma

membrane AngII receptors [8-11]. Intracellular application of AngII (AngIIi)

inhibited cell communication through gap-junctions in heart muscle [8], induced

[Ca2+]i increases in vascular smooth muscle cells [9], initiated tyrosine

phosphorylation in myocytes [10] and elicited contraction of rat aorta [11].

Crosstalk between different receptor systems is essential for physiological

functioning. Second messengers induced after receptor stimulation often

modulate other second messenger systems either activated by themselves or by

other receptor types. Crosstalk have been described at the level of cAMP,

Ins(1,4,5)P3, [Ca2+]i and/or contraction after stimulation of heterologous

receptors in smooth muscle [12-15]. With respect to AngII, only Ca2+ dependent

transactivation of EGF receptors was reported after stimulation of the plasma

membrane AT1-receptor [16].

There are no data published on a possible role of intracellular AngII in the

modulation of cellular responses induced after stimulation of different plasma

membrane receptors (heterologous receptor stimulation). Therefore, we

investigated the effects of intracellular application of AngII on [Ca2+]i in A7r5

smooth muscle cells after serotonin (5HT) and UTP receptor stimulation.

Methods

Cell culture. A7r5 vascular smooth muscle cells (kindly provided by Dr. H. De

Smedt, K. U. Leuven, Belgium) were grown in 75cm2 flasks in Dulbecco’s

Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum

(FCS), penicillin (50 'g ml-1) and streptomycin (50 units ml-1) at 37oC in a

humidified atmosphere (5 % CO2). The cells were subcultured at 95%

confluency by trypsinization. 45Ca2+ experiments were performed in 6 well

plates (Costar, 9.6 cm2 well-1) at a density of 105cells well-1. For fluorescence

Chapter IV

66

experiments the cells were plated 24-72 hours before the start of the experiment

in LabTek II (type 155382) chambers.

Chemicals. All culture media were obtained from Gibco BRL (U.S.A.). Inositol

1,4,5-trisphosphate sodium salt was obtained from Boehringer (Germany).

Fura2-AM and AngII-fluorescein were obtained from Molecular Probes

(U.S.A.). 45CaCl2 (specific activity: 19.3 Ci g-1) and D-[2-3H]inositol 1.4,5-

trisphosphate (specific activity: 3.3 Ci mmol-1) were obtained from Dupont-

NEN (U.S.A.). AngII was supplied by the Academic Hospital Pharmacy of the

University of Groningen. Losartan (2-n- butyl-4-chloro-5-hydroxymethyl-1-

[(2’-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole) was provided by Merck

Sharp & Dohme (U.S.A.) and PD123319 ( (s)-1-(4-[dimethylamino]-3-

methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-

c]pyridine-6-carboxylate) by Park Davis Company (U.S.A.). All other

compounds were obtained from Sigma (U.S.A.).

Liposomes preparation and intracellular application. Liposomes were

prepared as described previously [17, 18], using 10 mg phosphatidylcholine per

ml of solution containing the substance to be incorporated. The number of

lamellae was decreased by addition of diethyl ether in a ratio of 1/10 (v/v).

AngII was dissolved at a concentration of 10-6 M in 140 mM KCl solution (pH

7.0). Control liposomes contained only 140 mM KCl. In order to remove non-

incorporated solutes, liposomes were subjected to dialysis, 2 times during 120

min, against buffer solution in a ratio of 1/600 v/v. (Sigma dialysis tubing,

molecular weight cut-off: 12400 dalton). The buffer solution had the following

composition (mM): 145 NaCl, 5 KCl, 0.5 MgSO4, 0.5 CaCl2, 10 glucose and 10

HEPES (pH adjusted to 7.4 with NaOH). The amount of Ang II delivered into

the cells by this procedure was estimated by loading the cells with 10-6 M

fluorescein-AngII filled liposomes and subsequent cell permeabilization with

saponin comparable to the method described for intracellular adenosine

delivery [17]. Fluorescence was measured with excitation wavelength 470 nm,


67

emission wavelength 520 nm, and a bandpass filter of 4 nm (Aminco Bowman

LS Series 2). Liposome-incapsulated fluorescein-AngII amounted to 12.4 ± 0.9

% (n=4) of the initial amount in the aqueous phase. Angiotensin II incorporated

into the cells with an efficiency of 2.3 ± 0.4 % (n=4). The actual [AngIIi] can

be calculated if an estimation of the cell volume is established. The cellular

volume of A7r5 cells was estimated by determination of the maximal radius (r)

of spherical cells and calculation of the volume (V) according to V=4/3*%*r3.

Cells were slightly attached to the culture dish and the culture medium was

sequentially diluted with an equal volume of water. The osmotic swelling

process was followed under microscopy and the maximal diameter reached was

estimated with a standard micrometer in view. The maximal diameter was 13.4

& 0.2 'm (n=64), resulting in a volume of 1.26 & 0.02 pL cell-1. Therefore, if

the protocol is used to deliver liposomes filled with 10-6 M AngII to the cells,

this will result in an estimated intracellular [AngII] of 18 & 3 'mol/L.

45Ca2+ uptake by intact cells. These experiments were performed as described

previously (19). In brief, culture medium was replaced 1 hr before the start of

the experiment with a buffer solution containing (mM): 145 NaCl, 5 KCl, 0.5

MgSO4, 0.5 CaCl2, 10 glucose and 10 HEPES (pH adjusted to 7.4 with NaOH).

Uptake of 45Ca2+ was measured at room temperature (22-24 0C) and started by

removing the solution and replacing it by the same buffer (1 ml) supplemented

with 10 'Ci 45Ca2+ (specific activity 19.3 Ci g-1) and indicated compounds. The

liposomes were added at a ratio of 1/20 (v/v) in the buffer solution. Aspiration

of the solution and addition of 1ml ice-cold buffer stopped uptake of 45Ca2+ in

the absence of CaCl2 after 5 min. Thereafter, cells were washed 3 times with

buffer without CaCl2 and containing 2 mM EGTA. Cells were lysed in the

presence of NaOH (1 ml, 1 M) and radioactivity was measured by liquid

scintillation counting. Data were corrected for non-specific binding as

determined by adding buffer with 45Ca2+ and immediately terminating uptake.

Chapter IV

68

[Ca2+]i measurements. Cells were loaded with 5 'M fura2-AM for 45 min at

37oC. Calcium measurements were performed using a S100 Axiovert inverted

microscope (Zeiss). The 340/380 ratio was acquired at room temperature with

at a frequency by 1 Hz using a cooled CCD camera (SensiCam) and Imaging

Workbench 2.2. software (Axon Instruments). Liposomes were added at a ratio

of 1/20 vol/vol, 5 min before agonist addition. Ratio values were transformed

in [Ca2+]i at the end of the experiment [20].

Measurement of Ins (1,4,5)P3. Mass measurement of Ins (1,4,5)P3 was performed

using a ligand binding assay as described earlier in detail [15].

Statistics. All experiments were performed in series with n # 4. The results are

expressed as mean ± S.D. Statistical differences were tested either by ANOVA

analysis followed by Bonferroni post-test or by unpaired Student’s t-test

considering p ( 0.05 significantly different.

Results

A7r5 cells represent an attractive model to investigate the effects of intracellular

AngII. Since these cells do not respond to extracellular addition of AngII (10-6 M)

in terms of [Ca2+]i or Ins(1,4,5)P3 levels (Table 1) one can study the effects of

AngIIi on signal transduction processes without interference of cellular signalling

activated by plasma membrane AngII receptors.

Table 1 Effects of extracellular AngII on [Ca2+]i and Ins(1,4,5)P3 levels in A7r5

cells

[Ca2+]i (nM) Ins(1,4,5)P3 (pmol/105 cells)

Basal 57 & 6 0.23 & 006

AngII 58 & 6 0.22 & 0.04

Peak [Ca2+]i and Ins(1,4,5)P3 were measured 30 s after stimulation with extracellularAngII (10-6 M). n # 6 in each case.


69

We used liposomes to administer AngII intracellularly. Various compounds can

be delivered by liposomes, while plasma membrane integrity is maintained (17,

18, 21). Cells are also not metabolically compromised by liposome treatment,

since methylene blue is still excluded and incubation with control liposomes (24

hr) did not affect cell growth (data not shown). Moreover, pre-treatment with

control liposomes (filled with 140 mM KCl) did not affect Ca2+ increases induced

by either 5HT or UTP (Fig.1).

Influx of 45Ca2+ was increased about 2-fold after stimulation with 5HT or UTP for

5 min (Fig. 1). This increase was partially inhibited by liposomes containing 10-6

M AngII, whereas control liposomes were ineffective. The effect of AngII filled

liposomes was unaffected by the extracellular addition of the AT1-receptor

antagonist losartan (10-6 M) or the AT2-receptor antagonist PD123319 (10-6 M,

n=6, data not shown). In contrast, inclusion of losartan into liposomes together

with AngII reversed the inhibition of 45Ca2+ influx by AngII. Liposomes

100

120

140

160

180

200

* *

+Lcon+Llos +LPD

45C

a2+

upt

ake

(%

of c

ontr

ol)

100

120

140

160

180

200

* *

+Lcon

A

B

+LAngII +LAngII +LAngII

+LAngII +LAngII +LAngII

+Llos +LPD

Fig. 1. Effects of intracellularAngII on 45Ca2+ uptakeinduced by 5HT or UTP. PanelA: 45Ca2+ uptake induced byextracellular 5HT (10-5 M) for 5min (first bar), in the presence ofliposomes filled with 140 mMKCl (Lcon) or filled with either10-6 M Ang II(+LAngII) alone ortogether with 10-6 M losartan(+LAngII +Llos) or 10-6 M PD123319 (+LAngII +LPD). Panel B:idem for extracellular stimulationwith UTP (10-3 M). Data arepresented as mean ± S.D. (n=6)and expressed as % of uptake inthe absence of extracellular 5HTor UTP amounting 85 dpm/105

cells. * P<0.05 vs controlstimulation (5HT or UTP alone).

Chapter IV

70

containing both AngII and PD123319 were ineffective in reversal of the AngIIi

effect.

The effect of AngIIi on 5HT and UTP induced changes in [Ca2+]i homeostasis

was confirmed by single cell [Ca2+]i measurements using fura2 fluorescence.

Pre-incubation with Ang II containing liposomes reduced the 5HT and UTP

induced [Ca2+]i increases similarly as in the 45Ca2+ uptake experiments to

approximately 30 % (Fig. 2).

tim e (s)0 60 120 180

0

100

200

300

400

500

600

700

0 60 120

0

100

200

300

a

b

a

b

5HT UTP

[Ca2

+ ] i (

nM

)[C

a2+ ] i in

cre

as

es (n

M)

0

100

200

300

400

500

+Lcon +LAngII

*

0

20

40

60

80

100

120

*

tim e (s)

A B

+Lcon +LAngII

Fig. 2. Effects of intracellular AngII on [Ca2+]i induced by 5HT or UTP.Upper panels: Changes in [Ca2+]i as measured by single cell fura2 fluorescence induced byextracellular 5HT (A, 10-5 M) or UTP (B, 10-3 M) in the absence (a) or presence (b) ofliposomes filled with 10-6 M AngII.Lower panels: maximal [Ca2+]i increases induced by 10-5 M 5HT (left panel) or 10-3 M UTP(right panel) in the absence (first bar) or the presence of liposomes (5 min pre-incubation)filled with 140 mM KCl (Lcon) or with 10-6 M AngII (LAngII). Data are presented as mean ±S.D. (n=6). * P<0.05 vs control.


71

Control liposomes filled with 140 mM KCl were ineffective. The inhibitory

effect was reversed by losartan containing liposomes, but not by PD123319

containing liposomes (Table 2). These results show that intracellular AngII

exerts its effect through an AT1-like receptor, probably via inhibition of Ca2+

influx.

Table 2 Inhibition of 5HT induced [Ca2+]i increases by intracellular Ang II.

Effects of antagonists

[Ca2+]i increases

L control + 5HT 100 & 8 (%)

L AngII + 5HT 27 & 8 a

L(AngII + losartan) + 5HT 72 & 16 a, b

L(AngII + PD123319) + 5HT 30 & 10 a

Increases in peak [Ca2+]i were measured after stimulation with 5HT (10-5 M) andliposomes filled with AngII (10-6 M) either in the absence or presence of losartan (10-6

M) or PD123319 (10-6 M). n # 6 in each case. a) different from L control + 5HT(P<0.05). b) different from L AngII + 5HT (P<0.05).

To clarify which part of Ca2+ signalling is affected by intracellular AngII we

further investigated this effect on 5HT induced changes in [Ca2+]i. Using a Ca2+

chelation/readmission protocol, Ca2+ release and Ca2+ influx could be

discriminated.

Stimulation with 5HT predominantly elevates [Ca2+]i via Ca2+ influx, which is

largely mediated via voltage dependent Ca2+ channel, as demonstrated by the

almost complete inhibition by 10-6 M verapamil (Fig. 3). Intracellular application

of AngII inhibits this 5HT induced Ca2+ influx to a similar extent as verapamil,

possibly indicating that inhibition of this channel by AngIIi occurs, whereas it

does not affect Ca2+ release.

Chapter IV

72

Discussion

Effects of AngIIi can be mediated via mechanisms atypical for plasma membrane

AngII receptors in various cell types [8-11]. This implies that patho-physiological

actions of AngII might occur intracellularly and therefore not accessible to

classical pharmacological agents working at the plasma membrane. Many studies

described crosstalk between components of different receptor classes and at

different levels downstream receptor activation [22-24]. However, the novelty of

the present work is to show interactions between an agonist (AngII) and

Fig. 3. Differential effects of intracellular AngII on Ca2+ release and Ca2+ influx.Changes in intracellular Ca2+ release and Ca2+ influx were measured by single cell fura2fluorescence after stimulation with extracellular 5HT (10-5 M) under Ca2+ free conditionsand after subsequent restoration of the normal [Ca2+] in the buffer solution. Left panel:tracing a) control, b) in the presence of verapamil (verap, 10-6 M, 5 min pre-incubation),c) in the presence of liposomes (5 min pre-incubation) filled with 10-6 M AngII (LAngII),Right panel: maximal Ca2+ increase. Open bars: Ca2+ release, filled bars: Ca2+ influx.Data are presented as mean ± S.D. (n=6). * P<0.05 vs control.

t im e (s )

0 1 2 0 2 4 0 3 6 0 4 8 0 6 0 0

[Ca

2+

] i (n

M)

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

5 H T

C a 2 + fre e

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

4 0 0

co

ntr

ol

+ v

era

p

L a

ng

II

*

*

a

b

c


73

heterologous receptor stimulation not initiated by AngII at the plasma membrane

level, but by intracellular receptor stimulation.

In this study we have used A7r5 cells which cannot be stimulated by extracellular

AngII as concluded from the results on Ins(1,4,5)P3 formation and [Ca2+]i.

Therefore interference of the results obtained by AngIIi on Ca2+ homeostasis with

possible effects induced by plasma membrane AT-receptors receptor is excluded.

AngIIi potently reduced the Ca2+ signal evoked by 5HT and UTP stimulation. In

view of the experiments performed with the antagonist losartan and PD123319 it

is suggested that this effect of AngIIi be mediated by an AT1-like receptor, but the

ineffectiveness of AngI indicate that it might be different from common plasma

membrane AT1 receptors [25].

Reductions of Ca2+ increases induced by AngIIi are the not the consequence of

inhibition of Ca2+ release from intracellular stores, but can be totally attributed to

inhibition of Ca2+ influx. The parallel effect of AngIIi and verapamil indicates that

the L-type Ca2+ channel might be the target for this effect. This novel finding of

the AngIIi induced inhibition of heterologous receptor activated Ca2+ influx can

be supported by the observations made in rat cardiomyocytes showing reduction

in amplitude of inward voltage-dependent Ca2+ current after dialysing AngII into

the cell [26].

Inhibition of heterologous receptor activated Ca2+ influx might be of

physiological relevance in the process of fine-tuning of multiple receptor

signalling pathways. Also overstimulation due to exposure to different

mediators can be avoided by such a mechanism, particular beneficial under

patho-physiological conditions in which Ca2+ overload is implicated like e.g.

ischemia. For such a negative protective feedback mechanism availability of

AngIIi is essential. It is likely that AngIIi can fulfil this role, since intracellular

pools of AngII were observed in cardiomyocytes [5] and renal endosomes [27]

under physiological conditions.

In conclusion, AngIIi inhibited Ca2+ entry induced after heterologous receptor

stimulation with either 5HT or UTP via an AT1-like receptor in A7r5 smooth

muscle cells.

Chapter IV

74

Acknowledgements

C.M. Filipeanu is recipient of an Ubbo Emmius fellowship from the Groningen

University Institute for Drug Exploration (GUIDE).

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[1] Griendling KK, Ushio-Fukai M, Lassegue B, Alexander RW. Angiotensin IIsignaling in vascular smooth muscle. New concepts. Hypertension, 1997; 29: 366-73.

[2] Hunyady L, Balla T, Catt KJ. The ligand binding site of the angiotensin AT1receptor. Trends Pharmacol Sci, 1996; 17: 135-140.

[3] Horiuchi M, Akishita M, Dzau, V.J. Recent progress in angiotensin II type 2 receptorresearch in the cardiovascular system. Hypertension, 1999; 33: 613-21.

[4] Kiron MA, Soffer RL. Purification and properties of a soluble angiotensin II-bindingprotein from rabbit liver. J Biol Chem, 1989; 264: 4138-4142.

[5] Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin IImediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell, 1993; 75: 977-984.

[6] Mercure C, Ramla D, Garcia R., Thibault G., Deschepper CF., Reudelhuber TL.Evidence for intracellular generation of angiotensin II in rat juxtaglomerular cells. FEBSLett, 1998; 422: 395-399.

[7] Li X, Shams M, Zhu J, Khalig A, Wilkes M, Whittle M, Barnes N, Ahmed A.Cellular localization of AT1 receptor mRNA and protein in normal placenta and itsreduced expression in intrauterine growth restriction. Angiotensin II stimulates therelease of vasorelaxants. J Clin Invest, 1998; 101: 442-454.

[8] De Mello WC. Renin-angiotensin system and cell communication in the failing heart.Hypertension, 1996; 27: 1267-1272.

[9] Haller H, Lindschau C, Erdmann B, Quass P, Luft FC. Effects of intracellularangiotensin II in vascular smooth muscle cells. Circ Res, 1996; 79: 765-772.

[10] Haller H, Lindschau C, Quass P, Luft FC. Intracellular actions of angiotensin II invascular smooth muscle cells. J Am Soc Nephrol, 1999; Suppl 11:S75-83.

[11] Brailoiu E, Filipeanu CM, Tica A, Toma CP, de Zeeuw D, Nelemans SA.Contractile effects by intracellular angiotensin II via receptors with a distinctpharmacological profile in rat aorta. Br J Pharmacol, 1999; 126: 1133-1138.


75

[12] Dickenson JM, Hill SJ. Intracellular cross-talk between receptors coupled tophospholipase C via pertussis toxin-sensitive and insensitive G-proteins in DDT1MF-2cells. Br J Pharmacol, 1993; 109: 719-24.

[13] Manolopoulos VG, Pipili-Synetos E, Den Hertog A., Nelemans A. Inositolphosphates formed in rat aorta after alpha 1-adrenoceptor stimulation are inhibited byforskolin. Eur J Pharmacol, 1991; 207: 29-36.

[14] Gerwins P, Fredholm BB. Stimulation of adenosine A1 receptors and bradykininreceptors, which act via different G proteins, synergistically raises inositol 1,4,5-trisphosphate and intracellular free calcium in DDT1 MF-2 smooth muscle cells. ProcNatl Acad Sci U S A, 1992; 89: 7330-4.

[15] Sipma H, Duin M, Hoiting B, den Hertog A, Nelemans, A. Regulation ofhistamine- and UTP-induced increases in Ins(1,4,5)P3, Ins (1,3,4,5)P4 and Ca2+ bycyclic AMP in DDT1 MF-2 cells Br J Pharmacol, 1995; 114: 383-90.

[16] Eguchi, S., Numaguchi, K., Iwasaki, H., Matsumoto, T., Yamakawa, T.,Utsunomiya, H., Motley, E.D., Kawakatsu, H., Owada, K.M., Hirata, Y., Marumo, F.and Inagami, T. Calcium-dependent epidermal growth factor receptor transactivationmediates the angiotensin II-induced mitogen-activated protein kinase activation invascular smooth muscle cells. J Biol Chem, 1998; 273: 8890-8896.

[17] Brailoiu E, Serban DN, Slatineanu S, Filipeanu CM, Petrescu BC, Branisteanu DD.Effects of liposome-entrapped adenosine in the isolated rat aorta. Eur J Pharmacol, 1993;250: 489-492.

[18] Filipeanu CM, Brailoiu E, Petrescu G, Nelemans SA. Extracellular and intracellulararachidonic acid-induced contractions in rat aorta. Eur J Pharmacol 1998; 349: 67-73.

[19] Sipma H, van der Zee L, van den Akker J, den Hertog A, Nelemans A. The effect ofthe PKC inhibitor GF109203X on the release of Ca2+ from internal stores and Ca2+entry in DDT1 MF-2 cells. Br J Pharmacol, 1996; 119: 730-736.

[20] Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators withgreatly improved fluorescence properties. J Biol Chem, 1985; 260: 3440-3449.

[21] Brailoiu E, van der Kloot WV. Bromoacetylcholine and acetylcholinesteraseintroduced via liposomes into motor nerve endings block increases in quantal size.Pfluegers Arch-Eur J Physiol, 1996; 432: 413-418.

[22]. Luttrell LM, Daaka Y, Lefkowitz RJ. Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Current Opinion Cell Biol, 1999; 11: 177-183.

[23] Selbie LA, Hill SJ. G protein-coupled-receptor cross-talk: the fine-tuning ofmultiple receptor-signalling pathways. Trends Pharmacol Sci., 1998; 19: 87-93.

[24]. Bornfeldt KE, Krebs EG. Crosstalk between protein kinase A and growth factorreceptor-signalling pathways in arterial smooth muscle. Cell Signal, 1999; 11: 465-477.

Chapter IV

76

[25] Tschudi MR, Luscher TF. Age and hypertension differently affect coronarycontractions to endothelin-1, serotonin, and angiotensins. Circulation, 1995; 91: 2415-22.

[26] De Mello WC. Intracellular angiotensin II regulates the inward calcium current incardiac myocytes. Hypertension, 1998; 32: 976-82.

[27] Imig, J.D., Navar, G.L, Zou, L.X., O'Reilly, K.C., Allen, P.L., Kaysen, J.H.,Hammond, T.G. & Navar, L.G. Renal endosomes contain angiotensin peptides,converting enzyme, and AT(1A) receptors. Am J Physiol, 1999; 277, F303-311.

Chapter 5

Intracellular angiotensin II and cell growth of vascular

smooth muscle cells.

Catalin M. Filipeanu, Robert H. Henning, Dick de Zeeuw and Adriaan

Nelemans

British Journal of Pharmacology 132:1590-1596, 2001.

Chapter V

78

Summary

1.We recently demonstrated that intracellular application of angiotensin II

(AngIIintr) induces rat aorta contraction independent of plasma membrane AngII

receptors. In this study we investigated the effects of AngIIintr on cell growth in

A7r5 smooth muscle cells.

2. DNA-synthesis was increased dose-dependently by liposomes filled with

AngII as measured by [3H]thymidine incorporation at high (EC50=27&6 pM) and

low (EC50=14&5 nM) affinity binding sites with increases in Emax of 58&4 and

37&4 % above quiescent cells, respectively. Cell growth was corroborated by an

increase in cell number.

3. Extracellular AngII (10 pM-10 'M) did not modify [3H]thymidine

incorporation.

4. Growth effects of AngIIintr mediated via high affinity sites were inhibited by

liposomes filled with 1 'M of the non-peptidergic antagonists losartan (AT1-

receptor) or PD123319 (AT2-receptor) or with the peptidergic agonist

CGP42112A (AT2-receptor). Emax values were decreased to 30±3, 29±4 and

4±2 %, respectively, without changes in EC50. The AngIIintr effect via low

affinity sites was only antagonised by CGP42112A (Emax =11±3 %), while

losartan and PD123319 increased Emax to 69±4 %. Intracellular applications

were ineffective in the absence of AngIIintr.

5. Neither intracellular nor extracellular AngI (1 'M) were effective.

6. The AngIIintr induced growth response was blocked by selective inhibition of

phosphatidyl inositol 3-kinase (PI-3K) by wortmannin (1 'M) and of the

mitogen-activated protein kinase (MAPK/ERK) pathway by PD98059 (1 'M) to

61±14 and 4±8 % of control, respectively.

7. These data demonstrate that AngIIintr induces cell growth through atypical

AT-receptors via a PI-3K and MAPK/ERK -sensitive pathway.

Keywords: intracellular angiotensin II, growth, losartan, PD123319,

CGP42112A, PI-3 kinase, MAP kinase, A7r5 cells

Abbreviations: AngIIintr, intracellular angiotensin II; CGP42112A, nicotinic acid-Tyr-

(N-benzoylcarbonyl-Arg)-Lys-His-Pro-Ile-OH; DMEM, Dulbecco’s Modified Eagle’s

Intracellular AngII stimulates cell growth

79

Medium; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum;

Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; losartan, (2-n- butyl-4-chloro-5-

hydroxymethyl-1-[(2’-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole); MAPK,

mitogen-activated protein kinase; PI-3K, phosphatidyl inositol 3-kinase; PD98059, 2-

(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; PD123319, (s)-1-(4-

[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-

imidazo[4,5-c]pyridine-6-carboxylate

Introduction

It has been extensively documented that the renin-angiotensin system is a major

factor in the regulation of cardiovascular homeostasis, including blood pressure,

mineral balance and tissue remodelling (Weber, 1998). However, the beneficial

effects of ACE inhibitors on tissue remodelling appear to be independent, at least

in part, of their effects on blood pressure (Linz et al., 1996). In this respect,

AngII can be generated either in the kidney and released in the circulation

(circulating AngII) subsequently activating different plasma membrane receptors

or it can be produced in different tissues to exert its effects at the place of

production (local AngII; Danser & Schalekamp, 1996). To date, two different

receptors have been cloned; namely the AT1 and AT2-subtype receptor

(Griendling et al., 1996). These receptors are differently localised and have

different functions, among which is modulation of cellular growth. The AT1-

receptor, which is prominent in adult tissues, stimulates cell growth (Matsukada

& Ichikawa, 1997). In contrast, the AT2-receptor, which is mostly abundant in

fetal tissues, inhibits cell growth and promotes apoptosis (Xoriuchi et al., 1999).

There is growing evidence for intracellular actions of AngII not related to

activation of ‘classical’ plasma membrane receptors. We recently reported

effects of intracellular AngII (AngIIintr) on rat aorta contraction, independent of

activation of plasma membrane AngII receptors (Brailoiu et al., 1999).

Intracellular AngII was reported to increase cytosolic [Ca2+] in vascular smooth

muscle cells (Haller et al., 1996; 1999), to inhibit gap conductance in heart

muscle (De Mello, 1996) and to affect L-type Ca2+ channel in a specific manner

(De Mello 1998). Such changes in Ca2+ homeostasis are important for cell

Chapter V

80

growth, therefore we addressed the following questions in this investigation: 1)

Is there a role for an AngIIintr receptor in vascular smooth muscle cell growth, 2)

Is the receptor similar to the known subtypes based on its pharmacological

profile, and 3) Can we identify part of its signal transduction pathway leading to

cell growth.

Methods

Cell culture

A7r5 vascular smooth muscle cells were grown in 75cm2 flasks in Dulbecco’s

Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum

(FCS), penicillin (50 'g ml-1) and streptomycin (50 units ml-1) at 37 0C in a

humidified atmosphere (5 % CO2). The cells were subcultured at 95%

confluency by trypsinization. Cell number was established by counting dispersed

cells in a Bürker counter (Schreek, Germany). Experiments were performed in 6

well plates (Costar, 9.6 cm2 well-1) at a density of 105cells well-1, unless stated

otherwise.

Determination of DNA synthesis

To obtain quiescent cells, the medium was replaced with DMEM containing

0.1% fetal calf serum 24 h after plating. The intracellular additions of AngII by

liposomal delivery were performed one day after the switch to 0.1 % FCS

containing DMEM and lasted for another 24 h. Extracellular addition of various

compounds happened 1 min prior to liposomal addition. [3H]thymidine (0.5

'Ci, specific activity: 20 Ci mmol-1) was added on each well during the final 3 h

of incubation. The medium was withdrawn at the end of incubation period and

the cells were washed twice with ice-cold phosphate buffered saline (PBS). To

remove non-genomic [3H]thymidine, the cells were incubated in the presence of

400 'l trichloroacetic acid for 1 h on ice. Finally, the cells were digested with 1

ml of 1 M NaOH and the incorporated radioactivity was measured in a !-

scintillation counter. Quiescent cells incorporated 210 & 11 dpm well-1 (mean &


81

s.e.mean, n=24), whereas 10% FCS stimulated cells incorporated 1078 & 71 dpm

well-1 (n=24).

Liposomes preparation

Liposomes containing AngII or AngI and control liposomes containing 140 mM

KCl were prepared as described (Brailoiu et al., 1999) from egg phosphatidyl

choline, using 10 mg ml-1 of solution to be incorporated. Dialysis against PBS

solution was performed for 4 h in order to remove the non-incorporated

compounds. To maintain sterile cell culture conditions the liposomes solution

was filtrated (0.2 'm pore seize). Liposomes were added to the medium above

the cells in a ratio of 1 to 20 (v v-1). If other compounds were delivered

intracellularly, they were encapsulated together with AngII. The amount of

AngII delivered intracellularly was determined using 125I-angiotensin II filled

liposomes. The incorporation into liposomes after the filtration step was 7.2 &

0.2 % (n=8) of the initial amount of radioactive AngII added to the cells.

Recovery of incorporated 125I angiotensin II into the cells after incubation for 30

min amounted to 5.6 & 0.2 % (n=8) of the initial amount of radioactive AngII

added to the cells.

Measurement of inositol (1,4,5) trisphosphate (Ins(1,4,5)P3)

Mass measurements of Ins(1,4,5)P3 were performed as described earlier (Sipma

et al, 1995), using an isotope dilution ligand binding assay. In brief, samples

were assayed in 25 mM Tris/HCl (pH=9.0), 1 mM EDTA, 1 mg bovine serum

albumin, [1-3H(N)]- Ins(1,4,5)P3 (21.0 Ci mmol–1, 2000 c.p.m. assay-1) and 1 mg

binding protein isolated from beef liver. Bound and free radioactivity was

separated by centrifugation. The radioactivity in the pellet was determined by

scintillation counting.

Chapter V

82

Measurement of intracellular Ca2+

Intracellular [Ca2+] was measured using Fura-2 fluorometry as described

(Filipeanu et al., 1997). Cells were loaded with 5 'M Fura-2 acetoxymethyl

ester at 22 oC, for 45 min in the dark. Fluorescence was measured at 37 oC.

Chemicals

All cell culture media were purchased from Gibco BRL, phosphatidyl choline

type X-E, AngI, AngII, and wortmannin from Sigma Chemical Co, CGP

422112A (nicotinic acid-Tyr-(N-benzoylcarbonyl-Arg)-Lys-His-Pro-Ile-OH)

from RBI, and PD98059 (2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-

one) from Calbiochem. Fura 2-AM was obtained from Molecular Probes,

losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[( 2’ - ( 1H - tetrazol - 5-yl)

biphenyl-4-yl )methyl]imidazole) from Merck, Sharpe and Dohme, PD123319

((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-

tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) from Park-Davis, [6-3H]-thymidine from Amersham Int, [1-3H(N)]- Ins(1,4,5)P3 from NEN Life

Science Products, and all other agents from Merck.

Data analysis

Data are given as mean & s.e.mean. The results of the growth experiments are

expressed as percentage of the radioactivity incorporated by control quiescent

cells. Independent measurements were performed in at least 2 different passages.

Measurements were normalised against liposomes filled with 10-7 M AngII,

present in every experimental protocol. Statistical significance was tested by

one-way ANOVA followed by Bonferroni test. A value of P<0.05 was

considered statistically significant. Concentration response curves were fitted

and the corresponding parameters calculated using Multifit (Dr. J. H. Proost,

Dept. of Pharmacokinetics and Drug Delivery, University Centre for Pharmacy,

University of Groningen). Curve fitting was based on the following sigmoidal

model: Y = V1 + V2 x X^V3 / (X^V3 + V4^V3) + V5 x X^V6 / (X^V6 + V7^V6).

Fitting for a single binding site was performed after omission of the term


83

containing the parameters V5, V6 and V7. To determine if the data were fitted

significantly better with 1 or 2 binding sites the variance was calculated using

the F-test.

Results

Intracellular AngII stimulates cell growth in quiescent A7r5 cells

Addition of AngII filled liposomes increases DNA synthesis in a dose-dependent

fashion, as measured by [3H]thymidine incorporation into A7r5 cells (Fig 1). The

first observation above the background was obtained with liposomes containing

10 pM AngII, whereas the maximum effect was reached with liposomes

containing 0.1 'M AngII (doubling of [3H]thymidine incorporation compared to

quiescent cells).

l o g [ L A n g I I ] ( M )- 1 2 - 1 1 - 1 0 - 9 - 8 - 7 - 6

[3H

] th

ym

idin

e i

nc

orp

ora

tio

n

(%)

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

+ L P D 1 2 3 3 1 9

+ L lo s a r t a n

+ L C G P 4 2 1 1 2 A

L A n g I I

Figure 1. Effects of AngII filled liposomes on A7r5 cell growth. Effect curves ofAngII filled liposomes alone (n=12) and in the presence of liposomes encapsulatinglosartan, PD123319 or CGP42112A (all 1 'M, n=12). All results were reported asincreases (%) above [3H]thymidine incorporation in quiescent cells. The maximalvalue (100 % at LAngII = 0.1 'M) corresponds to 448 & 56 dpm well-1, n=24). Lineswere fitted according to the 2 binding site model given in Table 1.

Chapter V

84

Data analysis showed that the increases in [3H]thymidine incorporation were

better described using a model with 2 binding site kinetics. Parameters of the

dose-response curve are given in Table 1.

Table 1. Dose-response parameters of AngIIintr induced [3H]thymidine

incorporation

Control (LAngII)

Binding sites 1 2*

EmaxHill-coefficientEC50

EmaxHill-coefficientEC50

(V2)(V3)(V4)(V5)(V6)(V7)

93.5 & 2.5 (%)0.46 & 0.042.2 & 0.5 x 10-10 (M)---

58.3 & 3.7 (%)1.02 & 0.162.7 & 0.6 x 10-11 (M)36.9 & 3.8 (%)1 (fixed)1.4 & 0.5 x 10-8 (M)

Dose-effect curves of liposomes filled with AngII (LAngII) in the range of 1 pM to1 'Mwere fitted using the equation: Y = V1 + V2 x X^V3 / (X^V3 + V4^V3) + V5 x X^V6 /(X^V6 + V7^V6). The last term was omitted for the single site fit. Effects are expressedas increases (%) above [3H]thymidine incorporation in quiescent cells (V1=0) andpresented as mean & s.e.mean, n=12 for each concentration. Significance level: * F-value = 23.8, P < 0.0001 vs single site model.

The simplest model with V6=1 was used for further analysis since varying the

Hill-coefficient (V6) from 1 to 9 for the second binding site did not significantly

alter the fitting results. Neither ‘empty’ control liposomes, filled only with 140

mM KCl, nor liposomes filled with the parent peptide AngI (1 'M) affected

[3H]thymidine incorporation (102.7 & 3.8 %, n=36 and 102.6 & 0.8 %, n=12 of

control cells, respectively).

The growth stimulating effect of liposomes containing 0.1 'M AngII was

corroborated in experiments showing actual increases in cell number (Table 2).

The growth stimulating effect of AngII filled liposomes (0.1 'M AngII) was

unchanged by extracellular addition (1 'M) of the nonpeptidergic AT1-type

receptor antagonist losartan, the nonpeptidergic AT2-type receptor antagonist

PD123319, or the peptidergic AT2-type receptor agonist CGP42112A (97.1 & 4.0

%, 103.9 & 2.8 %, and 91.7 & 5.6 % of control cells, n=18, respectively).


85

Table 2. Effect of intracellular applied AngII on cell number

Cell number flask-1

Control 81 & 11 x 103

Lcontrol 75 & 6 x 103

LAngII 114 & 12 x 103 *

A7r5 cells were plated in 25 cm2 flasks at a density of 2.103 cells cm-2. After 24 hr themedium containing 10 % FCS was replaced for 24 hr with medium containing 0.1 %FCS. Then either intracellular AngII (LAngII) using liposomes filled with AngII (0.1'M) or control liposomes (Lcontrol) containing 140 mM KCl. were applied. The cellnumber was counted 24 hr after application of the liposomes (mean & s.e.mean, n=4,determination in triplicate). Significance level: * P< 0.05 vs. Lcontrol.

In contrast to intracellular delivered AngII, extracellular application of AngII (10

pM to 10 'M) did not change [3H]thymidine incorporation in quiescent A7r5

cells. A similar amount of radioactivity as in control cells was incorporated after

0.1 'M extracellular AngII compared to control cells (98.1 & 3.2 %, n=12). A7r5

cells apparently lack functional AT -receptors which is also evident from the

inability of extracellular AngII (10 'M) to change basal Ins(1,4,5)P3 formation

(2.3 & 0.6 vs. 2.1 & 0.8 pmol 105 cells-1, n=12) or basal intracellular Ca2+

concentration (57 & 6 vs. 58 & 7 nM, n=12), as reported before (Filipeanu et al.,

1998a,b). Furthermore, extracellular application of AngI (1 'M, 102 & 2.4 %,

n=12), losartan (1 'M, 95.6 & 4.7 %, n=24), PD123319 (1 'M, 98.7 & 5.8 %,

n=16) or CGP42112A (1 'M, 92.9 & 4.8 %, n=18) did not affect [3H]thymidine

incorporation.

Pharmacology of intracellular AngII

We next attempted to characterise pharmacologically the effects of AngIIintr.

Addition of liposomes filled with losartan (1 'M), PD123319 (1 'M) together

with various concentrations of AngII reduced [3H]thymidine incorporation (Fig

1). Abolition of the growth stimulating effect of AngII was obtained by

liposomes filled with the AT2-type receptor agonist CGP42112A (1 'M, Fig 1).

Chapter V

86

All 3 agents substantially reduced stimulation of the high affinity binding site.

Non-competitive inhibition is this receptor site is likely involved in view of the

decreasing Emax value without changes in EC50. The following rank order of

antagonist potencies was obtained: CGP42112A>PD123319=losartan (Fig 2,

Table 3).

In contrast, at the low affinity binding site these compounds elicited opposite

effects. Again strong inhibition was observed for CGP42112A, but losartan and

PD123319 significantly increased Emax as compared to control. It is noticeable

that maximal values obtained at this site are comparable to the control value at

the high affinity binding site (Fig 2, Table 3). To verify the nature of the

antagonism by the compounds studied additional experiments were performed at

Figure 2. Contribution of the different binding sites to growth induced by intracellularAngII in the absence or presence of antagonists. Dose-response curves were plottedusing the data of Fig 1 according to the model as given in Table 1. AngII filledliposomes were in the absence (control) or in the presence of liposomes encapsulatinglosartan (los), PD123319 (PD) or CGP42112A (CGP). Values obtained for Emax andEC50 are presented in Tabel 3.

lo g [L A n g II] (M )

-1 2 -1 1 -1 0 -9 -8 -7 -6

eff

ec

t (

%)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

co n tro l

cg p

lo s

P D

cg p

co n tro l

P D

lo s


87

other concentrations of the antagonists across a limited range of AngII

concentrations (Table 3). Although one should be cautious not to over interpret

the fitting results of the limited data, it is clear that no evidence of a parallel shift

of the log dose-response relationship was observed for both binding sites.

Table 3. Effect of LAngII on [3H]thymidine incorporation in the presence of

various agents on dose-response parameters

Emax (h) Emax (l) EC50 (h) EC50 (l) Data

(%) (%) (M) (M) (n)

LAngII +Llos7.5 68 & 8 42 & 10 3 & 2 10-11 2 & 2 10-8 24

LAngII +Llos6 30 & 3 * 69 & 4 * 3 & 1 10-11 1.5 & 0.3 10-8 84

LAngII +LPD6 29 & 4 * 69 & 4 * 2 & 1 10-11 1.0 & 0.2 10-8 84

LAngII +LPD4.5 40 & 8 * 61 & 10 * 5 & 4 10-11 2 & 1 10-8 24

LAngII +Llos6 +LPD6 17 & 2 * 65 & 3 * 1.5 & 0.9 10-11 3.2 & 0.7 10-8 84

LAngII +LCGP6 4 & 2 * 11 & 3 * 0.5 & 1.2 10-11* 0.1 & 0.1 10-8 84

Liposomes were filled with AngII (LAngII) in the range of 1 pM to1 'M and added inthe presence of losartan (Llos), PD123319 (LPD) or CGP42112A (LCGP) atconcentrations as indicated by its anti-log. Effects are expressed as increases (%)above [3H]thymidine incorporation in quiescent cells. Parameters were obtained afterfitting the 2 binding site model as given in Tabel 1, with the constraints V1=0,V3=V6=1 and presented for the high (h)- and low (l) affinity sites as mean & s.e.mean.Datapoints were obtained from either 7 or 4 different concentrations with n=12 or 6each. Significance level: * P < 0.05 vs LAngII (Tabel 1).

The lowest antagonist concentration used (30 nM losartan) was insufficient to

induce inhibition. At high antagonist concentrations (e.g. 30 'M PD123319)

further increases were not observed in the Emax of the low affinity binding site.

This was also concluded from the experiment in which losartan and PD123319

(both 1 'M) were given simultaneously, and from an experiment using losartan

(10 'M) or PD123319 (10 'M) at a single dose of AngII (100 nM), showing

maximal [3H]thymidine incorporation was maintained (98.2 & 4.1 %, n=6 and

109.0 & 6.5 %, n=6 for losartan- and PD123319 filled liposomes, respectively).

Chapter V

88

In the absence of AngII filled liposomes, losartan-, PD123319-, and CGP4112A-

filled liposomes did not modify basal [3H]thymidine incorporation into quiescent

cells (n=12; 99.1 & 2.9 %, 101.5 & 2.4 %, 98.3 & 5.5 % of control, respectively).

Signal transduction of intracellular AngII effects

Extracellular signals often use multiple pathways to modify cell growth. In order

to gain insight in the mechanism involved in growth stimulation by AngIIintr two

of these pathways were tested. Intracellular AngII induced cell growth was

totally abolished by inhibition of the mitogen-activated protein kinase

(MAPK)/extracellular signal-regulated kinase (ERK) pathway by co-incubation

ofthe cells with extracellular PD98059 (1 'M, Fig 3). Growth stimulation was

also reduced, but to a lesser extent by inhibition of the phosphatidyl inositol 3-

kinase (PI-3K) pathway with wortmannin (1 'M, Fig 3).

[3H

] th

ym

idin

e i

nc

orp

ora

tio

n (

%)

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0 L A n g II

+ P D 0 9 8 0 5 9

+ w o rtm a n n in

*

*

Figure 3. Involvement of MAPK and PI-3K in AngII induced cell growth. Cell growthinduced by AngII filled liposomes (LAngII, 0.1 'M) was inhibited by simultaneousextracellular treatment with PD98059 (1 'M, n=16) or wortmannin (1 'M, n=16).


89

Discussion

In the present study, we demonstrate that AngIIintr stimulates cell growth in

quiescent A7r5 cells. Although ‘classical’ plasma membrane AngII receptors

are involved in growth and apoptotic processes underlying cardiovascular

remodelling (Dzau & Horiuchi, 1998) our results suggest additional targets for

AngII at sites which have not previously been recognised. The growth response

was characterised by the presence of two distinct binding sites for AngIIintr. The

high affinity site (picomolar range) was sensitive to intracellular delivered

antagonists in the rank order of potencies, CGP42112A > PD123319 = losartan.

In contrast, only the peptidergic antagonist CGP42112A could inhibit the low

affinity site (nanomolar range). The other compounds even increased the

maximal effect to the value obtained by stimulation of the high affinity site,

indicating that both sites are possibly closely involved in the growth response.

Also important is the observation that all three compounds are ineffective in the

absence of AngIIintr, showing that occupation of the receptor site by AngII is

needed for their action. Although the nature and physiological significance of

these distinct sites needs further investigation, it is unlikely that ‘classical’ AT1-

or AT2-receptors (De Gasparo et al., 1998) mediate the AngIIintr effect on

[3H]thymidine incorporation in view of the different potencies and the lack of

competitive inhibition observed, and the growth inhibitory action of both

CGP42112A and PD123319. These compounds were described as agonist and

antagonist of the anti-proliferative AT2-receptor subtype, respectively

(Timmermans et al., 1992; De Gasparo et al., 1998). Antagonist activity of

CGP42112A on AT1- or AT2 -receptors has only been reported for extracellular

AngII mediated phospholipase A2 activation (Lokuta et al., 1994). The presence

of an unusual type of receptor in A7r5 cells becomes also apparent from the lack

of the growth response by extracellular addition of AngII and the ineffectiveness

of extracellular addition of AT1-or AT2-receptor antagonists. No functional

plasma membrane AT-receptors seems to be present in A7r5 cells, also in view

of the absence of other cellular responses to extracellular AngII like Ins(1,4,5)P3

formation and cytosolic [Ca2+] elevation. Therefore, the growth response elicited

Chapter V

90

by AngIIintr is likely mediated by atypical AT -receptors with distinct

pharmacology from AT1- or AT2 –receptors.

Liposomes filled with the parent peptide AngI did not affect DNA synthesis. In

contractility studies of adult rat aorta, we observed that both AngI and AngII

filled liposomes induced contraction (Brailoiu et al., 1999). Either the contractile

and growth responses are not intimately related or different pharmacological

profiles of AngIIintr-receptors are present in different cell types. Differences in

pharmacological receptor profiles among different cell types is supported by

recent observations that AngIIintr inhibited inward Ca2+ current in rat cardiac

myocytes, but stimulated this current in hamster cardiac myocytes (De Mello,

1998).

The presence of various atypical angiotensin receptors was reported previously

(Noble et al., 1993, 1996; Smith, 1995; Regitz-Zagrosek et al., 1996; Li et al.,

1998; Moriuchi et al., 1998). The pharmacological profile of one of those

atypical angiotensin receptors resembles the profile obtained in A7r5 cells.

Although observed in another species, this receptor mediates microvascular

network formation in chick embryo, has a low affinity for losartan and

PD123319 and is antagonised by CGP42112A (Noble et al., 1993, 1996).

Further studies are necessary to elucidate if the receptors activated by AngIIintr,

as observed by us, are related to one of those atypical receptors, and to establish

their existence and binding profiles in other tissues.

Extracellular AngII induces several effects commonly evoked by growth factor

receptor stimulation, such as tyrosine phosphorylation or activation of the

Ras/ERK pathway ultimately leading to protein synthesis and cell cycle

progression (Berk, 1999; Eguchi et al., 1999; Inagami et al., 1999). Stimulation

of plasma membrane AT1 -receptors activate the MAPK cascade in vascular

smooth muscle cells other than A7r5 cells (Ge & Anand-Srivastava, 1998; Li et

al., 1998; Moriuchi et al., 1998) and this pathway is inhibited by PD98059

(Servant et al., 1996; Ushio-Fukai et al., 1998). Our experiments showed that

inhibition of this pathway by PD98059 effectively blocked the growth response

to AngIIintr administration. Activation of the MAPK cascade can be achieved


91

via the PI-3K pathway, but a redundant pathway stimulates MAPK when large

numbers of receptors are activated (Duckworth & Cantley, 1997). Interestingly,

wortmannin only partially inhibited our AngIIintr effect, a finding also reported

for extracellular AngII induced growth (Berk, 1999) and for other stimuli or

cell types activated (Balla et al. 1998; Gutkind, 1998). This indicates that a

strong signal is evoked by the AngIIintr mediated stimulation, comparable to

activation of large number of ‘classical’ plasma membrane receptors.

The obvious physiological candidate to stimulate the AngIIintr-receptor is

AngII. Intracellular trafficking of AngII might be important for directing AngII

to certain cellular locations to fully express its biological response. Several

studies have demonstrated that AngII is internalised into the cells via an AT1-

but not AT2 -mediated process (Anderson et al., 1993; Hein et al., 1997).

Intracellular pools of AngII were noticed in cardiomyocytes (Sadoshima et al.,

1993) and recently angiotensin peptides, ACE-activity and AT1-receptors were

detected in a renal endosomal fraction (Imig et al., 1999). The functional

targets for AngIIintr are still unclear, but nuclear binding-proteins were reported

for AngII (Booz et al., 1992; Tang et al., 1992; Jimenez et al., 1994).

Interaction of AngIIintr with proteins at the cytosolic side of the plasma

membrane also occurs in view of the results of AngIIintr on Ca2+ channels and

gap junctions (De Mello 1996, 1998; Haller et al, 1996, 1999). This is possibly

only a secondary related phenomenon, since the MAP-kinase pathway shown

to be activated by AngIIintr in the present paper, modulates the opening of L-

type Ca2+ channels in cardiomyocytes (Murata et al., 1999).

In conclusion, these data demonstrate that intracellular delivered AngII induces

cell growth in A7r5 cells. Atypical AT-receptors are involved in view of the

ineffectiveness of extracellular addition and the rank order of antagonist

potencies obtained by intracellular application. The AngIIintr induced growth

response is mediated via a PI-3K and MAPK/ERK -sensitive pathway. AngIIintr

actions, inaccessible for common treatment, might open new views in

understanding and treatment of cardio-vascular related diseases.

Chapter V

92

Acknowledgements

Catalin M. Filipeanu is a recipient of an Ubbo Emmius fellowship from Groningen

University Institute of Drug Exploration (GUIDE). Disposition of the Multifit software

by Hans Proost and additional help with the fitting procedures is greatly appreciated.

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Balla, T., Varnai, P., Tian, Y. & Smith, R.D. (1998). Signaling events activated byangiotensin II receptors: what goes before and after the calcium signals. Endocr Res.24, 335-344.

Berk, B.C. (1999). Angiotensin II signal transduction in vascular smooth muscle:pathways activated by specific tyrosine kinases. J Am Soc Nephrol. 10, S62-68.

Booz, G.W., Conrad, K.M., Hess, A.L., Singer, H.A. & Baker, K.M. (1992).Angiotensin-II-binding sites on hepatocyte nuclei. Endocrinology. 130, 3641-3649.

Brailoiu, E., Filipeanu, C.M, Tica, A., Toma, C.P., de Zeeuw, D. & Nelemans, S.A.(1999) Contractile effects by intracellular angiotensin II via receptors with a distinctpharmacological profile(in rat aorta. Br J Pharmacol . 126, 1133-1138.

Danser, A.H. & Schalekamp, M.A.(1996). Is there an internal cardiac renin-angiotensin system? Heart. 76, 28-32.

De Gasparo, M., Catt, K.J. & Inagami, T. (1998). The IUPHAR Compendium ofReceptor Characterization and Classification. Angiotensin receptors. 1 th edition, 80-86.

De Mello, W.C. (1998). Intracellular Angiotensin regulates the inward calcium currentin cardiac myocytes. Hypertension. 32, 976-982.

De Mello, W.C. (1996). Renin-Angiotensin system and cell communication in the fallingheart. Hypertension. 27, 1267-1272.

Duckworth, B.C. & Cantley, L.C. (1997). Conditional inhibition of the mitogen-activated protein kinase cascade by wortmannin. Dependence on signal strength. JBiol Chem. 272, 27665-27670.

Dzau, V.J. & Horiuchi, M. (1998). Vascular remodeling--the emerging paradigm ofprogrammed cell death (apoptosis): the Francis B. Parker lectureship. Chest. 114,91S-99S.

Eguchi, S., Iwasaki, H., Ueno, H., Frank, G.D., Motley, E.D., Eguchi, K., Marumo,F., Hirata, Y. & Inagami, T. (1999). Intracellular signalling of angiotensin II-induced


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p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possiblerequirement of epidermal growth factor receptor, Ras, extracellular signal-regulatedkinase, and Akt. J Biol Chem. 274, 36843-36851.

Filipeanu, C.M., Henning, R.H., de Zeeuw, D. & Nelemans, S.A. (1998a). Functionalevidence for a role of intracellular Angiotensin II in A7r5 cells. Br J Pharmacol. 123,141P. Abstract.

Filipeanu, C.M., Nelemans, S.A., Henning, R.H., Brailoiu, E. & de Zeeuw, D. (1998b).Intracellular angiotensin II effects in vascular smooth muscle cells. J.Am.Soc.Nephrol.9, 422A. Abstract.

Filipeanu, C.M., de Zeeuw, D. & Nelemans, S.A. (1997). Delta9-Tetrahydrocannabinolactivates [Ca2+]i increases partly sensitive to capacitative store refilling . Eur JPharmacol. 336, R1-R3.

Ge, C., & Anand-Srivastava MB. (1998). Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in AII-mediated enhancedexpression of Gi proteins in vascular smooth muscle cells. Biochem Biophys ResCommun. 251, 570-575.

Griendling, K.K., Lassegue, B. & Alexander RW. (1996). Angiotensin receptors andtheir therapeutic implications. Annu Rev Pharmacol Toxicol. 36, 281-306.

Gutkind JS. (1998). The pathways connecting G protein-coupled receptors to thenucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem.273, 1839-1842.

Haller, H., Lindschau, C., Erdmann, B., Quass, P. & Luft, F.C. (1996). Effects ofintracellular Angiotensin II in vascular smooth muscle cells. Circ Res. 79, 765-772.

Haller, H., Lindschau, C., Quass, P. & Luft, F.C. (1999). Intracellular actions ofangiotensin II in vascular smooth muscle cells. J Am Soc Nephrol. 10, S75-S83.

Hein, L., Meinel, L., Pratt, R.E., Dzau, V.J. & Kobilka, B.K. (1997). Intracellulartrafficking of Angiotensin II and its AT1 and AT2 receptors: evidence for selectivesorting of receptor and ligand. Mol Endocrinol. 11, 1266-1277.

Imig, J.D., Navar, G.L,, Zou, L.X., O'Reilly, K.C., Allen, P.L., Kaysen, J.H.,Hammond, T.G. & Navar, L.G. (1999). Renal endosomes contain angiotensinpeptides, converting enzyme, and AT(1A) receptors. Am J Physiol. 277, F303-311.

Inagami, T., Eguchi, S., Numaguchi, K., Motley, E.D., Tang, H., Matsumoto, T. &Yamakawa T. (1999). Cross-talk between angiotensin II receptors and the tyrosinekinases and phosphatases. J Am Soc Nephrol. 10, S57-61.

Jimenez, E., Vinson, G.P. & Montiel M. (1994). Angiotensin II (AII)-binding sites innuclei from rat liver: partial characterization of the mechanism of AII accumulation innuclei. J Endocrinol. 143, 449-453.

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Li, X., Shams, M., Zhu, J., Khalig, A., Wilkes, M., Whittle, M., Barnes, N. & Ahmed, A.(1998). Cellular localization of AT1 receptor mRNA and protein in normal placenta andits reduced expression in intrauterine growth. Angiotensin II stimulates the release ofvasorelaxants. J Clin Invest. 101, 442-454.

Linz, W., Wiemer, G., Gohlke, P., Unger, T. & Scholkens, B.A. (1995). Contributionof kinins to the cardiovascular actions of angiotensin-converting enzyme inhibitors.Pharmacol Rev. 47, 25-49.

Lokuta, A.J., Cooper, C., Gaa, H.E., & Rogers, T.B. (1994). Angiotensin II stimulatesthe release of phospholipid-derived second messengers through multiple receptorsubtypes in heart cells. J Biol Chem. 269, 4832-38.

Matsusaka, T. & Ichikawa, I. (1997). Biological functions of angiotensin and itsreceptors. Annu Rev Physiol . 59, 395-412.

Moriuchi, R., Shibata, S., Himeno, A., Johren, O., Hoe, K.L. & Saavedra JM. (1998).Molecular cloning and pharmacological characterization of an atypical gerbilangiotensin II type-1 receptor and its mRNA expression in brain and peripheraltissues. Brain Res Mol Brain Res. 60, 234-246.

Murata, M., Fukuda, K., Ishida, H., Miyoshi, S., Koura, T., Kodama, H., Nakazawa,H.K., & Ogawa S. (1999). Leukemia inhibitory factor, a potent cardiac hypertrophiccytokine, enhances L-type Ca2+ current and [Ca2+]i transient in cardiomyocytes. JMol Cell Cardiol. 31, 237-245.

Noble, F.A. Le, Kessels-van Wylick, L.C., Hacking, W.J., Slaaf, D.W., oude Egbrink,M.G., & Struijker-Boudier, H.A. (1996). The role of angiotensin II and prostaglandinsin arcade formation in a developing microvascular network. J Vasc Res. 33, 480-488.

Noble, F.A. Le, Schreurs, N.H., van Straaten, H.W., Slaaf, D.W., Smits, J.F., Rogg,H. & Struijker-Boudier HA. (1993). Evidence for a novel angiotensin II receptorinvolved in angiogenesis in chick embryo chorioallantoic membrane. Am J Physiol.264, R460-465.

Regitz-Zagrosek, V., Neuss, M., Warnecke, C., Holzmeister, J., Hildebrandt, A.G. &Fleck E. (1996). Subtype 2 and atypical angiotensin receptors in the human heart.Basic Res Cardiol. 91, 73-77.

Sadoshima, J., Xu, Y., Slayter, H.S., & Izumo, S. (1993). Autocrine release ofangiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro.Cell, 75, 977-984.

Servant, M.J., Giasson, E. & Meloche, S. (1996). Inhibition of growth factor-inducedprotein synthesis by a selective MEK inhibitor in aortic smooth muscle cells. J BiolChem. 271, 16047-16052.

Sipma, H., Duin, M., Hoiting, B., den Hertog, A. & Nelemans, A. (1995). Regulationof histamine- and UTP-induced increases in Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ca2+ bycyclic AMP in DDT1 MF-2 cells. Br J Pharmacol. 114, 383-390.


95

Smith, R.D. (1995). Identification of atypical (non-AT1, non-AT2) angiotensinbinding sites with high affinity for angiotensin I on IEC-18 rat intestinal epithelialcells. FEBS Lett. 373, 199-202.

Tang, S.S., Rogg, H., Schumacher, R. & Dzau VJ. (1992). Characterization of nuclearangiotensin-II-binding sites in rat liver and comparison with plasma membranereceptors. Endocrinology. 131, 374-380.

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Chapter 6

Contribution of receptor internalization and recycling

to angiotensin AT1 receptor desensitization to rat

aorta contractility

Catalin M. Filipeanu a, Robert H. Henning a, Hendrik Buikema a, Azuwerus van

Buiten a, Sander Croes b, Dick de Zeeuw a and Adriaan Nelemans b, *

Submitted: Journal of Vascular Research.

Chapter VI

98

Abstract

The role of internalization and recycling of the AT1 subtype angiotensin II

receptor (AT1R) in its desensitization have been extensively studied in cell

cultures. However, their contribution to AT1 desensitization in intact tissue is

not known. We investigated this aspect by studying repetitive application of

angiotensin II (ang II) in rat aorta vascular smooth muscle rings using specific

blockers at concentrations not interfering with other functional aspects.

Desensitization was clearly shown by a reduction to 48.6 & 6.3 % of second

contractile response to ang II (10-6 M) at 45 min after the first exposure to ang

II. Desensitization was completely prevented (95.2 & 9.7 %) by inhibiting

internalization via clathrin-coated pits formation (hyperosmolar medium, 90

mM sucrose). Blocking internalization via caveolae (cadaverine 10-4 M) also

reduced desensitization, but to a lesser extent (65.2 & 6.7 %). In contrast,

inhibition of receptor recycling using brefeldin A (10-6 M) and nocodazole (10-6

M) enhanced desensitization (21.2 & 6.3 % and 16.8 & 6.6 %, respectively).

When a combination of treatments was used, brefeldin A was able to normalize

the desensitization in the presence of hyperosmolar medium or cadaverine,

whereas nocodazole was ineffective. This is the first demonstration, that

receptor internalization plays an important role in AT1R desensitization in rat

aorta. Clathrin coated pits and receptor recycling within the trans Golgi

network are the major cellular pathways involved in this process.

Key words: angiotensin II, rat aorta, desensitization, clathrin-coated pits, caveolae,

cell traffic, brefeldin A, nocodazole.

Introduction

Prolonged exposure of a G-protein coupled receptor to its agonist generally

induces desensitization of the evoked response. The steps involved in the

desensitization process can be defined as receptor phosphorylation (occurring

very fast), internalization (fast) and recycling (slow) [1]. Much knowledge on

Effects of receptor internalization and recycling on AT1 desensitization.

99

these processes has been gathered during the past decades. Dependent on the

receptor, phosphorylation may be mediated by specific receptor kinases, like

!ARK or by second messenger activated kinases such as PKA or PKC [1].

Receptor internalization may be accomplished via different routes: i.e. clathrin

coated vesicles (“coated pits”) and caveolae [2]. Once internalised the receptor

enters the tubulo-endosomal cellular compartment, from where it is either

dephosphorylised and recycled back to the plasma membrane or directed to

lysosomes for breakdown [3, 4]. Generally, these mechanisms appear to act in

concert, as e.g. phosphorylated receptors are internalized at a higher rate [5]

and their sorting differs from that of non-phosphorylated receptors [6].

However, dependent on the specific receptor, considerable differences exist as

to what extent the various processes contribute to the desensitization of the

functional response.

The angiotensin II type 1 receptor (AT1R) represents a particular receptor,

which is known to undergo rapid and extensive desensitization [7]. Various

studies, mainly in cell culture, have identified receptor phosphorylation to

augment AT1R desensitization [8]. In addition, cell culture studies show the

AT1R to undergo substantial internalization. It appears that the AT1R

internalization mainly proceeds through the clathrin coated vesicle pathway [9,

10], but may in addition also employ the caveolae pathway [11].

In contrast to these studies in cell culture, the role of receptor internalization on

AT1R desensitization in intact vascular smooth muscle has not been

investigated so far. Therefore, in the present work we address this question by

measuring contraction following repetitive administration of angiotensin II in

de-endothelised rat aortic rings, after specific pharmacological interruption of

receptor recycling pathways and internalization.

We demonstrate that receptor internalization followed by cellular cycling plays

a major role in the maintenance of the responsiveness of rat aorta contraction to

angiotensin II.

Chapter VI

100

Material and Methods

Tissue preparation.

The local animal ethical committee (University of Groningen) approved the

experiments. Males Wistar rats weighing 350-450 g (12-15 weeks old) were

killed by exsangination under general anesthesia. Their thoracic aorta was

rapidly removed and immersed in Krebs Henseleit solution of the following

composition (mM) NaCl 120.4; KCl 5.9; CaCl2 2.5; MgCl2 1.2; NaH2PO4 1.2;

glucose 11.5; NaHCO3 25. This solution was continuously aerated with 95%O2

/ 5%CO2 and kept at 37oC. The aorta was dissected free of surrounding

connective tissue and divided in 8 rings (~2 mm long). Endothelium was

removed by gently rubbing the intimate surface with a smooth softwood stick.

Experimental procedures.

The rings were connected to an isotonic displacement transducer, with a

preload of 1.4 g [12]. They were allowed to equilibrate for 60 minutes, during

which regular washing was performed. Then, rings were repeatedly (usually 2-

3 times) stimulated with 10-5 M phenylephrine until two successive

contractions differed less than 5 %. The amplitude of the last contraction was

considered 100 % for further comparisons. On the top of these contractions,

acetylcholine (10-5 M) was added, to verify the removal of endothelium. Rings

showing more than 5 % relaxation in response to acetylcholine were excluded.

Angiotensin II induced AT1R desensitization was studied according to the

protocol described in fig. 1. After the first stimulation with angiotensin II (10-6

M) lasting 15 min, the rings were washed three times every 15 min,

subsequently followed by the second angiotensin II stimulation. Compounds

interfering with receptor desensitization were added 15 min before the first

angiotensin II stimulation and maintained throughout the remaining of the

experiment. These compounds were used at concentrations not affecting the

first angiotensin II contraction (see results). Routinely, at the end of the

experiment an additional stimulation with phenylephrine was performed,

ensuring the viability of the aortic ring. Results from rings that failed to


101

respond with at least 90% of their initial phenylphrine contraction were

discarded.

For each experiment, the rings were randomly distributed in order to minimize

anatomical influence upon the final results, while one ring was always kept as

control. No more than two rings per rat were assigned to a specific treatment.

Drugs

Angiotensin II, phenylephrine, brefeldin A and cadaverine were obtained from

Sigma and nocodazole was from Calbiochem. Nocodazole and brefeldin A

were solved in dimethyl sulfoxide. The final concentration of dimethyl

sulfoxide in organ bath was 0.01 %, which was without an effect on

contraction. Other compounds used were of the highest purity grade

commercially available.

PE ‘Blockers’

Ang II Ang II1g

45 min

Fig. 1. The protocol used to study angiotensin AT1 receptor desensitization in rat aorta.Bars show the addition of various compounds; phenylephrine (PE, 10-5 M), angiotensinII (Ang II, 10-6 M), ‘Blockers’ denote the application of sucrose, 90 mM; cadaverine,10-4 M; brefeldin A, 10-6 M or nocodazole, 10-6 M.

Chapter VI

102

Statistics

All series were performed in at least four different animals with n # 8. Data are

presented as means ± SEM. Statistical differences were tested by one-way

analysis of variance (ANOVA) followed by Bonferroni post-test, considering

p<0.05 significantly different.

Results

In de-endothelised rat aorta, angiotensin II (10-6 M) induced a maximum

contraction amounting 44.2 & 6.1 % of the response to phenylephrine (PE; 10-5

M; n=78). Angiotensin II mediated contraction was completely prevented by

the AT1R antagonist, losartan (10-6 M, 2.6 & 4.8% of PE contraction, n=6) and

not affected by the AT2R antagonist, PD123319 (10-6 M, 41.0 & 6.7 % of PE

contraction, n=6). After washing for 45 min, the contraction in response to a

second addition of angiotensin II was approximately half of that obtained to the

first application of angiotensin II (48.6 & 6.3 % of first Ang II response, n=70).

Thus, these results demonstrate that the angiotensin II induced contraction is

exclusively mediated via the AT1R subtype and sensitive to desensitization.

To study the role of various AT1R desensitization pathways, we examined

different pharmacological agents interfering with internalization and recycling

of receptors. However, at high concentrations these treatments might directly

impede vascular smooth muscle contractility, thereby generating false results.

To circumvent this adverse effect, we determined the highest concentration of

each agent that did not affect contractions induced by phenylephrine or

angiotensin II. The values for these concentrations are presented in table 1. By

employing these concentrations throughout the study, their effect on the second

angiotensin II contraction can be exclusively attributed to actions on the

desensitization process. Receptor internalization was disrupted at the plasma

membrane by using high sucrose buffer, inhibiting the formation of coated-pits

[13] and by employing cadaverine, blocking caveolae-mediated receptor

internalization [14].


103

Table 1. Effects of the treatments interfering with receptor internalization

and trafficking on rat aorta contraction

Treatment Phenylephrine Angiotensin II

(% of phenylephrine)

Control 100 44.2 & 6.1

Sucrose 90 mM 96 & 6.5 41.2 & 6.0

Cadaverine 10-4 M 102 & 4.5 42.9 & 3.5

Brefeldin A 10-6 M 94 & 4.5 43.2 & 6.1

Nocodazole 10-6 M 95.5 & 6.0 47.2 & 4.8

Contraction was induced initially by phenylephrine (10-5 M) and subsequently byangiotensin II (10-6 M). Data are expressed as % of the control contractions obtainedby phenylehrine (n=8; means & SEM). Phenylephrine- or angiotensin II-inducedcontractions were not affected by the treatments (p * 0.05).

Sucrose (90 mM) treatment completely reversed the AT1R desensitization of

the second angiotensin II response to a level of 95.2 & 9.7 % of the first

response (fig. 2). Pretreatment with cadaverine (10-4 M) also counteracted

desensitization, although to a lesser extent than sucrose, as contraction of the

second response amounted 65.2 & 6.7 % of the initial response (fig. 2). Next,

trafficking inhibitors were used to study the intracellular pathways involved in

receptor desensitization. To this end we used nocodazole (10-6 M), an inhibitor

of microtubule polymerization [16], and brefeldin A (10-6 M), a compound that

disrupts Golgi structures [17]. Pre-treatment with nocodazole strongly

augmented the desensitization by reducing the second angiotensin II response

from 48.6 & 6.3 to 16.8 & 6.6 % (fig. 2). A similar effect was obtained using

brefeldin A (10-6 M), which reduced the second angiotensin II response to 21.2

& 6.6 % (fig. 2). Further, combined pretreatment with nocodazole and brefeldin

A resulted in a similar reduction of the second angiotensin II response as

observed for the individual compounds (22.6 ± 2.4 %). These experiments

demonstrate that blockade of internalization at the plasma membrane prevents

Chapter VI

104

AT1R desensitization, whereas disruption of intracellular trafficking pathways

enhances it.

To further explore to what extent replenishment of the plasma membrane

AT1Rs are involved in attenuation of desensitization, AT1R responses were

investigated using combinations of the above compounds. A marked difference

between nocodazole and brefeldin A was observed when applied in the

presence of either sucrose or cadaverine. Whereas the inhibition of

desensitization caused by sucrose or cadaverine was not affected by

nocodazole, brefeldin A restored a normal desensitization pattern (fig. 3).

Fig. 2. Effects of sucrose,cadaverine, brefeldin A andnocodazole on angiotensinAT1 receptor desensitizationin rat aorta. The experimentswere conducted as shown infig. 1, the agents studied(sucrose, 90 mM;cadaverine, 10-4 M; brefeldinA, BFA, 10-6 M; nocodazole10-6 M) were added 15 minbefore the first angiotensin II(10-6 M) stimulation and waspresent throughout theremaining of the experiment.Data of the secondangiotensin II (10-6 M)stimulation are expressed aspercentage of the firstresponse to angiotensin IIand reported as means &SEM, n=8-12. * Statisticallydifferent from control, p +0.05.

% o

f firs

t ang

II c

ontr

actio

n

0

30

60

90

120

0

10

20

30

40

50

60

cont

rol

cont

rol

cada

verin

e

sucr

ose

BFA

noco

dazo

le

*

*

**


105

Discussion

In the present study we investigated the contribution of receptor internalization

and recycling mechanisms to the desensitization of the angiotensin AT1

receptor by measuring contraction in rat aorta smooth muscle tissue.

Repetitiveapplication of angiotensin II resulted in a marked desensitization of

the contractile response. The desensitization was fully blocked by interference

with internalization through coated pits and partially blocked by interrupting

the caveolae pathway. In contrast, desensitization was markedly enhanced by

interference with cellular trafficking. Finally, in the presence of blockade of

receptor internalization, inhibition of cellular trafficking by brefeldin A

restored the desensitization of AT1R. Therefore, this study demonstrates that in

Fig. 3. Effects of nocodazoleand brefeldin A on theinhibition of angiotensin AT1

receptor desensitization bysucrose (upper panel) andcadaverine (lower panel). Theexperiments were conducted asshown in fig. 1, the agentsstudied (sucrose, 90 mM;cadaverine, 10-4 M; brefeldin A,BFA, 10-6 M; nocodazole 10-6

M) were added 15 min beforethe first angiotensin II (10-6 M)stimulation and was presentthroughout the remaining of theexperiment. Data of the secondangiotensin II (10-6 M)stimulation are expressed aspercentage of the first responseto angiotensin II and reported asmeans & SEM, n=8-12. *Statistically different fromcontrol, p + 0.05.

% o

f fi

rst

an

g I

I c

on

tra

cti

on

0

2 0

4 0

6 0

8 0

1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

s u c ro s e

c a d a v e rin e

+ n

oco

da

zole

+ B

FA

*

*

Chapter VI

106

intact vascular smooth muscle tissue (1) AT1R desensitization is to a large

extend dependent on receptor internalization and (2) extensive recycling of

receptors from the trans-Golgi network counteracts AT1R desensitization.

The contractile response evoked by angiotensin II in denuded rat aorta is

exclusively mediated via the AT1R subtype, as the response was fully blocked

by the AT1R antagonist losartan, while being insensitive to the AT2R

antagonist PD12319. Further, an about 50% desensitization of the contractile

response was found after repeated application of angiotensin II after 45 min.

This magnitude of desensitization is in agreement with data from vascular

smooth muscle cells, showing 40 % desensitization within 5 min after

angiotensin II treatment [18]. These results demonstrated that our protocol is

suitable to study the AT1R desensitization in intact tissue.

This is the first investigation of the contribution of receptor internalization and

recycling to AT1R desensitization in intact vascular tissue. Receptor

internalization is regarded as a principle step in the desensitization process of

plasma membrane receptors [1]. Our data indicate that internalization via

clathrin coated pits represent the predominant mechanism involved in the AT1R

desensitization of rat aorta smooth muscle, in view of the complete blockade of

desensitization by high sucrose. In addition, internalization via the caveolae

pathway contributes to AT1R desensitization, as shown by the partial blockade

by cadaverine. The present results are in agreement with those observed in

cultured cells overexpressing the AT1R [9-12]. There, AT1R internalization was

prevented by high sucrose conditions and by dominant negative isoforms of

dynamin, a GTPase regulating the internalization of the vesicle. Further, co-

existence of the clathrin and caveolae pathways was also observed in cultured

cells [10, 12]. Thus, our results extend these observations, showing that in

intact vascular smooth muscle the ‘default’ internalization pathway of AT1R

consists of clathrin coated pits and that internalization via the caveolae provide

an alternative pathway.

Further, it was found that, in view of the strong amplification in presence of

nocodazole and brefeldin A, trafficking of the intracellular AT1R receptor plays


107

an important role in its desensitization. As both compounds are interfering with

receptor recycling [19], their action demonstrates the importance of

intracellular trafficking in the maintenance of AT1R responsiveness. This

supposition fits the changing view on the role of receptor internalization from

serving to attenuate the cellular response and down-regulate the receptor [20] to

being considered a key process in receptor re-sensitization [1].

The importance of receptor trafficking is substantiated further by the

restoration of receptor desensitization by brefeldin A in the presence of

blockade of receptor internalization. Brefeldin A is known to merge Golgi

structures in the endoplasmic reticulum, thereby fully preventing the retrograde

transport of receptors to the plasma membrane [21, 22]. Our results

demonstrate that AT1R desensitization is counteracted by replenishment of

plasma membrane AT1Rs from the trans Golgi network, as has been implicated

recently for the neurotensin receptor [23]. Further, substantial desensitization of

AT1R response was observed in the presence of sucrose and brefeldin A, (i.e. in

the absence of any receptor transport), indicating that receptor phosphorylation

does contribute to the desensitization process in rat aorta smooth muscle. This

conclusion is supported by reports showing that AT1 receptor phosphorylation

resulted in a substantial desensitization of the receptor in human embryonic

kidney cells and chinese hamster ovary cells [8, 9, 11].

In contrast to brefeldin A, nocodazole did not restore the AT1R desensitization

under conditions of blockade of receptor internalization. This differential effect

of brefeldin A and nocodazole is most likely explained by differences in their

site of action. Nocodazole treatment has been found to collapse only the

superficial endoplasmic reticulum [24]. As replenishment of plasma membrane

receptors occurs from the “deeper” located trans-Golgi network, nocodazole is

not likely to interfere with this process, hence not reestablishing the

desensitization in the presence of a blockade of receptor internalization.

The extensive role of receptor internalization and intracellular trafficking in

AT1R desensitization may have pathophysiological implications. For instance,

cholesterol oxidation was found to induce a switch in the internalization

Chapter VI

108

pathway from the caveolae pathway to clathrin-coated pits in case of the

endothelin type A receptor [25]. Whether such mechanisms also apply to the

AT1R is unknown. Interestingly, hypercholesterolemia was reported to reduce

sensitivity to angiotensin II induced contractions in the aorta [26].

Further, the internalization of the AT1R may serve to cellular responses that go

beyond desensitization of the response to extracellular angiotensin II. As

angiotensin II is internalized together with the AT1R, this route may deliver

substantial amounts of the agonist intracellularly. Recent studies in vascular

smooth muscle have demonstrated that intracellular application of angiotensin

II evokes specific responses such as contraction and cell-growth, that are

unaffected by the extracellular application of AT1R or AT2R antagonists [27-

29]. Although its particular pharmacology precludes that the intracellular

angiotensin II receptor is a typical AT1R, the present work indicates that

interfering with receptor desensitization might influence the intracellular

angiotensin II actions. Accordingly, angiotensin II internalized together with

AT1R can be rapidly converted in the endosomal pathway into fragments as

observed in adrenal medullary cells [30], that can bind specifically to putative

intracellular angiotensin receptors.

In summary, our results demonstrate for the first time that receptor

internalization plays a major role in AT1R desensitization. Internalization

occurs predominantly via clathrin coated pits. Replenishment of the plasma

membrane receptor pool from the trans-Golgi network is of vital importance to

maintain angiotensin II responsiveness. Consequently, interference with AT1R

trafficking may provide an alternative pharmacological strategy to counteract

the actions of angiotensin II on vascular smooth muscle.

Acknowledgements

Catalin M. Filipeanu is recipient of an Ubbo Emmius fellowship from the Groningen

University Institute of Drug Exploration (GUIDE).


109

References1. Lefkowitz RJ G protein-coupled receptors. III. New roles for receptor kinases andbeta-arrestins in receptor signaling and desensitization. J Biol Chem 1998; 273:18677-80.

2. Hunyady L, Catt KJ, Clark AJ, Gaborik Z: Mechanisms and functions of AT1Rangiotensin receptor internalization. Regul Pept 2000; 91:29-44.

3. Tooze J, Hollinshead M: Tubular early endosomal networks in AtT20 and othercells. J Cell Biol 1991; 115(3):635-53.

4. Koenig JA, Edwardson JM: Kinetic analysis of the trafficking of muscarinicacetylcholine receptors between the plasma membrane and intracellularcompartments. J Biol Chem 1994; 269: 17174-82.

5. Anborgh PH, Seachrist JL, Dale LB, Ferguson SS Receptor/beta-arrestin complexformation and the differential trafficking and resensitization of beta2-adrenergic andangiotensin II type 1A receptors. Mol Endocrinol. 2000; 14: 2040-53.

6. Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG. Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosisdictates the profile of receptor resensitization. J Biol Chem 1999; 274:32248-57.

7. Thekkumkara TJ, Du J, Dostal DE, Motel TJ, Thomas WG, Baker KM. Stableexpression of a functional rat angiotensin II (AT1RA) receptor in CHO-K1 cells: rapiddesensitization by angiotensin II. Mol Cell Biochem. 1995; 146:79-89.

8. Oppermann M, Freedman NJ, Alexander RW, Lefkowitz RJ. Phosphorylation ofthe type 1A angiotensin II receptor by G protein-coupled receptor kinases and proteinkinase C. J Biol Chem 1996 May 31;271:13266-72.

9. Balmforth AJ, Shepherd FH, Warburton P, Ball SG. Evidence of an important anddirect role for protein kinase C in agonist-induced phosphorylation leading todesensitization of the angiotensin AT1A receptor. Br J Pharmacol. 1997; 122:1469-77.

10. Zhang J, Ferguson SS, Barak LS, Menard L, Caron MG. Dynamin and beta-arrestin reveal distinct mechanisms for G protein-coupled receptor internalization. JBiol Chem 1996; 271:18302-5.

11. Gaborik Z, Szaszak M, Szidonya L, Balla B, Paku S, Catt KJ, Clark AJ, HunyadyL beta-Arrestin- and Dynamin-Dependent Endocytosis of the AT1 AngiotensinReceptor. Mol Pharmacol. 2001; 59: 239-247.

12. Ishizaka N, Griendling KK, Lassegue B, Alexander RW. Angiotensin II type 1receptor: relationship with caveolae and caveolin after initial agonist stimulation.Hypertension 1998; 32: 459-66.

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13. Buikema H, Grandjean JG, van den Broek S, van Gilst WH, Lie KI, Wesseling H.Differences in vasomotor control between human gastroepiploic and left internalmammary artery. Circulation. 1992; 86:II205-9.

14. Buys SS, Novak JM, Gren LH, Kaplan J. Effect of volume and pH on surfacereceptor number in macrophages. J Cell Physiol 1989; 140:371-8.

15. Schutze S, Machleidt T, Adam D, Schwandner R, Wiegmann K, Kruse ML,Heinrich M, Wickel M, Kronke M. Inhibition of receptor internalization bymonodansylcadaverine selectively blocks p55 tumor necrosis factor receptor deathdomain signaling. J Biol Chem 1999; 274:10203-12.

16. Hoebeke J, Van Nijen G, De Brabander M. Interaction of oncodazole (R 17934), anew antitumoral drug, with rat brain tubulin. Biochem Biophys Res Commun 1976;69:319-24.

17. Fujiwara T, Oda K, Yokota S, Takatsuki A, Ikehara. Brefeldin A causesdisassembly of the Golgi complex and accumulation of secretory proteins in theendoplasmic reticulum. J Biol Chem 1988; 263:18545-52.

18. Tang H, Nishishita T, Fitzgerald T, Landon EJ, Inagami T. Inhibition of AT1receptor internalization by concanavalin A blocks angiotensin II-induced ERKactivation in vascular smooth muscle cells. Involvement of epidermal growth factorreceptor proteolysis but not AT1 receptor internalization. J Biol Chem 2000;275:13420-6.

19. Hay JC, Hirling H, Scheller RH. Mammalian vesicle trafficking proteins of theendoplasmic reticulum and Golgi apparatus. J Biol Chem 1996; 271:5671-9.

20. Bertaccini G, Coruzzi G. Receptors in the gastrointestinal tract. Pharmacol ResCommun 1987; 19:87-118.

21. Stoorvogel W, Oorschot V, Geuze HJ. A novel class of clathrin-coated vesiclesbudding from endosomes. J Cell Biol 1996; 132:21-33.

22. Kok JW, Babia T, Filipeanu CM, Nelemans A, Egea G, Hoekstra D. PDMPblocks brefeldin A-induced retrograde membrane transport from golgi to ER:evidence for involvement of calcium homeostasis and dissociation from sphingolipidmetabolism. J Cell Biol 1998; 142:25-38.

23. Vandenbulcke F, Nouel D, Vincent JP, Mazella J, Beaudet A. Ligand-inducedinternalization of neurotensin in transfected COS-7 cells: differential intracellulartrafficking of ligand and receptor. J Cell Sci. 2000; 113: 2963-75.

24. Paltauf-Doburzynska J, Frieden M, Spitaler M, Graier WF Histamine-inducedCa2+ oscillations in a human endothelial cell line depend on transmembrane ion flux,ryanodine receptors and endoplasmic reticulum Ca2+-ATPase. J Physiol 2000; 524:701-13.


111

25. Okamoto Y, Ninomiya H, Miwa S, Masaki T. Cholesterol oxidation switches theinternalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells. J Biol Chem 2000; 275: 6439-46.

26. Dam JP, Vleeming W, Riezebos J, Post MJ, Porsius AJ, Wemer J. Effects ofhypercholesterolemia on the contractions to angiotensin II in the isolated aorta andiliac artery of the rabbit: role of arachidonic acid metabolites. J CardiovascPharmacol. 1997; 30:118-23.

27. Haller H, Lindschau C, Erdmann B, Quass P, Luft FC. Effects of intracellularangiotensin II in vascular smooth muscle cells. Circ Res 1996; 79:765-72.

28. Brailoiu E, Filipeanu CM, Tica A, Toma CP, de Zeeuw D, Nelemans SA.Contractile effects by intracellular angiotensin II via receptors with a distinctpharmacological profile in rat aorta. Br J Pharmacol 1999; 126:1133-8.

29. Filipeanu CM, Henning RH, de Zeeuw D, Nelemans A. Intracellular angiotensinII and cell growth of vascular smooth muscle cells. British Journal of Pharmacology132:1590-1596.

30. Wang JM, Baudhuin P, Courtoy PJ, de Potter W. Conversion of angiotensin II intoactive fragments by an endosomal pathway in bovine adrenal medullary cells inprimary culture. Endocrinology 1995; 136:5274-82.

Chapter 7

Intracellular angiotensin II: from myth to reality.

Catalin M. Filipeanu, Robert H. Henning, S. Adriaan Nelemans and Dick de

Zeeuw

Submitted to Journal of the Renin-Angiotensin-Aldosterone System

Chapter VII

114

Introduction

The importance of the renin-angiotensin-aldosteron system (RAAS) was

first recognized in 1934 (Goldbatt et al., 1934). Extensive research started in

the mid 50’s by Skeggs and co-workers, who identified transformation of a

decapeptide into an octapeptide by a factor from horse blood plasma (Lenz et

al., 1996; Skeggs et al., 1956). By then, the two peptides were called

hypertensin I and hypertensin II. In the same year, the laboratory synthesis of

the octapeptide was reported (Bumpus et al., 1957), who named the peptide

angiotonin. Soon, researchers made a compromise and the peptides were called

angiotensin I and angiotensin II.

For a long period, the renin-angiotensin system was primarily viewed as

a circulating endocrine system, in which renin released by the kidney cleaves

angiotensinogen produced by the liver into angiotensin I. Angiotensin I is

converted to angiotensin II by angiotensin converting enzyme (ACE) while

passing the pulmonary vasculature (Catt et al., 1970). However, it was

observed that the levels of angiotensin II in the venous compartment were

much larger that depicted from clearance calculation (Campbell et al., 1985)

and renin isoforms were found outside the kidney (Ganten et al., 1975). Thus,

angiotensin II might be synthesized in various tissues, either from local sources

or from components derived from the circulation. Local angiotensin II

production was found to correlate with the patho-physiological status in rat

myocardial infarction (Ruzicka et al., 1993), as well, high levels of locally

produced angiotensin II were accompanied by cardiac hypertrophy in mice

(Mazzolai et al., 2000). The findings further evolved in the past decade leading

to the concept of concept of local renin angiotensin system, as a determinant of

progression of organ failure further evolved in the past decade (Dzau, 1987).

Today, the important question is, should we take the concept of the local

RAAS system a step further. There is accumulating evidence, that next to the

plasma membrane localized RAAS system, various cell-types are responsive to

the intracellular application of angiotensin II. These observations suggest the

existence of an intracellular RAAS system. The first published support for the

Intracellular angiotensin II from myth to reality

115

effect of intracellularly applied angiotensin II originated from De Mello (1994),

showing that intracellular injection of angiotensin II is accompanied by a

decrease in cellular junction conductance.

The purpose of this review is to examine whether the intracellular

system is a myth or if it should acknowledged its reality as a new entity

completing the circulating and/or local RAAS system. To this end we present

the limited evidence of angiotensin II presence within the cell. Further, the

possible origin of intracellular angiotensin II receptors is discussed. In the next

section the known data about their pharmacology are presented and compared to

that of plasma membrane receptors. Next, we identify intracellular Ca2+ ([Ca2+]i)

as the major signal transduction pathway modulated by intracellular angiotensin

II. As a consequence of these changes in [Ca2+]i, intracellular angiotensin II

induces effects such as vascular smooth muscle contraction, as well as cell

growth. These effects are apparently similar to the effects obtained after AT1

receptor stimulation, but they are insensitive to current therapeutic and blocking

agents, suggesting that a sustained investigation of intracellular angiotensin II

effects is necessary for a better understanding of the renin-angiotensin-

aldosteron system.

Intracellular levels of angiotensin II

Measurable angiotensin II levels ranging from 5 to 20 fmol/g wet tissue

have been reported in various tissues (van Kats et al., 1998; Imig et al., 1999,

Herman and Ring, 1995 De Mello and Danser, 2000). However, how

angiotensin II accumulates into the cell is still unclear at the present. Its

intracellular production is under strong debate (De Mello and Danser, 2000). It

has been reported that intracellularly produced angiotensin II can be released

by stretching cardiac myocytes (Sadoshima et al., 1993; Schunkert et al., 1995)

and in mesangial cells (Becker et al., 1998), while other authors report opposite

results (van Kesteren et al., 1999). The main technical problem may be due to

contamination of the samples with culture medium containing angiotensin II

and other related molecules (De Mello and Danser, 2000). Thus, although

Chapter VII

116

components of the of the RAAS system were identified in cardiac (Passier et

al., 1996) and smooth muscle cells (Kato et al., 1993), we are still searching for

evidence of intracellular synthesis of angiotensin II.

An alternative source for intracellular angiotensin II can be the

internalization of the circulating angiotensin II. Within 60 min, plasma-

administered angiotensin II accumulates within the heart, kidney and adrenal

glands. Presence of an AT1 antagonist prevented this process (van Kats et al.,

1997). In the endosomes, due to the low pH environment (Krueger et al., 1997),

internalized AT1 receptor releases the bound angiotensin. Next, the molecule

can be degraded or used to stimulate the local intracellular receptor.

This pathway requires existence of plasma membrane receptors able to

be internalized in order to release angiotensin II intracellularly. The A7r5 cell

line, isolated from rat fetal aorta (Kimes and Brandt, 1976), is devoid of

functional plasma membrane angiotensin receptors (Adams et al., 1999;

Filipeanu et al., 2001 a,b). However, we identified a basal pool of angiotensin

II in these cells and a large increase in its level was observed after extracellular

addition of angiotensin II (fig 1). These results suggest that uptake of

angiotensin II can occur via AT1 receptor independent process. As expected,

both AT1 and AT2 receptor antagonists were ineffective in preventing

angiotensin II accumulation in these cells (fig 1).

In conclusion, even if the origin of intracellular angiotensin is not

clarified in all cases, enough angiotensin II is present intracellularly within

different cell types to activate a putative intracellular angiotensin II receptor.

Importantly, variations in intracellular angiotensin II levels may be involved in

patho-physiological situations since it has been shown to be changed in certain

conditions like high salt diet (Imig et al., 1999) or anaphylaxis (Herman and

Ring, 1995).

The origin of intracellular angiotensin receptors

The existence of intracellular angiotensin II effects independent of

plasma membrane receptor activation demands existence of an intracellular


117

effector protein. Although data are accumulating about such a receptor, as in

the case of intracellular levels of angiotensin II, still we cannot yet precisely

define it.

An obvious candidate is the internalized plasma membrane angiotensin

receptor (fig 2B), which may fully maintain its signaling mechanisms within

the cell as has been reported for the insulin receptor (Bevan et al., 1995). Of the

two well-characterized plasma membrane angiotensin II receptors, only AT1

undergoes extensive recycling (Ullian and Linas, 1989), based on a highly

conserved Ser-Thr-Leu motif (Hunyady et al., 1994). The AT2 receptor is

devoid of this motif and therefore not internalized (Hein et al., 1998).

Plasma membrane AT1 receptor internalization is triggered by the

presence of an agonist, although our preliminary results suggest an intense

receptor recycling even in non-stimulated rat aortic rings (Filipeanu et al.,

unpublished). Internalized plasma membrane receptors are replaced rapidly by

newly synthesized and recycled receptors. Continuous presence of angiotensin

Figure 1.Intracellular levels ofangiotensin II in A7r5 cells.Values are reported underbasal conditions (control) andafter extracellular incubationswith angiotensin II (ang II, 10-

7 M) or in combination witheither losartan (10-6 M) orPD123319 (10-6 M). Thedeterminations were made incells plated at a density of 105

cells in 6 well plates for 24hours in DMEM with 0.1 %FCS. The protein content wasextracted in 98 % ethanol andresuspended in 100 'l assaybuffer. Then, the peptidecontent was determined usinga peptide enzymeimmunoassay kit (PeninsulaLaboratories Inc) followingthe manufacturer’s protocol.

intr

acel

lula

r le

vels

of a

ng

iote

nsi

n II

in A

7r5

cells

(fm

ol/l)

10

100

1000

3000

cont

rol

+ 10

-7 M a

ng II

+ 10

-7 M a

ng II

+ 10

-7 M a

ng II

+ 10

-6 M lo

sarta

n

10-6 M

PD

1233

19

Chapter VII

118

II is accompanied by the reduction of cellular AT1 mRNA (Adams et al., 1999).

The internalization process occurs via two distinct mechanisms: clathrin coated

pits and caveolae. Once internalized in the tubulo-endosomal compartment, the

AT1 receptor can be recycled to the plasma membrane or directed into

lysosomes for degradation (Koenig and Edwardson, 1994). Interestingly, this

may be highly dependent on the activation state of the receptors, as shown for

the thrombin receptor (Hein et al., 1994). During its passage through

endosomal/lysosomal compartment, the activated receptor may interact with

various locally available signal transduction mechanisms available locally.

Thus, the availability of various kinases/phospholipases dependent on the prior

status of the cell could also contribute to the biological response induced by

internalized receptor. No data are available on the internalization process of

nonAT1/nonAT2 binding sites reported to be present at the plasma membrane of

intestinal epithelium (Smith, 1995) and human heart (Regitz- Zagrosek et al.,

1996). Nevertheless, several observations question whether intracellular

angiotensin II exerts its effect via the internalized AT1 receptor. The effects of

intracellular angiotensin II are unaltered by previous stimulation of plasma

membrane AT1 receptors, which drastically increasing the amount of

intracellular AT1 receptors in cultured VSM cells (Haller et al, 1996) and in

adult rat aorta (Brailoiu et al., 1999). Moreover, A7r5 cells display a variety of

physiological responses to intracellularly applied angiotensin II, yet they are

devoid of functional plasma membrane AT1 receptors (Adams et al., 1999,

Filipeanu et al., 2001a). The independence of intracellular angiotensin II effects

from receptor internalization in three different model systems suggests that

intracellular angiotensin receptors probably represent a separate entity. Further

investigation should focus on whether it consists of a processed AT1 receptor

e.g. defective in plasma membrane insertion, as shown for the vasopressin

receptor (Morello et al., 2000), or represents a yet unidentified gene product.

In this respect, several studies have described intracellular binding sites

for the peptide. Cytosolic binding sites for angiotensin II were identified in

heart and smooth muscle as early as 1971 (Robertson and Khairalah, 1971).


119

More recently, angiotensin II binding sites were identified in liver nuclei

(Kiron and Soffer, 1989) and a particular protein was cloned as a ‘soluble

angiotensin binding protein’ (McKie et al., 1993). No data are available about

the antagonist binding for these sites, hampering the comparison to functional

studies. However, existence of binding sites demonstrates the heterogeneity of

angiotensin II binding proteins and provides indirect support for intracellular

angiotensin II actions.

Figure 2.Proposed scheme for origin of intracellular angiotensin II, its signal transduction,pharmacology and functional effects. See the text for detailed explanation.Abbreviations: AT1- angiotensin type 1 receptor; AT2 – angiotensin type 2 receptor;ER – endoplasmic reticulum; IP3 – inositol(1,4,5)P3 trisphosphate.

AT1

• contraction• growth

losartan

• apoptosis• cell differentiation

CGP42112AAT2

PD123319

plasma membrane effects

intracellularproduction

ang II

sources of intracellular ang II

AT1

MAPk PI3K

losartanPD123319CGP42112A

intracellular ang II receptors and signal transduction

losartan

IP3

ER

Ca2+

functional effects of intracellular ang II

ang II ang IIalternativeuptake

• UTP• 5-HT

Ca2+

AT1

CGP42112A

Ca2+,, MAPk, PI3K• contraction

losartan

• growth

PD123319

CGP42112A

losartan

PD123319

Chapter VII

120

Intracellular delivery of plasma membrane impermeable compounds

Obviously, the biggest challenge in investigating the intracellular effects

of a compound is its intracellular delivery, with simultaneous preservation of

cellular function. Common techniques employed consist of cellular injection or

delivery with liposomes. The main limitation of cellular injection is that the

effect can be measured only at the single cell level and the plasma membrane is

damaged. Liposomes have been successfully employed to deliver their content

intracellularly. However their use may change the membrane fluidity and

permeability. For this reasons the concentration of angiotensin II and its related

peptides in the liposomes should be kept below 10-4 M (Brailoiu et al., 1997).

Both techniques experience additional limitations. Firstly, the amount of the

delivered compound is difficult to control. Secondly, plasma membrane

receptor activation may result from leakage of the agonist during application.

Also, excretion of the agonist to the outside may occur after intracellular

delivery. Often, this problem is circumvented by the extracellular co-

application of impermeable antagonists together with intracellular delivery of

angiotensin II. The main disadvantage of this strategy is that these antagonists

can block receptor internalization (Zou et al., 1998), which may interfere with

intracellular receptor activation or signal transduction.

A different strategy to circumvent plasma membrane receptor activation

consists of the controlled removal of the plasma membrane. This procedure,

cell permeabilization, will result in equilibrium between extracellular and

intracellular concentrations of the desired compound. However, cell

permeabilization will also block the plasma membrane dependent processes

and only organelles-dependent processes (e.g. Ca2+ release from the

endoplasmic reticulum) can be studied. Furthermore, the cell architecture

changes such as re-arrangement of intracellular organelles, induced by this

technique may also report false results (Xu et al., 1990).

Intracellularly delivered angiotensin II was found to accumulate in

different cellular compartments, depending on the technique used. Gold-

coupled angiotensin II internalized by the AT1 receptor, accumulates in the


121

‘deep’ endosomal compartment (Anderson and Peach 1994). Injected

fluorescent angiotensin II localizes in the nuclei (Haller et al., 1996), whereas it

has a relative uniform cytosolic distribution if delivered by liposomes

(Filipeanu et al., 2001b). Thus, observed differences of intracellular angiotensin

II in signalling mechanisms and/or functional responses might arise from the

technique used. Ideally, the same process affected by intracellular angiotensin

II should be studied using all techniques available.

Pharmacology of intracellular angiotensin receptors

One of the most striking features of the effects evoked by intracellular

angiotensin II is the difference in sensitivity to AT receptor antagonists, even

within the same model (table 1). Unfortunately, because of the technical

problems outlined above, all the pharmacological characterizations made to

date have been on antagonist effects on functional parameters and not on

receptor binding.

Still, different subtypes can be demonstrated, one with AT1

characteristics, a high affinity nonAT1/nonAT2 receptor, and a low affinity

nonAT1/nonAT2 receptor. The receptor that modulates cell to cell

communication in heart muscle was blocked by intracellular losartan (De Mello

1994, 1996). In contrast, neither AT1 nor AT2 antagonists prevented the effects

of intracellular angiotensin II on inward Ca2+ current in cardiac myocytes (De

Mello, 1998).

In cultured vascular smooth muscle the effects of intracellular

angiotensin II were also entirely blocked by the AT1 antagonist, CV11947. The

contractile actions of intracellular angiotensin II in rat aorta showed an EC50 of

~2 nM/mg-tissue. This value was shifted to ~2 'M/mg-tissue by the AT1

antagonist CV11947 indicating an AT1 like receptor. However, this is not the

case because the AT2 antagonist antagonist PD123319 also shifted the EC50 to

~40 nM/mg-tissue, (Brailoiu et al., 1999, fig 3A).

Most of our investigations were conducted in the convenient model of

A7r5 smooth muscle cell line devoid of extracellular angiotensin II receptors.

Chapter VII

122

Among the variety of intracellular angiotensin II effects in this cell line, only

inhibition of the [Ca2+]i increase evoked by the extracellular agonists serotonin

or UTP has been found to be sensitive to losartan (Filipeanu et al., 2001c).

Other effects, like cellular growth showed a limited sensitivity to AT1 and AT2

antagonists (fig 3B), but were totally blocked by CGP42112A, previously

described as a partial AT2 agonist (Tsuzuki et al., 1996). Yet, stimulation of an

intracellular AT2 like receptor is unlikely to explain the intracellular

angiotensin II effect, as the AT2 antagonist, PD123319 also inhibited

angiotensin II induced cell-growth (Filipeanu et al., 2001a). Apparently,

multiple intracellular angiotensin receptors are present in A7r5 cells.

The high affinity type receptor being sensitive to CGP42112A and both

AT1 and AT2 antagonists is apparently involved in growth (A7r5 cells) and

contraction (rat aorta). The low affinity type receptor, being sensitive to

CGP42112A and not to AT1 and AT2 antagonists, is involved in growth and

contraction, as well as in the intracellular angiotensin II-induced stimulation of

Figure 3Dose effects of intracellular angiotensin and its antagonists on rat aorta contraction (A) andA7r5 cell growth (B). Angiotensin II and the antagonists were applied intracellularly usingliposomes. Adapted from Brailoiu et al., 1999 and Filipeanu et al., 2001a.

incorporated Ang II nmol/mg

0.01 0.1 1 10 100 1000

cont

ract

ion

(% o

f con

trol)

0

10

20

30

40

50

60

70

*

**

* ** *

*

log [LAngII] (M)

-12 -11 -10 -9 -8 -7 -6

effe

ct (

%)

0

10

20

30

40

50

60

70

80

90

100

control

cgplos

PD cgp

control

PD

los


123

Ca2+ influx in A7r5 cells. At present it is unclear whether different receptors are

involved or whether the effects are produced by a single entity with a different

subcellular localization, explaining the differences in pharmacological

properties and coupling to various signaling mechanisms (fig 2C).

Related angiotensin II peptides may activate some plasma membrane

angiotensin II receptors (Champion et al., 1998). The intracellular angiotensin

receptor showed differential sensitivity to these peptides. Most of these

experiments were performed using angiotensin I, the normal precursor peptide

of angiotensin II. This decapeptide may substitute for angiotensin II in the

inhibition of junctional conductance in heart muscle, and its effect was entirely

abolished by the ACE inhibitor enaprilat, demonstrating dependence on

intracellular ACE activity (De Mello, 1995). However, the angiotensin I

contractile effects in rat aorta were not affected by captopril, suggesting either

angiotensin I binding to intracellular angiotensin II receptor or the existence of

an alternative angiotensin I splicing pathway. Chymase involvement seems

unlikely in light of its absence in rat tissues (Urata and Ganten, 1993), but other

metalloproteinase activity cannot be excluded.

Angiotensin I cannot substitute for angiotensin II in any of the

parameters investigated in A7r5 cells and no ACE activity was measurable

(Filipeanu et al., 2001b). Among smaller peptides only angiotensin IV

(angiotensin 3-8) could mimic the effects of angiotensin II on [Ca2+]i in

cultured cells (Haller et al., 1996). In conclusion, the intracellular effects of

angiotensin II are mediated by receptors with a pharmacology that is distinct of

the classical plasma membrane receptors.

Signal transduction pathways modulated by intracellular angiotensin II

Identification of the signal transduction pathways for any hormone is

important in order to understand the function of its receptors. From the

multitude of possible signalling pathways, the published results indicate that

intracellular angiotensin II regulates intracellular Ca2+ levels. In addition

kinase(s) activation has been also reported (table 2).

Chapter VII

124

N.D

. – n

ot d

eter

min

ed; A

T1R

– ty

pe 1

ang

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nsin

II

rece

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2R –

type

2 a

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ecep

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AC

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ang

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me

Act

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nsP

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-tr

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125

[Ca2+]i is a crucial cellular parameter, its increase being the initial signal

for many cellular functions such as contraction or secretion (Putney and

Ribeiro, 2000). The intracellular Ca2+ concentration is regulated by various

mechanisms, like: (i) Ca2+ entry from the extracellular space through either

voltage dependent or -independent Ca2+ channels, (ii) Ca2+ release from

intracellular stores and (iii) Ca2+ extrusion via Ca2+ pumps or Na+/Ca2+

exchange towards other organelles or to the extracellular space.

Extracellular angiotensin II effects on vascular smooth muscle

contraction are dependent on both Ca2+ release from intracellular stores and

Ca2+ entry from the extracellular space (van Heiningen et al., 1991). In contrast,

the effects of intracellular angiotensin II on vascular smooth muscle contraction

are solely dependent on Ca2+ influx from the extracellular space (fig 4),

demonstrating that the intracellular angiotensin II receptor from adult rat aorta

is not able to induce Ca2+ release from intracellular stores. Also, in cultured rat

aortic vascular smooth muscle cells, Ca2+ influx from the extracellular space

represents the main component of [Ca2+]i raise, although a small Ca2+ release

from intracellular stores was also detectable (Haller et al., 1996). Likewise, we

found that angiotensin II filled liposomes induced a modest production of InsP3

in A7r5 cells, a second messenger that releases Ca2+ from intracellular stores

(Filipeanu et al., 2001b).

Figure 4.Ca2+ dependency of angiotensinII filled liposomes induced rataorta contraction. In Ca2+ freeextracellular-mediumintracellular angiotensin II haveno effect, but contractionoccurred following restorationof normal Ca2+ concentration.Reproduced from Brailoiu et al.,1999.

Chapter VII

126

In A7r5 cells, intracellular angiotensin II delivered by liposomes induced

Ca2+ influx via voltage independent Ca2+ channels (fig 5). However, intracellular

angiotensin did not stimulate Ca2+ release in permeabilized cells but its presence

increased the size of the InsP3 releasable Ca2+ pool (Filipeanu et al., 2001b). This

enhancement is of similar size as the synergism reported for other compounds,

such as IP4, ATP or GTP (van der Zee et al., 95; Missiaen et al., 97; Loomis-

Husselbee et al 1996) suggesting that intracellular angiotensin II may substitute for

one of these compounds in cell signalling. A mechanism often used to increase the

size of the IP3 releasable Ca2+ pool is activation of small G proteins (Xu et al.1995,

Loomis-Husselbee et al 1996). Such G-proteins are not involved in the potentiation

induced by intracellular angiotensin II, as shown by the experiments using GDP!S,

a blocker of these proteins (Filipeanu et al., 2001b). Intracellular application of

angiotensin II also modulates Ca2+ signaling evoked by heterologous stimulation.

Intracellular angiotensin II inhibited Ca2+ influx stimulated by extracellular

hormones like serotonin and UTP (Filipeanu et al., 2001c). An inhibition of the

Ca2+ inward Ca2+ current was also observed in rat cardiac myocytes (De Mello,

1998). The latter effect is species dependent, since stimulation of a Ca2+ current

was observed in hamster myocytes. The effect on the inward Ca2+ current appeared

to be independent of cyclic AMP levels or protein kinase C stimulation, but was

enhanced after increases in cyclic GMP levels. Noticeable, the effects of

intracellular angiotensin II on inward current in different species mirror the effects

of angiotensin II on heart contractility: positive inotropic effect in hamsters and

shortening of action potential in rats (De Mello, 1998).

In conclusion, intracellular angiotensin II stimulates [Ca2+]i when applied in

non-stimulated cells and inhibits Ca2+ entry evoked by extracellular stimulation. In

virtue of its ability to modulate [Ca2+]i levels in different manners, intracellular

angiotensin II may play an important role in regulation of cardio-vascular muscle

contractility.

In addition to modulation of [Ca2+]i, intracellular angiotensin II has been

found to modulate phosphorylation of various cytoskeletal proteins (Haller et

al., 1999). In vascular smooth muscle cells, the [Ca2+]i increase in a single cell


127

was propagated to the adjacent cells, whereas tyrosine phosphorylation was not

(Haller et al., 1999). Thus, it is unclear at the moment if the stimulatory effect

of intracellular angiotensin II on kinase activity is related to a prior [Ca2+]i

raise, or is a direct effect. However, specific inhibitors of MAPK/ERK and PI-

3K prevented its functional effects in A7r5 cells, indicating that these pathways

are involved in long-term cellular effects.

Figure 5A: effects of angiotensin II filledliposomes on [Ca2+]i in A7r5vascular smooth muscle cells.The onset of the response is slow,but is followed by a moreproeminent sustained Ca2+ entry.B: dose-effect curve forangiotensin II filled liposomes inA7r5 cell. Reproduced fromFilipeanu et al., 2001.C. microinjection of angiotensinII into the cytoplasm of culturedvascular smooth muscle cellsproduced a rapid increase in[Ca2+]i. The increase occurred notonly in injected cell, but in theadjacent cells as well.Experiments adapted from Halleret al., 1996.

t im e ( s )

0 6 0 1 2 0 1 8 0 2 4 0

0

2 0

4 0

6 0

8 0

1 0 0

Lang II

A

lo g L [A n g II] (M )

-1 0 -9 -8 -7 -6

0

1 0

2 0

3 0

4 0

5 0

incr

ea

se in

[C

a2+] i

(n

M)

[Ca2

+ ] i (

nM

)

Injected cell

Adjacent cell

Adjacent cell

B

C

time (s)

Chapter VII

128

Table 2

Signal transduction pathways known to be modulated by intracellular

angiotensin II.

Signaling pathways Intracellular angiotensinII effects

Reference

Voltage dependent Ca2+

channel

Inhibition Filipeanu et al., 2001c

Receptor operated Ca2+

channels

Stimulation Brailoiu et al., 1999

Filipeanu et al., 2001b

Phospholipase C weak stimulation Haller et al., 1996

Filipeanu et al., 2001b

MAP kinase Stimulation Filipeanu et al., 2001b

Cell communication Inhibition de Mello, 1994

Cell communication Stimulation Haller et al., 1996; 1999

Functional effects of intracellular angiotensin II

Extracellular angiotensin II exerts its hypertensive actions mainly by

acting on AT1 subtype receptors, that trigger vascular smooth muscle

contraction and cell proliferation (Griendling et al., 1997). Results from our

laboratory demonstrate that angiotensin II may exert similar actions from

within the cell, independently of plasma membrane receptor stimulation.

Intracellular angiotensin II induced contractions of de-endothelised rat

aortic rings, with amplitude similar to the contractions elicited by extracellular

angiotensin II (fig 4). The contractile response is subject to desensitization, as

is the case for the plasma membrane AT1 receptor, but extracellular pre-

treatment with the AT1 receptor antagonist CV11947 or AT2 receptor

antagonist PD123319 does not prevent the contractile actions of intracellular

angiotensin II.

Further, A7r5 vascular smooth muscle cells are devoid of any functional

response to extracellular applied angiotensin II (Adams et al., 1999; Filipeanu

et al., 2001a,b,c). However, angiotensin II filled liposomes stimulate cell


129

proliferation and increase [3H] thymidine incorporation dose-dependently (fig

3).

Finally, intracellular angiotensin II also modulates cell to cell

communication. This process ensures a uniform propagation of cellular stimuli

and a unitary biological response (i.e. smooth muscle contraction).

Intracellularly applied angiotensin II inhibited gap junction communication in

rat or hamster heart (De Mello, 1994; 1996). An opposite effect was observed

in cultured vascular smooth muscle cells, where the [Ca2+]i raise induced by

intracellular angiotensin II was propagated to adjacent cells via gap junctions

(Haller et al., 1999).

Role of intracellular angiotensin II

Results from the last five years univocally show that angiotensin II has

complex actions from within the cell, unrelated to the activation of typical

plasma membrane receptors. Although our knowledge about these intracellular

angiotensin II receptors is only at beginning, some important aspects can

already be defined. In contrast to the AT3 angiotensin receptor subtype (Unger

et al., 1997), intracellular angiotensin receptor distribution is not limited to

specific cell lines, but are also present in heart and smooth muscle. At present,

it is unclear if this system is totally independent, or just complementary, to the

local renin angiotensin system. However, the effects on vascular smooth

muscle growth and contraction, two processes involved in hypertension

(Touyz, 2000) suggest that intracellular angiotensin further contributes to the

physio-pathological actions of the hormone.

Extracellular angiotensin II may rapidly degrade under actions of

different angiotensinases, whereas AT1 subtype receptor may undergo rapid

desensitization during continuous stimulation (van Kats et al., 1997,

Griendling et al., 1997). The intracellular receptors may serve to prolongation

the effects of angiotensin II. Although it is expected that additional signal

transduction pathways will be identified in the future, we have shown that

intracellular angiotensin II modulates crucial processes in cellular

Chapter VII

130

compartments not accessible to the classical therapeutic agents like AT1

antagonists and/or ACE inhibitors. AT1 antagonists are not internalized

together with AT1 receptors (Zhou et al., 1998), whereas an internalization

pathway for ACE inhibitors has not yet been proposed yet. Furthermore, the

effects of intracellular angiotensin II on modulation of [Ca2+]i, cell to cell

communication and tyrosine phosphorylation showed a different sensitivity to

that of commonly used AT1 or AT2 antagonists.

Existence of the intracellular effects of angiotensin II and the presence

of intracellular angiotensin II binding proteins demonstrate the reality of an

intracellular angiotensin system. In order to avoid confusions, these new facts

should lead to the redefinition of the local renin angiotensin system. The renin-

angiotensin system independent of the circulating renin angiotensin system but

localized outside the cell (i.e. plasma membrane angiotensin II receptors,

angiotensin converting enzyme, etc) should be called the interstitial renin

angiotensin system. The other renin-angiotensin system, taking place within the

cell should be assigned as the intracellular renin angiotensin system (fig 6).

In the light of the results summarized here, the absence of a detectable

cellular response after extracellular stimulation does not preclude an important

Figure 6Proposed nomenclature of various renin angiotensin system. The “local” reninangiotensin system is independent from circulating renin angiotensin system and itis composed from interstitial and intracellular system

circulating angiotensin system

intracellular angiotensin system

interstitial angiotensin system

local angiotensin system

blood vesselcell

cell

interstitial space


131

role of angiotensin II within the cell. Therefore, when considering clinical

paradigms, one should consider the inclusion of pharmacological modulators of

the renin angiotensin system in cellular compartments where their activity is

not yet evaluated. The last ten years has contributed decisively to our

understanding of plasma membrane angiotensin receptors. The next decade will

most likely reveal the importance of intracellular angiotensin receptors in the

patho-physiological activities of the ‘old’ hypertensin (angiotensin II)

molecule.

Acknowledgements

Catalin M. Filipeanu is a recipient of an Ubbo Emmius fellowship from Groningen

University Institute of Drug Exploration (GUIDE).

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Summary

137

Summary

Angiotensin II is an important physiological and pathological hormone

involved in multiple cardio-vascular diseases. The investigation of the renin

angiotensin system started in the fifties and it was considered to represent a

circulating endocrine system, in which renin released from the kidney cleaves

angiotensinogen produced by the liver into the decapeptide called angiotensin I.

Angiotensin I is converted to the octapeptide angiotensin II by angiotensin

converting enzyme (ACE) while passing the pulmonary vasculature. However,

research from the last two decades demonstrated the existence of a tissue or a

local renin angiotensin system, which may produce angiotensin II within the

tissue. As many other hormones, angiotensin II exerts its biological effects after

interaction with specific receptors, localized at the level of the cellular plasma

membrane. Among these receptors AT1 and AT2 subtypes are the best

characterized at the moment.

The aim of the present thesis was to investigate whether angiotensin II can have

intracellular effects, independent of plasma membrane receptor activation.

The investigations were conducted in vascular smooth muscle, aiming to find

alternative cellular pathways, not considered yet in cardio-vascular therapy. In

chapter 1 a general overview of the renin-angiotensin system is presented,

together with the existent support for the intracellular angiotensin II hypothesis.

The few data about intracellular angiotensin II reported before the work

presented in this thesis was started were obtained in cultured cells. As an

alternative, in chapter 2 the effects of intracellular angiotensin II were studied

in rat aortic rings. Angiotensin II was delivered intracellularly by means of

liposomes. It has been found that intracellular angiotensin II induced smooth

muscle contractions, which were insensitive to the extracellular application of

losartan, an AT1 receptor antagonist. However, intracellularly administered

losartan abolished the contractions evoked by intracellular angiotensin II. In

contrast to the typical plasma membrane AT1 receptor, the intracellular receptor

was also antagonized, but to a lesser extent, by an AT2 receptor antagonist,

PD123319. Intracellularly applied angiotensin I also induced rat aorta smooth

Summary

138

muscle contraction and its effects were insensitive to an angiotensin converting

enzyme inhibitor, indicating alternative generating pathways. These

experiments showed the existence of pathological angiotensin II effects in

cellular compartments not investigated before.

In chapter 3 the signal transduction mechanisms activated by intracellular

angiotensin II were investigated. These experiments were conducted in A7r5

vascular smooth muscle cell line, in which extracellular angiotensin II do not

have functional responses. Still, intracellular angiotensin II applied by

liposomes or after controlled removal of the plasma membrane, induced influx

of extracellular Ca2+ ions, stimulated IP3 production and potentiated IP3

induced Ca2+-release from intracellular stores. These effects were insensitive to

AT1 or AT2 receptor blockers, demonstrating that although intracellular

angiotensin II is activating common signal transduction pathways, the

intracellular receptors are different from the typical plasma membrane

angiotensin II receptors.

In chapter 4, the interaction between intracellular angiotensin II and other

extracellular hormones was investigated. Intracellular angiotensin II inhibited

the effects of extracellular serotonin and UTP in A7r5 cells. In contrast with the

results described previously, an AT1 antagonist blocked these effects of

angiotensin II from inside the cell, indicating that multiple intracellular

angiotensin II receptors may exist.

In the chapter 5, this conclusion is further substantiated by investigating the

effects of intracellular angiotensin II on cell growth in A7r5 cell line. Data

analysis showed that the stimulatory effect of angiotensin II on cell growth is

mediated by two receptors with different affinities for the hormone. AT1 and

AT2 receptor antagonists inhibit the low affinity receptor, whereas the high

affinity site is insensitive to these agents. Both receptors are inhibited by

CGP42112A, a compound previously described as a (partial) AT2 receptor

agonist. Phosphatidyl 3-kinase and mitogen activated protein kinase stimulation

were the signal transduction pathways activated by intracellular angiotensin II

for stimulation of cell growth.

Summary

139

In chapter 6, the desensitization of the extracellular AT1 receptor in rat aorta

was studied, in order to investigate the mechanisms of AT1 receptor

internalization, which can be involved in intracellular delivery of angiotensin

II. We detected an important desensitization of the angiotensin II induced

contractile response after repetitive application of the agonist. It was found that

the AT1 receptor is internalized prominently via clathrin coated pits, but in part

also via caveolae. Further, extensive trafficking of AT1 receptors take place in

the tubulo-endosomal compartment. Brefeldin A, an agent that disrupts Golgi

structures, is able to enhance the desensitization even after blockade of

internalization pathways. In contrast, nocodazole, an inhibitor of microtubule

polymerization, was able to increase desensitization only under control

conditions, but not after blockade of the internalization. Thus, our results reveal

the subcellular pathways followed by AT1 receptor desensitization and indicate

an additional target for attenuating the contractile actions of angiotensin II.

A review of the present knowledge on the intracellular angiotensin II receptors

and their effects is presented in chapter 7. The potential clinical relevance of

intracellular angiotensin II for cardio-vascular pathology, together with the

pharmacological implications and the possible directions of future research are

discussed.

The balance between the different plasma membrane angiotensin II receptor

subtypes depends on the physiological and pathological conditions. The

existence of intracellular angiotensin II receptors is adding a new dimension to

this process. Intracellular angiotensin II induces growth in vascular smooth

muscle and may play a role in remodeling processes. Further, it may also be

involved in sustained vascular contractility by bypassing the rapid

desensitization of extracellular angiotensin II-induced signaling. Therefore, the

existence of intracellular angiotensin II receptors and possible of other

components of the renin-angiotensin system as well, may contribute to novel

pharmacological approaches in the treatment of renin-angiotensin system

mediated diseases.

Samenvatting

141

Samenvatting

Angiotensine II is een hormoon, dat een belangrijke pathofysiologische rol

speelt bij hart- en vaatziekten. Het onderzoek naar het renine-angiotensine-

systeem begon in de jaren vijftig; het systeem werd beschouwd als een

circulerend endocrien systeem, waarin renine, afgegeven door de nier, het

angiotensinogeen, gevormd in de lever, omzet in het decapeptide angiotensine

I. Tijdens passage in het longvaatbed wordt angiotensine I omgezet in

angiotensine II door het angiotensine-converterend enzym (ACE). Onderzoek

van de laatste twintig jaar heeft echter het bestaan van een weefselgebonden of

een locaal renine-angiotensine-systeem aangetoond, dat angiotensine II in het

weefsel produceert. Zoals veel andere hormonen, oefent angiotensine II zijn

biologische effecten uit via specifieke receptoren, gelokaliseerd in de cellulaire

plasmamembraan.

Dit promotie-onderzoek had ten doel om te onderzoeken of angiotensine II ook

intracellulaire effecten heeft, welke onafhankelijk zijn van activering van de

receptoren in de plasmamembraan. Het onderzoek werd uitgevoerd in gladde

spiercellen van de vaatwand, met als belangrijk doel om alternatieve cellulaire

signaaltransductie-routes op te sporen, waarmee in cardiovasculaire

therapeutische strategieën nog geen rekening gehouden wordt.

In hoofdstuk 1 wordt een algemeen overzicht van het renine-angiotensine-

systeem gegeven, inclusief recent gerapporteerde gegevens die wijzen op een

rol voor intracellulair angiotensine II.

De schaarse gegevens met betrekking tot intracellulair angiotensine II waren

verkregen in gekweekte cellen. In hoofstuk 2 is het effect van intracellulair

angiotensine II daarentegen onderzocht in ring-preparaten van de aorta van de

rat. Angiotensine II werd intracellulair afgeleverd door middel van liposomen.

Intracellulair angiotensine II bleek contracties van de gladde spiercellen te

bewerkstelligen, die niet beïnvloed werden door extracellulair toegediend

losartan, een AT1-receptor antagonist. Intracellulair toegediend losartan deed

de door intracellulair angiotensine II geïnduceerde contracties daarentegen

teniet. In tegenstelling tot de membraan gebonden AT1-receptor werden de

Samenvatting

142

effecten van intracellulair angiotensine II ook geblokkeerd door een AT2-

receptor antagonist PD123319, zij het in mindere mate. Intracellulair

toegediend angiotensine I veroorzaakte ook contracties van de rattenaorta; dit

effect werd niet beïnvloed door een remmer van het angiotensine-converterend

enzym, hetgeen mogelijk wijst op het bestaan van andere wijzen van

angiotensine II vorming of op een direct angiotensine I effect. Deze proeven

tonen het bestaan van patho-fysiologische effecten van angiotensine II aan in

cellulaire compartimenten, welke nog niet eerder onderzocht zijn.

In hoofdstuk 3 zijn de signaaltransductie-mechanismen, die geactiveerd

worden door intracellulair angiotensine II, onderzocht. Deze experimenten

werden uitgevoerd in de A7r5 gladde spiercellijn, die stamt uit de

aortavaatwand. In deze A7r5-cellen induceert extracellulair toegediend

angiotensine II geen functionele responsen. Intracellulair angiotensine II

daarentegen, toegediend via liposomen of na gecontroleerde permeabilisatie

van de plasmamembraan, induceerde een influx van extracellulaire Ca2+ ionen,

stimuleerde IP3-productie en versterkte de IP3-geinduceerde mobilisatie van

intracellulair opgeslagen Ca2+. Deze effecten werden niet beïnvloed door AT1-

en AT2-receptor antagonisten, hetgeen aantoont dat de intracellulaire

receptoren verschillen van de AT1-receptor op de plasmamembraan, hoewel

intracellulair angiotensine II wel de gebruikelijke signaaltransductie-routes

activeert.

In hoofdstuk 4 is de interactie tussen intracellulair angiotensine II en andere

extracellulair toegediende hormonen onderzocht in A7r5 cellen. Intracellulair

afgeleverd angiotensine II remde de effecten (Ca2+-influx) van extracellulair

serotonine en UTP. In tegenstelling tot de hierboven beschreven resultaten,

werden deze effecten van angiotensine II wel geblokkeerd door een

intracellulair toegediende AT1-receptor antagonist. Dit wijst erop, dat er

mogelijk meerdere typen intracellulaire angiotensine II-receptoren bestaan.

In hoofdstuk 5 is deze conclusie verder onderbouwd door onderzoek naar de

effecten van intracellulair angiotensine II op de celgroei van A7r5-cellen.

Analyse van de gegevens liet een stimulerend effect van angiotensine II op

Samenvatting

143

celgroei zien waarbij twee receptoren met verschillende affiniteit voor

angiotensin II betrokken waren. AT1- en AT2-receptor antagonisten

blokkeerden het hoog-affine effect van angiotensine II, terwijl de

bindingsplaats met lage affiniteit niet beïnvloed werd. Beide receptoren werden

echter geblokkeerd door CGP42112A. Deze stof stond tot nu toe bekend als

een (partiële) AT2-receptor agonist. Experimenten met selective remmers lieten

zien dat de stimulatie van de celgroei afhankelijk is van activering van de

signaaltransductie-routes fosfatidyl-3-kinase (PI-3K) en mitogen activated

protein kinase (MAPK/ERK).

In hoofdstuk 6 is de desensitisatie van de extracellulaire AT1-receptor in de

aorta van ratten onderzocht, teneinde de rol en de mechanismen van

internalisering van AT1-receptoren te ontrafelen. Deze internalisering kan een

rol spelen bij het intracellulair afleveren van angiotensine II. Wij ontdekten een

belangrijke mate van desensitisatie van de door angiotensine II geïnduceerde

contractiele respons na herhaaldelijke toediening van de agonist. Het bleek dat

internalisering van de AT1-receptor met name plaatsvond via z.g. clathrin-

coated pits, maar deels ook via een alternatieve route, de caveolae. Er vond

tevens een uitgebreid transport van geinternaliseerde AT1-receptoren plaats in

het tubulo-endosomale compartiment dat bij receptor-recycling betrokken is.

Brefeldine A, een stof die structuren van het Golgi-systeem verandert, is zelfs

na blokkade van internaliseringroutes in staat om de desensitisatie toe te laten

nemen. Nocodazole, een remmer van de polymerisatie van microtubuli, was

daarentegen alleen onder controle omstandigheden in staat om de desensitisatie

toe te laten nemen, maar niet na blokkade van de internalisering. Onze

resultaten brengen daarmee de desensensitisatie-gerelateerde, sub-cellulaire

“trafficking routes” in kaart die mogelijk een extra aangrijpingspunt vormen

voor het verminderen van de contractiele effecten van angiotensine II.

Een overzicht van de huidige inzichten in de intracellulaire angiotensine II-

receptoren is weergegeven in hoofdstuk 7. De potentiële klinische relevantie

van intracellulair angiotensine II in hart- en vaatziekten, de farmacologische

Samenvatting

144

implicaties en mogelijke toekomstige onderzoeksontwikkelingen worden

besproken.

De relatieve rol van de verschillende subtypen van de

plasmamembraangebonden angiotensine II-receptor past zich aan fysiologische

en pathologische omstandigheden aan. Het bestaan van intracellulaire

angiotensine II-receptoren voegt hier een nieuwe dimensie aan toe.

Intracellulair angiotensine II induceert groei in gladde spiercellen van de

vaatwand en speelt mogelijk een rol in remodeling-processen. Intracellulair

angiotensine II zou voorts betrokken kunnen zijn bij de aanhoudende vasculaire

contractiliteit door het omzeilen van de snelle desensitisatie van door

extracellulair angiotensine II geactiveerde signaalroutes. Het bestaan van

intracellulaire angiotensine II-receptoren en mogelijk ook andere

componenten van het renine-angiotensine-systeem kan daarom bijdragen aan

nieuwe farmacologische benaderingen in de behandeling van ziekten, waarbij

het renine-angiotensine-systeem een rol speelt.

Sumarul în limba română

145

Sumarul in limba român!Angiotensina II este un important hormon cu roluri fiziologice şi patologice în multipleafecţiuni cardio-vasculare. Investigarea sistemului renină-angiotensină a început înceea de a cincea decada a secolului trecut. Pentru multă vreme, sistemul renină-angiotensină a fost considerat un sistem endocrin circulant, în care renina eliberata derinichi acţionează asupra angiotensinogenului hepatic, producând un decapeptid numitangiotensină I. Angiotensina I este transformată într-un octapeptid numit angiotensină IIla trecerea prin vasculatura pulmonară de catre enzima de conversie a angiotensinei(ACE). Acest concept a fost contrazis de cercetări efectuate in ultimii douăzeci de ani,care au demonstrat existenţa unui sistem renină-angiotensină la nivelul ţesuturilor(sistemul renină-angiotensină local). La fel ca mulţi alţi hormoni, angiotensina II îşiexercită acţiunile biologice prin intermediul unor receptori specifici situaţi la nivelulmembranei celulare, dintre care cei mai bine caracterizaţi sunt receptorii de tip AT1 şiAT2.Scopul acestei teze a fost investigarea ipotezei că angiotensin II poate exercita acţiuniintracelulare specifice, independente de activarea receptorilor membranari. Cercetărileau fost efectuate in celule din musculatura netedă, una dintre principalele ţinte aleacţiunii hormonilor hipertensivi. În capitolul " este prezentat un sumar al cunoştinţeloractuale despre sistemul renină-angiotensină împreună cu bazele ipotezei privindacţiunile intracelulare ale angiotensinei II.Puţinele date despre angiotensina II intracelulară existente înaintea începeriiexperimentelor descrise în această teză, au fost obţinute în culturi celulare. De aceea,in capitolul 2, efectele angiotensinei intracelulare au fost studiate în inele de aortă deşobolan. Angiotensina II a fost livrată intracelular folosind lipozomi. S-a constatat căangiotensina II intracelulară induce contracţia muşschiului neted vascular, contracţiecare nu este blocată de aplicarea extracelulară a unui antagonist al receptorilor de tipAT1, losartan. Totusi, atunci când antagonistul a fost livrat intracelular, acţiuneaangiotensinei II a fost blocată în întregime. În contradicţie cu acţiunile angiotensinei IIextracelulare, efectele intracelulare ale acesteia au fost inhibate şi de catre PD123319,un antagonist al receptorilor de tip AT2. Angiotensina I livrată intracelular a avut aceleaşiefecte ca ale angiotensinei II. Captoprilul, un inhibitor al enzimei de conversie a


146

angiotensinei, nu a avut nici un efect atunci când a fost livrat intracelular asupraacţiunilor determinate de angiotensina I, sugerând că aceasta poate acţiona directasupra receptorului intracelular din aortă sau existenţa unor mecanisme alternative deformare. Aceste experimente demonstrează existenţa unor acţiuni patologice aleangiotensinei II in compartimente celulare necercetate până in prezent.În capitolul 3 s-au investigat căile de semnalizarea intracelulară activate deangiotensina II din interiorul celulei. Aceste experimente au fost efectuate în A7r5, olinie de celule vasculare netede în care aplicarea extracelulară a angiotensinei II nudetermina răspunsuri funcţionale. Totuşi, aplicarea intracelulară a angiotensinei II prinlipozomi sau dupa permeabilzarea selectivă a membranei celulare, determină un influxde calciu din mediul extracelular, stimuleaza producerea de inozitol trifosfat şipotenţează efectele acestuia asupra eliberării de calciu din rezervele intracelulare.Aceste efecte nu sunt influenţate de către antagoniştii receptorilor de tip AT1 sau AT2,demonstrând că deşi angiotensina II intracelulară activează căi de semnalizareintracelulară comune cu cele activate de angiotensina II extracelulară, receptoriiintracelulari au o farmacologie diferită.În capitolul 4 au fost studiate interacţiile dintre angiotensina II intracelulară şi diverşihormoni extracelulari. S-a demonstrat că angiotensina II intracelulară inhibă efecteledeclanşate de serotonină sau UTP in celulele A7r5. Diferit faţă de cele constatateanterior, un antagonist al receptorilor de tip AT1 blochează aceste acţiuni, indicândexistenţa unor multipli receptori intracelulari pentru angiotensina II.În capitolul 5 aceasta concluzie este susţinută prin demonstrarea efectelor intracelulareale angiotensinei II asupra creşterii celulare in linia de celule vasculare A7r5. Analizastatistică a rezultatelor demonstrează că efectul stimulator al angiotensinei IIintracelulare este mediat de doua tipuri distincte de receptori, având afinităţi diferitepentru hormon. Antagoniştii receptorilor de tip AT1 şi AT2 inhibă numai unul dintreacestea, cel cu afinitate scăzută pentru angiotensina II, şi nu au nici un efect asuprareceptorului cu afinitate ridicată. Aplicarea intracelulară a CGP42112A, un compusdescris anterior ca un antagonist (parţial) al receptorilor de tip AT2 blocheaza amăndouatipurile de receptor. Stimularea creşterii celulare de către angiotensina II intracelularăeste determinată de activarea fosfatidil 3-kinazei şi a protein kinazei dependentă de


147

mitogen, fapt demonstrat de blocarea creşterii celulare în experimentele efectuate înprezenţa unor inhibitori specifici.În capitolol 6 a fost investigat procesul de desensitizare a receptorilor de tip AT1,pentru a verifica posibilul rol al acestora în livrarea intracelulară a angiotensinei II. S-aobservat o reducere importantă a efectului contractil al angiotensinei II dupa aplicarearepetată a acesteia. Aceasta se datorează în primul rând internalizării receptorilor de tipAT1 prin "clathrin coated pits", dar există şi o altă cale secundară de internalizare prin"caveolae". Brefeldina A, un agent care distruge structura aparatului Golgi, accentueazăprocesul de desensitizare, chiar şi în condiţiile blocării procesului de internalizare prinultilzarea mediului hiperosmolar (care blochează formarea de "clathrin coated pits", ori acadaverinei (care blocheză internalizarea prin "caveolae"). Spre deosebire de brefeldinaA, nocodazolul, un inhibitor al polimerzării microtubulilor, stimulează desensitizareanumai în condiţii normale şi nu după blocarea internalizării receptorilor de tip AT1.Aceste experimente arată căile subcelulare folosite în internalizarea receptorilor de tipAT1 şi sugerează noi metode pentru reducerea efectelor contractile ale angiotensinei II.O privire de ansamblu a cunoştinţelor actuale despre efectele şi receptorii intracelularide angiotensină II este prezentată în capitoloul 7. Sunt discutate posibila importanţăclinică, implicaţiile farmacologice şi direcţiile viitoare de cercetare.Echilibrul relativ dintre diverşii receptori ai angiotensinei II depinde de condiţiilefiziologice şi patologice la care este expus organismul. Prezenţa receptorilorintracelulari pentru angiotensina II adaugă o nouă latură acestui proces. Angiotensina IIintracelulară modifică creşterea celulară si poate fi implicată în remodelarea vasculară.Mai mult, angiotensina II intracelulară poate determina creşterea tonusului musculaturiivasculare chiar şi după desensitizarea efectelor extracelulare ale hormonului. Deaceea, existenţa receptorilor intracelulari pentru angiotensina II şi posibil al altorcomponente a sistemului renină-angiotensină deschide noi perspective în investigareabolilor cardio-vasculare.

149

Curriculum Vitae.

The author of this thesis was born on 11 October 1968, in Vaslui, a small town

in the east of Romania. After graduating from the "College of Informatics" in

Iasi in 1987, he did his military service in the Romanian Army between 1987

and 1989. In 1990 he was admitted to the University of Medicine "Gr. T. Popa"

in Iasi. Since his second year at the university (1991), he started working in the

Department of Physiology under auspices of Prof. Dr. D.D. Branisteanu. This

period initiated his interest not in clinical medicine, but in the physio-

pharmacological research field. Two research projects in the Department of

Physiology, Katholieke Universiteit Leuven, Belgium (Prof. Dr. Bernard

Himpens) and the Section of Molecular Neuropharmacology, Department of

Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (Prof.

Dr. Bertil Freholm) helped him to focus his interest on intracellular signal

transduction. After graduating as a Medical Doctor (October 1996), he started

his Ph.D. study, which is presented in this book on intracellular angiotensin II

in the Department of Clinical Pharmacology, University of Groningen, working

with dr. Adriaan Nelemans, dr Rob Henning and Prof. Dr. Dick de Zeeuw. In

parallel, he was involved in a separate study on the interaction between Ca2+

homostasis and Multidrug Resistance Proteins with Dr. Jan Willem Kok from

Department of Physiological Chemistry, University of Groningen. After

completing this thesis, he will continue his work on intracellular signal

transduction at the Department of Pharmacology, East Tennessee State

University, Johnson City, United States with Prof. Dr. Nae J. Dun.

151

Publication list.

1. Brailoiu E, Serban DN, Slatineanu S, Filipeanu CM, Petrescu BC,

Branisteanu DD. Effects of liposome-entrapped adenosine in the isolated rat

aorta. Eur J Pharmacol. 250(3): 489-92, 1993.

2. Brailoiu E, Serban DN, Popescu LM, Slatineanu S, Filipeanu CM,

Branisteanu DD.Effects of liposome-entrapped D-myo-inositol 1,4,5-

trisphosphate and D-myo-inositol 1,3,4,5-tetrakisphosphate in the isolated rat

aorta. Eur J Pharmacol. 250(3): 493-5, 1993.

3. Brailoiu E, Saila L, Huhurez G, Costuleanu M, Filipeanu CM, Slatineanu S,

Cotrutz C, Branisteanu DD. TLC--a rapid method for liposome

characterization. Biomed Chromatogr. 8(4): 193-5, 1994.

4. Filipeanu CM, Brailoiu E, Huhurez G, Slatineanu S, Baltatu O, Branisteanu

DD. Multiple effects of tyrosine kinase inhibitors on vascular smooth muscle

contraction. Eur J Pharmacol. 281(1): 29-35, 1995.

5. Brailoiu E, Huhurez G, Slatineanu S, Filipeanu CM, Costuleanu M,

Branisteanu DD. TLC characterization of liposomes containing D-myo-inositol

derivatives. Biomed Chromatogr. 9(4): 175-8, 1995.

6. Costuleanu M, Brailoiu E, Filipeanu CM, Baltatu O, Slatineanu S, Saila L,

Nechifor M, Branisteanu DD. Effects of liposome-entrapped platelet-activating

factor in the isolated rat trachea. Eur J Pharmacol. 281(1): 89-92, 1995.

7. Brailoiu E, Baltatu O, Costuleanu M, Slatineanu S, Filipeanu CM,

Branisteanu DD. Effects of alpha-trinositol administered extra- and

intracellularly (using liposomes) on rat aorta rings. Eur J Pharmacol. 281(2):

209-12, 1995.

152

8. Brailoiu E, Beschea C, Brailoiu C, Costuleanu A, Filipeanu CM,

Costuleanu M, Fallgren B, Branisteanu DD. TLC characterization of small

unilamellar liposomes containing D-myo-inositol derivatives. Biomed

Chromatogr. 10(5): 233-6, 1995.

9. Brailoiu E, Todiras M, Margineanu A, Costuleanu M, Brailoiu C, Filipeanu

C, Costuleanu A, Rusu V, Petrescu G. TLC characterization of liposomes

containing angiotensinogen, angiotensine I, angiotensine II and saralazin.

Biomed Chromatogr. 11(3):160-3, 1997.

10. Filipeanu CM, Brailoiu E, Costuleanu M, Costuleanu A, Toma CP,

Branisteanu DD. Vasorelaxant properties of brefeldin A in rat aorta. Eur J

Pharmacol. 332(1): 71-6, 1997.

11. Filipeanu CM, de Zeeuw D, Nelemans SA. Delta9-tetrahydrocannabinol

activates [Ca2+]i increases partly sensitive to capacitative store refilling. Eur J

Pharmacol. 336(1): R1-3, 1997.

12. Brailoiu E, Margineanu A, Toma CP, Filipeanu CM, Rusu V, Branisteanu

DD. D-myo-inositol derivatives alter liposomal membrane fluidity. Biochem

Mol Biol Int. 44(1): 195-201, 1998.

13. Kok JW, Babia T, Filipeanu CM, Nelemans A, Egea G, Hoekstra D.

PDMP blocks brefeldin A-induced retrograde membrane transport from golgi

to ER: evidence for involvement of calcium homeostasis and dissociation from

sphingolipid metabolism. J Cell Biol. 142(1): 25-38, 1998.

14. Filipeanu CM, Brailoiu E, Petrescu G, Nelemans SA. Extracellular and

intracellular arachidonic acid-induced contractions in rat aorta. Eur J

Pharmacol. 349(1): 67-73, 1998.

153

15. Brailoiu E, Filipeanu CM, Tica A, Toma CP, de Zeeuw D, Nelemans SA.

Contractile effects by intracellular angiotensin II via receptors with a distinct

pharmacological profile in rat aorta. Br J Pharmacol. 126(5): 1133-8, 1999.

16. Arslan G, Filipeanu CM, Irenius E, Kull B, Clementi E, Allgaier C,

Erlinge D, Fredholm BB. P2Y receptors contribute to ATP-induced increases

in intracellular calcium in differentiated but not undifferentiated PC12 cells.

Neuropharmacology. 39(3): 482-96, 2000.

17. Filipeanu CM, Nelemans A, Veldman RJ, de Zeeuw D, Kok JW.

Regulation of [Ca2+]i homeostasis in MRP1 overexpressing cells. FEBS Lett.

474(1): 107-10, 2000.

18. Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu CM, Muller M.

Differential expression of sphingolipids in MRP1 overexpressing HT29 cells.

Int J Cancer. 87(2): 172-8, 2000.

19. Filipeanu CM, Henning RH, de Zeeuw D, Nelemans A. Intracellular

angiotensin II and cell growth of vascular smooth muscle cells. Br. J.

Pharmacol. 132(7): 1590-1596, 2001.

20. Filipeanu CM, Brailoiu E, Kok JW, Henning RH, de Zeeuw D, Nelemans

S.A. Intracellular angiotensin II elicits Ca2+ increases in A7r5 vascular smooth

muscle cells. Eur J Pharmacol. In press.

155

Acknowledgements

Although I always admired The Netherlands, in September 1996 when I learnt

that I would spend the next four years as an “Ubbo Emmius” Ph.D. student

within the Medical University in Groningen, I didn’t know very much about the

Dutch people. I am still missing much of the ‘Nederlandse’ spirit (I certainly

didn't learn the Dutch language) but after 54 months working in “Clinical

Pharmacology” group I feel very sad to leave. It was a great period and I want

to say “thank you” to everyone who contributed to my enjoying experience in

The Netherlands. It is very difficult to find the right words to express my

gratitude, so please try to read between lines and be sure that what is missing in

my words is present in my heart. I hope that the world is small enough for our

ways to cross again in the future.

So, first of all, thanks to you, Ad Nelemans, I had the opportunity to do my

Ph.D. in Groningen and during these four and a half years you contributed to

all my scientific achievements. It was a great pleasure working with you,

thanks for showing me that is still possible to do “pure” science and for

everything else that you taught me. Thanks for taking care of all other problems

besides work. Thanks to Michelle and to you for helping me to adapt to The

Netherlands, and be assured that I will remember you.

Next, I have to say the most sincere “thank you” to Rob Henning. Thank you

Rob for your contaminating optimism, for your continuous moral support and

most importantly, thanks for teaching me how to keep working even if the

outcome was sometimes beyond our expectations. I will miss our discussions

about football, history or everything else very much. From now on, I will try to

remember “thus, we are the first to show…”, on each paper that I will write.

And if smoking again, “Gauloises Blondes” will be my favorite brand of

cigarettes.

Naturally, performing the experiments and writing this thesis was not possible

without my promoter, Dick de Zeeuw. So, Dick thanks for all your patience,

thanks for our “spicy” discussions and thanks for trying and (sometimes)

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succeeding to teach me the pragmatism I am sure that will help me in the

future.

I am most grateful to my reading committee composed of Prof. dr. J. Zaagsma,

Prof. dr. H. Haller and Prof. dr. J. de Mey for commenting on my thesis and

giving me valuable comments that helped me to improve it. One more word of

thanks to Professor Zaagsma for his encouraging words and his help with the

“Nederlandse Samenvatting”. Also I want to thank the others members of my

promotion committee.

I wish to express my gratitude to Prof. Dr. D.D. Branisteanu (Department of

Physiology, University of Medicine and Pharmacy Gr. T. Popa Iasi, Romania)

who supervised my first steps in physio-pharmacological science and made me

determined to follow this career. Also, I would like to express my special

thanks to Dr. Eugen Brailoiu for all his support and friendship since I was a

second year medical student.

A special help during these four years was Dr. Jan Willem Kok. Thank you Jan

Willem for enlarging my scientific interests, for our fine collaboration and for

all our conversations. Thanks to Roekie and to you for all your support and for

your friendship. Also many thanks to Karin Klappe and Robert Jan Veldman

for their help in the “Multidrug resistence and intracellular calcium” project.

Technical support from Jan van der Akker, Mary Duin, Mirian Wietses and

Ursula Kleef in various stages of this thesis is gratefully acknowledged. I owe a

special word of thanks to Jan Roggeveld, Azuwerus van Buiten, Alex Kluppel

and Jacko Dukker. Without their help certain parts of this thesis would not have

been written and without their jokes some of my experiments would have been

boring. Also, I want to thank two people at the starting point of their scientific

careers for helping me with the contractile experiments, Sander Croes and

Marcel Visser.

I have to say many thanks to Alexandra Doeglas, Ardy Kupperus and Paula

Hooghiemstra for their excellent secretarial assistance, for helping me with all

sort of problems and for their nice words. Thanks for helping me with

computer problems and for the nice “chats” to Wessel Sloof and to Yunus

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Kocabasoglu for his help on various occasions. A special word of thanks is

addressed to Riekje Banus and to all people from GUIDE Bureau for all their

help with the official procedures.

My thanks also to Prof. dr. Wiek van Gilst and to dr. Cees de Langen for their

kind help whenever I needed it.

These last years I spent more time with people from the sixth floor and

especially from room 608 than with my parents. So thanks to everyone there

for the friendly atmosphere. Thanks to Peter Paul van Geel, Marijke Haas, Ard

Teisman, Menno Kocks, Arnold Boonstra, Soesja Hu and Bianca Brundel for

being around when I needed them. Thanks to Lisa Pont for her very kind help

with correcting language of various chapters of this book.

Special thanks to Simone Gschwend for all her help. I wish you and Thomas all

the best for the future. Thank you, Annemarieke Loot, for your suggestions and

for all our jokes. And thank you, Hendrik Buikema for all our “political”

discussions and for all the cigarettes that we smoked together and all the beers

that we drank together.

Now, I have to thank some very “unique” persons. Thanks Anton Roks for

trying to teach me how to ride a bike and for helping me fall off twice. You

have been my last teacher in this aspect. Thanks for being a friend all these

years and for being my third “paranimf” for the preparation of my defense. I

address all my best wishes to Edith and you for the future.

I have to thank many times Leo Deelman for his invaluable help with

experiments and all other problems, and naturally for our drinking in Miami. I

wish him ‘good luck’ in his new position of ‘respectable’ father and wish the

happiest life to Mathijs Louwe, to Cecile and to you.

Finally I have to express all my gratitude to the person that started his Ph.D. on

the same day as me and was my closest friend during this time. Thank you

Henk Bos for being an excellent friend, for showing me how beautiful The

Netherlands it is and thank you for all that you have done for me.

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Before finishing I wish to express my gratitude to Mihaiela and Crizantema just

for being such lovely sisters and to Adela and Ionel Dominte for being such

gracious parents-in- law.

This thesis is dedicated to the people who mean everything to me and to whom

I was more like a rare guest during all these years. All my love and respect to

my parents, Margareta and Mihai Filipeanu, and to my fiancée, Gabriela. They

and only they know how much time is comprised between these covers: hours,

days, months, years…

C!t!lin29.04.2001

Intracellular angiotensin II inhibits heterologous receptor stimulated Ca2+ entry

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