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Angiotensin II signal transduction pathways in chick cardiomyocytes: involvement of tyrosine phosphorylation Goutsouliak, Valeri 1994

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ANGIOTENSIN II SIGNAL TRANSDUCTION PATHWAYS IN CHICK CARDIOMYOCYTES: INVOLVEMENT OF TYROSINE PHOSPHORYLATION by VALERI GOUTSOULIAK Doctor of Medicine (Hon), University of Chernovtsy, Ukraine, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Medicine)  We accept this thesis as conforming to the required standard  tMM.  OL£&^^THE UNIVERSITY OF BRITISH COLUMBIA August, 1994 @ Valeri Goutsouliak, 1994  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department  of A / ^ ^ c y k ^  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  @&f. 12AH  22^  a ABSTRACT The general subject of these studies is the cellular action of angiotensin II (All) on cardiac myocytes. The present work was based on the hypothesis that several intracellular messengers or processes mediated by both AT] and AT2 receptors, underlie the response of cardiomyocytes to All and that protein tyrosine phosphorylation plays an important role in All-mediated signal transduction in the cardiomyocyte. The aim of this work was to elucidate some aspects of the effect of All on cardiac myocytes. Cardiomyocytes from 7 day old chick embryo were selected for investigations. The ability of All to induce several different and important second messengers, namely hydrolysis of phosphatidylinositol 4,5biphosphate and phosphatidylcholine were examined The involvement of protein tyrosine phosphorylation in signal transduction activated by All in chick cardiomyocytes as well as the effect of All on Ptdlns 3-kinase were also assessed The role of AT] and AT2 subtypes of All receptors in complex signaling mechanisms following All stimulation of chick cardiomyocytes was investigated using relatively selective ATj and AT2 receptor antagonists. All stimulation of cardiac cells produced phosphoinositide hydrolysis and accumulation of inositol phosphates, mainly InsP^. The kinetics of InsP$ production, a hallmark of PtdInsP2~PLC activation, suggest that PtdIns2~PLC activation by All in chick cardiomyocytes occurs within I minute. The All-stimulated production oflnsP], InsP2, and InsP^ was partially but not completely blocked by the AT] receptor antagonist. Forty percent of the increase in InsP^ after All stimulation was blocked by AT2 receptor antagonism while both AT] and AT2 receptor blockade completely prevented IP3 formation. Thus, activation of PLC by All in chick cardiac myocytes is not solely mediated by AT] receptors, as has been previously proposed, but rather occurs through both receptor subtypes. The hypothesis that protein tyrosine phosphorylation induced by All might play an important role in early activation of PtdInsP2 hydrolysis and formation oflnsP^ in cardiac cells was tested. Genistein, a tyrosine kinase inhibitor, partially reduced All-induced accumulation oflnsP^. Consequently, not only PLC-/3 is activated in response to All cell stimulation but PLC-y is also likely activated. In chick cardiomyocytes, All produced phosphatidylcholine (PC) hydrolysis. However, the maximal effect, in contrast to PtdInsP2 hydrolysis, was late as it was observed at 30 minutes and remained increased over the I hour observation period One minute exposure to All induced tyrosine phosphorylation of proteins in chick cardiac myocytes. The most prominent of these proteins were proteins with approximate molecular weights 70 kDa and 195 kDa. All-induced protein tyrosine phosphorylation was almost completely blocked by pretreatment of cells with Lavendustin A, a tyrosine kinase inhibitor. Both AT] and AT2 are involved, to different extent, in Allinduced tyrosine phosphorylation in chick cardiac myocytes. Ptdlns 3-kinase was  iii activated in response to AH stimulation of chick cardiac myocytes and was blocked by tyrosine kinase inhibitor. This suggests that Ptdlns 3-kinase might play an important role in All-induced signal transduction in cardiomyocytes. In summary, All produces immediate, within 1 minute, activation of PLC to produce InsP$ through AT} and AT2 receptors. All produces immediate activation of Ptdlns 3-kinase while PC hydrolysis is a late event. These data also show that tyrosine phosphorylation induced by All plays an important role in InsP$ formation and Ptdlns 3-kinase activation in the heart.  iv Table of Contents ABSTRACT  u  Table of Contents  j  List of Figures  YJJ  List of Abbreviations  JX  Acknowledgements CHAPTERI  v  x  Introduction  l.Angiotensin II and the heart a) Sources of Angiotensin: Renin-Angiotensin System...., i. Circulating RAS ii. Tissue RAS b) All Receptors c) Direct Physiological Effects of Angiotensin on the Heart 2. Angiotensin II and Inositol Triphosphate signaling pathway a) Signaling Pathways and Molecular Heterogeneity t b) G protein-linked Receptors i. Types of G Proteins and Signal Transduction in Myocardial Cells c) Tyrosine Kinase-linked Receptors d) An Stimulation of PtdIns(4,5)P2 Hydrolysis  .".  1 1 2 3 4 7 8 8 9  11 13 ....13  3. Angiotensin II and PLD Activity a) Signaling Trough Phosphatidylcholine Breakdown b) All and PC Metabolism  14 14 16  4. Angiotensin II and Protein Tyrosine Phosphorylation  17  5. Angiotensin II, Phosphatidyl inositol 3-Kinase and Signal Transduction  21  V  6. Choice of 7-day Chick Embryo Ventricular Myocytes as a Culture Model a) Histogenesis of the Embryonic Chick Ventricular Myocardium  24 24  i. Ventricular Myocytes of the 7-day Old Chick Embryo 7. Hypotheses and Objectives CHAPTER H  25 26  Materials and Methods  1. Isolation of Chick Embryonic Cardiac Cells  28  2. Polyphosphoinositide Turnover Studies a) Incorporation of myo-[2-^H] inositol b) Challenge ("Cell Activation") c) Column Chromatography i. Preparing Dowex 1-8X Columns.. ii. Loading and Running Columns  29 29 29 30 30 30  3. Phosphatidylcholine Degradation and Choline Efflux  31  4. Preparation of Cell Lysates and Immunoprecipitation  .'.  33  5. SDS Polyacrylamide Gel Electrophoresis  34  6. Assay for Associated Phosphoinositide 3-Kinase Activity a) Preparation of Phosphatidylinositol  34 35  b) Ptdlns 3-Kinase Reaction  35  7. Densitometrical Analysis  36  8. Methods of Statistical Analysis  36  9. Materials a) Radiochemicals b) Antibodies  36 36 37  vi CHAPTER III  Results  1. All Induces Phosphoinositide Hydrolysis  38  2. All Stimulates Phosphatidylcholine Hydrolysis  52  3. All Induces Protein Tyrosine Phosphorylation in Chick Cardiomyocytes  57  .4. All Stimulates Phosphatidylinositol 3-Kinase Activity in Chick Embryo Cardiomyocytes  64  CHAPTER IV  Discussion  68  CHAPTER V  Summary and Conclusions  83  References  86  vu  List of figures Figure number  Figure Title  Page  1.  The Renin Angiotensin System  4  2.  Summary of the two major receptor-mediated pathways for stimulating the formation of PtdInsP3 and DAG  10  3.  MAP Kinase Activating Pathways  19  4.  Time-dependence of All-induced accumulation of InsP3  39  5.  Effect of Aflon InsP2 formation (time course)  40  6.  Time course for All-induced InsP3 release  41  7.  Dose-dependence ofAH-induced accumulation of InsP3  43  8.  All-induced accumulation of InsPi andInsP2 (concentration-dependence)  44  Concentration-dependence of All-stimulated Inositol accumulation  45  Effect of AT j and AT2 receptor blockers on Allinduced InsP3 accumulation  46  All-induced formation of InsP2 ^ d effect of AT 1 and AT2 receptor blockers on it  47  Dose-inhibition curves of All receptor subtype-specific antagonists on All-induced InsPi release  48  13.  Effect of AQ receptor blockers on All-induced InsP3  50  14  Effect of genistein on AH-stimulated production of  9. 10. 11. 12.  inositol phosphates  51  15.  All stimulation and intracellular free choline  53  16.  % of intracellular free choline in All-stimulated cells  54  viii 17.  18.  19.  20.  21.  22.  23.  Time course for All-induced release of choline into the medium by cardiomyocytes  55  Intracellular phospholipid in All-stimulated cardiac cells (time course)  56  Time course for All-stimulated protein tyrosine phosphorylation  59  Effect of tyrosine kinase inhibitor Lavendustin A on All-induced protein tyrosine phosphorylation  60-61  Effects of AT i and AT2 receptor blockers on Allstimulated protein tyrosine phosphorylation  62-63  In vivo stimulation of Ptdlns 3-kinase by All in chick cardiac cells (time course)  65  Effect of tyrosine kinase inhibitor on All-induced Ptdlns 3-kinase  66-67  List of Abbreviations ACE Angiotensin converting enzyme ACS Aqueous Counting Scintillant ADH Antidiuretic hormone AI Angiotensin I AH Angiotensin II ARA Arachidonic acid ATI Angiotensin II receptor, type 1 AT2 Angiotensin II receptor, type 2 cAMP Cyclic AMP DAG 1,2 diacylglycerol EGF Epidermal growth factor G proteins Guanine triphosphate proteins Gi Inhibitory G protein Gs Stimulatory G protein InsP 3 Inositol triphosphate Inositol triphosphate receptor IP3R MAP Mitogen-activated protein PA Phosphatidic acid PAGE Polyacrylamide gel electrophoresis PC N Phosphatidylcholine PDGF Platelet-derived growth factor PKC Protein kinase C Phospholipase A2 PLA 2 PLC Phospholipase C PLD Phospholipase D PMSF Phenylmethylsulfonyl fluoride Ptdlns 3-kinase Phosphatidylinositol 3-kinase Phosphatidylinositol 4,5-biphosphate PtdInsP2 RAS Renin-angiotensin system SDS Sodium dodecyl sulfate TLC Thin layer chromatography VSMC Vascular smooth muscle cells  X  A cknowledgements This work would not have been possible without Dr. Simon Rabkin, whose kind support and thoughtful advice throughout this project is greatly appreciated I especially enjoyed our frequent debates on the current research findings from our lab and others. It is my hope that some of the work presented now will be useful as the basis for further experimental inquiries in his lab. I am thankful to Carol Smithe who provided logistic and technical help. Others in Dr. Rabkin's lab who helped me were Danny Villacrusis, Nemer Dabage Forsoli, Frederico Carranza, and Jennifer Kong. During this research, I had the opportunity to work in Dr. Gerald Krystal's laboratory at the Terry Fox Laboratory. This added an exciting scientific and educational dimension to this project; and I am grateful to Dr. Gerald Krystal, Dr. Jacqueline E. Damenfor advice and practical help. Lastly, I wish to thank my family, Valentino, Arthur, and Kristina, for their enduring support and patience.  CHAPTER I INTRODUCTION 1. ANGIOTENSIN H AND THE HEART It is now recognized that cardiac myocytes are the target for a variety of growth factors and trophic hormones (Schneider and Parker, 1990; Long et al., 1990; Parker and Schneider, 1991). The octapeptide angiotensin II (AH) has emerged as an important hormone that regulates cardiac growth in vivo (Morgan and Baker, 1991; Nagano et al., 1992); as well as affecting cardiac contractility (Peach, 1977); yet, at present, the precise intracellular signaling messengers or processes underlying the response of cardiac cells to AH remain to be defined at the molecular level. AH plays an important role in cardiovascular regulation, fluid volume homeostasis, and neuroendocrine regulation (Peach, 1981; Catt et al., 1987; Timmermans et al., 1993). AH is the major effector molecule of the reninangiotensin system. It maintains vascular tone by interacting with vascular smooth muscle cells (VSMC) causing vasoconstriction. AH has also many other effects including induction of hypertrophic growth (Berk et al., 1989), stimulation of platelet-derived growth factor (PDGF) A-chain expression (Naftilan et al., 1989), induction of proto-oncogenes c-fos and c-jun (Taubman et al., 1989), and stimulation of protein tyrosine phosphorylation (Tsuda et al., 1991; Marrero et al., 1994). a) Sources of Angiotensin: Renin-Angiotensin Systems The classically held view of the renin-angiotensin system (RAS) revolves around the juxtaglomerular cells of the kidney where renin, an aspartyl protease, is released into the blood in response to lowering of pressure in the afferent arteriole. Renin release, which is also stimulated by the baroreceptor reflex, low sodium l  concentrations at the macula densa, and sympathetic stimulation of the juxtaglomerular  cells,  enzymatically  converts  the  hepatically-synthesized  angiotensinogen, a 55 kDa glycoprotein of the 0:2 globulin class, into the decapeptide angiotensin I (AT). Conversion of AI into All, an octapeptide, occurs during passage, through the pulmonary circulation, where it encounters the dipeptidyl carboxypeptidase, angiotensin converting enzyme (ACE). All is one of the most potent vasoconstrictors known. It also stimulates the release of Antidiuretic Hormone (ADH) from the posterior pituitary and aldosterone from the adrenal cortex. These actions serve to maintain perfusion pressure (Peach, 1977). However, evidence has mounted over the last two decades which indicates that the RAS is rather more diverse than described above. Two general foci of evidence add significantly to the classically held view of RAS: firstly, that RAS is present as an autocrine or paracrine system in a number of tissues and that this accounts for AH in these tissues. Secondly, AH and to a lesser extent AI, have broad and important biological actions other than vasoconstriction and stimulation of hormone release. The widened concept of this system has implications in physiology and pathology. i. Circulating RAS Whereas the classically held notion is that AI is converted to All and then conveyed to the peripheral tissues, evidence suggests that the production of AI and AQ occurs at peripheral tissues in the following way: plasma renin (from the kidneys) acts on plasma angiotensinogen (from the liver) at the peripheral tissue to generate AI which is then converted to AQ by tissue converting enzyme (Campbell, 1985). Thus, in one revised model, the RAS does not result in the systemic delivery of AH to tissues, but in the delivery of renin and angiotensinogen to tissues. In this model, plasma angiotensin is thought to be the result of spillover from the site of production at the tissues (Campbell, 1987). 2  Plasma angiotensin levels are known to drop following nephrectomy, supporting the importance of kidney renin in angiotensin production (Catt et al., 1967). One of the important aspects of this decentralized mechanism of plasma angiotensin production may be in the diversity of control mechanisms that are brought to bear on AH generation. ii. Tissue RAS The results of several studies have led to speculation that a complete RAS exists within specific tissues, or even within cells. These studies have employed a host of inhibitors of the RAS, such as the All receptor antagonist saralasin, enzyme inhibitors of ACE, as well as immunological detection of RAS components. However, immunological detection of elements of the RAS system is insufficient to draw conclusion about a local autocrine system in operation since spillover and accumulation from circulating RAS can confound the results. The existence of local angiotensin generating systems which operate independently of circulating RAS has been suggested by studies in brain, kidney, uterus, adrenal, testis, arterial wall, and heart.  Messenger RNA's encoding  angiotensinogen, which is the only known precursor of AH, have been detected in kidney, brain, spinal cord, aorta, mesentery, adrenal, atria, stomach, colon, spleen, and ovary (Campbell and Habener, 1986). However, comparisons of angiotensinogen and renin from the same tissue show that angiotensinogen mRNA is far more abundant, suggesting that renin is the primary enzyme active in converting angiotensinogen, and renin is the rate limiting step in these local systems (Dzau, 1987). Another group in a recent comprehensive review of physiological and molecular biological evidence refutes claims for synthesis of renin by cardiac and vascular tissues (von Lutterotti et al., 1994).  3  A growing number of investigators have concluded that the kidney is the only source of cardiovascular tissue renin. Although prorenin is secreted from extrarenal tissues as well as from kidney, there is no evidence that it is ever converted to renin in the circulation.  :  Tissue Synthesis  Converting  Tissue  ATI!  Angkrtensinogen  Renin Angiotensinogen  \  1 ATII  ATI  Converting Enzyme  Blood  Figure 1. The Renin Angiotensin System Scheme indicating the movement of renin and angiotensinogen, angiotensin I and angiotensin II between tissue and blood and the conversion to angiotensin II (From P. Sunga PhD Thesis, U.B.C., 1992). b) All Receptors AH receptors have been characterized from cardiac tissue from chick (Peach, 1981), rat (Rogers et al., 1986), as well as rabbit (Wright et al., 1983; Baker et al., 1984), calf (Rogers, 1984) and human (Urata et al., 1989). Most of these studies found low-affinity, low-binding capacity, as well as high affinity high binding capacity components. The All binding sites are both saturable, specific and reversible, fulfilling key requirements of a hormone receptor. The difference in affinities between the two receptors is from 10- to 100-fold, with a 34  to 20-fold difference in binding capacities. The receptor density on the neonatal rat myocyte was estimated at 45,000 per cell (Rogers et al., 1986). Autoradiographic localization of receptors shows them on the myocardium and coronary vessels. Similar receptor density was observed between right and left ventricles (Urata et al., 1989). The bovine All receptor has been identified as a 116 kDa protein by affinity cross-linking studies (Rogers, 1984) which is suggested to be a dimer whose subunits aggregate upon binding (Peach, 1981). Receptor sites were shown to decrease between day 1 and day 10 after birth in rats (Urata, 1989). This regulation appears to differ between species since All receptor sites on chick hearts increase in number during development (Peach, 1981). The highly diverse nature of All functions suggests the presence of multiple isoforms of its receptor. Recent pharmacological studies with isoform-selective antagonists uncovered the presence of two major receptor types, type 1 (ATj) and type 2 (AT2) (Chiu et al., 1989; Whitebread et al., 1989; Dubley et al., 1990). Studies with the ATj-specific antagonist losartan (Chiu et al., 1989) and the cloned and expressed ATj (Murphy et al., 1991; Sasaki et al., 1991; Iwai et al., 1991) indicated that ATi mediates many of the biological responses hitherto attributed to All (Peach, 1981; Catt et al., 1987; Timmermans et al., 1993). However, little is known on the structure and the signaling mechanism of the second major receptor isoform, AT2. Its abundance in the mesenchymal tissues of a developing fetus (Grady et al., 1991; Tsutsumi and Saavedra, 1991), uterus (Streuli, 1990), adrenal medulla (Chiu et al., 1989), pheochromocytoma (Speth and Kim, 1990), and specific brain regions (Rowe, 1992; Tsutsumi and Saavedra, 1992) suggests a hitherto unidentified role of the type 2 receptor in neuronal and developmental function. Recently, cDNA's for the rat and mouse AT2 have been cloned (Kambayashi et al., 1993; Mukoyama et al., 1993; Ichiki et al., 1994; Nakajima et 5  al., 1993). AT2 receptor is a receptor with seven membrane-spanning domains. The gene of human AT2 receptor was isolated from a genomic DNA library prepared from human placenta (Tsuzuki et al., 1994). Two AH receptor subtypes have been identified on isolated cardiac membranes as well (Sechi et al., 1992; Scott et al., 1992). The molecular significance of multiple All receptors in cardiac cells or in other tissues is not well defined. The angiotensin receptor and its functional correlates have been redefined by the cloning of angiotensin receptors and the discovery and widespread use of highly specific nonpeptide Ang IIreceptor antagonists losartan (ATi selective) and PD123177 (AT2 selective). The discovery of losartan has been reviewed in detail by Duncia et al., 1992; and Timmermans et al.,1991. In brief, the initial lead for losartan, S-8307, was chemically modified to yield a series of progressively more potent and orally active nonpeptide All antagonists. Losartan has 10,000-fold greater affinity for ATi receptor than S-8307 but, like all the phenyl- or biphenyl-substituted imidazoles, losartan did not totally displace radiolabeled All, suggesting that there were multiple receptor subtypes. The disclosure of a series of imidazopyridines, exemplified by PD123177 and PD123319 (Blankley et al., 1991), provided the necessary additional tools to complete the current concept that All has two distinct binding sites (Chiu et al., 1989). With the antagonists, it has been possible to extend the concept of All-receptor heterogeneity to virtually every tissue and species. The losartan-sensitive sites have been shown to mediate all of the major All-induced biologic effects, including vasoconstriction, aldosterone release, and central, All-induced drinking behavior. The function of the AT2 sites is not fully understood; however, the presence of AT2 sites in fetal tissues and in discrete locations in the brain has encouraged continued research.  6  c)Direct Physiological Effects of Angiotensin n on the Heart One of the first recognized direct effects of All on the heart was a positive inotropic action in several species including cat (Koch-Weser, 1964), chick, rabbit (Freer et al., 1976), and others (Peach, 1977). A number of methods were used to demonstrate that the effect was due to All and not through modulation of sympathetic nervous function.  After experimentally eliminating sympathetic  effects, All displayed a dose-dependent, positive inotropic action on the myocardium. Another effect of All on the heart that has received much attention recently is hypertrophy. Cardiac hypertrophy is based mainly on increase in myocyte size, as opposed to myocyte proliferation. Part of the process of growth of myocytes involves increased macromolecular turnover.  Angiotensin is emerging as a  potential player in the regulation of this process. Thus, whether or not All is generated within the heart, All has a number of actions on the heart, some of which may be central to the control of growth, and others which are certainly important in cardiac contractility. Increasing evidence suggests that All acts as a direct growth factor for cardiac myocytes (Baker and Aceto, 1990; Sadoshima and Izumo, 1993). There is emerging evidence that many peptide growth factors activate multiple second- messenger systems (Rozengurt, 1991). These peptide growth factors bind to G protein-coupled receptors and activate phospholipase C (PLC), phospholipase D (PLD), phospholipase A2 (PLA2), adenylate cyclase, tyrosine kinases, and their downstream protein kinases (Rozengurt, 1991; Liu et al., 1992). These second-messenger systems are thought to act together and induce the complex mitogenic response. It has been reported that AQ activates PLC through a G protein-coupled receptor, liberates inositol triphosphate (UISP3), and induces Ca 2+ release from InsP3-sensitive Ca 2+ storage sites in cardiac myocytes (Baker et al., 1989). In other cell types, All has also 7  been shown to activate protein kinase C (PKC), PLA2, PLD, adenylate cyclase, and arachidonic acid metabolism (Kojima et al., 1985; Lassegue et al., 1991; Hassid, 1986; Madhun et al., 1991). Emerging indirect evidence suggests that All may be one of the most important growth factors for the heart in humans and animals (Lindpainter and Ganten, 1991; Baker et al., 1992; Sadoshima and Izumo, 1993).  2. ANGIOTENSIN II AND INOSITOL TRIPHOSPHATE SIGNALING PATHWAYS Inositol triphosphate (InsP3) is a second messenger that controls many cellular processes by stimulation of the intracellular calcium release (Berridge and Irvine, 1989; Rana and Hokin, 1990; Henzi and MacDermott, 1992). Its receptors closely resemble the calcium-mobilizing ryanodine receptors of muscle (Henzi and MacDermott, 1992; Tsien, 1990). This family of intracellular calcium channels displays the regenerative process of calcium waves and oscillations (Meyer and Stryer, 1991; Boitano et al., 1992). This signaling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception, and neuronal signaling (Carroll and Swann, 1992; Homa et al., 1991; Poussegur and Seuwen, 1992; Gutkind et al., 1991; Nishizuka, 1988).  y  a) Signaling Pathways and Molecular Heterogeneity External signals activate cell surface receptors to initiate signaling pathways whereby information flows to the final effector system. The formation of InsP3 is the major point for two pathways, one initiated by a family of G protein-linked receptors and the other by receptors linked by tyrosine kinase either directly or 8  indirectly (Fig. 2) (Berridge, 1993)  These separate receptor mechanisms are  coupled to GTP or ATP transducing mechanisms that activate PLC.  PLC  hydrolyses the lipid precursor phosphatidylinositol 4,5-biphosphate (Ptdlns(4,5) p  2)  t0  yield both diacylglycerol (DAG) and InsP3. InsP3 binds to an InsP3  receptor (IP3R) to mobilize stored calcium, whereas DAG activates PKC (Nishizuka, 1988; Irvine, 1990; Irvine, 1992). Each of the stimuli (listed in Fig. 2) acts through a separate receptor. There is an extensive heterogeneity of the downstream elements, such as the G proteins, PLC, IP3R.  However, the commonallity of external signals is to utilize the  InsP3/calcium and DAG/PKC pathways to regulate a wide range of cellular activities.  b) G protein-linked Receptors Activation of receptors that stimulate PLC operate through a G protein (Berridge, 1993).  Most of the identified G protein-linked receptors are  characterized by seven membrane-spanning domains connected by extracellular and intracellular loops. The transmembrane columns interact to form a pocket where the agonist binds and induce the conformation that can activate the G protein, the next component of the signaling pathway.  9  G PROTEIN-LINKED RECEPTORS Acetylcholine, histamine NA. 5-HT. ATP. PAF. TXA 2 . Glulamale. Angiotensin 8. Vasopressin. Bradykimn. Substance P. Bombesin. Neuropeptide Y . Thrombin Cholecystokinin. EndotheSn Neuromedin. TRH. GnRH. PTH Odorants. Light  lnsP4R  Ca 2»  TYROSINE KINASE-LINKEO RECEPTORS POGF. EGF  Cellular  ) £}  activity :  Antigen  @ « &  TCR  fo  y\ ATP  PI-3K  mitogenosis  PIP-  Ick  | G A P | ( j a s ) — > • ral -1  Figure 2.  MAP-2 kinase  Summary of two major receptor-mediated pathways for stimulating theformation oflnsP^ and DAG. (from Berridge, M, Nature. 361:p.315, 1993)  Many agonists bind to 7-membrane-spanning receptors (R), which use a GTP-binding protein (G) to activate phospholipase C~pi (PLC-/31), whereas PLC-yl is stimulated by thetyrosinekinase— linked receptors. The latter activate other effectors such as the Ptdlns 3-kinase (PI-3K), which generates the putative lipid messenger Ptdlns (3,4,5)-triphosphate (PIP3) and the GTPaseactivating protein (GAP) that regulates ras.  InsP^R, InsP^  receptor; PKC, protein kinase C; NA, noradrenaline; 5-HT, 5hydroxytryptamine; PAF, platelet-activating factor;  TXA2,  thromboxane A2; TRH, thyrotropin-releasing hormone; GnRH, gonadotrophin-releasing hormone; PTH, parathyroid hormone.  10  i. Types of G Proteins and Signal Transduction in Myocardial Cells Guanine triphosphate binding proteins (G proteins) are key components in signal transduction pathways in myocardial cells, since they are associated with most, if not all, membrane-bound receptors (Fleming et al., 1992). The G proteins play a key role in the regulation of the myocardium since they link receptors to adenylyl cyclase, ion channels and phospholipid hydrolysis. The generation of cyclic AMP (cAMP) via the activity of adenylyl cyclase controls the contractile capacity of the cardiomyocyte through a number of actions, and also may play a mediating role in macromolecular turnover associated with hypertrophy (Morgan and Baker, 1991). Thus, the activity of both stimulatory G proteins (Gs) and inhibitory G proteins (Gi), by governing adenylyl cyclase activity, are centrally located in the constellation of factors controlling the myocardium. G proteins are heterotrimers consisting of a, P, and y subunits. The a subunit confers the specific functional characteristics of the whole protein by virtue of structural heterogeneity. The P and y subunits of the different functional classes of G proteins are relatively homogeneous. Characteristics that are specific to the a subunit are the GTP binding site and GTPase activity, which is activated by the presence of the p-y complex (Gilman,1987). There are several types of G proteins which have been identified in cardiac tissue (Robishaw and Foster, 1989): Gs, Gi, and Go while other types such as Gq also exist. In the heart, Gs acts as a mediator of adenylyl cyclase stimulation by the P1 and p-2 adrenergic receptors. Gs also mediates beta adrenergic regulation of voltage-dependent ion channels, such as the cardiac Ca^ + channel (Birnbaumer et al., 1990) by a mechanism which operates independently of cAMP changes (Trautwein and Hescheler, 1990). Gs-alpha exists as four isoforms that migrate on  n  SDS gel electrophoresis as two bands of molecular weight 42-45 kDa and differ by 15 amino acids (Bray et al., 1986). Gi mediates the inhibition of adenylyl cyclase activity via muscarinic, o> adrenergic (Allen et al., 1988, Brown et al., 1985) as well as other factors. The mechanism of the inhibitory action of Gi on adenylyl cyclase is controversial. Gilman (1984) has presented evidence supporting the hypothesis that activation of inhibitory receptors causes dissociation of Gi into Gi-a and p/y subunits, thereby allowing excess p/y subunits to associate to Gs (by mass action), which inhibits Gs (Gilman, 1984).  However, other hypotheses have been proposed based on  . experiments in which Gi activation depressed forskolin-stimulated adenylyl cyclase activity, without involvement of Gs, (Hildebrant et al., 1984). The function of the third type of G protein in the heart, Go, has not been completely elucidated, although in the brain, where Go is abundant, it regulates Ca2 + channels (Birnbaumer et al., 1989). A fourth G protein in cardiac tissue is Gq, a 42 kDa protein linked to activation of phospholipase C (Pappano et al., 1988). Extracellular signaling of the normal myocardial cell is primarily achieved through the P-adrenergic and muscarinic cholinergic receptors that are respectively regulated by the sympathetic and parasympathetic division of the autonomic nervous system. However, the contribution of All (Baker et al., 1990) and a-1 adrenergic (Buxton and Brunton, 1985) receptors may be important in pathological conditions.  The muscarinic and a-adrenergic receptors are linked through  pertussis toxin- and cholera toxin-sensitive G proteins to PLC (Birnbaumer et al., 1990), the activation of which results in the generation of the second messengers InsP3 and DAG releasing Ca 2 + from sarcoplasmatic reticulum and activating PKC (Berridge, 1987).  12  ^Tyrosine Kinase-linked Receptors Many cell-surface receptors convert an extracellular signal into an intracellular one by regulating the activity of a G protein, but some signal the cell more directly. The direct pathway responsible for stimulating the release of I11SP3 involves tyrosine kinase receptors, stimulation of which produces a direct interaction between the receptor and the y-form of PLC. Tyrosine kinase-linked receptors are catalytic receptor proteins. Their actions have been outlined by Berridge (1993). Briefly, in this family of receptors are those for insulin and for a number of growth factors, including PDGF and epidermal growth factor (EGF). PDGF and EGF act by bringing two receptors together, that enables their cytoplasmic kinase domains to phosphorylate each other on tyrosine residues to create docking sites to bind PLC-yl. ATP is consumed not only for receptor autophosphorylation, but also during subsequent phosphorylation of PLCyl. In unstimulated cells, PLC-yl is largely cytosolic but translocates to the membrane as its SIC domain binds to the activated receptor. This is important for the activation of PLC-yl; it is phosphorylated by the receptor on specific tyrosine residues. In addition, its membrane translocation brings it into contact with its substrate PtdIns(4,5)P2 (Berridge, 1993).  d) All Stimulation of P t d l n s f ^ P ? Hydrolysis All stimulation of PtdInsP2 hydrolysis is mediated by the phosphoinositidespecific phospholipase C. i. Types of PLC Three major families of mammalian PLC isozymes designated P, y, and 8 have been described based on their molecular structures  and mechanism of  regulation (Rhee and Choi, 1992). The PLC-p group of isozymes have a mass of 150 kDa as determined by SDS-polyacrylamide gel electrophoresis (PAGE), and 13  the members of this family have been shown to be regulated by the Gq class of heterotrimeric GTP-binding proteins (Taylor et al., 1991). The PLC-y family of iso2ymes migrates on SDS-PAGE gels with an apparent mass of 145 kDa (Kim et al., 1989) and appears to be regulated by tyrosine phosphorylation (Kim et al., 1991). Finally, PLC-8 isozymes have a molecular mass of 85 kDa (Meldrum et al., 1991); Their regulatory mechanism has not yet been elucidated although they do not appear to be stimulated by G proteins or tyrosine phosphorylation (Rhee and Choi, 1992). All three types of PLC isozymes display calcium-dependent activity and phosphoinositide selectivity. Little information is available concerning the regulation of PLC isozymes by All (Marrero et al., 1994). In neonatal rat cardiac myocytes, AH was found to produce hydrolysis of PtdInsP2 and production of InsPj and InsP2 but not InsP3 by Allen et al., 1988, whereas in another study All did cause an increase in InsP3 (Abdellatif et al., 1991).  Moreover, it has been suggested that the InsP3  production induced by All is ATj receptor-mediated (Sadoshima and Izumo, 1993). Recently, more information is available about the role of PLC in mediating All-induced c-fos expression as well (Sadoshima and Izumo, 1993).. 3. ANGIOTENSIN H AND PLD ACTIVITY  a) Signaling Through Phosphatidylcholine Breakdown Although there is abundant evidence that many hormones and neurotransmitters cause some of their effects through the hydrolysis of PtdInsP2 to InsP3 and DAG in their target cells, it is becoming clear that many of them also stimulate the breakdown of phosphatidylcholine (PC) (Exton, 1990).  The  hydrolysis of PC by PLA2 is an important source of arachidonic acid which is subsequently metabolized to a variety of functionally significant eicosanoids, but 14  the phospholipid can also be hydrolyzed by phospholipases of C and D types to yield DAG and phosphatidic acid (PA), respectively. More direct evidence that certain agonists induce PC hydrolysis has come from studies in which various cells and tissues have been labeled with [^H]choline and then exposed to agonists (Cabot et al., 1988; Uhing et al., 1989; Martin and Michaelis, 1989). These experiments have shown increased release of labeled choline, phosphocholine, or choline metabolites, consistent with PC hydrolysis by PLC or PLD. It has also been reported that stimulation of perfused hearts in vitro and of rat brains in vivo with muscarinic agonists causes the release of choline and has presented indirect evidence that this is due to the hydrolysis of choline phospholipids (Brehm et al., 1987; Lindmar et al., 1988). There is indirect evidence that activation of PC hydrolysis by PLD or PLC involves a G protein (Bocckino et al., 1987; Martin and Michaelis, 1989; Irving and Exton, 1987).  Another mechanism of activation of PC phospholipases  involves PKC. A role of PKC is shown by the observations that inactive phorbol esters do not cause the response (Mufson et al., 1981; Guy and Murray, 1982), that inhibitors of PKC block the effects of agonists and phorbol esters (Uhing et al., 1989; Slivka et al., 1988; Liscovitch and Amsterdam, 1989), and that downregulation of PKC attenuates the response (Cabot et al., 1989; Uhing et al., 1989). There is evidence that PC hydrolysis in some cells may be partly secondary to the rise in cytosolic Ca 2+ induced by agonists (Polverino and Barritt, 1988; Augert et al., 1989; Bocckino et al., 1987). Thus, agonist control of PC hydrolysis may involve several mechanisms: control by G proteins, PKC, Ca 24 , and tyrosine kinases. The importance of each of these mechanisms remains to be demonstrated. Another major point is the physiological significance of PC hydrolysis. The prolonged formation of DAG from PC may be important in cellular control mechanisms that require long term 15  activation of PKC.  It is obvious that much work remains to define the  physiological significance of agonist-stimulated PC breakdown. b) An and PC Metabolism PtdInsP2-PLC activation by AH is rapid and occurs within 1 minute and subsides within 3 minutes (Sadoshhna and Izumo, 1993). On the other hand, DAG production occurs within 1 minute but is much more sustained. This suggests the existence of other mechanisms to generate DAG after AH stimulation. Increasing evidence suggests that another major membrane phospholipid, PC is also hydrolyzed by PLD, PLC, and PLA2 in response to various growth factors including AH (Exton, 1990). PA, produced through activation of PLD or PLA2 is metabolized into DAG by PA phosphohydrolyse, and can be a major pathway for the activation of PKC in some systems. Sadoshhna and Izumo, 1993, suggested that activation of PLD is ATi receptor-mediated. AH activates multiple secondmessenger system in neonatal rat cardiac myocytes. It is known that there is a "cross talk" between the second-messenger system; activity of one secondmessenger system is regulated by another (Rozengurt, 1991; Nishizuka, 1992). PKC activation could, in theory, cause both an increase in PLD activity and release of arachidonic acid, because they can be regulated by an increase in PKC activity and intracellular Ca2+, respectively (Exton, 1990; Piomelli and Greengard, 1990). However, they can also be activated independently through the receptor-coupled G proteins.  PLD activation is ATj receptor-mediated and  induced by both PKC-dependent and -independent mechanisms (Sadoshhna and Izumo, 1993). The cardiac myocyte is known to express the AT2-type receptors as well, but the physiological functions of the AT2 receptor, ie. whether or not it is involved in PC hydrolysis, and its second messengers are not known (Sechi et al., 1992). 16  4 ANGIOTENSIN II AND PROTEIN TYROSINE PHOSPHORYLATION Protein tyrosine phosphorylation is induced by different growth factors and is thought to play an important role in signal transduction leading to a autogenic response. Several proteins have been identified as potential substrates and are phosphorylated at tyrosine residues by receptor tyrosine kinase. Among them are: PLC-y, Ras GTPase activating protein, and Ptdlns 3-kinase. These proteins take part in signaling mechanisms for cell growth (Cantley et al., 1991). PLC catalyzes a critical step in signal transduction through the hydrolysis of phosphatidylinositol 4,5-biphosphate to InsP3 and DAG, both of which have second messenger roles in cells. Subsequently, InsP3 mobilizes Ca^+ from intracellular Ca2+ stores and DAG activates PKC. Several PLC isozymes have been identified (Rhee and Choi, 1992); among them , PLC-y has been demonstrated to couple with the tyrosine kinase signaling mechanism. AH is a potent growth factor and shares many signal transduction mechanisms with mitogens, including stimulation of mitogen-activated protein (MAP) kinases and protein tyrosine phosphorylation (Duff, 1993). Support for the role of protein tyrosine phosphorylation in the regulation of cell growth has been derived from the identification and characterization of a number of growth factor receptors possessing this activity, as well as the involvement of constitutively active tyrosine kinases in tumorogenesis. Investigations, both in rat liver epithelial cells (Huckle et al., 1990) and rat renal mesangial cells (Force et al., 1991), have demonstrated that All stimulates rapid protein tyrosine phosphorylation, and this pathway may be important in cell growth stimulated by All. It has been shown that All treatment of quiescent smooth muscle cells stimulates rapid protein tyrosine phosphorylation of a distinct set of cellular proteins when compared with polypeptide growth factors such as PDGF (MoUoy et al., 1992). All-stimulated protein tyrosine phosphorylation is 17  accompanied by the activation of several endogenous serine/threonine protein kinases, suggesting that activation of a complex protein kinases cascade is involved in All-mediated signal transduction (Fig. 3). Activation of one or more specific protein kinases may be critical to All stimulation of cell growth. There is a similarity of the tyrosine phosphorylation pattern in response to All and EGF stimulation (Force et al., 1991). This similarity of the tyrosine phosphorylation pattern not only suggests common intermediates in the signalling pathways for growth factors and phospholipase C-linked mitogenic peptides but also suggests activation of a common tyrosine kinase which enhances phosphorylation of these intermediates. Tyrosine kinase inhibitors are very useful in the investigation of the role of agonist-induced tyrosine phosphorylation. Several of them have been synthesized and applied to study the role of tyrosine phosphorylation in signal transduction. Among them are genistein and Lavendustin A.  Genistein, an isoflavone  compound, is a highly specific inhibitor of tyrosine kinases (Akiyama et al., 1987; Akiyama and Ogawara, 1991). Genistein inhibits EGF-stimulated phosphorylation with an IC50 of 2.6 JJM. For PKC and PICA (cAMP-dependent protein kinase) inhibition, the IC50 of genistein is much higher (>100 pM) (Akiyama et al., 1987). Genistein blocked fibroblast growth factor (FGF)-mediated signaling in coronary endothelial cells, i.e. tyrosine phosphorylation of cellular proteins, FGF nuclear translocation, and initiation of DNA synthesis (Hawker and Granger, 1994). Lavendustin A has been considered a potent protein kinase inhibitor as well with an IC50 for EGF receptor kinase of 1 InM.  IS  Figure 3.  MAP Kinase A ctivating Pathways  (From Pelech et al., 1993, Upstate Biotechnology Cat.)  To our knowledge, there have not been any reports on whether or not AH induces tyrosine phosphorylation in cardiac muscle cells. Thus, the activity of tyrosine kinases in cardiomyocytes needs further investigation.  The precise  mechanism responsible for functional coupling of activated All receptors and protein tyrosine kinase activity are presently unknown.  Since All-stimulated  protein tyrosine phosphorylation in vascular smooth muscle cells was blocked by losartan, activation of the ATj receptor is required for this activity (Chiu et al., 1991; Cohen et al., 1993). The ATj receptor has been cloned and identified as a member of the 7-transmembrane domain-containing receptor class thought to be functionally coupled to G proteins (Sasaki et al., 1991; Murphy et al., 1991). These receptors do not have intrinsic tyrosine kinase activity.  Therefore, All  binding to the ATj receptors may rapidly induce additional coupling to an intracellular protein tyrosine kinase. Evidence supporting a pathway involving 19  ATi receptor activation and subsequent G protein coupling leads to signal "crosstalk", resulting in modulation of intracellular protein tyrosine kinases in cardiac myocytes, is one of the aims of our investigation. In vascular smooth muscle cells, All activation of endogenous protein kinase  C  was  required  for  the  activation  of  MAP  kinases  and  hyperphosphorylation of Raf-1 (Molloy et al., 1993). MAP kinases, such as P42DBpk, P44'Ml*, and others, have been identified as a family of protein Ser /Thrkinases that are activated rapidly in response to diverse stimuli (Pelech and Sanghera, 1992; Nishida and Gotoh, 1993; Leevers and Marshall, 1992). This activation results from phosphorylation of Tyr and Thr residues within MAP kinase (Granot et al., 1993). There is much evidence that MAP kinases are involved in the control of gene expression and mitogenesis.  The role that MAP kinases may play in  terminally differentiated nondividing cells, such as cardiomyocytes, has not been fully addressed. In vivo, in response to a variety of stimuli, cardiac myocytes respond with an increase in cell size, rather than by cell division. Thus, MAP kinases likely represent secondary or tertiary signaling molecules in the AH-initiated cascade. In this regard, All signal transduction shares common features with other mitogenic factors, including growth factors and vasoconstrictors, such as PDGF and endothelin (Cantley et al., 1991; Force et al., 1991; Bogoyevich et al., 1994). The precise role of this common signaling kinase cascade is not clear. Additional signaling mechanisms, or convergent modifying pathways, specifically regulated by individual agonists, may be required to coordinate cellular responses to a specific agonist. For example, although All has been reported to have immediate effects on smooth muscle cell protein synthesis and gene expression, its mitogenic effects are delayed by several hours when compared with protein polypeptide growth factors 20  such as PDGF (Stouffer and Owens, 1992; Gibbons et al., 1992). Therefore, early biochemical signaling events triggered by both All and PDGF, including protein tyrosine phosphorylation and the coordinate activation of PKC, Raf-1, and MAP kinases, may not be sufficient to induce DNA synthesis.  Additional events  required for All-stimulated mitogenesis may include the delayed activation of molecules such as PLC-y, p21ras GTPase-activating protein, p21ras, and/or phosphatidylinositol 3-kinase (Ptdlns 3-kinase), implicated in PDGF-initiated signaling. Future studies, should be aimed at identification of activated intracellular protein kinase(s) and their role in All-initiated signal transduction. 5. ANGIOTENSIN II, PHOSPHATIDYLINOSITOL 3-KINASE AND SIGNAL TRANSDUCTION The metabolism of inositolphospholipids is thought to be an essential part of the receptor-mediated signal transduction pathways in response to various hormones and growth factors. As mentioned above, two intracellular second messengers, InsP3 and DAG, are generated through the hydrolysis of PtdInsP2 by PLC.  Following breakdown, PtdInsP2 is rapidly resynthesized by stepwise  phosphorylation of Ptdlns by Ptdlns 4-kinase and PtdIns-4-phosphate (PtdIns4P) kinase. These two kinases appear to play important roles in the production of second messengers. However, several studies have identified the existence of a unique Ptdlns kinase associated with a certain activated tyrosine kinase (Courtneidge and Heber, 1987; Kaplan et al., 1987). The discovery of a Ptdlns kinase that phosphorylates Ptdlns at the D-3 position of the inositol ring uncovered a new pathway of Ptdlns metabolism and potential intracellular signals. This pathway is distinct from the pathway which leads from Ptdlns to PtdIns-4,5-P2 and then to DAG and InsP3 through the action of PLC. 21  Ptdlns 3-kinase  acts as a biochemical link between a novel  phosphatidylinositol pathway and a number of proteins containing intrinsic or associated tyrosine kinase activities, such as the receptor for PDGF (Kazlauskas and Cooper, 1990; Escobido et al., 1991), insulin (Endeman et al., 1990; Rudermann et al., 1990), colony-stimulating factor-1 (Varticovski et al., 1989; Shurtleff et al., 1990), and the products of oncogenes v-src (Fukui et al., 1991), vyes (Fului et al.,1991), and v-abl (Varticovski, et al., 1991), as well as the polyomavirus middle T antigen/pp60c-5rc complex (Whitman et al., 1985). Ptdlns 3-kinase is a heterodimer composed of an 85-kDa (p85) and a 110-kDa (pi 10) subunit (Escobedo et al., 1991; Otsu et al., 1991; Skolnik et al., 1991). Cloning and sequencing of the 85-kDa subunit revealed that there are two different forms of this subunit, p85a and p850. Both p85 isoforms contain two SIC domains and appear to play a regulatory role in the enzymatic activity contained in the pi 10 subunit. Recently, this subunit has been cloned and sequenced, and it has been shown to possess enzymatic activity in the presence of the p85 subunit (Hiles et al., 1992). Growth factor stimulation of the associated tyrosine kinases results in phosphorylation of the 85-subunit of Ptdlns 3-kinase; whereas the mechanism for activation of Ptdlns 3-kinase is not well understood, it is thought that phosphorylation of Ptdlns 3-kinase is important for activation of Ptdlns 3-kinase activity and the subsequent mitogenesis observed in stimulated cells (Cohen et al., 1990). However, Ptdlns 3-kinase activity has also been identified with G proteinassociated receptors such as, for example, the thrombin receptor in platelets (Sultan et al., 1990; Kucera and Rittenhouse, 1990; Huang et al., 1991) and the formyl peptide receptor in neutrophils (Eberle et al., 1990).  Stimulation of  neutrophils with formyl peptide results in activation of Ptdlns 3-kinase independent of tyrosine phosphorylation (Vlahos and Matter, 1992; Stephens et 22  al., 1993), suggesting an alternative pathway for Ptdlns 3-kinase activation in association with G proteins. Ptdlns 3-kinase transfers the terminal phosphate of ATP to the D-3 position of Ptdlns, PtdIns-4-monophosphate, or PtdIns-4,5-biphosphate to yield the products PtdIns-3-P, PtdTns-3,4-P2, or PtdIns-3,4,5-P3, respectively (Carpenter and Cantley,1990). The biological role of Ptdlns 3-kinase or its products has not been established. It is clear that these novel products are not directly involved in the traditional pathway for generating the second messenger InsP3 or for generating substrates for PLC (Serunian et al., 1989; Lips et al., 1989; Vlahos et al., 1994). The kinase has been suggested to play a role in cell proliferation and/or motility in response to growth factors and chemotactic agents (Carpenter and Cantley, 1990; Williams, 1988; Dobos et al., 1992). The importance of Ptdlns 3-kinase as a downstream mediator of tyrosine kinase action has been inferred from studies of mutant tyrosine kinases. The two src homology 2 domains of the 85-kDa regulatory subunit of Ptdlns 3-kinasemediate the binding of Ptdlns 3-kinase to activated tyrosine kinases. Mutated versions of the pp60v-src (Fukui and Hanafusa, 1989) and abl (Varricovski et al., 1991) tyrosine kinases that cannot bind or activate Ptdlns 3-kinase do not transform cells. Similarly, site-directed mutagenesis of the PDGF receptor has revealed that mutations that prevent the binding of Ptdlns 3-kinase to this receptor abrogate the ability of PDGF to stimulate cell division (Fantl et al., 1992). In contrast, removal of the binding site on the PDGF receptor for the p21ras GTPaseactivating protein does not affect the mitogenic response to PDGF (Fantl et al., 1992). Thus, Ptdlns 3-kinase appears to be a key component of tyrosine kinaseregulated signaling pathways that lead to cell proliferation. Until recently, the signal transduction functions of Ptdlns 3-kinase and its products were not known. The Ptdlns 3-kinase products are not substrates for 23  phospholipase C, which preferentially cleaves Ptdlns 4,5-P2 (Serunian et al., 1989; Lips et al., 1989). Nakanishi et al. (1993) have shown that Ptdlns 3,4,5-P3 and to a lesser extent Ptdlns 3,4-P2 can activate the ^-isoform of protein kinase C in vitro. Protein kinase C£ differs from all the other protein kinase C isoforms in that it is not stimulated by phorbol esters or DAG (Nakanishi and Exton, 1992; Liyanage et al., 1992). Thus, Ptdlns 3-kinase and protein kinase C£ comprise a new signal transduction pathway. The role of All in activation of Ptdlns 3-kinase needs further investigation, particularly in cardiac cells.  6 CHOICE OF 7-DAY CHICK EMBRYO VENTRICULAR MYOCYTES AS A CULTURE MODEL It has been shown that cultured chick heart cells are a good model to provide a means by which the biochemical and molecular mechanisms of action of All on cardiac tissue can be elucidated. Some aspects of All receptor-mediated stimulation of inositol phosphates in chick cardiomyocytes have been described previously (Baker et al., 1989). Chick cardiac myocytes were a good model for investigation  of the  effect  of  some drugs on choline uptake and  phosphatidylcholine biosynthesis (Rabkin, 1988).  a) Histogenesis of the Embryonic Chick Ventricular Myocardium The ultrastructure of the developing chick ventricular myocardium was examined between Hamburger-Hamilton stage (45 hours incubation) and the time of hatching (about 21 days incubation). The myocardium is an epithelial tissue initially containing only developing myocytes. No epicardium is present and no mesenchymal cells such as fibroblasts are seen. By the third day of development, portions of the ventricular endocardium invade the myocardium. After this event 24  and the development of the epicardium from an extramyocardial source, mesenchymal cells are seen within the myocardium.  These cells, the first  nonmuscular components seen within the myocardium, are probably fibroblasts derivedfromthe endocardium or the epicardium. Early cardiac myocytes contain few fibrils and large amounts of cytoplasm. The fibrils are not regularly oriented within the young cells. As development proceeds, more fibrils are formed; the cytoplasm decreases and the fibrils become aligned in the mature orderly pattern. i. Ventricular Myocytes of the 7 Day Old Embryo Ventricular myocytes of the 7 day old embryo contain many myofibrils, but they often still lack alignment. Whereas myofibrils insert into intercalated discs nearly at right angles in the mature heart, the random fibrils of these embryonic myocytes often insert at an acute angle. Similar configurations have been seen as late as the tenth day of development (Noble and Cocchi, 1990). Thus, monolayer culture of beating 7 day embryonic chick myocytes is an appropriate model for the investigation of signaling in cardiomyocytes induced by All. Our studies were performed on 4th to 7th incubation day ilsing confluent monolayers of spontaneously beating myocytes. 7. HYPOTHESES AND OBJECTIVES There is a considerable amount known about the action of All on different cell types, but mainly in VSMC, hepatocytes, and rat renal mesangial cells. There are also several studies reporting the relationship between All and the myocardium. AH has various effects on the myocardiocytes (Schelling et al., 1991; Neyses and Vetter, 1989; Baker and Aceto, 1990; Sadoshima and Izumo, 1993; Sunga and Rabkin, 1991; Lokuta et al., 1994), In addition, the effect of 25  inhibitors of All production, such as ACE inhibitors, has been studied. However, the signal transduction pathways activated by All in cardiac myocytes have not been examined fully, despite its potential importance in our understanding of the integrated cellular function of All and All antagonists in cardiovascular diseases. All activates PLC through a G protein-coupled receptor (Berridge, 1993). It leads to InsP3 liberation and induces Ca^ + release (Nishizuka, 1988; Irvine, 1990; Irvine, 1992; Birnbaumer et al., 1990). In other cell types, All has also been shown to activate PLA2 and PLD (Exton, 1990). Moreover, some investigations have reported that All stimulates rapid tyrosine phosphorylation, and this pathway is important in cell growth stimulated by AIL However, in cardiac myocytes, this action of All has not been studied well. In addition, the role of Ptdlns 3-kinase which is implicated in growth factor signal transduction, needs further investigation in myocardial cells, particularly in response to All stimulation. In our laboratory, we have established a 7 day chick embryo cardiac cell culture, which can serve as an effective tool for further investigation of the biochemical and molecular mechanisms of action of All on cardiac tissue.  In the present study, the principal hypotheses were that 1. there are several intracellular messengers or processes underlying the response of cardiac cells to All, namely hydrolysis of phosphatidylinositol 4,5-biphosphate and phosphatidylcholine, protein tyrosine phosphorylation, and activation of Ptdlns 3kinase; 2. ATj and AT2 receptors are involved in AH-induced signaling mechanisms in chick cardiomyocytes; 3. Protein tyrosine phosphorylation may play an important role in All-stimulated signal transduction in cardiac myocytes; 26  The specific objectives were to 1.  determine the All receptor-mediated stimulation of inositol phosphates in 7 day chick embryo cardiomyocytes;  2.  investigate the potential role of AQ in generation of DAG by stimulation of phosphatidylcholine hydrolysis;  3.  study the involvement of protein tyrosine phosphorylation in signal transduction activated by All in cardiac muscle cells;  4.  define if All affects Ptdlns 3-kinase activity in cardiac myocytes;  5.  examine the ATj and/or AT2 receptor involvement in complex signaling mechanisms following All stimulation of chick cardiac cells;  27  CHAPTER II MA TERIALS AND METHODS 1. Isolation of Chick Embryonic Cardiac Cells Monolayer culture of beating 7 day embryonic chick ventricular cells were prepared using previously described methods (Rabkin and Sunga, 1987). Fertilized White Leghron eggs were incubated in an automatic incubator for 7 days at 37.8 C and 87% humidity. Hearts were then removed under sterile conditions from the 7 day chick embryo (all procedures were performed in the tissue culture hood). Ventricles were isolated from atria and used for further culturing. Then, ventricles were cut into 0.5-mm fragments under dissecting microscope in a solution of balanced salts (DMS8) with the following composition (in mM): NaCl 170, KC1 5.4, NaH2PC>4 4.3, Na2HPC>4 l,dextrose 5.6. Disaggregation was carried  out by 5 minute digestions in 0.005% trypsin, 0.1% bovine serum  albumin, and DNAase lxlO-? Dornase units/mL, DMS8 at 37.8°C This procedure was repeated 5 times, each time with replacement of disaggregation solution. After 5 digestions, the digests were diluted 1:5 in culture medium 818A (73.5% of DBSK which contains (in g/L): NaCl 6.8; MgS047H20 0.2; NaH 2 P04H 2 0 0.13; dextrose 1; NaHCC>3 2.2, 20% of M199, 6% of fetal calf serum, and 0.5% of antibiotic-antimycotic (10,000 units/ml penicillin G sodium, 10,000 jog/ml streptomycin sulfate, and 25 fig/ml amphotericin B), and the cells centrifuged for 3 minutes at 1000 x g. Cells were plated into 35-mm culture plates at 1x10^ cells per culture dish. Cultures were incubated in a humidified 5% C02-95% air atmosphere at 37 C. Studies were performed on the 4th to 7th incubation day using confluent monolayers of spontaneously beating myocytes.  28  2. Polyphosphoinisitide Turnover Studies Polyphosphoinositide turnover studies were performed using previously. described methods (Watson et al., 1984; Salari et al., 1990) with some modifications. a) Incorporation of myo-f2-lH| inositol Cardiac myocytes were maintained in culture in medium 818A (73.5% DBSK, 20% M199,6% fetal calf serum, and 0.5% antibiotic-antimycotic) for 72 hours prior to being labeled with myo pH] inositol. Thereafter, dishes with cells were removed from the incubator; the old medium was replaced with the myo [^H] inositol medium The final concentration of myo pH] inositol was 2 iiCi/ml (specific activity: 629 GBq/mmol, 17 Ci/mmol). Cells were placed back into the incubator and incubated for 18-24 hours. b) Challenge ("Cell Activation") After being labeled wih pH] inositol, cells were washed twice with medium 818A, 37 C to remove all traces of unincorporated inositol. Then, cells were incubated for 20 minutes in medium 818A containing 10 mM LiCl. LiCl was present to inhibit inositol 1-phosphate phosphatase, to allow accumulation of inositol phosphates. Cells were removed from the incubator and treated with All using various concentrations and times. For the control condition, parallel myocyte cultures were harvested at the same time course without stimulation. At predetermined times, the medium was removed, cells were washed with ice cold PBS (to make 500 mL were disolved: 4 g NaCl, O.lg KC1, 0.6 g Na 2 HP0 4 , 0.1 g KH 2 P0 4 , 0.0915 g MgCl2, 0.5 g D-glucose, 0.5 g BSA, and 0.05 g CaCl2. pH 7.4).  29  Thereafter, 2 mL of cold methanol/water (1:0.8) were added. Cells were scraped and placed on top of 2 mL of chloroform. The tubes were kept overnight at -4 C. Next day, the tubes were vortexed at 3,000 rpm for 10 minutes. The aqueous layer was removed and used for column chromatography. Samples were loaded on Dowex 1-8X columns (Biorad) packed with resin and ammonium formate.  c) Column Chromatography i.Preparing Dowex 1-8X Columns 50 g of Dowex 1-8X were disolved in 1 litre of distilled water and stirred overnight at 4°C, very slowly. Next day, the solution was filtered , and resin, yellowish powder, collected.  Then, the resin was suspended in 1 litre of IM  ammonium formate and stirred again very slowly overnight at 4 C. Once complete the resin was stored at 4 C in 1 M ammonium formate, until needed. For 1 M NH4 formate 63.06 g were dissolved in 1 litre of (IH2O). Thereafter, 1 mL of Dowex mixture was loaded into each biorad poly-prop chromatography column.  The bed volume was 1 mL.  Columns were pre-  equilibrated by washing several times (approximately 10X) with distilled water to remove traces of free ammonium formate. At this point, columns were ready to be loaded. Loading and Running Columns The samples were loaded onto columns and allowed to elute out by gravity. Then, columns were washed with different solutions to separate inositides. Inositol was separated with 8 mL of 60 mM ammonium formate and 5 mM disodium tetraborate solution (3.785 g NH4 formate and 1.905 g disodium tetraborate in 1 litre of distilled water).  30  InsPj was eluted using 8 mL of a 200 mM ammonium formate and 100 mM formic acid solution (12.613 g NH4 formate and 3.795 mL formic acid in 1 litre of distilled water). InsP2 was recovered with 10 mL of a 400 mM ammonium formate and 100 mM formic acid solution (25.223 g NH4 formate and 3.795 mL formic acid in 1 litre of distilled water). InsP^ was released with 6 mL of ammonium formate 1 M and 100 mM formic acid (63.04 g NH4 formate and 3.795 mL formic acid in 1 litre of distilled water). The eluents were collected in separate vials of which 1 mL mixed with 10 mL of aqueous counting scintillant (ACS) (Amersham). Samples were counted in a Beckman liquid scintilation counter.  3. Phosphatidylcholine Degradation and Choline Efflux Myocytes maintained in culture medium 818A in petri dishes for 72 hours were studied. Cells were incubated in the 818A medium containing [methyl-^H] choline chloride, 1.25 nCi/mL (specific activity: 3.18 TBq/mmol, 86Ci/mmol), for 18-24 hours. Thereafter, the radioactive medium was removed, and the cells washed twice in 818A medium (37°C) to remove excess and unincorporated label. The medium then was replaced by fresh 818A medium, 2 mL in each dish. All at different concentrations was added to the medium, and dishes were returned to the incubator. At predetermined times, aliquots of the medium were removed and the amount of tritium counted. Control myocytes were treated in an identical fashion, except for stimulation with All. It was previously determined that over 80% of the [3H] label that effluxes out of the cardiomyocytes is choline (Rabkin, 1989). At predetermined times, the growth medium was removed, the cells were washed with ice cold PBS, pH 7.4, and 2mL of cold methanol/water (1:0.8) solution was 31  added. Subsequently, cells were scraped and placed on top of 2 mL of chloroform. Then, the tubes were vortexed for 10 minutes at 3,000 rpm.The aqueous layer was separated, middle layer discarded, and 100 nL of top aqueous layer were counted for radioactivity. 0.5 mL of chloroform layer was placed in counting vials and allowed to dry; and thereafter, the radioactivity of it was determined using the scintilation counter. Following extraction of lipids, the recovery of [^H] choline in choline, phosphocholine, and phospholipids was determined using thin layer chromatography.  a) Thin Layer Chromatography (TLQ Thin layer chromatography was performed following previously described methods (Sunga et al., 1988) with some modifications.  The TLC procedure  consisted of extraction of lipids from the cells using ice-cold methanol/water (1:1, v/v) and added to chloroform. The water-soluble and lipid-soluble fractions were separated by centrifugation. Samples of each phase were placed on TLC plates of silica gel G (BDH, Toronto, Canada). The carrier mixture contained choline (0.3 mg/|il), phosphocholine (0.9 mg/|il), cytidine diphosphocholine (CDP-choline) (0.15 mg/|il), and glycerophosphocholine (GP-choline) (0.45 mg/jil). The plates were developed in methanol/1.2% NaCl/ammonia (10:10:1 by vol). Spots were made visible with Iodine vapour; and areas of the plates were scraped into counting vials. lmL of 1 N HC1 was added to each vial. Next day, they were assayed for radioactivity in the scintillation counter using 10 mL aqueous counting scintillant in each vial.  32  4. Preparation of Cell Lysates and Immunoprecipitation The cardiomyocytes were cultured on 100-mm dishes in 818A medium, containing 2% fetal calf serum, for 4 days prior to the experiment. On the day of experiment, various concentrations of drugs were added to the dish for predetermined times. The media were removed; and the culture dishes placed on ice. After washing them twice with PBS, cell pellets were lysed by a buffer containing 137 mM NaCl, 20 mM Tris pH 7.4, 1 mM MgCl2, ImM CaCl2, 1% Nonidet P-40, 1 mM phenylmethylsulphonylfluoride (PMSF),and 2 mM sodium orthovanadate. One millilitre of the lysis buffer was added to each dish, and the plates rocked for 20 minutes at 4°C. Thereafter, the cells and debris were scraped from the plates with a rubber policeman; and the lysis buffer and cell debris were transferred to 1.5 mL conical tubes. At this point, the supernatant was ready for the addition of antibody against phosphotyrosine (PY-69, mouse Mab IgG2a)In some experiments RIPA buffer was used to lyse the cells; RIPA (150 mM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 7.5). Then, the supernatants were incubated for 1 hour with antibody, approximately 10 pL (i. e. 1.0 ng), (for anti-P-Tyr immunoprecipitations we used mouse monoclonal IgG). The incubation was performed at 4 C on a rotating device. Twenty microlitres of agarose conjugate (Protein A) were added to each sample: and incubation was continued for 24 hours under the same conditions. After  overnight incubation,  centrifugation in a microfuge  immunoprecipitates  were collected  by  at 2,500 rpm for 10 minutes. The pellets were  washed 3 times in freshly prepared Wash Buffer (50 mM HEPES pH7.4, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 100 mM NaF, 2 mM sodium orthovanadate). After the final wash, supernatants were aspirated and discarded; pellets were resuspended in 40 \xL of the electrophoresis sample buffer (125 mM Tris/HCl pH 6.8, 4% SDS, 10% beta-mercaptophenol, 20% glycerol, 0.01% 33  bromophenol blue), boiled for 4 minutes, and analysed by SDS-PAGE, or stored at -20°C. 5. SDS Polyacrylamide Gel Electrophoresis SDS PAGE of extracts was performed according to the method of Laemmli (1970) on 7.5% separating gels containing 4.85 mL of distilled water, 2.5 mL of 1.5 M Tris/HCl pH 8.8, 100 uL of 10% (w/v) of SDS stock, 2.5 mL of Acrylamide /Bis (30% stock), 50 \xL of 10% ammonium persulfate (freshly prepared), and 5 uL of TEMED. Samples were dissolved in SDS gel electrophoresis buffer, boiled for 4 minutes, and equal amounts of protein loaded onto 4% stacking gels (distilled water - 6.1 mL, 0.5 M Tris/HCl pH 6.8 - 2.5 mL, 10% (w/v) SDS - 100 uL, Acrylamide/Bis (30% stock) - 1.3 mL, 10% ammonium persulfate - 50 uL, and TEMED - 10 uL) overlying 7.5% polyacrylamide separating gels. Following electrophoresis, gels were stained with Coomassie Brilliant Blue R250, destained, and dried onto paper. i,  6. Assay for Associated Phosphoinositide 3-Kinase activity Chick cardiac cells were cultured on 10-mm dishes in 818A medium containing 2% fetal calf serum at a density of approximately 6x10^ per culture dish for 4 days proir to the experiment. Growing cells were stimulated with drugs for the indicated times. After stimulation, the cells were cooled to 4°C and washed with ice-cold phosphate-buffered saline (140 mM NaCl, 3 mM KC1, 6mM Na2HP04, ImM KH2PO4, pH 7.4) containing ImM CaCl2, 1 mM MgCl2, and 100 |iM sodium orthovanadate. Thereafter, cells were washed gently (2x5 mis) with freshly prepared 'Buffer-A' (137 mM NaCl, 20 mM Tris pH 7.4, 1 mM MgCl2, 1 mM CaCl2 100 nM sodium orthovanadate). Then, the Wash Buffer was shaken from the plates; and the cells were lysed by adding 1 mL of 'Buffer-A' 34  containing 1% NP-40, 1 mM PMSF, 100 pM sodium orthovanadate and rocking the plates for 20 minutes at 4°C. After scraping the cells from the dishes, the cells and debris were transfered to 1.5 mL microfuge tubes. Insoluble material was removed by centrifuging for 10 minutes at 4 C, 16,000g. Immunoprecipitation was performed with 5 uL PtdIns-3 kinase p85 antibody (rabbit polyclonal IgG) for 90 minutes at 4°C (Giorgetti et al., 1992) followed by addition of Protein A-Agarose beads (20 pL) and incubation overnight at 4°C on a rocker platform. The next day, the immunoprecipitates were washed twice by centrifuging briefly (5 seconds) in a microfuge with each of the following buffers: (i) phosphate-buffered saline (pH 7.4) containing 1% NP-40; (ii) lOOmM Tris, 0.5 M LiCl, pH7.4; and (iii) 10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4 (TNE) (Whitman et al., 1985; Endemann et al., 1990).  The above buffers contained 100 pM sodium  orthovanadate and were freshly prepared.  The last wash was removed as  completely as possible and added to each pellet: 50 pL TNE, 10 pL phosphatidylinositol (20 pg), and 10 pL 100 mM MgCl2-  a) Preparation of Phosphatidylinositol Twenty microlitres of a chloroform solution of Ptdlns (10 mg/ml) were dried in a 1.5 mL Eppendorf tube, and 10 mM Tris pH 7.4, 1 mM EGTA added to bring the Ptdlns to a concentration of 2 pg/mL. Lipids were resuspended by sonicating in an ice bath for 10 minutes.  b) Ptdlns 3-kinase Reaction The reaction was initiated by adding 30 pCi of [y-32p]-ATP. After 10 minutes with constant agitation, the reaction was stopped by addition of 20 fiL of 6 N  HC1.  The phosphoinositides  35  were extracted with  160 pL of  chloroform/methanol (1:1).  The phases were separated by centrifuging  10  minutes in a microcentrifuge. The phospholipids were analysed by thin-layer chromatography (TLC) on oxalate-treated silica plates in l-propanol/2 M acetic acid (13.7:7) as previously described (Remillard et al., 1991; Damen et al., 1993). Unlabeled lipid standards (PI and PI-4-P) were chromatographed with the samples and visualized by exposure to iodine vapor. 7. Densitometric Analysis For densitometric studies a Model 620 Video Densitometer (Bio-Rad, Mississauga, Ontario) was used. Transmission measurements were used during the analysis as well as reflectance measurements.  8. Methods of Statistical Analysis All results were expressed as the mean +standard error of the mean (SEM). Unpaired Student's t-test was used to determine the significance of difference between two means. Analysis of variance was used to examine for group comparison followed by Tukey test. The null hypothesis was rejected if the probability of a Type I error was less than 5% (p<0.05).  9. Materials a) Radiochemicals [Methyl-^H] choline (specific activity: 3.18 TBq/mmol, 86 Ci/mmol) and myo-[2-3H] inositol (specific activity: 629 GBq/mmol, 17 Ci/mmol) were obtained from Amersham. [y-32P]ATP (specific activity: 111 TBq/mmol, 3000 Ci/mmol) wasfromNew England Nuclear Research Products, Boston, MA.  36  b) Biochemicals Culture media (DBS8, DBSK, and M199) and serum were obtained from GIBCO  (Burlington,  Ontario,  Canada).  L-a-Phpsphatidylinositol,  phenylmethylsulfonyl fluoride, and other drugs were from Sigma Chemicals (St. Louis, Missouri, USA).  Angiotensin II was from Peninsula Laboratories  (Belmont, CA, USA). 1-X8 Resin, Prestained SDS-PAGE Standards, and other chemicals were from BIO-RAD (Mississauga, Ontario, Canada). obtained from Amersham (Oakville, Ontario, Canada).  ACS was  Lavendustin A,  Lavendustin B, Daidzein and Genistein were from LC Laboratories (Woburn, Mass., USA). Losartan was a gift from DuPont (Wilmington, Delaware, USA). PD123319 was from Park Davis (Ann Arbor, Michigan, USA). c) Antibodies Antibodies against phosphotyrosine (PY-69, mouse Mab IgG2a), Ptdlns 3kinase p85 a (rabbit polyclonal IgG), Protein A-Agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA).  37  CHAPTER III RESULTS  1. All INDUCES PHOSPHOINOSITIDE HYDROLYSIS InsP3 production is a hallmark of PtdInsP2-PLC activation. To determine whether PLC is activated by All in our system, All-induced accumulation of InsPi, InsP2,  an  d InsP3 was measured. After 24 hours of incubation with [3H]  myoinositol (2 nCi/mL), cardiac myocytes were stimulated with All in the presence of lithium chloride, an inhibitor of inositol phosphatase; and the inositol phosphates were measured on a Dowex column. The time course of All induced phosphoinositide hydrolysis was examined. All produced significant (p<0.01) increases in the levels of InsPi (Fig.4), Ins P2 (Fig.5), and InsP3 (Fig.6) within 1 minute of incubation of cells and was sustained for 5 minutes. In 10 minutes, we did not observe an increased level of InsP3 (Fig. 6). The levels of InsP2 and InsPj were not significantly increased at 10 minutes of incubation with All (Figs. 4-6). Thus, the kinetics of InsP3 production shown in Figs. 4-6 suggest that PtdInsP2-PLC activation by All in chick cardiomyocytes occurs within 1 minute and subsides within 5 minutes. Therefore, a 1-minute All stimulation of our cells was chosen for a concentration-response analysis as it represented the best response in the time course. Concentration-dependence  studies  of All  induced  phosphoinositide  hydrolysis showed that All caused a rapid increase in InsP2 and InsP3 formation at 100 nM and 1000 nM respectively (Figs. 7 and 8). All induced InsP3 production showed a direct dependence on All concentration (Fig. 7). The best response was obtained at 1000 nM of All; the amount of InsP3 (in cpm/ml) increased from 1,500 to 2,350 cpm/ml. 38  3,500  Time (min)  Figure 4.  Time-dependence of All-induced accumulation of InsPi  Cardiac myocytes were prelabeled with [3HJ myoinositol for 24 hours and treated subsequently with All (1000 nM) in the presence of LiCl (10 mM) for the time indicated. InsP] was separated by Dowex column chromatography.  The results were compared with  the counts obtained in nonstimulated myocytes (control), which were harvested at the same time as stimulated cells. Data were obtained from three separate experiments performed in triplicate. Results represent mean ±SEM. * *P<. 01 39  1,000  800 E Q.  CM CD  600  +->  (0  sz a. tn o sz  400  CL  oto c  200  Time (min)  Figure 5.  Effect of All on InsP2 formation (time course)  Chick cardiomyocytes were labeled with f^HJ inositol and incubated in the presence or absence of All (1000 nM) for predetermined times. Cells were treated in the presence of 10 mMLiCl. InsP2 was extracted by Dowex-1 column chromatography. Results represent mean±SEM(n=3).  *P<.05, **P<.01  40  * *  400  Time (min)  Figure 6.  Time course for All-induced I11SP3 release  Cultures were prelabeled with f^HJ inositol for 24 hours and subsequently treated with All (1000 nM) for 1, 5, and 10 minutes in the presence of LiCl (10 mM). InsP$ was separated by Dowex column chromatography. The counts oflnsP^ were compared with the counts obtained in nonstimulated cells which were harvested by the same time course as stimulated cells. Results represent mean +SEM obtained from three experiments done in triplicate. *P<.05, **P<.01 41  In cells incubated with All at a concentration of 100 nM, the accumulation of InsP3 was around 65% of maximal, and only 18% increase was observed at stimulation of cells with 1 nM All. However, accumulation of InsP2 (Fig. 8) was more prominent with All (100 nM). InsP2 was increased from 2,620 to 3,933 cpm/ml at this concentration of All, and a slight decrease in InsP2 formation was observed at 1000 nM. Production of inositol, as expected (presence of LiCl), was not increased (Fig. 9). It went down from 6331 cpm/ml in control to 5549 cpm/ml in All (1000 nM) stimulated cells. InsPi formation did not show any obvious dose dependent response (Fig. 8).  Since InsP3 production is the best indicator of  PtdInsP2-PLC activity, we decided to use a concentration of All of 1000 nM for further investigations. To investigate the involvement of ATj and/or AT2 receptors in this pathway, the ATj receptor antagonist losartan and AT2 receptor antagonist PD123319 were used. The All stimulated production of InsPj, InsP2, and InsP3 was inhibited by losartan (Figs. 10-12). We have studied the dose response of the effect of losartan (Figs. 10-12). However, the inhibition was not complete even at 10'^M.  It leads us to suggest that not only ATj receptors were involved in  activation of PLC by Ang II in 7 day chick embryo cardiomyocytes. We investigated the possibility of inhibition of All-induced PtdInsP2 hydrolysis by PD123319, an AT2 receptor antagonist (Fig. 12). The InsPi, InsP2, and InsP3 production were partially inhibited by this blocker, mostly production of UISP3. These results suggest that the PtdInsP2 hydrolysis and the formation of InsP3 induced by All is mostly, but not only, ATj receptor-mediated.  42  100  1  10  100  1000  Angiotensin II (nM)  2,400 £=-2,200 Q. 2,000  1  10  100  1000  Angiotensin II (nM)  Figure 7.  Concentration-dependence of All-induced accumulation of InsP3  f^HJ inositol-prelabeled myocytes were stimulated for 1 minute with All at indicated doses in the presence of LiCl (10 mM). The accumulation of InsPj was quantitated by Dowex column chromatography as described under "Materials and Methods". The results were compared with the counts obtained from unstimulated cells (control). Values are the means +SD of three determinations. 43  3,500  1,000  1  10  100  1000  Angiotensin II (nM) 4,500 p  1,000  1  10  100  1000  Angiotensin li (nM)  Figure 8.  All-induced accumulation of InsPj and InsP2 (concentration-dependence)  Cardiac myocytes were prelabeled with f^HJ myoinositol for 24 hours and treated subsequently with All (1 nM, 10 nM. 100 nM,and WOO nM) for 1 minute in the presence ofLiCI ( 10 mM). Inositol phosphates were separated by Dowex column chromatography. Nonstimulated cells were used as a control. Results represent mean ±SD of three determinations. A. Accumulation of InsPj. stimulated accumulation ofInsP244  B.Angll"  .  1.1  0  1  1  1  1  1  10  100  1000  Angiotensin li (nM)  Figure 9.  Concentration dependence of All-stimulated Inositol accumulation  pH] inositol prelabeled chick cardiomyocytes were treated with All at different doses for 1 minute in the presence of LiCl (10 mM). Inositol was extracted by column chromatography as described previously.  For the control condition, myocyte cultures were  harvested at the same time without All stimulation. representative of three determinations (means +SD).  45  Results are  120  J.10O X (0  jl 80 o 0  6 0  s 1 40 o o  <C CO  c 0  0  9  7  5.  -log [ANTAGONIST] nM Figure 10.  Effect of ATj and AT2 receptor blockers on Allinduced InsP3 accumulation  [3HJ inositol-prelabeled myocytes were stimulated with All (J 000 nM, 1 minute); and the accumulation of InsP$ was quantitated as : described above. The maximal response to All (the 100% value) was 439 cpm/ml; in nonstimulated cells the response was 335cpm/ml. A 30-min preincubation with increasing concentrations of antagonist (10~9, 10~7, 10~5 M) in medium prior to the 1 minute 1000 nM All stimulation was included  Stimulation values were  compared directly to parallel experiments in which antagonist was not added. Shown are the dose-response curves for losartan and PD123319. Results represent mean +SEM of five experiments each done in triplicate. 46  120  9  7  -log [ANTAGONIST] hM  Figure 11.  AH induced formation of InsP2 and effect of ATj and AT2 receptor blockers on it  Cardiac myocytes prelabeled with [3HJ inositol were pretreated with losartan or PD123319 at indicated doses for 30 minutes and subsequently stimulated with All (1000 nM) for 1 min. The level of InsP2 was determined by Dowex column chromatography and compared with the level ofInsP2 in the All stimulated cells which were not pretreated with blockers. The actual counts ofInsP2 in All stimulated cells vs. nonstimulated were 1187 cpm/ml vs. 938 cpm/ml. The maximal response to All in the absence of antagonist was defined as 100%. Each point represents the mean +SEM of five experiments performed in triplicate. 47  120 Losartan ^PD123319  9  7  -log[ANTAGONIST] nM  Figure 12.  Dose-inhibition curves of All receptor subtype-specific antagonists on AIHnduced InsPj release  Cells were incubated with increasing concentrations of losartan or PD123319 for 30 minutes, and the response to stimulation with All 1000 nM for 1 minute was measured as described for Fig. 10 and under "Materials and Methods".  The actual counts of IP] were  5195 cpm /ml in All stimulated cells and 3456 cpm/ml in nonstimulated.  This increase was defined as 100% value.  All  experiments were done in triplicate. Values are the means ±SEM of five experiments. 48  Moreover, we studied the effect of losartan and PD123319 on InsP3 production in nonstimulated cells. Neither ATj nor AT2 receptor blocker by themselves affected the PtdInsP2 hydrolysis and formation of InsP3 (Fig. 13). The All-induced phosphatidylinositol turnover and formation of InsP3 was blocked by losartan (10 - 5 M), but not completely (71%). In addition, 40% of the increase in IP3 by AQ stimulation was blocked by PD 123319. Combination of both blockers led to almost complete inhibition of IP3 formation in All-stimulated myocardial cells. Taken together, the data from Figs. 10-13 reveal that All-stimulated accumulation of inositol phosphates in 7 day chick embryo cardiomyocytes was mainly mediated by ATj receptor subtype as expected.  However, the AT2  receptor subtype is also likely to be involved in this pathway. Consequently, we suggest multiple receptor subtypes mediated All-stimulated phosphatidylinositol hydrolysis in chick embryo cardiac cells. We decided to investigate whether or not All-stimulated phosphoinositide hydrolysis was associated with protein tyrosine phosphorylation in chick cardiomyocytes and whether or not All-stimulated tyrosine phosphorylation is responsible for early signal transduction events in our cells.  Specifically, we  wanted to know whether or not All-stimulated phosphatidylinositol 4,5biphosphate breakdown, generating l,4,5-InsP3 w a s correlated with an increase in protein tyrosine phosphorylation.  To investigate the relationship between All  mediated protein phosphorylation and InsP3 formation, we used genistein, a tyrosine kinase inhibitor, and daidzein as a negative control for genistein. Genistein, 10 JIM, reduced All-induced formation of InsP3 by 65% (Fig. 14). The genistein concentration selected was found previously to be the minimal concentration that gave maximal inhibition of InsP3 formation when cells were preincubated for 1 hour with this tyrosine kinase inhibitor (Akiyama et al., 1987; 49  150  _J40 o *  I 130 o  X  — 120 c o  *  I 110 o m 100  Q.  X  CO  c -  90 80  • / '/ / ^  <* ^  v° Ang Figure 13.  II  Effect of All receptor blockers on All-induced InsP3  Cardiomyocytes prelabeled with f^HJ inositol were stimulated with All (1000 nMjfor 1 minute in the presence ofLiCl (10 mM). The accumulation  of  chromatography.  LnsP$ was For  measured  antagonist  by  Dowex  experiments,  cells  column. were  preincubated for 30 minutes with losartan (10r$ M), or PD123319 (10~5 M), or losartan and PD123319 (both 10~5 M) prior to the stimulation by AIL  Some cells were incubated with blockers and  harvested without being stimulated with ALL The amount oflnsP$ in control was defined as a 100% value. Actual counts of LnsP^ in control and ALL stimulated cells were 439±18 and 335+14 cpm/ml, respectively. All values reflect the mean ±SEM of three to five experiments performed in triplicate. *P<. 05 50  Figure 14.  Effect of genistein on All-stimulated production of inositol phosphates  Cardiac muscle cells were pretreated for 24 hours with f^HJ inositol and consequently incubated with 1000 nM All for 1 minute. Inositol phosphates were extracted as described above. For tyrosine kinase inhibitor experiments, cardiomyocytes were preincubated for 1 hour in the presence of genistein (10 iM) and thereafter with or without All (1000 nM).  Daidzein (same kind of molecular structure but  different IC$ofor tyrosine phosphorylation) was used as a negative control for genistein. Amount (cpm/ml) oflnsP], InsP2, and InsP$ were compared with inositol phosphates  in nontreated cells  (control), which were defined as 100%. Results represent mean +SEM(n=3). *P<.05  51  Marrero et al., 1994). IC50 for genistein, for instance, varies from 2.6 joM for EGF receptor kinase to >100 JJM for other kinases. These results suggest that protein tyrosine phosphorylation induced by All plays an important role in early activation of PtdInsP2 hydrolysis and formation of InsP3.  2. A n STIMULATES PHOSPHATIDYLCHOLINE HYDROLYSIS It is known that All-stimulated PtdIP2-PLC activation and production of MP3 occurs within 1 minute and subsides within 3-5 minutes. On the other hand, DAG production occurs within 1 minute but is much more sustained. This suggests the existence of other mechanisms to generate DAG after AH stimulation. Increasing evidence suggests that another major membrane phospholipid, PC, is also hydrolyzed (Sadoshima and Izumo, 1993; Exton, 1990). We examined the possibility that All also stimulates the hydrolysis of PC in cardiomyocytes. In chick cardiac cells prelabeled with [^H] choline for 24 hours, All increased intracellular free choline after exposure for 1 minute to 1000 nM All (Fig. 15). The maximal and significant (P<.05) increase was observed at 30 minutes and remained increased over a one hour observation period. However, the increase at 1 hour was not statistically significant. The intracellular distribution of the [^H] choline was as follows: choline 3.78%, phosphocholine 18.13%, phospholipid 76.46% with the remainder in CDP-choline and glycerophosphocholine.  All  produced a significant increase (P<.05) in the percent of label in free choline (Fig. 16) after stimulation for 30 minutes. The increase at 10 and 60 minutes was not significant. The previous observations in our lab showed that at shorter periods of  52  3,000  I a  1,500  1,000 0  5  10  15  20  25  30  35  40  45  50  55  60  TIME (min)  Figure 15.  All stimulation and intracellular free choline  Chick cardiomyocyles were prelabeled for 24 hours with f^HJ choline and consequently treated with All  (1000 nM).  At  predetermined times, the cells were harvested, the lipids extracted, and  the  recovery  phosphochoiine,  of  and  f^HJ  was  determined  phospholipids  using  in thin  choline, layer  chromatography. Spots were made visible with iodine vapor; and areas of the plates were scraped and assayed for radioactivity. The results were compared with counts in nontreated cells. Each point represents the mean ±SEM (n=3). *P<. 05 53  o  D Control EJAng II  *  i  w6 z _l o X  nr  ,T  T  N>  1  D  PERCENT [31  o  §  30  10  %  60  TIME (min)  Figure 16.  % of intracellular free choline in All-stimulated cells  Cardiac myocytes prelabeled with pH] choline for 24 hours were: treated with All (1000 nM) for indicated times. Then, cells were harvested; and the lipids extracted. Lipids were separated by thin layer chromatography. Amount of free choline was determined by assay for radioactivity as described previously. The values were compared directly to parallel experiments in which cells were not stimulated with All.  Results represent % of intracellular free  choline in All stimulated and nonstimulated cardiomyocytes,(% of total counts), mean +SEM(n=3), *P<.05  54  10 15 20 25 30 35 40 45 50 55 60 TIME (min)  Figure 17.  Time course for the All-induced release of choline into the medium by cardiomyocytes  7 day chick embryo cardiomyocytes were pretreated with f^HJ choline for 24 hours. Subsequently, cells were stimulated with 1000 nM AH.  At predetermined times, aliquots of the medium were  removed and amount of tritium counted  The cells were harvested;  and experiment continued as described for Figs. 15 and 16. Aliquots of medium were exposed to TLC and assayed for radioactivity. The results represent mean +SEM (n=3). *P<.05, **P<.01. 55  600' 0  Figure 18.  5  . 10 15 20 25 30 35 40 45 50 55 . 60 TIME (min)  Intracellular phospholipid in All induced cardiac cells (time course)  Chick cardiomyocytes pretreated -with [$H] choline were exposed to AH (1000 nM) for indicated times.  Then , cells were harvested,  lipids extracted, and the recovery of[3H] choline was determined as described under "Materials and Methods".  The amount of f^HJ  choline in the intracellular phospholipid was compared with the counts obtained in nonstimulated cells (control), which were harvested by the same time courses as stimulated cardiomyocytes. Results represent mean +SEM (n=3).  56  incubation (1, 5, 10 minutes) All did not affect the hydrolysis of PC. The intracellular choline label effluxes out of the cardiomyocytes into the medium. All, 1000 nM, produced a significant (P<.01) increase in amount of [^H] choline in the medium after incubation of cardiac cells for 30 minutes (Fig. 17). The increase was sustained significantly (P<.05) for the one hour observation period. TLC showed that most of the label in the medium, 80%, was choline. The increase in free choline arises from phosphatidylcholine hydrolysis. The amount of label in phospholipid, which is over 90% phosphatidylcholine was decreased after incubation of cells with All for 10 and 30 minutes (Fig. 18). Thus, these results suggest that All induced PC hydrolysis; However, the best response, in contrast to PtdInsP2 hydrolysis, was obtained at 30 minutes of stimulation of chick embryo cardiomyocytes.  3. An INDUCES PROTEIN TYROSINE PHOSPHORYLATION IN CHICK CARDIOMYOCYTES In previous experiments, we demonstrated that All induces PtdInsP2 breakdown and formation of InsP3, and suggested that this is correlated with tyrosine phosphorylation (genistein blocked this action). For further investigation, we evaluated the ability of All to stimulate protein tyrosine phosphorylation in our cells. Chick cardiomyocytes were incubated with All before the cells were lysed and  phosphotyrosine-containing  proteins  immunoprecipitated  with  antiphosphotyrosine monoclonal antibodies. Phosphoproteins were examined by SDS-PAGE. To investigate the effect of All on protein tyrosine phosphorylation, cells were stimulated for different times with All (1 jiM) before lysing them. Timedependence studies of All-stimulated chick cardiomyocytes showed a rapid 57  increase of protein phosphorylation in the immunoprecipitates reaching a maximum at 1 minute after All addition to the cells. To illustrate the effect of All on chick cardiomyocytes, the time course of tyrosine phosphorylation of a 70 kDa protein is shown (Fig. 19). Increased tyrosine phosphorylation of a major protein of approximately 70 kDa occurred within 1 minute of All exposure. After longer incubation  of  cells  with  All,  protein  tyrosine  phosphorylation  in  immunoprecipitates decreased reaching values slightly above the controls at 30 minutes. Protein tyrosine phosphorylation stimulated by All was almost completely blocked by pretreatment of cells for 30 minutes with Lavendustin A (10 nM), a tyrosine kinase inhibitor (Fig. 20). Lavendustin B was used as a negative control for Lavendustin A. The results for two peptides, pi95 and p70, are shown as representative of the All-stimulated immunoprecipitated proteins. The role of ATj and AT2 receptors in All-induced protein tyrosine phosphorylation was investigated as well. For these studies, cardiomyocytes were pretreated with losartan or PD 123319 prior to stimulation with All. Pretreatment of cardiomyocytes with losartan (10"5 M) substantially reduced All-induced protein tyrosine phosphorylation (84% for pi95 and 80% for p70) as shown in Fig 21. These results suggest that All-induced protein tyrosine phosphorylation in our cells is mainly mediated by ATi receptors. However, pretreatment of cardiac cells with PD123319 (10"^ M) prior to All stimulation decreased All-induced tyrosine phosphorylation (40% for pl95 and 26% for p70) (Fig. 21). Thus, a role for AT2 receptors in All-stimulated protein tyrosine phosphorylation in cardiac cells is suggested.  58  200  100 0  Figure 19.  2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 TIME (min) Time course for All-stimulated protein tyrosine phosphorylation  Chick embryo cardiomyocytes, cultured in 818A medium containing 2% FBS, were stimulatedfor the indicated times with All (1000 nM) ' and harvested with lysis buffer. Proteins were immunoprecipitated with an antiphosphotyrosine monoclonal antibody. Subsequently, proteins were resolved by SDS-polyacrylamide gel electrophoresis. Immunoreactive bands were visualized using the color reaction with Coomassie Blue. The bands were quantitated by densitometry. For the control, parallel myocyte cultures were harvested at the same times without All stimulation. The results shown are expressed as a percentage of control levels. Control values at time 0 was defined as 100%. 59  P195-*  _  pl94  pll6  p85  p70 +  SB o £  + + -<«  s •-< > >  U < .J iJ  Figure 20 a. Effect of tyrosine kinase inhibitor Lavendustin A on Allinduced protein tyrosine phosphorylation Cardiomyocytes were cultured in 818A medium containing 2% FBS. On the day of experiment, cells were left either untreated (lane 1, see inserts), or stimulated for 1 minute with 1000 nM All (lane 2), Lavendustin A (10 nM, 30 minutes) followed by All, (lane 3), or Lavendustin B (10 nM, 30 minutes) followed by All 1000 nM for 1 minute (lane 4), prior to lysis.  Then, proteins were  immunoprecipitated with anti-P-Tyr antibody and exposed to SDSPAGE. The bands were visualized by Coomassie Blue and 60  300  p195—>[gi--~i 250  1 234  Control  Angll  Lav A  LavB  Angl! 250  Control  Figure 20 b.  Angll  Lav A+Ang II  Effect of tyrosine kinase inhibitor Lavendustin A on Allinduced protein tyrosine phosphorylation (con't)  quantitaled by densitometry.  The results are expressed as a  percentage of control level (untreated cells) defined as 100%. This experiment has been repeated two times. A.Tyrosine phosphorylation of a protein of approximately 195 kDa. B. Tyrosine phosphorylation of p70. 61  p195  — — P194  pU6  p8S  p70 1P49  "2? + + o\  C »-<  o 5 t-i o Q o H  Figure 21 a.  Effects of ATj and AT2 receptor blockers on Allstimulated protein tyrosine phosphorylation  Cardiac cells, incubated in 818A medium containing 2% FBS, were treated with All 1000 nMfor 1 minute. For inhibition experiments, the cells were preincubatedfor 1 hour in the presence oflosartan or PD123319 (both at 10~5 M). Proteins were immunoprecipitated 62  250 lp 195 E2p70  =^200 o c o o *-•  2 150 c CO  < 100  50  Control  Angll  Losartan  PD123319  Angll Figure 21b.  Effects of ATj and AT2 receptor blockers on AIIstimulated protein tyrosine phosphorylation (con't)  with anti-P-Tyr antibody, separated by SDS-PAGE, visualized, and quantitated as described under "Materials and Methods".  The  results were compared with control (untreated cells) which was defined as 100%.  Data are representative of independent  experiments that were performed at least three times.  (On the  insert: 1-control; 2-AII alone; 3-AII with losartan; 4-AII with PD123319). 63  4. All STIMULATES PHOSPHATIDYLINOSITOL 3-KINASE ACTIVITY IN CHICK EMBRYO CARDIAC CELLS Ptdlns 3-kinase is thought to participate in the signal transduction cascade used by several tyrosine kinase receptors. However, Ptdlns 3-kinase activity has also been identified with G protein-associated receptors. We investigated the capacity of All to stimulate the Ptdlns 3-kinase activity in chick embryo cardiomyocytes. Cells were stimulated with AH; and the activation of Ptdlns 3kinase was measured after immunoprecipitation with antibodies to the p85 subunit of Ptdlns 3-kinase. Immunoprecipitation with an antibody to p85 showed that in All-treated chick cardiomyocytes Ptdlns  3-kinase activity was dramatically  stimulated (Fig. 22). The time course for All-induced Ptdlns 3-kinase activity revealed that after 1 rftinute of stimulation of cells with AIT the activity of Ptdlns 3-kinase was 4.5 fold increased. All stimulation of cardiomyocytes for 5 minutes produced 3.5 fold increase in the activity of Ptdlns 3-kinase. We studied the effect of a tyrosine kinase inhibitor, Lavendustin A, which was used in our previous experiments.  Pretreatment of cardiac cells with  Lavendustin A (10 nM) for 30 minutes dramatically inhibited the All-stimulated Ptdlns 3-kinase activity in our cells (Fig. 23). Thus, these results are important since they lead us to suggest that All stimulates the activity of Ptdlns 3-kinase in chick cardiomyocytes. Moreover, this activation occurs rapidly within 1 minute of incubation with AIL In addition, we can propose that tyrosine phosphorylation plays an important role in the Allinduced stimulated Ptdlns 3-kinase activity.  64  500  Control Fig. 22.  All 1 min  AH 5 mir  In vivo stimulation of Ptdlns 3-kinase by All in chick cardiac cells (time course)  Chick cardiomyocytes were cultured in 818A medium containing 2% FBS. Subsequently, cells were incubated in the presence or absence of All (1000 nM) for indicated times. Cells were .solubilized; and homogenates were immunoprecipitated with antibodies against the p85 subunit of Ptdlns 3-kinase.  The Ptdlns 3-kinase activity was  measured as described under "Materials and Methods". was  analyzed  by  autoradiography  following  Reaction  thin  layer  chromatography. The autoradiogram was exposedfor 12 hours at -80 C  [32P]PtdIns  3-monophosphate  was  quantitated  by  densitometry and using liquid scintillation counting, and is presented as percent activity compared with control in the absence of All which was defined as 100%. The actual counts for control, Ang II stimulated cells for 1 minute, and 5 minutes were 230, 1072, and800 cp.m. respectively, (autoradiograph on the insert) 65  500 CPM IS] Densitometry 400  300 c o o 200  100  Angll  Control  Fig. 23.  Lav A + Ang II  Effect of a tyrosine kinase inhibitor on All-induced Ptdlns 3-kinase activity  Cardiac cells incubated in 818A medium containing 2% FBS were stimulated with AH (1000 nM) for 1 minute.  For inhibition  experiments, cells were pre treated with Lavendustin A (10 nM) for 1 hour prior to stimulation with All.  Then, cells were lyzed,  homogenates immunoprecipitated with p85 antibodies, and the Ptdlns 3-kinase activity measured as described previously. Reaction 66  B  PIP  f  CONTROL  ANG II  LAV. A + ANG  was analyzed by autoradiography following TLC. [^PJPtdlns  3-  monophosphate was quantitated by densitometry and using liquid scintillation counting.  The results were compared with control  (nonstimulated cells), which was defined as 100%. A. Bar graph showing the effect of Lav A on All-induced Ptdlns 3kinase activity; B. Autoradiograph  67  CHAPTER IV DISCUSSION AQ is one of the most important hormones in regulation of the function of the cardiovascular system.  Its action on cardiac growth has been recently  identified. However at present, the intracellular signal transduction mechanisms or processes that occur in cardiac cells in response to All have not been fully described.  Therefore, we examined signal transduction of AQ in cardiac  myocytes, using 7 day chick embryo cardiomyocytes. The molecular action of All in many tissues involves multiple distinct processes. This hormone is a potent stimulator of the phosphoinositide pathway, consistent with its role as a calciummobilizing factor (Garcia-Sainz and Macias-Silva, 1990; Baker et al., 1989; Berridge, 1993; Marrero et al., 1994; Sadoshima and Izumo, 1993). Through a G protein-coupled receptor, All activates PLC that leads to formation of InsP3 and induces calcium release from InsP3-sensitive calcium storage sites in cardiac myocytes (Baker et al., 1989).  However, the phosphoinositide response  desensitizes within minutes in cardiac cells. It has been proposed that this is not consistent with the role of this pathway in long-term regulation of cell growth (Abdellatifetal., 1991). Beside  the  phosphatidylinositol/phospholipase  C  pathway,  other  intracellular messengers may underlie the response of cardiac cells to AIL AQ is a potent factor in the release of arachidonic acid (ARA), a messenger derived from the phospholipase A2-catalyzed hydrolysis of membrane phospholipids, in cultured cardiomyocytes (Lokuta et al., 1994). Inositol phosphates and ARA are produced by independent signaling pathways; and distinctly different All receptor subtypes mediate the production of inositol phosphates and ARA (Lokuta et al., 1994). 68  Moreover, it has been described that another major phospholipid, PC, is also hydrolyzed by PLD, PLC, and PLA2 in response to various growth factors including All, and that their breakdown products play role as second messengers (Exton, 1990; Sadoshima and Izumo, 1993). Thus, there is a complex signalling mechanisms following All stimulation in many tissues including heart. Protein tyrosine phosphorylation in response to variety of growth factors is thought to play an important role in signal transduction leading to a mitogenic response. It has not been considered to play the major role for cellular activation by PLC-linked  vasoactive  peptides.  All rapidly  enhanced  tyrosine  phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat mesangial cells (Force et al., 1991). Interaction of an agonist with its cell surface receptor frequently initiates protein tyrosine phosphorylation within that cell.  Tyrosine phosphorylation is recognized as an important  mechanism for the control of metabolism and growth in many cell types. The MAP kinases, such as p42ma , p44map, and others, are activated rapidly in response to diverse stimuli (Pelech and Sanghera, 1992; Nishida and Gotoh, 1993). However, there is a very little information about the consequences of activation of the MAP kinase cascade in cardiac myocytes. Moreover, the effect of All on protein tyrosine phosphorylation in cardiomyocytes and its role in signaling leading to the hypertrophic response remains to be studied. The enzyme which phosphorylates PtdInsP2 to PtdInsP3, Ptdlns 3-kinase, is frequently associated with receptor protein kinases. It has been suggested that the tyrosine phosphorylation and activation of the MAP kinase cascade occurs through a pathway that initially involves Ptdlns 3-kinase, PtdInsP3, and PKC-£ (or possibly other members of the atypical PKC subfamily). Even though the AQ receptors are not tyrosine kinase-linked, the ability of All to induce tyrosine phosphorylation led to our investigations that All activates Ptdlns 3-kinase and 69  suggest that subsequent MAP kinase cascade may be involved in the control of cell growth and initiation of the hypertrophic response. Studies presented here were designed to determine some aspects of the complex signal transduction pathways initiated by All in chick cardiomyocytes. First of all, the phosphoinositide hydrolysis induced by All was studied.  We  continued our studies defining the role of AH in PC breakdown and how it is related to the signaling mechanisms in our cells. Protein tyrosine phosphorylation attracted major attention in our investigations since, at present, its role in Allinduced signaling in cardiomyocytes has been poorly studied. In the present work, we studied Ptdlns 3-kinase activity in All-stimulated cardiomyocytes as well. In addition, the involvement of ATj and AT2 receptors in the complex signaling mechanisms induced by All in chick cardiac myocytes was investigated.  1. All-induced phosphoinositide hydrolysis In neonatal rat cardiac myocytes, Allen et al. reported that AH treatment leads to production of InsPj and InsP2 but not InsP3 (Allen et al., 1988), whereas another study reported that All did cause an increase in InsP3 (Abdellatif et al., 1991). InsP3 is the best indicator of Ptdlns-PLC activity. In our studies, we investigated  the  cardiomyocytes.  All-induced  accumulation  of  inositol  phosphates  in  It was found that in our system, All produced a significant  increase in the levels of InsPj, InsP2, and InsP3 within 1 minute, that was sustained for 5 minutes. These results are in agreement with the results reported by Allen et al, 1988 who showed that in neonatal rat cardiomyocytes All treatment leads to production of InsPj and InsP2, and with data reported by Abdellatif et al., 1991 showing All-induced formation of InsP3. Thus, it can be suggested that PtdInsP2-PLC activation is produced by All in chick cardiac myocytes.  In  response to All-stimulation both UISP3 and DAG are formed. The InsP3 released 70  into the cytoplasm mobilizes calcium from internal stores, whereas DAG activates PKC. Although it was shown previously that All causes hydrolysis of PtdInsP2 and production of InsP3 in chick cardiomycytes (Baker et al., 1989), we decided to repeat these experiments to make sure that this pathway is activated in our cells and to use these data for our further investigations. Since recent pharmacological studies indicated the presence of at least two isoforms of All receptors, ATi and AT2 (Dubley et al., 1991; Timmermans et al., 1993; Kambayashi et al., 1993; Mukoyama et al., 1993; Ichiki et al., 1994; Nakajima et al., 1993; Tsuzuki et al., 1994), we investigated the role of both All receptor types in this pathway using the ATj receptor antagonist losartan and AT2 receptor antagonist PD 123319. previously,  a  reasonable  From the results in many tissues described hypothesis  was  that  the  All-stimulated  phosphatidylinositol turnover is mediated by ATi receptors.  However, All-  induced InsP3 formation was not inhibited completely by losartan even at 10"^ M. Moreover, inositol phosphate formation, mostly production of InsP3, was inhibited by PD 123319 at 10"^ M. These results suggest that All-induced accumulation of InsP3 in 7 day chick embryo cardiomyocytes is mediated mainly, but not exclusively, by the ATj receptor subtype. AT2 receptors are involved in this pathway as well. Since losartan and PD 123319 are highly specific All receptor blockers, for ATj and AT2 respectively, (Duncia et al., 1992; Chiu et al., 1989; Timmermans et al., 1991; Blankley et al. 1991); and since a combination of losartan and PD 123319 almost completely blocked UISP3 formation, it can be suggested that PtdInsP2 hydrolysis in chick cardiomyocytes is mediated by both ATi and AT2 receptors. PtdInsP2 turnover is mediated by phosphoinositide-specific PLC. Three major families of PLC isozymes, P, y, and 8, have been described (Rhee and Choi, 1992). The PLC-P group of isozymes have been shown to be regulated by the Gq 71  proteins (Taylor et al., 1991).  In contrast, the PLC-y family appears to be  regulated by tyrosine phosphorylation (Kim et al., 1989).  G-protein-coupled  receptors such as the All receptors (AT2 receptor is found to be a receptor with seven membrane-spanning domains as well, Tsuzuki et al., 1994) have been thought to activate only PLC-P (Rhee and Choi, 1992). To study the possibility of All-induced tyrosine phosphorylation in chick cardiomyocytes, perhaps via activation of PLC-y by its phosphorylation, we examined whether or not All-stimulated tyrosine phosphorylation is responsible for early signal transduction events in our cells. Specifically, we wanted to know whether or not All-stimulated PtdInsP2 hydrolysis, generating InsP3 was correlated with increase in protein tyrosine phosphorylation. Genistein, a tyrosine kinase inhibitor, was used to investigate the relationship between All-mediated protein tyrosine phosphorylation and InsP3 formation. To my knowledge, tyrosine kinase inhibitors have not been used to study signal transduction in cardiomyocytes. Genistein, 10 |iM, reduced All-induced formation of InsP3 by 65%. Thus, the most important finding of these experiments is that All-stimulated phosphatidylinositol 4,5-biphosphate breakdown, generating InsP3 and stimulating calcium  mobilization,  is  temporally  correlated  with  increased  tyrosine  phosphorylation which follows a time course similar to the formation of InsP3. It can be suggested that perhaps tyrosine phosphorylation of PLC-yl occurs that leads to PtdInsP2 hydrolysis and formation of InsP3 in chick cardiomyocytes. It is difficult to explain precisely whether PLC-p, or PLC-yl, or both were activated in All-stimulated chick cardiac cells to cause increase in InsP3 accumulation since little information is available concerning which PLC isozimes are present in chick cardiac muscle cells. Thus, a novel finding of the present work is that All-induced InsP3 formation is mediated by the activation of tyrosine phosphorylation in chick 72  cardiomyocytes. This strengthens the concept that activation of G-protein-coupled receptors can lead to growth since many receptors that activate tyrosine phosphorylation are mitogens (e.g. PDGF, EGF, fibroblast growth factor). This part of our work was aimed at investigating the relationship between All-induced tyrosine phosphorylation and increased formation of a second messenger InsP3 caused by All-stimulated PtdInsP2 hydrolysis. More information about tyrosine phosphorylation in All-stimulated chick cardiomyocytes was obtained from other experiments.  2. All stimulates phosphatidylcholine hydrolysis The kinetics of All-induced InsP3 production suggest that PtdInsP2-PLC activation occurs within 1 minute and subsides within 3-5 minutes. As described previously (Sadoshima and Izumo, 1993), production of DAG occurs within 1 minute but is sustained much longer. It suggests that there is a possibility of existence of other sources of DAG after All stimulation.  Increasing evidence  suggests that another major membrane phospholipid, PC, is also hydrolyzed (Sadoshima and Izumo, 1993; Exton, 1990). Our research focused on this signal transduction pathway and demonstrated that AH stimulation produced a significant increase in intracellular choline in chick cardiomyocytes concomitant with a decrease in phospholipid indicating a substantial effect on PC hydrolysis. All increased  intracellular  choline and choline  efflux  from  the  cardiomyocytes after 30 minutes of incubation. These results are in agreement with the previously described (Sadoshima and Izumo, 1993) All-induced activation of PLD in cardiac myocytes. The prolonged formation of DAG from PC may be important in cellular mechanisms that require long term activation of PKC. All-mediated PC hydrolysis may operate through different phospholipases. PC can be hydrolyzed by PC-specific PLC, PLD, and PLA2. Increasing evidence 73  suggests that their breakdown products also act as second messengers (Exton, 1990).  PLD catalyzes the breakdown of PC yielding phosphatidic acid and  choline. PA, produced through activation of PLD or PLA2, is metabolized into DAG by PA phosphohydrolase, and can be the major pathway for the activation of PKC in some systems. PA can also be generated from PC hydrolysis by PCspecific PLC and subsequent phosphorylation by DAG kinase. However, the Allinduced formation of PA was also observed in the presence of a DAG kinase inhibitor, R59022 (Fukami and Takenawa, 1992). This suggests that the PA is mainly produced by PLD activation. The increase in production of choline in our experiments after All stimulation of chick cardiomyocytes indicated that All activated PLD and consequently the formation of PA would be increased as well. Moreover, PC-specific PLC catalyzes PC hydrolysis yielding DAG and phosphocholine.  DAG activates PKC which in turn activates PLD and PLC.  However, it has been shown that PLD and PLC can be activated independently of PKC activity through receptor-coupled G proteins.  Thus, there are two  mechanisms for activation of PLD, PKC-dependent and PKC-independent. Sadoshima and Izumo showed that PKC-independent mechanisms for All-induced PLD activation exist in cardiac myocytes.  In addition, calcium may play a  permissive or potentiating function in activating various second messenger systems, including PLC, PKC, PLD, and PLA2. We have not studied the involvement of ATi and AT2 receptors in the activation of PLD in our cells. It has been suggested that activation of PLD in cardiac cells is ATj receptor-mediated (Sadoshima and Izumo, 1993). However, as we showed in the study of inositol metabolism, All induced PtdInsP2 hydrolysis and formation of InsP3. The accumulation of I11SP3 due to AQ stimulation is an indicator of increased activity of PLC and, consequently, not only InsP3 accumulation but DAG as well. It is known that DAG is a major activator of PKC 74  which subsequently activates PLD. Thus, this long pathway can be started with All-induced activity of PtdInsP2-PLC.  As we suggested above, in chick  cardiomyocytes, the All signaling is not mediated only through AT\ receptors. Therefore, it can be suggested that in our cells, activation of PLD and subsequent phosphatidylcholine breakdown is mediated not only by the ATi receptor subtype. AT2 and perhaps other unknown All receptor types may be involved as well.  3. All induces protein tyrosine phosphorylation Recent data obtained with different cell types have demonstrated that All causes a number of short term effects which are often observed in the signaling events leading to cell proliferation in response to growth factors: G-proteincoupled PLC-mediated increase in InsP3 (Gaul et al., 1988), InsP3-induced calcium release (Ehrlich and Watras, 1988), activation of PKC (Kawahara et al., 1988), and induction of the proto-oncogenes c-fos,c-jun, c-myc (Taubman et al., 1989; Baker et al., 1989; Ji et al., 1991; Sadoshima and Izumo, 1993; Naftilan et al., 1990; Naftilan et al., 1989). However, an important feature of a growth factorinduced response, such as protein tyrosine phosphorylation, has not been described in cultured cardiac myocytes in response to AIL It is known that protein tyrosine phosphorylation is a critical component of the mitogenic response to various growth factors (Ullrich and Schlessinger, 1990; Yarden and Ullrich, 1988). The autophosphorylation on tyrosine residues of growth factor receptors enhances tyrosine kinase activity of the receptor toward other proteins which may be activated by the phosphorylation. PDGF, EGF, and colony-stimulating factor-1 receptors may phosphorylate several proteins, among them phosphatidylinositol 3kinase PLC-y, the ras GTPase-activating protein, and possibly the serine-threonine kinase. In cardiac myocytes, tyrosine kinase activation has not been considered to be a component of the response of cells to a vasoactive peptide such as AIL 75  The studies herein were designed to determine if AH induces cellular protein tyrosine phosphorylation. Stimulation of cells with All led to enhanced tyrosine phosphorylation of different  proteins, among them proteins of  approximate molecular mass 195 and 70 kDa. Phosphorylation of the two proteins was observed in chick cardiomyocytes with the peak at 1 minute and persisted for about 30 minutes.  At 30 minutes after exposure to AQ, intensity of  phosphorylation declined to near-base-line levels. A low level of constitutive tyrosine phosphorylation of a 49 kDa protein was also observed. Thus, although tyrosine kinases have not been considered to be important components of the signaling pathways associated with different phospholipase C-linked mitogenic and vasoactive peptides including All, our data revealed a characteristic pattern of immediate protein tyrosine phosphorylation after stimulation of cardiomyocytes with All. We cannot determine whether All-enhanced tyrosine phosphorylation by activating some kinases, by inactivating a phosphatase(s), or both. However, we assume it is the former since it was blocked by tyrosine kinase inhibitors. In addition, sodium orthovanadate, which was used in our experiments, inhibits cellular tyrosine phosphatases. The role  of ATj  and  AT2  receptors  in  All-induced  tyrosine  phosphorylation was investigated in our studies as well. In other cell types, it has been shown that All stimulates protein tyrosine phosphorylation via AT\ receptors (Molloy et al., 1993).  Our results showed that All-induced protein tyrosine  phosphorylation was not completely inhibited with losartan (ATj receptor blocker)  76  nor PD1213319 (AT2 receptor blocker). Thus, we can propose that All-stimulated tyrosine phosphorylation in chick cardiac myocytes is not mediated solely by AT1. The role of AT2 or other types of All receptors cannot be excluded. Our experiments were not aimed at identification of the phosphorylated proteins. However, we can form some speculations about the results. Since AQ induces tyrosine phosphorylation of a 195 kDa protein in our cells, All may activate an intermediate kinase(s) which phosphorylates the EGF receptor, HER2 (erb-B2, neu) subfamily, 190 kDa; and it can be a common pathway for signaling from receptors to the nucleus. The tyrosine phosphorylated protein that we found with apparent mass 70 kDa could be Raf-1, the 74 kDa cytoplasmic serinethreonine kinase (Morrison et al., 1989) or 75 kDa PKC< (Bogoyevich et al., 1994). PLC-P activity is modulated by G-protein. All has been reported to induce G-protein interaction in cardiac cells. As we indicated above, protein tyrosine phosphorylation  is involved  in All-stimulated  PtdInsP2  hydrolysis  and  accumulation of InsP3 in chick cardiomyocytes. We hypothesized that not only activation of PLC-P but activation of PLC-y 1 through its tyrosine phosphorylation occurs in All-stimulated cells and both of them can be involved in stimulation of" PtdInsP2 breakdown and formation of InsP3. This contention is supported by the fact that Lavendustin A partially inhibited inositol phosphates accumulation in our experiments.  This hypothesis can raise a reasonable question, why tyrosine  phosphorylation of a 145 kDa protein (molecular mass of PLC-y, Kim et al., 1989) did not show up in our experiments.  It can be explained by the activity of  phosphatases which dephosphorylate tyrosine phosphorylated proteins. Moreover, tyrosine phosphorylation of PLC-y in chick cardiomyocytes may follow another time course. In VSMC, All-induced tyrosine phosphorylation of PLC-y follows a  77  time course similar to the formation of I11SP3. This needs further investigation in cardiac myocytes. At present, the biochemical link between All receptor and protein tyrosine phosphorylation in cardiac cells is not known.  All receptors are not protein  kinases. Consequently, direct protein phosphorylation, of PLC-yl for example is not possible. It has been reported that tyrosine phosphorylation occurs in growth factor receptors that are themselves kinases such as the PDGF receptor and EGF receptor. However, tyrosine phosphorylation has also been reported for agonists that stimulate receptors that lack kinase activity. For example, All stimulates tyrosine phosphorylation of many proteins in VSMC (Tsuda et al., 1991; Marrero et al., 1993) and liver (Carter et al., 1991).  From the data obtained in our  experiments, we can suggest that one or more intracellular tyrosine kinases must be stimulated by AH-AII receptor interaction in chick cardiomyocytes.  All  binding to the All receptors may rapidly induce additional coupling to an intracellular protein tyrosine kinase. In this regard, a study by Hausdorff et al. (1992) has shown that purified pp60c"s,c can phosphorylate the a-subunit of several heterotrimeric G-proteins in vitro. Consequently, there is a possibility that AIIreceptor activation and subsequent G-protein coupling leads to signal "cross-talk", resulting in modulation of intracellular protein tyrosine kinases. Since the time course of All-induced InsP3 formation in our cells corresponds  with  the  time  course  of  All-stimulated  protein  tyrosine  phosphorylation, it can be postulated that some products of the activation of PLC by All (InsP3 and subsequent rise in [Ca?+]j, or DAG and the resulting activation of PKC) might be the cellular intermediate necessary for transduction of the initial AH-cardiomyocytes interaction into protein tyrosine phosphorylation. Although the role of PKC in tyrosine phosphorylation has not been fully clarified, it has been suggested that PKC may be an important part of the signaling pathway 78  activating tyrosine kinases in response to a phospholipase C-linked agonist such as All (Force, 1991). Furthermore, although most growth factor-induced tyrosine phosphorylation events are independent of PKC, the growth factor-induced tyrosine phosphorylation of the 42- kDa MAP2 kinase appears to be dependent upon PKC. It has been shown, for example in rat aortic smooth muscle cells, that PKC activation plays only a partial role in All-mediated protein tyrosine phosphorylation and may not be sufficient to account for AH effects on tyrosine phosphorylation (Molloy et al., 1993).  Moreover, PKC-dependent and PKC-  independent pathways of activation of the kinase(s) or inactivation of the phosphatase(s) responsible for All-enhanced tyrosine phosphorylation were confirmed previously (Force, 1991).  All appears to be able to utilize either  pathway. It has been suggested that PKC-independent pathway is modulated by the PLC A2/arachidonic acid/eicosanoid cascade. As it was proposed above, All might enhance tyrosine phosphorylation of Raf-1 in chick cardiac cells. In other cells, All activation of endogenous PKC was required for the activation of MAP kinases and hyperphosphorylation of Raf-1 (Molloy et al., 1993). Therefore, these kinases likely represent secondary or tertiary signaling molecules in the All-initiated cascade. In this regard, the Allstimulated signal transduction pathway shares common features with other mitogenic factors, including growth factors. regard  to tyrosine phosphorylation  This supports our supposition in  of pl95  that All-induced  tyrosine  phosphorylation and EGF receptor-induced signaling may share a common pathway. All has been considered as a potent hypertrophic agonist (Berk et al., 1989). Recent investigations in rat ventricular cardiac myocytes showed activation of MAP kinase by hypertrophic agonists (Bogoyevitch et al., 1994). It was shown that two isotypes of MAP kinases, p42map and 044"" , were coordinately activated 79  and suggested that this activation may be important in the hypertrophic response. The MAP-activating activity is MAPKK. In cardiomyocytes, the phosphoinositide hydrolysis stimulated by All may play a role in the upstream regulation of MAP kinases and activation of the MAP kinase cascade. So far, there is relatively little information about the events connecting transmembrane signaling, activation of the MAP kinase cascade, and the hypertrophic response in cardiac myocytes. Thus, we have shown the involvement of protein tyrosine phosphorylation in signaling mechanisms stimulated by All in cultured chick cardiomyocytes. However, future studies should be aimed at identification of activated intracellular protein tyrosine kinase(s) and their role in All-induced signal transduction. i  4. All stimulates the phosphatidylinositol 3-kinase activity Several proteins that are phosphorylated at tyrosine residues have been identified as substrate candidates for receptor tyrosine kinase; these are PLC-y, Ras GTPase activating protein, and phosphatidylinositol 3-kinase (Cantley et al., 1991). All these proteins are important in signaling systems for cell growth; however, the increased Ptdlns 3-kinase activity has been associated with G protein associated receptors (Sultan et al., 1990; Huang et al., 1991; Kucera and Rittenhouse, 1990). Phosphoinositide metabolism is an essential part of the All-induced signaling in cardiac cells. The capacity of All to increase Ptdlns 3-kinase activity was investigated in chick cardiomyocytes. In our cell type, All dramatically induced Ptdlns 3-kinase activity after 1 minute of cell stimulation that was inhibited by Lavendustin A pretreatment. Thus, we can suggest that tyrosine phosphorylation plays an important role in the induction of Ptdlns 3-kinase activity by AIL To our knowledge,this is a new pathway of signaling mechanisms in All-stimulated cardiomyocytes. 80  Ptdlns 3-kinase is implicated and appears to be centrally involved in growth factor signal transduction by association not only with receptor tyrosine kinases but with nonreceptor ones as well. It may be an alternative pathway for Ptdlns 3kinase activation in association with G proteins. Although the AH receptors do not themselves contain tyrosine kinase domains, ligand binding of these receptors can result in activation of discrete tyrosine kinases. Activation of Ptdlns 3-kinase may also involve translocation of Ptdlns 3-kinase from the cytosol to the plasma membrane where its substrates reside. As mentioned previously, Ptdlns 3-kinase phosphorylates the membrane lipids Ptdlns, Ptdlns 4-P, and Ptdlns 4,5-P2 on the third position of the inositol ring yielding Ptdlns 3-P, Ptdlns 3,4-P, and Ptdlns 3,4,5-P3 (Auger and Cantley, 1991). By phosphorylating the 3 position rather than 4 position, this kinase generates new phosphoinositides, apparently not on the pathway for production of inositol 1,4,5-triphosphate (InsP3). The role of these putative second messengers has not been yet determined completely. Recently, PKC-C, has been shown to be activated by the putative second messenger phosphatidylinositol 3,4,5-triphosphate (Nakanishi et al., 1993). Consequently, we can suggest that in cardiomyocytes All might activate the MAP kinase cascade through a pathway that initially involves phosphatidylinositol-4,5biphosphate 3-kinase, phosphatidylinositol-3,4,5-triphosphate, and PKC-£ (or possibly other members of the "atypical" PKC subfamily.  This contention is  supported by data that many growth factors, possibly including All, stimulate MAP kinases by a mechanism dependent on phosphoinositide hydrolysis. In addition, stimulation of MAP kinases might be consistent with PKC-dependent response.  Several PKC families have been described.  The "classical" PKC  subfamily (PKC-a, -pi, -p2, and -y) require Ca 2 + , phosphatidylserine, and DAG for activation. The activity of the "novel" PKC subfamily (PKC-8, -8, -n, and -0) is independent of Ca 2 + but is probably still activated by DAG. The activity of the 81  "atypical" PKC subfamily (PKC-£ and -A.) is also independent of Ca£+, and the physiological activators) of these isotypes is unclear. This subfamily differs from all the other protein kinase C isoforms in that it is not stimulated by phorbol esters or diacylglycerol (Nakanishi and Exton, 1992; Liyanage et al., 1992). Thus, PtdTns 3-kinase and PKC-£ comprise a new signal transduction pathway.  82  CHAPTER V SUMMARY AND CONCLUSIONS This work was aimed at elucidating the effect of All on cardiac cells from 7 day chick embryo cardiomyocytes. We evaluated the ability of AH to induce the formation of several different and important second messengers, namely hydrolysis of phosphatidylinositol 4,5-biphosphate and stimulation of the phosphatidylcholine  pathway.  The  involvement  of  protein  tyrosine  phosphorylation in signal transduction activated by All in chick cardiac cells as well as the effects of AH on Ptdlns 3-kinase were examined. Using ATi and AT2 receptor blockers, we tried to define the role of these two different types of All receptors in complex signaling mechanisms following All stimulation of chick cardiomyocytes. The results obtained in the present study suggest following conclusions: 1.  All stimulation of 7 day chick embryo cardiac myocytes  produced phosphoinositide hydrolysis and accumulation of inositol phosphates, mainly InsP3.  2. The kinetics of L1SP3 production, a hallmark of PtdInsP2-PLC activation, suggest that PtdInsP2-PLC activation by All in chick cardiomyocytes occurs with 1 minute.  3. The All-stimulated production of InsPi, InsP2, and InsP3 was blunted by ATj receptor blockade. However, All-induced PtdInsP2 breakdown was not inhibited completely even at 10"5 M.  In  addition, 40% of the increase in InsP3 after AH stimulation was blocked by AT2 receptor antagonist. Thus, we can suggest that 83  activation of PLC by All in chick cardiac cells is not solely mediated by ATj receptors. 4. Protein tyrosine phosphorylation induced by All might play an important role in early activation of PtdInsP2 hydrolysis and formation of L1SP3 in cardiomyocytes. Genistein, a tyrosine kinase inhibitor, partially reduced All-induced accumulation of L1SP3. Consequently, we can suppose that not only PLC-P is activated in response to All cell stimulation but PLC-yl as well.  5. In chick cardiomyocytes, All plays a potential role in stimulation of phosphatidylcholine hydrolysis.  However, the maximal  stimulation, in contrast to PtdInsP2 hydrolysis was observed at 30 minutes and remained increased over 1 hour observation period.  6. AQ induced tyrosine phosphorylation of proteins in chick cardiac myocytes with 1 minute exposure to AH. The most prominent of them were proteins with approximate molecular mass 195 kDa and 70 kDa.  7. Protein tyrosine phosphorylation induced by All was almost completely blocked by pretreatment of cells with Lavendustin A, a tyrosine kinase inhibitor.  8. Both ATi and AT2 are involved, to different extents, in AQstimulated protein tyrosine phosphorylation in chick cardiac myocytes. 84  9. Ptdlns 3-kinase activation might play an important role in signal transduction induced by All in cardiomyocytes. Its products, as a new family of second messengers, may be crucial in activation of other kinases such as, perhaps, MAP kinase activation and signaling to the nucleus. Ptdlns 3-kinase may be a key element in a new Allinduced signaling pathway connecting transmembrane signaling, activation of the MAP kinase cascade, and the hypertrophic response in cardiac muscle cells.  In summary, we have studied some aspects of signaling mechanisms stimulated by All in 7 day chick embryo cardiomyocytes. Future studies aimed at characterization of additional pathways or different parts of this complicated network of All-induced signaling mechanisms, identification of activated intracellular protein tyrosine kinase(s) and their role in All-initiated signal transduction, may provide novel cellular targets for development of specific functional antagonists.  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