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Cardiac renin-angiotensin system
Cardiac Renin-Angiotensin System Molecular and Functional Aspects
Current data support the existence of an endogenous renin-angiotensin system in the heart. Vascular angiotensin may contribute to the regulation of coronary vascular tone. Enhanced local angiotensin production in areas of vascular injury or inflammation may result in increased vasoconstriction or vasospasm. Cardiac angiotensin may adversely influence myocardial metabolism and provoke ventricular arrhythmia during ischeniia and reperfusion-induced myocardial injury. Local angiotensin may stimulate cardiac contractility. In addition, angiotensin may influence cardiac myocyte growth and niay contribute to the development of cardiac hypertrophy in hypertension. Recent data show that the pharmacologic inhibition of cardiac angiotensin converting enzyme may have important therapeutic consequences for the ischemic, hypertrophic, or failing heart.
VICTOR J. DZAU, M.D. Boston,
Massachusetts
Molecular biologic technology has led to an improved understanding of the renin-angiotensin system. It is now established that renin and angiotensinogen genes are co-expressed in many tissues [l-5]. Angiotensin produced by endogenous tissue renin-angiotensin system may exert autocrine and/or paracrine influences on local tissue function [6,7]. The vascular, adrenal, and renal renin-angiotensin systems have already been described in some detail [7,8]. The functional significances of angiotensin production in these tissue? have been examined. This report focuses on the cardiac system and (1) provides evidence for the existence of the renin-angiotensin system in cardiac tissue, (2) examines the factors that regulate its expression, (3) discusses the possible functional significance of local angiotensin synthesis in th,e heart, and (4) examines the pharmacologic effects of the inhibition of cardiac angiotensin converting enzyme.
EVIDENCE OF CARDIAC RENIN-ANGIOTENSIN From the Molecular and Cellular Vascular Research Laboratory, Division of Vascular Medicine and Atherosclerosis, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. This work was supported by NIH Grants HL35610, HL-35792, HL-19259, and HL-35252, and NIH Specialized Center of Research in Hypertension Grant HL-36568. Dr. Dzau is an Established Investigator of the American Heart Association. Requests for reprints should be addressed to Dr. Victor J. Dzau, Brigham and Women’s Hospital, Division of Vascular Medicine and Atherosclerosis, 75 Francis Street, Boston, Massachusetts 02115.
22
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11, 1988
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The evidence is derived from several lines of research: biochemical characterization of renin-angiotensin components in the heart; biochemical studies of isolated cardiac myocytes; demonstration of renin and angiotensinogen mRNAs in the heart; and production of angiotensin II by isolated perfused hearts. Renin enzymatic activity can be detected in the homogenates of mouse and rat hearts [9,10]. The optimal pH of cardiac renin is identical to that of renal renin (i.e., 6.5). We have characterized further the identity of the cardiac enzyme using renin specific antibody. The I& of the antiserum for cardiac renin is identical to the I&, for renal and submandibular gland renins (i.e., 1:50,000). Taken together, these data suggest that the car-
of Medicine
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TABLE
disc and renin enzymes are identical. In addition to renin, other components, i.e., angiotensin, its binding site, as well as angiotensin converting enzyme, have all been demonstrated in the intact heart [I I-131. To localize the presence of renin in the heart, its enzymatic activity was sought in isolated cardiac myocytes isolated from male Wistar-Kyoto rats using the collagenous cell dispersion method [9]. Sonicated myocytes contain renin activity whose authenticity was verified with reninspecific antibody. These cells also contain angiotensin and angiotensin converting enzyme. In addition, Rogers et al [ll] reported that cardiac myocytes in culture contained angiotensin II binding sites. Thus, it appears that cardiac myocytes contain an endogenous renin-angiotensin system. Biochemical detection of renin-angiotensin components does not distinguish between local synthesis versus uptake from circulation. To address this question, Northern blot hybridization was performed using renin- and angiotensinogen-complementary DNAs on RNA extracted from mouse and rat hearts. Renin RNA sequences can be detected in low quantities in the hearts of both species [2,9,10]. Cardiac renin mRNA is indistinguishable in size from renal renin mRNA (approximately 1,550 bases) [14] according to migration in agarose gel electrophoresis. Similarly, angiotensinogen RNA sequences identical in size to the liver counterpart [3,15] are present in mouse and rat hearts. In both species, cardiac angiotensinogen mRNA is expressed in greater abundance than is renin mRNA. The co-expression of renin and angiotensinogen genes is strong evidence for the existence of a local endogenous renin-angiotensin system in the heart. Recently, using in situ hybridization histochemistry, it was demonstrated that both renin and angiotensinogen mRNAs are expressed in the atrial and ventricular myocytes [16]. These preliminary results suggest that the expressions of both mRNAs is higher in the atrium than in the ventricle. Isolated perfused rat hearts have been recently employed to examine whether local angiotensin II production can occur in the beating heart. Angiotensin I was added to the perfusate, which was serum-free, and the coronary sinus effluent was collected for analysis. Angiotensin I was promptly converted to angiotensin II in the isolated heart [17], and the addition of angiotensin converting enzyme inhibitors prevented this conversion. These data support the existence of a functionally active cardiac angiotensin converting enzyme. Taken together, the foregoing findings present multiple lines of evidence supporting the existence of an endogenous cardiac renin angiotensin system. REGULATION
OF CARDIAC
Cardiac renin Cardiac angiotensinogen
SYSTEM-DZAU
Cardiac Expression Beta Adrenoceptor Stimulation
Increased
Increased
Increased
Not studied
Androgen Increased Not studied
this problem using two general approaches: (1) determination of renin and angiotensinogen mRNA levels as well as renin enzymatic activity in intact hearts of animals after various in vivo physiologic perturbations; and (2) measurement of renin activity in isolated cardiac myocytes after in vitro or in vivo perturbations. The influences of sodium status on cardiac renin and angiotensinogen expressions was examined. The hearts of sodium-depleted animals contained significantly higher renin and angiotensinogen mRNA levels than those of sodium-loaded animals [10,18]. Measurement of cardiac renin activity in sodium-depleted animals demonstrated a similar increase in enzymatic activity. Renal and adrenal renin expressions were also stimulated by sodium depletion, but submandibular or testicular renin expressions were not [lo]. Sodium depletion also activated renal and cardiac angiotensinogen mRNA expressions but not hepatic angiotensinogen [18]. Taken together, these data demonstrate that the regulation of renin-angiotensin expression is tissue-specific. This hypothesis is supported further by recent work showing that stimulation of beta adrenoceptors with isoproterenol increased cardiac and renal renin expressions but not submandibular, testicular, or adrenal renins 1191. The effect of beta adrenoceptor activation on tissue angiotensinogen expression has not been examined. My co-workers and I [20,21] have reported that submandibular gland renin levels in male mice increased markedly with puberty. This effect appears to be androgen-regulated, since castration of adult male mice results in decreases in submandibular gland renin mRNA and enzymatic activity. Recently, the role of androgen in renin expression in extrarenal tissues, including the hear-l, was examined. The results demonstrated that castration of male mice resulted in significant reductions in cardiac, adrenal, and submandibular gland renin levels [lo]. The factors that influence cardiac renin and angiotensinogen gene expressions are summarized in Table I. We [9,22] have studied the influence of a variety of antihypertensive treatments on cardiac myocyte renin activity. Rats were treated with a calcium antagonist (nifedipine), sympatholytic agent (alpha methyldopa), angiotensin converting enzyme inhibitors (CGS 14831, 14824A, and 16617), and a nonspecific vasodilator (hydralazine) for seven days. Myocytes were then isolated from rat ventri-
Not much is known about the regulation of cardiac reninangiotensin. My co-workers and I have begun research on
11, 1988
Factors Influencing Renin-Angiotensin Low Sodium
RENIN-ANGIOTENSIN
March
I
ON THE RENIN-ANGIOTENSIN
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SYMPOSIUM
TABLE
ON THE RENIN-ANGIOTENSIN
II
Functions System
of Cardiac
SYSTEM-DZAU
Renin-Angiotensin
The possible functions of the cardiac renin-angiotensin system are summarized in Table II. Local angiotensin may influence cardiac function directly (via activation of myocyte and/or coronary angiotensin receptor) or indirectly (via an enhancement of norepinephrine release from noradrenergic nerve endings) [24]. Angiotensin II’s Effect on Coronary Circulation. Angiotensin II causes intense coronary vasoconstriction. Angiotensin converting enzyme inhibitors cause vasodilation in vivo by reducing circulating angiotensin II. In addition, vasodilatory effects independent of circulating angiotensin II have been demonstrated in isolated perfused rat hearts [17,25]. This effect may be due to the blockade of tissue (coronary vascular) angiotensin II production, to the activation of a non-angiotensin-mediated mechanism such as vasodilatory prostaglandin (prostaglandin I*) biosynthesis [26], or to the inhibition of the lipoxygenase pathway [25]. Recently, it has been shown that captopril can potentiate the vasodilatory response to isosorbide dinitrate [27]. This effect appears to be due to the sulfhydryl group, which can
reverse nitrate tolerance, like the effect of N-acetyl cysteine. Angiotensin II as a Regulator of Cardiac Contractility. Previous studies demonstrated that angiotensin II possesses positive inotropic properties [28,29]. In intact animals, this effect is frequently masked by the vasoconstrictor (afterload) effect of angiotensin II during systemic administration. In support of angiotensin’s inotropic effect, Hartley and co-workers [30] observed that inhibition of the renin-angiotensin system by the renin inhibitory peptide resulted in depression of cardiac contractility in conscious monkeys. Hence, such a local system may be involved with the autocrine or paracrine control of cardiac contractility. Since angiotensin II has been shown to facilitate norepinephrine release from sympathetic nerve endings, an influence of local angiotensin on cardiac contractility via enhanced sympathetic discharge is possible. Hence, the inotropic effect of cardiac angiotensin in vivo may also be adrenergically mediated. Angiotensin II as a Regulator of Myocyte Growth. Angiotensin II has been reported to have mitogenic effects on cultured 3TB fibroblasts [31]. Recent data from Campbell-Boswell and Robertson [32] demonstrated that angiotensin II selectively increased growth and proliferation of cultured vascular myocytes grown in serum. Geisterfer and Owens [33] observed that angiotensin increased protein synthesis and cell mass of quiescent confluent vascular myocytes grown in serum-free medium. Taken together, these data suggest that angiotensin can induce hypertrophy and perhaps hyperplasia of vascular myocytes. Robertson and Khairallah [34] and Khairallah et al [35] observed that angiotensin II can be internalized by target cells with subsequent localization in mitochondria and nuclei. A recent study has suggested the existence of specific saturable receptors for angiotensin II on cell nuclei [36]. At least part of this binding site results from the presence of a chromatin acceptor for angiotensin II [37]. Direct effects of angiotensin II on nuclei to provide enhanced transcription [38] and increased solubilization of chromatin by endococcal nuclease [39] have also been suggested. Both effects point to a possible direct action of angiotensin II on gene activation and mRNA synthesis. Intracellular or local angiotensin II production may be involved with myocardial protein synthesis, cell growth, and the development of ventricular hypertrophy. To this end, this putative cardiac action of angiotensin II can potentially explain the observation that treatment with angiotensin converting enzyme inhibitors can lead to the regression of hypertensive myocardial hypertrophy [23]. Angiotensin II’s Influence on Myocardial Metabolism and Ventricular Arrhythmia during lschemia and Reperfusion Injury. Data suggesting that angiotensin II
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Coronary vasoconstriction Increased cardiac contractility Stimulation of cardiac myocyte growth (hypettrophy) Influence on myocardial metabolism during ischemia reperfusion injury Influence on ventricular arrhythmias during ischemia reperfusion injury
and and
cles, sonicated, and assayed for renin activity. Nifedipine reduced myocyte renin activity as compared with that in vehicle-treated animals. Alpha methyldopa and angiotensin converting enzyme inhibitors had no effect on myocyte renin level. In contrast, hydralazine increased myocyte renin activity. These findings may have pharmacologic implications for the mechanisms of antihypertensive drug effect on ventricular function. As will be discussed later, local angiotensin may influence the inotropic state of the myocyte and stimulate cardiac hypertrophy. Hydralazine activates cardiac renin-angiotensin. In previous reports, this drug has been shown to lower blood pressure but increase inotropy, with no effect on ventricular hypertrophy [23]. In contrast, calcium antagonists, sympathetics, and angiotensin converting enzyme inhibitors, which have been shown to reduce cardiac hypertrophy and systemic blood pressure, do not activate cardiac renin-angiotensin activity. These data suggest that cardiac renin-angiotensin may in some ways be associated with cardiac hypertrophy. FUNCTIONAL ANGIOTENSIN
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IMPLICATIONS SYSTEM
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may have deleterious effects on the ischemic heart are derived from experiments utilizing angiotensin converting enzyme inhibitors. Et-tl et al [40] demonstrated that angiotensin converting enzyme inhibition significantly reduced myocardial infarct size produced by coronary ligation in dogs. In studies of reperfusion-induced myocardial injury after coronary ligation in dogs [41], myocardial segmental function in vivo was altered from active shortening to passive lengthening despite reperfusion. Pretreatment of dogs with captopril resulted in a 40 to 60 percent return to active shortening within 60 to 120 minutes of reperfusion. Ventricular fibrillation provoked by reperfusion was prevented with captopril therapy. These data can be interpreted to indicate that angiotensin contributes to the reperfusion injury and that blockade of its production reduces the injury, or that captopril has a unique protective effect on reperfusion-induced myocardial injury. It has been postulated that the sulfhydryl groups may function as scavengers of oxygen-derived free radicals and thus reduce injury associated with reperfusion. Current data support both possibilities. In isolated perfused rat hearts, Linz and co-workers [17] demonstrated that reperfusion ischemia was aggravated by perfusion with angiotensin I and angiotensin II. Angiotensin l-enhanced ventricular fibrillation was completely eliminated by angiotensin converting enzyme inhibition, either by pretreatment with oral ramipril or by perfusion with ramiprilate. Angiotensin converting enzyme inhibition also improved cardiac hemodynamics, dp/d&,, left ventricular pressure, myocardial oxygen consumption, and coronary flow. In the perfusate of angiotensin converting enzyme inhibitor-treated hearts, lactate dehydrogenase and creatinine kinase activities and lactate production were reduced. Myocardial tissue levels of glycogen, ATP, and creatinine phosphate were increased, while lactate was decreased in the ramipril-pretreated hearts. Since ramipril does not contain a sulfhydryl group, it may be deduced that the cardioprotective effects of ramipril are due to local angiotensin converting enzyme inhibition. This is in contradistinction to the data of Westlin and Mullane [41], who were unable to observe a cardioprotective effect by teprotide or enalaprilate during reperfusion injury in open-chest anesthetized dogs. However, Westlin and Mullane did observe cardioprotective effects with other sulfhydryl-containing compounds, N-acetyl cysteine and N-2 mercaptopropionyl glycine, which do not have angiotensin converting enzyme inhibitory properties. PHARMACOLOGIC SIGNIFICANCE OF CARDIAC ANGIOTENSIN CONVERTING ENZYME INHIBITION To a large extent, this has been reviewed in the previous section. Table Ill summarizes the pharmacologic effects and the mechanisms of action of angiotensin converting enzyme inhibitors in the heart. These can be divided ac-
March
11, 1988
TABLE
Ill
ON THE RENIN-ANGIOTENSIN
SYSTEM-DZAU
Effects of Angiotensin Converting Enzyme Inhibitors on the Heart
Vascular effects Coronary vasodilafion Systemic arterial and arteriolar
dilation (afterload reduction) Increase in venous capacitance (preload reduction) Prevention of nitrate tolerance and potentiation of nitrate effect Cardiac effects Negative inotropism (?) Negative chronotropism (?) Regression of ventricular hypertrophy (cardioprotection) Attenuation of reperfusion injury-induced ventricular arrhythmias (cardioprotection) Prevention of ventricular enlargement (cardioprotection)
cording to the site of action-i.e., vascular versus myocardial-and the mechanism of action. Angiotensin converting enzyme inhibitors may affect cardiac function by inhibiting circulating angiotensin or tissue angiotensin production, by activating prostaglandin biosynthesis, by preventing bradykinin degradation, or by inhibiting catecholamine release. In addition, certain angiotensin converting enzyme inhibitors may have unique effects attributable to a unique property of the specific drug, e.g., the presence of the sulfhydryl group. On the basis of the foregoing data, differential effects of various angiotensin converting enzyme inhibitors may be expected in the treatment of various cardiac diseases such as ischemic heart disease, congestive heart failure, and cardiac hypet-trophy [42]. In addition, differences in penetration, distribution, and duration of action of different angiotensin converting enzyme inhibitors in the heart, blood vessels, and other tissues may also differentiate the various agents. A given drug’s chemical structure and physiochemical properties may play an important role in determining these effects. For example, it is conceivable that a lipophilic angiotensin converting enzyme inhibitor penetrates and accumulates in the central nervous system more readily and exerts a greater effect on the brain renin-angiotensin system than a hydrophilic compound. Similarly, other physicochemical properties may influence the distribution of these drugs in other tissues. indeed, captopril and enalapril appear to differ in the relative concentration and duration of actions in certain tissues. Cohen and Kurz [I 31 demonstrated that, although the duration of inhibition of vascular angiotensin converting enzyme is comparable with both agents, enalapril has a larger duration of inhibition of cardiac and renal angiotensin converting enzyme activities. Are these differences clinically relevant? Although a clinical study designed specifically to compare the relative tissue effects of various angiotensin converting enzyme inhibitors has not been performed, the recent results of Packer et al [43] suggest that such differences may exist.
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SYMPOSIUM
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These investigators compared the effects of captopril and enalapril in patients with congestive heart failure. Both drugs appeared to produce comparable reductions in systemic vascular resistance, but the cardiac indexes of patients receiving captoprii were significantly higher than those of patients receiving enalapril when these parameters were measured immediately before the administration of the next dose of the drugs. Considering the differences in the duration of cardiac angiotensin converting enzyme inhibitory effect between these two agents, the differences in cardiac index may be due to captopril’s shorter duration of action on cardiac angiotensin converting enzyme as compared with enalapril’s duration [16]. Taken together, these data suggest that angiotensin converting enzyme inhibitors exert their principal action at local tissue sites. Local tissue action of an angiotensin converting enzyme inhibitor can influence the activity profile of that agent. Furthermore, additional effects at tissue sites may be influenced by certain unique structural properties of the compounds.
COMMENTS Current data support the existence of an endogenous renin-angiotensin system in the heart. Local angiotensin may influence myocardial function. Local angiotensin may adversely influence myocardial metabolism and provoke ventricular arrhythmia during ischemia and reperfusioninduced myocardial injury. Cardiac angiotensin may also stimulate cardiac contractility and contribute to the development of cardiac hypertrophy in hypertension. Angiotensin may contribute to the regulation of coronary vascular tone and flow. Enhanced vascular angiotensin production in areas of injury or inflammation may result in increased vasoconstriction or vasospasm. Inhibition of cardiac angiotensin converting enzyme may have important pharmacologic consequences in the ischemic, hypertrophic, or failing heart. ACKNOWLEDGMENT I thank Ms. Donna
MacDonald
for secretarial
assistance.
REFERENCES 1.
Field LJ, McGowan gene specificity
2.
Dzau VJ, Ellison KE, Brody T, lngelfinger J, Pratt RE: A comparative study of the distributions of renin and angiotensin messenger ribonucleic acids in rat and mouse tissues. Endocrinology 1987; 120: 6: 2334-2338. Ohkubo H, Nakayama K, Tanaka T, Nakanishi S: Tissue distribution of rat angiotensinogen mRNA and structural analysis of its heterogeneity. J Biol Chem 1986; 261: 319-323. Dzau VJ, lngelfinger J, Pratt RE, Ellison KE: Identification of renin and angiotensinogen messenger RNA sequences in mouse and rat brains. Hypertension 1986; 8: 544-548. Campbell DJ: The site of angiotensin production. J Hypertens 1985; 3: 199-207. Dzau VJ: Significance of vascular renin angiotensin pathways. Hypertension 1986; 8: 553-559. Campbell DJ: Circulating and tissue angiotensin systems. J Clin Invest 1987; 79: l-6. Dzau VJ: Implications of local angiotensin production in cardiovascular physiology and pharmacology. Am J Cardiol 1987; 59 (suppl A): 59A-65A. Dzau VJ, Re RN: Evidence for the existence of renin in the heart. Circulation 1987; 73 (suppl I): l-134-1-136. Dzau VJ, Brody T, Ellison KE, Pratt RE, lngelfinger JR: Tissuespecific regulation of renin expression in the mouse. Hypertension 1987; 9 (suppl Ill): 111-36-111-41. Rogers TB, Gaa SH, Allen IS: Identification and characterization of functional angiotensin II receptors on cultured heart myocytes. J Pharmacol Exp Ther 1986; 236: 438-444. Baker KM, Campanile CP, Traclite GJ, Peach MJ: Identification and characterization of the rabbit angiotensin II myocardial receptor. Circ Res 1984; 54: 286-293. Cohen ML, Kurz KD: Angiotensin converting enzyme inhibition in tissues from spontaneously hypertensive rats after treatment with captopril or MK421. J Pharmacol Exp Ther 1982;
1984;
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RA, Dickinson DP, Gross KW: Tissue and of mouse renin expression. Hypertension 15.
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26.
27.
254-255.
63-69.
N, Knopf JL, Gross
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with gene duplications, correlates with renin levels in the mouse submaxillary gland. Cell 1982; 30: 205. Lynch KR, Simnad VT, Ben-Ari ET, Maniatis T, Zinn K, Garrison JC: Localization of preangiotensinogen messenger RNA sequences in the rat brain. Hypertension 1986; 8: 540-543. Dzau VJ, Fon E, lngelfinger JR: In situ hybridization localization of renin and angiotensinogen mRNA in the rat heart (in preparation). Linz W, Scholkens BA, Han YF: Beneficial effects of the converting enzyme inhibitor, ramipril, in ischemic rat hearts. J Cardiovasc Pharmacol 1986; 8: (suppl 10) S91-S99. Dzau VJ, Zou, Pratt RE: Unpublished data. Dzau VJ, Ellison KE, Ouellette AJ: Expression and regulation of renin in the mouse heart (abstr). Clin Res 1985; 33: 181A. Dzau VJ, lngelfinger JR, Pratt RE: Regulation of tissue renin and angiotensin gene expressions, J Cardiovasc Pharmacol 1986; 8 (suppl 10): Sll-S16. Pratt RE, Dzau VJ, Ouellette AJ: Influence of androgen on translatable renin mRNA in mouse submandibular gland. Hypertension 1984; 6: 615-621. Dzau VJ, Brody T, Re R: Unpublished data. Tarazi RC, Fouad FM: Reversal of cardiac hypertrophy. Hypertension 1984; 6 (suppl Ill): 111-140-111-145. Zimmerman BG: Adrenergic facilitation by angiotensin: does it serve a physiological function? Clin Sci 1981; 60: 343-348. Van Gilst WH, deGraeff PA, Wessling H, deLangen CDJ: Reduction of reperfusion arrhythmias in the ischemic isolated rat heart by angiotensin converting enzyme inhibitors: a comparison of captopril, enalapril and HOE 498. J Cardiovasc Pharmacol 1986; 8: 722-726. Zusman RM: Renin and non-renin-mediated antihypertensive action of converting enzyme inhibition. Kidney Int 1984; 25: 969-983. Van Gilst WH, deGraeff PA, Scholtens E, deLangen CDJ, Wessling H: Potentiation of isosorbide dinitrate-induced coronary dilation by captopril. J Cardiovasc Pharmacol 1987; 9:
The
KW: A DNA polmorphism,
American
Journal
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of Medicine
28.
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Baker
84 (suppl
KM,
3A)
Khosla
MC:
Cardiac
and
vascular
actions
of
SYMPOSIUM
29. 30. 31.
32.
33.
34.
35.
36.
decapeptide angiotensin analogs. J Pharmacol Exp Ther 1986; 239: 790-796. Koch-Weser J: Nature of the inotropic action of angiotensin on ventricular myocardium. Circ Res 1965; 16: 230-237. Hartley H, Kwan H, Zusman R, Haber E: Personal communication. Ganten D, Schelling P, Flugel RM, Ganten U: Effect of angiotensin and an angiotensin antagonist on iso-renin and cell growth in 3TB mouse cells. Int Res Commun Med Sci 1975; 3: 327333. Campbell-Boswell M, Robertson AL: Effects of angiotensin II and vasopressin on human smooth muscle cells in vitro. Exp Mol Pathol 1981; 35: 265-276. Geisterfer AAT, Owens GK: Hypertrophic response of cultured vascular smooth muscle cells to angiotensin II (abstr). Fed Proc 1986; 45: 584. Robertson AL, Khairallah PA: Angiotensin: rapid localization in nuclei of smooth and cardiac muscle. Science 1971; 172: 1138-1140. Khairallah PA, Robertson AL, Davil OD: Effect of angiotensin II on DNA, RNA and protein synthesis. IN: Genest J, Koiw E, eds. Hypertension. New York: Springer-Verlag, 1972; 212220.
37.
38. 39.
40.
41.
42.
43.
ON THE RENIN-ANGIOTENSIN
SYSTEM-DZAU
Re RN, MacPhee A, Fallon J: Specific nuclear binding of angiotensin II by rat liver and spleen nuclei. Clin Sci 1981; 61: S245-S250. Re RN, Vizardi DL, Brown J, Bryan SE: Angiotensin II receptors in chromatin fragments generated by micrococcal nuclease. Biochem Biophys Res Commun 1984; 119: 220-227. Re RN, Parab M: Effect of angiotensin II on RNA synthesis by isolated nuclei. Life Sci 1984; 34: 647-651. Re RN, LaBiche RA, Bryan SE: Nuclear hormone-mediated changes in chromatin solubility. Biochem Biophys Res Commun 1983; 110: 61-66. Ertl G, Alexander RW, Kloner RA: Interactions between coronary occlusion and the renin-angiotensin system in the dog. Basic Res Cardiol 1983; 78: 515-533. Westlin W, Mullane K: Does captopril attenuate reperfusioninduced myocardial dysfunction by scavenging free radicals? Circulation (in press). Dzau VJ: Tissue renin-angiotensin system: potential basis for differentiation of converting enzyme inhibitors. Circulation (in press). Packer M, Lee WH, Yshak M, Medina N: Comparison of captopril and enalapril in patients with severe chronic heart failure. N Engl J Med 1986; 315: 847-853.
Discussion have been identified that will cause hypertrophy of a single myocyte. Are you aware of any studies in which angiotensin converting enzyme has been inhibited and then stimulators of hypertrophy have been placed in the medium? Does angiotensin converting enzyme inhibition block the hypertrophy of a single cell or not? Dr. Dzau: I am not aware of any such studies. I am not aware of any measurements in isolated neonatal myocytes. Recently, Starksen et al showed that norepinephrine stimulates neonatal myocyte hypertrophy and at the same time increases c-myc proto-oncogene expression. But converting enzyme and angiotensin II have not been examined. It should be done.
Dr. Swynghedauw:
Could you tell us how you can get 100 pg of RNA for your Northern blot? Could you give us details of the oncogene expression induced by angiotensin? Dr. Diau: In our early experiments, we believed that there was a need to increase the total RNA in our Northern blots. We increased the glyoxyl concentration and solubilized the RNA. The transfer is probably not complete. Recently, with smaller quantities of RNA, we can demonstrate the same findings. Dr. Kokko: A number of laboratories have developed a heart hypertrophy model using an isolated culture cell. The beauty of that is that it removes the afterload effect. By recombinant technology, a number of compounds
March
11, 1988
The
American
Journal
of Medicine
Volume
84
(suppl 3A)
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Current data support the existence of an endogenous renin-angiotensin system in the heart. Vascular angiotensin may contribute to the regulation of coronary vascular tone. Enhanced local angiotensin production in areas of vascular injury or inflammation may result in increased vasoconstriction or vasospasm. Cardiac angiotensin may adversely influence myocardial metabolism and provoke ventricular arrhythmia during ischeniia and reperfusion-induced myocardial injury. Local angiotensin may stimulate cardiac contractility. In addition, angiotensin may influence cardiac myocyte growth and niay contribute to the development of cardiac hypertrophy in hypertension. Recent data show that the pharmacologic inhibition of cardiac angiotensin converting enzyme may have important therapeutic consequences for the ischemic, hypertrophic, or failing heart.
VICTOR J. DZAU, M.D. Boston,
Massachusetts
Molecular biologic technology has led to an improved understanding of the renin-angiotensin system. It is now established that renin and angiotensinogen genes are co-expressed in many tissues [l-5]. Angiotensin produced by endogenous tissue renin-angiotensin system may exert autocrine and/or paracrine influences on local tissue function [6,7]. The vascular, adrenal, and renal renin-angiotensin systems have already been described in some detail [7,8]. The functional significances of angiotensin production in these tissue? have been examined. This report focuses on the cardiac system and (1) provides evidence for the existence of the renin-angiotensin system in cardiac tissue, (2) examines the factors that regulate its expression, (3) discusses the possible functional significance of local angiotensin synthesis in th,e heart, and (4) examines the pharmacologic effects of the inhibition of cardiac angiotensin converting enzyme.
EVIDENCE OF CARDIAC RENIN-ANGIOTENSIN From the Molecular and Cellular Vascular Research Laboratory, Division of Vascular Medicine and Atherosclerosis, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. This work was supported by NIH Grants HL35610, HL-35792, HL-19259, and HL-35252, and NIH Specialized Center of Research in Hypertension Grant HL-36568. Dr. Dzau is an Established Investigator of the American Heart Association. Requests for reprints should be addressed to Dr. Victor J. Dzau, Brigham and Women’s Hospital, Division of Vascular Medicine and Atherosclerosis, 75 Francis Street, Boston, Massachusetts 02115.
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March
11, 1988
The
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The evidence is derived from several lines of research: biochemical characterization of renin-angiotensin components in the heart; biochemical studies of isolated cardiac myocytes; demonstration of renin and angiotensinogen mRNAs in the heart; and production of angiotensin II by isolated perfused hearts. Renin enzymatic activity can be detected in the homogenates of mouse and rat hearts [9,10]. The optimal pH of cardiac renin is identical to that of renal renin (i.e., 6.5). We have characterized further the identity of the cardiac enzyme using renin specific antibody. The I& of the antiserum for cardiac renin is identical to the I&, for renal and submandibular gland renins (i.e., 1:50,000). Taken together, these data suggest that the car-
of Medicine
Volume
84
(suppl 3A)
SYMPOSIUM
TABLE
disc and renin enzymes are identical. In addition to renin, other components, i.e., angiotensin, its binding site, as well as angiotensin converting enzyme, have all been demonstrated in the intact heart [I I-131. To localize the presence of renin in the heart, its enzymatic activity was sought in isolated cardiac myocytes isolated from male Wistar-Kyoto rats using the collagenous cell dispersion method [9]. Sonicated myocytes contain renin activity whose authenticity was verified with reninspecific antibody. These cells also contain angiotensin and angiotensin converting enzyme. In addition, Rogers et al [ll] reported that cardiac myocytes in culture contained angiotensin II binding sites. Thus, it appears that cardiac myocytes contain an endogenous renin-angiotensin system. Biochemical detection of renin-angiotensin components does not distinguish between local synthesis versus uptake from circulation. To address this question, Northern blot hybridization was performed using renin- and angiotensinogen-complementary DNAs on RNA extracted from mouse and rat hearts. Renin RNA sequences can be detected in low quantities in the hearts of both species [2,9,10]. Cardiac renin mRNA is indistinguishable in size from renal renin mRNA (approximately 1,550 bases) [14] according to migration in agarose gel electrophoresis. Similarly, angiotensinogen RNA sequences identical in size to the liver counterpart [3,15] are present in mouse and rat hearts. In both species, cardiac angiotensinogen mRNA is expressed in greater abundance than is renin mRNA. The co-expression of renin and angiotensinogen genes is strong evidence for the existence of a local endogenous renin-angiotensin system in the heart. Recently, using in situ hybridization histochemistry, it was demonstrated that both renin and angiotensinogen mRNAs are expressed in the atrial and ventricular myocytes [16]. These preliminary results suggest that the expressions of both mRNAs is higher in the atrium than in the ventricle. Isolated perfused rat hearts have been recently employed to examine whether local angiotensin II production can occur in the beating heart. Angiotensin I was added to the perfusate, which was serum-free, and the coronary sinus effluent was collected for analysis. Angiotensin I was promptly converted to angiotensin II in the isolated heart [17], and the addition of angiotensin converting enzyme inhibitors prevented this conversion. These data support the existence of a functionally active cardiac angiotensin converting enzyme. Taken together, the foregoing findings present multiple lines of evidence supporting the existence of an endogenous cardiac renin angiotensin system. REGULATION
OF CARDIAC
Cardiac renin Cardiac angiotensinogen
SYSTEM-DZAU
Cardiac Expression Beta Adrenoceptor Stimulation
Increased
Increased
Increased
Not studied
Androgen Increased Not studied
this problem using two general approaches: (1) determination of renin and angiotensinogen mRNA levels as well as renin enzymatic activity in intact hearts of animals after various in vivo physiologic perturbations; and (2) measurement of renin activity in isolated cardiac myocytes after in vitro or in vivo perturbations. The influences of sodium status on cardiac renin and angiotensinogen expressions was examined. The hearts of sodium-depleted animals contained significantly higher renin and angiotensinogen mRNA levels than those of sodium-loaded animals [10,18]. Measurement of cardiac renin activity in sodium-depleted animals demonstrated a similar increase in enzymatic activity. Renal and adrenal renin expressions were also stimulated by sodium depletion, but submandibular or testicular renin expressions were not [lo]. Sodium depletion also activated renal and cardiac angiotensinogen mRNA expressions but not hepatic angiotensinogen [18]. Taken together, these data demonstrate that the regulation of renin-angiotensin expression is tissue-specific. This hypothesis is supported further by recent work showing that stimulation of beta adrenoceptors with isoproterenol increased cardiac and renal renin expressions but not submandibular, testicular, or adrenal renins 1191. The effect of beta adrenoceptor activation on tissue angiotensinogen expression has not been examined. My co-workers and I [20,21] have reported that submandibular gland renin levels in male mice increased markedly with puberty. This effect appears to be androgen-regulated, since castration of adult male mice results in decreases in submandibular gland renin mRNA and enzymatic activity. Recently, the role of androgen in renin expression in extrarenal tissues, including the hear-l, was examined. The results demonstrated that castration of male mice resulted in significant reductions in cardiac, adrenal, and submandibular gland renin levels [lo]. The factors that influence cardiac renin and angiotensinogen gene expressions are summarized in Table I. We [9,22] have studied the influence of a variety of antihypertensive treatments on cardiac myocyte renin activity. Rats were treated with a calcium antagonist (nifedipine), sympatholytic agent (alpha methyldopa), angiotensin converting enzyme inhibitors (CGS 14831, 14824A, and 16617), and a nonspecific vasodilator (hydralazine) for seven days. Myocytes were then isolated from rat ventri-
Not much is known about the regulation of cardiac reninangiotensin. My co-workers and I have begun research on
11, 1988
Factors Influencing Renin-Angiotensin Low Sodium
RENIN-ANGIOTENSIN
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ON THE RENIN-ANGIOTENSIN
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TABLE
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II
Functions System
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SYSTEM-DZAU
Renin-Angiotensin
The possible functions of the cardiac renin-angiotensin system are summarized in Table II. Local angiotensin may influence cardiac function directly (via activation of myocyte and/or coronary angiotensin receptor) or indirectly (via an enhancement of norepinephrine release from noradrenergic nerve endings) [24]. Angiotensin II’s Effect on Coronary Circulation. Angiotensin II causes intense coronary vasoconstriction. Angiotensin converting enzyme inhibitors cause vasodilation in vivo by reducing circulating angiotensin II. In addition, vasodilatory effects independent of circulating angiotensin II have been demonstrated in isolated perfused rat hearts [17,25]. This effect may be due to the blockade of tissue (coronary vascular) angiotensin II production, to the activation of a non-angiotensin-mediated mechanism such as vasodilatory prostaglandin (prostaglandin I*) biosynthesis [26], or to the inhibition of the lipoxygenase pathway [25]. Recently, it has been shown that captopril can potentiate the vasodilatory response to isosorbide dinitrate [27]. This effect appears to be due to the sulfhydryl group, which can
reverse nitrate tolerance, like the effect of N-acetyl cysteine. Angiotensin II as a Regulator of Cardiac Contractility. Previous studies demonstrated that angiotensin II possesses positive inotropic properties [28,29]. In intact animals, this effect is frequently masked by the vasoconstrictor (afterload) effect of angiotensin II during systemic administration. In support of angiotensin’s inotropic effect, Hartley and co-workers [30] observed that inhibition of the renin-angiotensin system by the renin inhibitory peptide resulted in depression of cardiac contractility in conscious monkeys. Hence, such a local system may be involved with the autocrine or paracrine control of cardiac contractility. Since angiotensin II has been shown to facilitate norepinephrine release from sympathetic nerve endings, an influence of local angiotensin on cardiac contractility via enhanced sympathetic discharge is possible. Hence, the inotropic effect of cardiac angiotensin in vivo may also be adrenergically mediated. Angiotensin II as a Regulator of Myocyte Growth. Angiotensin II has been reported to have mitogenic effects on cultured 3TB fibroblasts [31]. Recent data from Campbell-Boswell and Robertson [32] demonstrated that angiotensin II selectively increased growth and proliferation of cultured vascular myocytes grown in serum. Geisterfer and Owens [33] observed that angiotensin increased protein synthesis and cell mass of quiescent confluent vascular myocytes grown in serum-free medium. Taken together, these data suggest that angiotensin can induce hypertrophy and perhaps hyperplasia of vascular myocytes. Robertson and Khairallah [34] and Khairallah et al [35] observed that angiotensin II can be internalized by target cells with subsequent localization in mitochondria and nuclei. A recent study has suggested the existence of specific saturable receptors for angiotensin II on cell nuclei [36]. At least part of this binding site results from the presence of a chromatin acceptor for angiotensin II [37]. Direct effects of angiotensin II on nuclei to provide enhanced transcription [38] and increased solubilization of chromatin by endococcal nuclease [39] have also been suggested. Both effects point to a possible direct action of angiotensin II on gene activation and mRNA synthesis. Intracellular or local angiotensin II production may be involved with myocardial protein synthesis, cell growth, and the development of ventricular hypertrophy. To this end, this putative cardiac action of angiotensin II can potentially explain the observation that treatment with angiotensin converting enzyme inhibitors can lead to the regression of hypertensive myocardial hypertrophy [23]. Angiotensin II’s Influence on Myocardial Metabolism and Ventricular Arrhythmia during lschemia and Reperfusion Injury. Data suggesting that angiotensin II
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Coronary vasoconstriction Increased cardiac contractility Stimulation of cardiac myocyte growth (hypettrophy) Influence on myocardial metabolism during ischemia reperfusion injury Influence on ventricular arrhythmias during ischemia reperfusion injury
and and
cles, sonicated, and assayed for renin activity. Nifedipine reduced myocyte renin activity as compared with that in vehicle-treated animals. Alpha methyldopa and angiotensin converting enzyme inhibitors had no effect on myocyte renin level. In contrast, hydralazine increased myocyte renin activity. These findings may have pharmacologic implications for the mechanisms of antihypertensive drug effect on ventricular function. As will be discussed later, local angiotensin may influence the inotropic state of the myocyte and stimulate cardiac hypertrophy. Hydralazine activates cardiac renin-angiotensin. In previous reports, this drug has been shown to lower blood pressure but increase inotropy, with no effect on ventricular hypertrophy [23]. In contrast, calcium antagonists, sympathetics, and angiotensin converting enzyme inhibitors, which have been shown to reduce cardiac hypertrophy and systemic blood pressure, do not activate cardiac renin-angiotensin activity. These data suggest that cardiac renin-angiotensin may in some ways be associated with cardiac hypertrophy. FUNCTIONAL ANGIOTENSIN
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IMPLICATIONS SYSTEM
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may have deleterious effects on the ischemic heart are derived from experiments utilizing angiotensin converting enzyme inhibitors. Et-tl et al [40] demonstrated that angiotensin converting enzyme inhibition significantly reduced myocardial infarct size produced by coronary ligation in dogs. In studies of reperfusion-induced myocardial injury after coronary ligation in dogs [41], myocardial segmental function in vivo was altered from active shortening to passive lengthening despite reperfusion. Pretreatment of dogs with captopril resulted in a 40 to 60 percent return to active shortening within 60 to 120 minutes of reperfusion. Ventricular fibrillation provoked by reperfusion was prevented with captopril therapy. These data can be interpreted to indicate that angiotensin contributes to the reperfusion injury and that blockade of its production reduces the injury, or that captopril has a unique protective effect on reperfusion-induced myocardial injury. It has been postulated that the sulfhydryl groups may function as scavengers of oxygen-derived free radicals and thus reduce injury associated with reperfusion. Current data support both possibilities. In isolated perfused rat hearts, Linz and co-workers [17] demonstrated that reperfusion ischemia was aggravated by perfusion with angiotensin I and angiotensin II. Angiotensin l-enhanced ventricular fibrillation was completely eliminated by angiotensin converting enzyme inhibition, either by pretreatment with oral ramipril or by perfusion with ramiprilate. Angiotensin converting enzyme inhibition also improved cardiac hemodynamics, dp/d&,, left ventricular pressure, myocardial oxygen consumption, and coronary flow. In the perfusate of angiotensin converting enzyme inhibitor-treated hearts, lactate dehydrogenase and creatinine kinase activities and lactate production were reduced. Myocardial tissue levels of glycogen, ATP, and creatinine phosphate were increased, while lactate was decreased in the ramipril-pretreated hearts. Since ramipril does not contain a sulfhydryl group, it may be deduced that the cardioprotective effects of ramipril are due to local angiotensin converting enzyme inhibition. This is in contradistinction to the data of Westlin and Mullane [41], who were unable to observe a cardioprotective effect by teprotide or enalaprilate during reperfusion injury in open-chest anesthetized dogs. However, Westlin and Mullane did observe cardioprotective effects with other sulfhydryl-containing compounds, N-acetyl cysteine and N-2 mercaptopropionyl glycine, which do not have angiotensin converting enzyme inhibitory properties. PHARMACOLOGIC SIGNIFICANCE OF CARDIAC ANGIOTENSIN CONVERTING ENZYME INHIBITION To a large extent, this has been reviewed in the previous section. Table Ill summarizes the pharmacologic effects and the mechanisms of action of angiotensin converting enzyme inhibitors in the heart. These can be divided ac-
March
11, 1988
TABLE
Ill
ON THE RENIN-ANGIOTENSIN
SYSTEM-DZAU
Effects of Angiotensin Converting Enzyme Inhibitors on the Heart
Vascular effects Coronary vasodilafion Systemic arterial and arteriolar
dilation (afterload reduction) Increase in venous capacitance (preload reduction) Prevention of nitrate tolerance and potentiation of nitrate effect Cardiac effects Negative inotropism (?) Negative chronotropism (?) Regression of ventricular hypertrophy (cardioprotection) Attenuation of reperfusion injury-induced ventricular arrhythmias (cardioprotection) Prevention of ventricular enlargement (cardioprotection)
cording to the site of action-i.e., vascular versus myocardial-and the mechanism of action. Angiotensin converting enzyme inhibitors may affect cardiac function by inhibiting circulating angiotensin or tissue angiotensin production, by activating prostaglandin biosynthesis, by preventing bradykinin degradation, or by inhibiting catecholamine release. In addition, certain angiotensin converting enzyme inhibitors may have unique effects attributable to a unique property of the specific drug, e.g., the presence of the sulfhydryl group. On the basis of the foregoing data, differential effects of various angiotensin converting enzyme inhibitors may be expected in the treatment of various cardiac diseases such as ischemic heart disease, congestive heart failure, and cardiac hypet-trophy [42]. In addition, differences in penetration, distribution, and duration of action of different angiotensin converting enzyme inhibitors in the heart, blood vessels, and other tissues may also differentiate the various agents. A given drug’s chemical structure and physiochemical properties may play an important role in determining these effects. For example, it is conceivable that a lipophilic angiotensin converting enzyme inhibitor penetrates and accumulates in the central nervous system more readily and exerts a greater effect on the brain renin-angiotensin system than a hydrophilic compound. Similarly, other physicochemical properties may influence the distribution of these drugs in other tissues. indeed, captopril and enalapril appear to differ in the relative concentration and duration of actions in certain tissues. Cohen and Kurz [I 31 demonstrated that, although the duration of inhibition of vascular angiotensin converting enzyme is comparable with both agents, enalapril has a larger duration of inhibition of cardiac and renal angiotensin converting enzyme activities. Are these differences clinically relevant? Although a clinical study designed specifically to compare the relative tissue effects of various angiotensin converting enzyme inhibitors has not been performed, the recent results of Packer et al [43] suggest that such differences may exist.
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These investigators compared the effects of captopril and enalapril in patients with congestive heart failure. Both drugs appeared to produce comparable reductions in systemic vascular resistance, but the cardiac indexes of patients receiving captoprii were significantly higher than those of patients receiving enalapril when these parameters were measured immediately before the administration of the next dose of the drugs. Considering the differences in the duration of cardiac angiotensin converting enzyme inhibitory effect between these two agents, the differences in cardiac index may be due to captopril’s shorter duration of action on cardiac angiotensin converting enzyme as compared with enalapril’s duration [16]. Taken together, these data suggest that angiotensin converting enzyme inhibitors exert their principal action at local tissue sites. Local tissue action of an angiotensin converting enzyme inhibitor can influence the activity profile of that agent. Furthermore, additional effects at tissue sites may be influenced by certain unique structural properties of the compounds.
COMMENTS Current data support the existence of an endogenous renin-angiotensin system in the heart. Local angiotensin may influence myocardial function. Local angiotensin may adversely influence myocardial metabolism and provoke ventricular arrhythmia during ischemia and reperfusioninduced myocardial injury. Cardiac angiotensin may also stimulate cardiac contractility and contribute to the development of cardiac hypertrophy in hypertension. Angiotensin may contribute to the regulation of coronary vascular tone and flow. Enhanced vascular angiotensin production in areas of injury or inflammation may result in increased vasoconstriction or vasospasm. Inhibition of cardiac angiotensin converting enzyme may have important pharmacologic consequences in the ischemic, hypertrophic, or failing heart. ACKNOWLEDGMENT I thank Ms. Donna
MacDonald
for secretarial
assistance.
REFERENCES 1.
Field LJ, McGowan gene specificity
2.
Dzau VJ, Ellison KE, Brody T, lngelfinger J, Pratt RE: A comparative study of the distributions of renin and angiotensin messenger ribonucleic acids in rat and mouse tissues. Endocrinology 1987; 120: 6: 2334-2338. Ohkubo H, Nakayama K, Tanaka T, Nakanishi S: Tissue distribution of rat angiotensinogen mRNA and structural analysis of its heterogeneity. J Biol Chem 1986; 261: 319-323. Dzau VJ, lngelfinger J, Pratt RE, Ellison KE: Identification of renin and angiotensinogen messenger RNA sequences in mouse and rat brains. Hypertension 1986; 8: 544-548. Campbell DJ: The site of angiotensin production. J Hypertens 1985; 3: 199-207. Dzau VJ: Significance of vascular renin angiotensin pathways. Hypertension 1986; 8: 553-559. Campbell DJ: Circulating and tissue angiotensin systems. J Clin Invest 1987; 79: l-6. Dzau VJ: Implications of local angiotensin production in cardiovascular physiology and pharmacology. Am J Cardiol 1987; 59 (suppl A): 59A-65A. Dzau VJ, Re RN: Evidence for the existence of renin in the heart. Circulation 1987; 73 (suppl I): l-134-1-136. Dzau VJ, Brody T, Ellison KE, Pratt RE, lngelfinger JR: Tissuespecific regulation of renin expression in the mouse. Hypertension 1987; 9 (suppl Ill): 111-36-111-41. Rogers TB, Gaa SH, Allen IS: Identification and characterization of functional angiotensin II receptors on cultured heart myocytes. J Pharmacol Exp Ther 1986; 236: 438-444. Baker KM, Campanile CP, Traclite GJ, Peach MJ: Identification and characterization of the rabbit angiotensin II myocardial receptor. Circ Res 1984; 54: 286-293. Cohen ML, Kurz KD: Angiotensin converting enzyme inhibition in tissues from spontaneously hypertensive rats after treatment with captopril or MK421. J Pharmacol Exp Ther 1982;
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with gene duplications, correlates with renin levels in the mouse submaxillary gland. Cell 1982; 30: 205. Lynch KR, Simnad VT, Ben-Ari ET, Maniatis T, Zinn K, Garrison JC: Localization of preangiotensinogen messenger RNA sequences in the rat brain. Hypertension 1986; 8: 540-543. Dzau VJ, Fon E, lngelfinger JR: In situ hybridization localization of renin and angiotensinogen mRNA in the rat heart (in preparation). Linz W, Scholkens BA, Han YF: Beneficial effects of the converting enzyme inhibitor, ramipril, in ischemic rat hearts. J Cardiovasc Pharmacol 1986; 8: (suppl 10) S91-S99. Dzau VJ, Zou, Pratt RE: Unpublished data. Dzau VJ, Ellison KE, Ouellette AJ: Expression and regulation of renin in the mouse heart (abstr). Clin Res 1985; 33: 181A. Dzau VJ, lngelfinger JR, Pratt RE: Regulation of tissue renin and angiotensin gene expressions, J Cardiovasc Pharmacol 1986; 8 (suppl 10): Sll-S16. Pratt RE, Dzau VJ, Ouellette AJ: Influence of androgen on translatable renin mRNA in mouse submandibular gland. Hypertension 1984; 6: 615-621. Dzau VJ, Brody T, Re R: Unpublished data. Tarazi RC, Fouad FM: Reversal of cardiac hypertrophy. Hypertension 1984; 6 (suppl Ill): 111-140-111-145. Zimmerman BG: Adrenergic facilitation by angiotensin: does it serve a physiological function? Clin Sci 1981; 60: 343-348. Van Gilst WH, deGraeff PA, Wessling H, deLangen CDJ: Reduction of reperfusion arrhythmias in the ischemic isolated rat heart by angiotensin converting enzyme inhibitors: a comparison of captopril, enalapril and HOE 498. J Cardiovasc Pharmacol 1986; 8: 722-726. Zusman RM: Renin and non-renin-mediated antihypertensive action of converting enzyme inhibition. Kidney Int 1984; 25: 969-983. Van Gilst WH, deGraeff PA, Scholtens E, deLangen CDJ, Wessling H: Potentiation of isosorbide dinitrate-induced coronary dilation by captopril. J Cardiovasc Pharmacol 1987; 9:
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KW: A DNA polmorphism,
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KM,
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Khosla
MC:
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and
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decapeptide angiotensin analogs. J Pharmacol Exp Ther 1986; 239: 790-796. Koch-Weser J: Nature of the inotropic action of angiotensin on ventricular myocardium. Circ Res 1965; 16: 230-237. Hartley H, Kwan H, Zusman R, Haber E: Personal communication. Ganten D, Schelling P, Flugel RM, Ganten U: Effect of angiotensin and an angiotensin antagonist on iso-renin and cell growth in 3TB mouse cells. Int Res Commun Med Sci 1975; 3: 327333. Campbell-Boswell M, Robertson AL: Effects of angiotensin II and vasopressin on human smooth muscle cells in vitro. Exp Mol Pathol 1981; 35: 265-276. Geisterfer AAT, Owens GK: Hypertrophic response of cultured vascular smooth muscle cells to angiotensin II (abstr). Fed Proc 1986; 45: 584. Robertson AL, Khairallah PA: Angiotensin: rapid localization in nuclei of smooth and cardiac muscle. Science 1971; 172: 1138-1140. Khairallah PA, Robertson AL, Davil OD: Effect of angiotensin II on DNA, RNA and protein synthesis. IN: Genest J, Koiw E, eds. Hypertension. New York: Springer-Verlag, 1972; 212220.
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Re RN, MacPhee A, Fallon J: Specific nuclear binding of angiotensin II by rat liver and spleen nuclei. Clin Sci 1981; 61: S245-S250. Re RN, Vizardi DL, Brown J, Bryan SE: Angiotensin II receptors in chromatin fragments generated by micrococcal nuclease. Biochem Biophys Res Commun 1984; 119: 220-227. Re RN, Parab M: Effect of angiotensin II on RNA synthesis by isolated nuclei. Life Sci 1984; 34: 647-651. Re RN, LaBiche RA, Bryan SE: Nuclear hormone-mediated changes in chromatin solubility. Biochem Biophys Res Commun 1983; 110: 61-66. Ertl G, Alexander RW, Kloner RA: Interactions between coronary occlusion and the renin-angiotensin system in the dog. Basic Res Cardiol 1983; 78: 515-533. Westlin W, Mullane K: Does captopril attenuate reperfusioninduced myocardial dysfunction by scavenging free radicals? Circulation (in press). Dzau VJ: Tissue renin-angiotensin system: potential basis for differentiation of converting enzyme inhibitors. Circulation (in press). Packer M, Lee WH, Yshak M, Medina N: Comparison of captopril and enalapril in patients with severe chronic heart failure. N Engl J Med 1986; 315: 847-853.
Discussion have been identified that will cause hypertrophy of a single myocyte. Are you aware of any studies in which angiotensin converting enzyme has been inhibited and then stimulators of hypertrophy have been placed in the medium? Does angiotensin converting enzyme inhibition block the hypertrophy of a single cell or not? Dr. Dzau: I am not aware of any such studies. I am not aware of any measurements in isolated neonatal myocytes. Recently, Starksen et al showed that norepinephrine stimulates neonatal myocyte hypertrophy and at the same time increases c-myc proto-oncogene expression. But converting enzyme and angiotensin II have not been examined. It should be done.
Dr. Swynghedauw:
Could you tell us how you can get 100 pg of RNA for your Northern blot? Could you give us details of the oncogene expression induced by angiotensin? Dr. Diau: In our early experiments, we believed that there was a need to increase the total RNA in our Northern blots. We increased the glyoxyl concentration and solubilized the RNA. The transfer is probably not complete. Recently, with smaller quantities of RNA, we can demonstrate the same findings. Dr. Kokko: A number of laboratories have developed a heart hypertrophy model using an isolated culture cell. The beauty of that is that it removes the afterload effect. By recombinant technology, a number of compounds
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