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The distribution of angiotensin II type 1 receptors, and the tissue renin-angiotensin systems
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The distribution of angiotensin I1 type 1 receptors, and the tissue renin-angiotensin systems Since its discovery, the functions of the renin-angiotensin system (RAS) have attracted a great deal of attention, and the roles it plays under rmal conditions, and in disease, acquire a epening significance with every year. In general, .=RAS has been considered largely in terms of its es in sodium and potassium homeostasis and the ]ulation of blood pressure. The continued ~uisition of information on the distribution of giotensin receptors, however, emphasizes that r interpretation needs to be widened, and it is w clear that angiotensin II has an array of lctions in the tissues, which are unrelated to its ~temic roles. This paracrine function is brought )out by the existence of complete, localized tissue ',ASs, which respond to physiological demand independently from the systemic system.
Gavin P. Vinson Department of Biochemistry, St. Bartholomew's and The London School of Medicine and Dentistry, Queen Mary and Westfield College, Mile End Road, London,
The renin-angiotensin system (RAS) is the name given to the system of substrates and enzymes that gives rise to the active circulating hormone, angiotensin II (Fig. 1). Renin is a proteolytic enzyme secreted into the blood stream by the juxtaglomerular cells of the kidney. It cleaves a substrate, angiotensinogen, which is a component of the a2-globulin fraction of the plasma proteins, to yield a decapeptide called angiotensin I. Two amino acids from the carboxyl terminus of this peptide are, in turn, cleaved by angiotensin converting enzyme (ACE) to produce the active octapeptide angiotensin II. Other enzymes may act on angiotensin I or II to yield angiotensin III (des-Aspl-angiotensin II) r angiotensin 1-7, but it is generally thought that angiotensin II has e greatest biological significance.
stribution and functions of angiotensin II receptors -Iistofically, angiotensin II has been recognized for its central role nammalian electrolyte homeostasis and haemodynamics 1, particuy through the regulation of aldosterone secretion and cardiovascufunction. At a pathological level, the RAS has a significant role lypertension: ACE antagonists are a valuable tool in the treatment :his disease 2, and both ACE and the angiotensinogen genes are . . . . . . . . . . . . . . . . . .
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Elsevier Science Lid 1357 - 4310/95/$9.50
35
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MOI~ECI~JI_AR MEDICINE
TODAY
and AT2 receptors are present in rat brain, but the concentration of AT~ receptors is particularly high in newborn and young animals 6. AT2, but not AT 1, receptors are expressed in rat ovary 7, while other tissues conAntagonist - A drug that blocks the actions of a hormone or other tain both AT l and A T 2 subtypes in varying proportions 4. agent, usually by preventing binding to its receptor. The use of a recently developed monoclonal antibody to the AT~ receptor in immunohistochemical studies has contributed to the emergHaemodynamics- The dynamics blood flow and blood pressure. ing evidence indicating that the AT t receptor is widespread ~. It is presMyocardiac hypertropy - A condition characterized by an increase in ent in the adrenal zona glomerulosa, where most of the receptor is heart muscle mass, internalized within the cell (Fig. 2), and in the vasculature, where it is particularly prominent on the luminal surface of the endothelium (Fig. Zona glomerulosa - The site of aldosterone secretion in the adrenal 3). However, AT 1 receptors are also widely present in secretory epigland. thelia; for example, in the rat uterus or human breast (Fig. 4). Another novel finding was the localization of the AT~ receptor in the tails of developing sperm in rat testis, and in the tails of rat and human ejacuconsidered to be candidates contributing to the pathogenesis of hyper- lated sperm9, as well as in Leydig cells (Fig. 5). tension and cardiovascular disease 3. The seemingly ubiquitous presence of the AT~ receptor in the Many studies have contributed to the characterization of angiotensin endothelium and in secretory epithelia conveys further messages, receptors, and the two main subtypes are classified as AT t and AT2, exemplified by the comparison of samples of normal breast tissue and according to their sensitivity to the angiotensin II antagonists, Dup753 turnouts. Normal breast, which is well supplied with blood vessels and (AT~ selective) and CGP42112A and PD123177 (AT2 selective)4. Most ductal tissue is, clearly, also abundantly provided with AT~ receptors. of the known physiological functions of angiotensin II appear to be The development of breast turnours clearly involves both of these cell mediated via the AT~ receptor, but the widespread incidence of the AT2 types, and so such tumours exhibit a massive density of AT~ receptor receptor suggests that it has specific roles. staining: the MCF-7 breast tumour cell line also contains AT, receptors. There are significant variations in the tissue distribution of the AT~ That AT~ receptors may have a functional bearing on the development and AT2 receptors. In the rat adrenal, most of the angiotensin receptors of the disease is obvious: the role of angiotensin as a purely trophic in the zona glomerulosa are of the AT] type, and those in the medulla hormone, stimulating tissue growth, surely deserves more attention 1°. are predominantly AT2: only AT~ receptors were detected in the bovine Taken together, all of these data support earlier work in suggesting adrenal cortex 5. The human uterus expresses predominantly AT 2 multiple functions for angiotensin. This, in turn, suggests that angioreceptors, while the rat uterus also contains AT~ receptors. Both AT~ tensin II should be made available in the tissues in a manner that is independent from the systemic RAS. Localized tissue renin-angiotensin systems have been found in the adrenal gland and other organs. The Angiotensinogen existence of such systems in addition to the systemic system makes sense if the generation Asp-Arg-VaI-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-VaI-Tyr-Ser-globulin of angiotensin locally in proximity to its receptors ensures that the tissue-specific functions - Renin are regulated precisely, without concomitant inappropriate actions in unrelated tissues. For these reasons, the tissue RAS now attract Angiotensin I a great deal of interest, but continue to present problems in interpretation. The physiological Asp-Arg-VaI-Tyr-Ile-His-Pro-Phe-His-Leu significance of the localized tissue generation of angiotensin II is sometimes unclear. For example, why is the adrenal tissue RAS Angiotensin converting enzyme required in addition to systemically available angiotensin II, as both appear to respond to the same stimuli? In addition, it remains to be Angiotensin II determined how the tissue distinguishes between angiotensin from these two sources. Asp-Arg-VaI-Tyr-Ile-His-Pro-Phe
Glossary
~
~.~LPeP
Angiotensin III Arg-VaI-Tyr-Ile-His-Pro-Phe Figure 1. The renin-angiotensin system.
i
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tidases
Angiotensin 1-7 Asp-Arg-VaI-Tyr-Ile-His-P
Distribution of the tissue renin-angiotensin system Perhaps because of the problems outlined above, the concept of tissue RAS has met with some resistance. Currently, however, there is a great deal of evidence in support of the existence of these systems, particularly in the adrenal gland, gonads, kidney, heart, pituitary and brain ~°. Some brief examples are discussed below.
MOLEC'[JI.ARMEDICINETODAY
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Figure 2. Immunofluorescence staining of AT1 receptors in rat glomeruIosa cells using monoclonal antibody 6313/G2. Scale bar = 25 I~m.
Adrenal gland Renin and angiotensin are formed primarily in the zona glomerulosa n. The amount of both renin mRNA and angiotensin 1I are regulated by dietary sodium t2, and the latter also by potassium t3. The local RAS is thought to have trophic effects on the glomerulosa and, in particular, to play a significant role in the regulation of aldosterone synthase L4.Similar systems exist in human adrenal glands ~.
Reproductive organs Renin has been identified in the Leydig cells of the rat testis, and renin and low levels of angiotensinogen mRNAs have been isolated from rat and mouse testes t6. Angiotensin converting enzyme is also present, and the generation of angiotensin II has been shown, for example, in the human testis and the rat epididymis ~7. In the ovary, prorenin, renin and ACE have been identified by hybridization and immunological and enzyme assay methods 18. In humans, ovarian prorenin and renin originate in the thecal cells ~9. In rats, angiotensin II immunostaining has been detected in the corpora lutea, and the stromal and thecal cells of larger follicles 2°. Evidence also exists for a discrete uterine RAS (Ref. 21).
Cardiovascular system Genes for all of the elements of the RAS are expressed in the heart 22"23,and angiotensinogen and renin have both been detected in neonatal rat heart 24. Both angiotensins I and II are secreted by cultured rat cardiac myocytesz~, and experiments on the expression of the renin gene in various tissues show that it is regulated in the heart in a specific manner 25. The importance of the tissue RAS in relation to cardiovascular disease has been discussed, and one possibility is that it is specifically involved in myocardiac hypertrophy and heart failure 26. The concept has been challenged however, and, according to other researchers, heart tissue does not produce renin, and the results of others are attributed to methodological failure 27.
Brain Renin gene expression has been described in rat, mouse and human brain tissue ~6'28, and its regulation is independent from kidney renin production25. The angiotensinogen gene is also expressed in rat brain 29, and renin, angiotensinogen and ACE have all been shown to be
Figure 3. Immunofluorescence staining of AT 1 receptors, using monoclonal antibody 6313/G2, in endothelial cells (EC) of: (a) bovine adrenal arteriole; and (b) human renal artery. The brightness of the lamina elastica (le) in (b) is attributable to autofluorescence. Abbreviation: L, lumen. Scale bar = 25 ~m. Reproduced, with permission, from Ref. 8.
present s°. Brain angiotensin is regulated in rats independently of circulating levels 3~, although not every study has shown this 32. It is difficult to dismiss the weight of these data as artefactual, as some have done27. In particular, the identification of specific mRNAs for the RAS components in tissues cannot be disregarded lightly. Nevertheless, some authors have expressed reservations because of the practical difficulties associated with distinguishing between systemic and tissue components of the system, and the failure, in some cases, to identify critical specific functions for the tissue RAS (Refs 10,1 1). However, on this point too, there is now convincing evidence (see below).
Specific roles for tissue renin-angiotensin system The t~tsk here is to identify functions that would be inappropriately met by the systemic RAS. The difficulty has been recognized, and in the case of both the adrenal and the cardiovascular tissue systems, it has been proposed that the systemic RAS is concerned with acute homeostatic functions, whereas the tissue system controls longer-term trophic events n°'26"33.It is thought this would be consistent with the likely dynamics of angiotensin availability from the two sources. Other quite different functions have been described, however. For example, angiotensin II stimulates ovarian oestrogen production 34, while m
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RAS present in both male and female reproductive tracts is that it exists to provide angiotensin II to sperm throughout their transit through the reproductive tract. Clearly, this is an angiotensin II function that requires the regulated production of angiotensin II in an entirely independent manner from the systemic source.
Uterus
Figure 4. Immunofluorescence staining of AT 1 receptors, using monoclonal antibody 6313/G2, in the secretory epithelium of human breast tissue. Scale bar = 50 Ixm.
The presence of angiotensin II receptors in the uterus has long been recognized, although only AT2 type receptors are found in humans 36. However, the role of angiotensin II has not been clearly defined. Angiotensin II receptors are present in uterine smooth muscle cells and, in rat uterus, angiotensin II induces uterine contractions 37, in addition to its vasoactive effects38. Uterine contractions are inhibited both by the specific AT1 antagonist, DuP753, and by monoclonal antibody 6313/G2. The antibody identifies the receptor in the luminal epithelium of the uterus, as well as in muscle and vascular sites, and it is possible that angiotensin II has specific actions on the functions of these cells as well.
Brain In brain, one set of actions of angiotensin lI is concerned with drinking behaviour. Intracerebrospinal injection of angiotensin lI rapidly induces increased water intake in rats, and a slower increase in NaCl intake 39, which is blocked by AT 1, but not by AT2, receptor antagonists 4°. All of these examples clearly indicate the requirement for tissue renin-angiotensin systems to be regulated independently from the systemic system.
Compartmentalization of tissue renin-angiotensin systems
Figure 5. Immunofluorescence staining of AT 1 receptors, using monoclonal antibody 6313/G2, in Leydig cells, primary spermatogonia and spermatid tails of rat testis. Scale bar = 25 }~m. Reproduced, with permission, from Ref. 9.
the angiotensin II antagonist saralasin inhibited ovulation in perfused rat ovaries35. We can add three examples of tissue differentiated angiotensin II functions that are more clearly distinguishable from those of systemic angiotensin II, and which evidently demand independent regulation.
Spermatozoa Following our development of a specific monoclonal antibody for the angiotensin II AT~ receptor (6313/G2), we identified AT 1 receptors in several components of the reproductive tract, including vascular and secretory epithelial components. In addition, however, we also identified the receptor in the tails of developing rat spermatozoa (Fig. 5), and ejaculated rat and human sperm. These receptors are indeed functional: our data show increased sperm motility in human ejaculates after five minutes exposure to angiotensin II (Ref. 9). Immunoblotting of solubilized sperm components after sodium dodecylsulphate (SDS) gel electrophoresis confirms the specificity of the immunorecognition: a single sperm component reacts with the antibody, and this component has a molecular weight that is identical with that of the angiotensin AT~ receptor from the adrenal cortex. Accordingly, an interpretation of the tissue m
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Clearly, it has to be established how the tissue systems, and the responses of the angiotensin receptors, are maintained separately from systemic events: in some cases there may be physical barriers; for example, the blood-brain or blood-testis barriers. In addition, we need to know how the delivery of angiotensin II to its receptors is regulated in the paracrine situation. There are several aspects to this. One is that the acquisition of precise data on the cellular localization of the sites of production of the components of the renin-angiotensin system, their regulation, and their relationship to the sites of angiotensin II action forms an absolute requirement for the interpretation of these systems, and this has not been achieved in most cases. As an example, it is clear that, in the adrenal cortex, the glomerulosa itself is the site of angiotensin II production. The question arises as to the relationship between the cellular production of angiotensin II and its receptors. If both occur in the same cell type, what implications does this hold for receptor occupancy, and the capacity of the adrenal gland to respond to physiological demand? One possibility is that angiotensin receptors are in some way sequestrated intracellularly so that their availability to locally generated angiotensin II can be regulated. However, the existence of non-receptor-binding proteins, as well as different receptor isoforms, their internalization, processing and recycling, and different mechanisms of intracellular signalling may all contribute to this separation of function. These questions need to be addressed.
Conclusions Our understanding of the role and functions of the RAS in the tissues needs to be widened significantly. • Angiotensin receptors are ubiquitous, and are widespread in many tissues that are not historically associated with angiotensin II action. In addition, several tissues contain all of the elements required for the
M O L E C U L A R MEDICINE T O D A Y
generation of angiotensin II in situ, and these systems are regulated separately f r o m the systemic system. It therefore follows that the locally generated angiotensin II acts in a paracrine fashion to elicit responses that m a y be c o n c e r n e d with tissue g r o w t h and repair, or with other functions that are highly specific, and which are not necessarily related to the functions of the systemic RAS. • M e c h a n i s m s must exist to ensure that the paracrine target cells respond in a specific m a n n e r to locally generated angiotensin II, and differently, if at all, to the systemic system. Such m e c h a n i s m s m a y include sequestration of the receptors f r o m systemic angiotensin II, partly by m e a n s of physical barriers, such as the b l o o d - b r a i n , or b l o o d - t e s t i s barriers, and also p e r h a p s by local regulation of angiotensin II receptor expression, and internalization, or by differential responses of different receptor subtypes. By analogy with other paracrine hormones, such as the insulin-like growth factors, it is also possible that there are angioter~sin II-binding proteins that regulate h o r m o n e delivery to the sites of action. • G i v e n the complexity of these mechanisms, it is conceivable that the system can be perturbed in disease. From the examples we give here, it is obvious that malfunction of tissue RAS could give rise to any of a spectrum of quite unrelated problems. In view of the widespread incidence of the AT~ receptor in epithelial tissue, and the potential for i n v o l v e m e n t of the tissue system in g r o w t h - p r o m o t i n g and t i s s u e - m o d e l l i n g events, one further striking possibility is that the R A S m a y be i n v o l v e d in cancer. The presence of AT~ receptors in breast tumours is consistent with this view. Future work should address these hypotheses.
The outstanding questions • Why do tissues generate the hormone angiotensin II in addition to receiving it from the bloodstream? • How do target tissues distinguish between tissue-generated and circulating angiotensin II? • What specific functions do the tissue-generated angiotensin II perform? • What factors regulate tissue generation of angiotensin 1I, and how is this different from the regulation of the circulating hormone?
References 1 Peach. M.T. (1977) Renin-angiotensin system: biochemistry and mechanism of action, Physiol. Rev. 57,313-370 2 Ferrario, C.M. (1990) The renin-angiotensin system: importance in physiology and pharmacology, J. Cardiovasc. Pharmacol. 15 (Suppl. 3), S 1-$5 3 Jeunemaitre. X. et al. (1992) Molecular basis of human hypertension: role of angiotensinogen, Cell 71, 169-180 4 Smith, R.D. et al. (1992) Analysis of angiotensin-II receptor antagonists. Annu. Rev Pharmacol. 58.1883-1888 5 Balla, I". et al. (1991) Angiotensin II receptor subtypes and biological responses in the adrenal cortex and medulla, Mol. Pharmacol. 40, 401-406 a Millan. M.A. et al. (1991) Differential distribution of AT~ and AT, receptor subtypes in the rat brain during development. Proc. Natl Acad. Sci. USA 88. 11440-11444 7 Pucell, A.G. etal. (1991) Biochemical properties of the ovarian granulosa-cell type2-angiotensin-II receptor, Endocrinology 128, 1947-1959 8 Barker, S. et al. (1993) A mouoclonal antibody to a conserved sequence in the extracellular domain recognizes the angiotensin II AT, receptor in mammalian tissues. J. Mol. Endocrinol. 11,241-245 9 Vinson. G.R et al. (1995) Type I angiotensin II (ATe) receptors in sperm. J. Endocrinol. 144, 36%378
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10 Phillips, M.1. et al. (1993) Levels of angiotensin and molecular biology of the tissue renin-angiotensin systems, Regul. Peptides 43, 1-20 II Mulrow, RJ. (1992) Adrenal renin: regulation and function, Fmnt. Neumendocrinol. 13, 47~50 12 Kilbr, I. et al. (1991) The effect of sodium intake on angiotensin content of the rat adrenal gland, Endocrinology 128, 1277-1284 13 Nakamaru, M. et al. (1985) A role for the adrenal renin-angiotensin system in the regulation of potassium-stimulated aldosterone production, Endocrinology 117. 1772-1778 14 Sander, M. et al. (1994) Role of adrenal renin in the regulation of adrenal steroidogenesis by corticotropin, Prec. Natl Acad. Sci. USA 91,148-152 15 Wang, Y. et al. (1992) Regulation of renin gene-expression in rat adrenal zona glomerulosa cells, tfypertension 20, 776-781 16 Dzau, V.J. et al. (1987) A comparative study of the distributions of renin and angiotensin messenger ribonucleic acids in rat and mouse, Endocrinology 120. 2334-2338 17 Okuyama, A. ctal. (1988) Renin-angiotensin system, Alzh. Androl. 21,169-180 18 Howard, R.B. et al. (1992) Rat ovarian angiotensin 11 receptors, renin, and angiotensin I-converting enzyme during pregnancy and the postpartum period, Biol. Reprod. 47,925-930 19 Paulson, R.J. et al. (1989) Ovarian renin production in vitro and in vivo characterization and clinical correlation, Fertil. Steril. 51,634-638 20 Lightman, A. et al. (1988) Immunocytochemical localization of angiotensin II immunoreactivity and demonstration of angiotensin II binding in the rat ovary, Am. J. Obstet. Gynecol. 159, 526-530 21 Squires, EM. et al. (1992) Evidence for a role for a uterine renin-angiotensin system in decidualization in rats, J. Reprod. Fertil. 95,791-802 22 Lee, Y.A. et al. (1993) The cardiac renin angiotensin system. From basic research to clinical relevance, Arzneimittelforschung 43,201-206 23 Sawa et al. (1992) Expression of the angiotensinogen gene and localization of its protein in the human heart, Circulation 86. 138-146 24 Dostal, D.E. et al. (1992) lntracardiac detection of angiotensinogen and renin: a localized renin-angiotensin system in neonatal heart, Am. J. Physiol. 263, C838~2850 25 Okura, T. et al. (1992) Renal and extra-renal renin gene expression in spontaneously hypertensive rats, Blood Pressure (Suppl.) 3.6-11 26 MacFadyen, R.J. (1993) Role of the circulating and tissue-based renin angiotensin system in the development of heart failure: implications for therapy, Cardiology 83, 38-48 27 yon Lutteroni, N. et al. (1994) Renin is not synthesized by cardiac and extrarenal vascular tissues. A review of experimental evidence, Circulation 89, 458-470 28 Dzau, V.J. et al. (1987) Tissue-specific regulation of renin expression in the mouse, Hypertension 9, III36-11141 29 Gyurko, R. et al. (1993) Antisense inhibition of AT, receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin, Regul. Peptides 49, 167-I 74 30 Chai, S.Y. etal. (1993) Localization of components of the renin-angiotensin system and site of action of inhibitors, Arzneimittelforschung 43, 214-221 31 Trolliet, M.R. et al. (1992) The effect of chronic bilateral nephrectomy on plasma and brain angioteusin, .L Hypertension 10, 2%36 32 Morishita, R. et al. (1993) Role of tissue renin-angiotensin system in two-kidney. one-clip hypertensive rats, Am. J. Physiol. 264, F510-F514 33 Dzan, V.J. (1993) Tissue renin-angiotensin system in myocardial hypertrophy and failure, Arch. Intern. Med. 53,937-942 34 Speth, R.C. et al. (1988) Distribution of angiotensin converting enzyme and angiotensin ll-receptor binding sites in the rat ovary, Biol. Reprod. 38,695-702 35 Peterson, C.M et al. (1993) The angiotensin II antagonist saralasin inhibits ovulation in the perfused rat ovary, Am. J. Obstet. Gynecol. 168,242-245 36 Whitebread, S. et al. (1989) Preliminary biochemical characterization of two angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun. 163,284-291 37 Calixto, J.B. et al. (1991) Effects of endothelins, Bay K 8644 and other oxytocics in non-pregnant and late pregnant rat isolated uterus, Eu~ J. Pharmacol. 192, 10%116 38 Allen, J. et al. (1991) Effects of vasodilators on isolated human uteroplacental arteries, Obstet. Gynecol. 77,765-771 39 Fitzsimons, J.T. (1972) Thirst, Physiol. Rev. 52, 468 561 40 Beresford, M.J. et al. (1992) Intracerebroventricular angiotensin II-induced thirst and sodium appetite in rat are blocked by the AT, receptor antogonist, Losartan t DuP753), but not by the AT2 antagonist, CGP42112B, Exp. Physiol. 77, 761-764 l
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'U,J.. MEO, _',NE I"ODAY
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w
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The distribution of angiotensin I1 type 1 receptors, and the tissue renin-angiotensin systems Since its discovery, the functions of the renin-angiotensin system (RAS) have attracted a great deal of attention, and the roles it plays under rmal conditions, and in disease, acquire a epening significance with every year. In general, .=RAS has been considered largely in terms of its es in sodium and potassium homeostasis and the ]ulation of blood pressure. The continued ~uisition of information on the distribution of giotensin receptors, however, emphasizes that r interpretation needs to be widened, and it is w clear that angiotensin II has an array of lctions in the tissues, which are unrelated to its ~temic roles. This paracrine function is brought )out by the existence of complete, localized tissue ',ASs, which respond to physiological demand independently from the systemic system.
Gavin P. Vinson Department of Biochemistry, St. Bartholomew's and The London School of Medicine and Dentistry, Queen Mary and Westfield College, Mile End Road, London,
The renin-angiotensin system (RAS) is the name given to the system of substrates and enzymes that gives rise to the active circulating hormone, angiotensin II (Fig. 1). Renin is a proteolytic enzyme secreted into the blood stream by the juxtaglomerular cells of the kidney. It cleaves a substrate, angiotensinogen, which is a component of the a2-globulin fraction of the plasma proteins, to yield a decapeptide called angiotensin I. Two amino acids from the carboxyl terminus of this peptide are, in turn, cleaved by angiotensin converting enzyme (ACE) to produce the active octapeptide angiotensin II. Other enzymes may act on angiotensin I or II to yield angiotensin III (des-Aspl-angiotensin II) r angiotensin 1-7, but it is generally thought that angiotensin II has e greatest biological significance.
stribution and functions of angiotensin II receptors -Iistofically, angiotensin II has been recognized for its central role nammalian electrolyte homeostasis and haemodynamics 1, particuy through the regulation of aldosterone secretion and cardiovascufunction. At a pathological level, the RAS has a significant role lypertension: ACE antagonists are a valuable tool in the treatment :his disease 2, and both ACE and the angiotensinogen genes are . . . . . . . . . . . . . . . . . .
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Elsevier Science Lid 1357 - 4310/95/$9.50
35
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e v i
e
"I~, ~
MOI~ECI~JI_AR MEDICINE
TODAY
and AT2 receptors are present in rat brain, but the concentration of AT~ receptors is particularly high in newborn and young animals 6. AT2, but not AT 1, receptors are expressed in rat ovary 7, while other tissues conAntagonist - A drug that blocks the actions of a hormone or other tain both AT l and A T 2 subtypes in varying proportions 4. agent, usually by preventing binding to its receptor. The use of a recently developed monoclonal antibody to the AT~ receptor in immunohistochemical studies has contributed to the emergHaemodynamics- The dynamics blood flow and blood pressure. ing evidence indicating that the AT t receptor is widespread ~. It is presMyocardiac hypertropy - A condition characterized by an increase in ent in the adrenal zona glomerulosa, where most of the receptor is heart muscle mass, internalized within the cell (Fig. 2), and in the vasculature, where it is particularly prominent on the luminal surface of the endothelium (Fig. Zona glomerulosa - The site of aldosterone secretion in the adrenal 3). However, AT 1 receptors are also widely present in secretory epigland. thelia; for example, in the rat uterus or human breast (Fig. 4). Another novel finding was the localization of the AT~ receptor in the tails of developing sperm in rat testis, and in the tails of rat and human ejacuconsidered to be candidates contributing to the pathogenesis of hyper- lated sperm9, as well as in Leydig cells (Fig. 5). tension and cardiovascular disease 3. The seemingly ubiquitous presence of the AT~ receptor in the Many studies have contributed to the characterization of angiotensin endothelium and in secretory epithelia conveys further messages, receptors, and the two main subtypes are classified as AT t and AT2, exemplified by the comparison of samples of normal breast tissue and according to their sensitivity to the angiotensin II antagonists, Dup753 turnouts. Normal breast, which is well supplied with blood vessels and (AT~ selective) and CGP42112A and PD123177 (AT2 selective)4. Most ductal tissue is, clearly, also abundantly provided with AT~ receptors. of the known physiological functions of angiotensin II appear to be The development of breast turnours clearly involves both of these cell mediated via the AT~ receptor, but the widespread incidence of the AT2 types, and so such tumours exhibit a massive density of AT~ receptor receptor suggests that it has specific roles. staining: the MCF-7 breast tumour cell line also contains AT, receptors. There are significant variations in the tissue distribution of the AT~ That AT~ receptors may have a functional bearing on the development and AT2 receptors. In the rat adrenal, most of the angiotensin receptors of the disease is obvious: the role of angiotensin as a purely trophic in the zona glomerulosa are of the AT] type, and those in the medulla hormone, stimulating tissue growth, surely deserves more attention 1°. are predominantly AT2: only AT~ receptors were detected in the bovine Taken together, all of these data support earlier work in suggesting adrenal cortex 5. The human uterus expresses predominantly AT 2 multiple functions for angiotensin. This, in turn, suggests that angioreceptors, while the rat uterus also contains AT~ receptors. Both AT~ tensin II should be made available in the tissues in a manner that is independent from the systemic RAS. Localized tissue renin-angiotensin systems have been found in the adrenal gland and other organs. The Angiotensinogen existence of such systems in addition to the systemic system makes sense if the generation Asp-Arg-VaI-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-VaI-Tyr-Ser-globulin of angiotensin locally in proximity to its receptors ensures that the tissue-specific functions - Renin are regulated precisely, without concomitant inappropriate actions in unrelated tissues. For these reasons, the tissue RAS now attract Angiotensin I a great deal of interest, but continue to present problems in interpretation. The physiological Asp-Arg-VaI-Tyr-Ile-His-Pro-Phe-His-Leu significance of the localized tissue generation of angiotensin II is sometimes unclear. For example, why is the adrenal tissue RAS Angiotensin converting enzyme required in addition to systemically available angiotensin II, as both appear to respond to the same stimuli? In addition, it remains to be Angiotensin II determined how the tissue distinguishes between angiotensin from these two sources. Asp-Arg-VaI-Tyr-Ile-His-Pro-Phe
Glossary
~
~.~LPeP
Angiotensin III Arg-VaI-Tyr-Ile-His-Pro-Phe Figure 1. The renin-angiotensin system.
i
36
tidases
Angiotensin 1-7 Asp-Arg-VaI-Tyr-Ile-His-P
Distribution of the tissue renin-angiotensin system Perhaps because of the problems outlined above, the concept of tissue RAS has met with some resistance. Currently, however, there is a great deal of evidence in support of the existence of these systems, particularly in the adrenal gland, gonads, kidney, heart, pituitary and brain ~°. Some brief examples are discussed below.
MOLEC'[JI.ARMEDICINETODAY
R
e v i e w, s
Figure 2. Immunofluorescence staining of AT1 receptors in rat glomeruIosa cells using monoclonal antibody 6313/G2. Scale bar = 25 I~m.
Adrenal gland Renin and angiotensin are formed primarily in the zona glomerulosa n. The amount of both renin mRNA and angiotensin 1I are regulated by dietary sodium t2, and the latter also by potassium t3. The local RAS is thought to have trophic effects on the glomerulosa and, in particular, to play a significant role in the regulation of aldosterone synthase L4.Similar systems exist in human adrenal glands ~.
Reproductive organs Renin has been identified in the Leydig cells of the rat testis, and renin and low levels of angiotensinogen mRNAs have been isolated from rat and mouse testes t6. Angiotensin converting enzyme is also present, and the generation of angiotensin II has been shown, for example, in the human testis and the rat epididymis ~7. In the ovary, prorenin, renin and ACE have been identified by hybridization and immunological and enzyme assay methods 18. In humans, ovarian prorenin and renin originate in the thecal cells ~9. In rats, angiotensin II immunostaining has been detected in the corpora lutea, and the stromal and thecal cells of larger follicles 2°. Evidence also exists for a discrete uterine RAS (Ref. 21).
Cardiovascular system Genes for all of the elements of the RAS are expressed in the heart 22"23,and angiotensinogen and renin have both been detected in neonatal rat heart 24. Both angiotensins I and II are secreted by cultured rat cardiac myocytesz~, and experiments on the expression of the renin gene in various tissues show that it is regulated in the heart in a specific manner 25. The importance of the tissue RAS in relation to cardiovascular disease has been discussed, and one possibility is that it is specifically involved in myocardiac hypertrophy and heart failure 26. The concept has been challenged however, and, according to other researchers, heart tissue does not produce renin, and the results of others are attributed to methodological failure 27.
Brain Renin gene expression has been described in rat, mouse and human brain tissue ~6'28, and its regulation is independent from kidney renin production25. The angiotensinogen gene is also expressed in rat brain 29, and renin, angiotensinogen and ACE have all been shown to be
Figure 3. Immunofluorescence staining of AT 1 receptors, using monoclonal antibody 6313/G2, in endothelial cells (EC) of: (a) bovine adrenal arteriole; and (b) human renal artery. The brightness of the lamina elastica (le) in (b) is attributable to autofluorescence. Abbreviation: L, lumen. Scale bar = 25 ~m. Reproduced, with permission, from Ref. 8.
present s°. Brain angiotensin is regulated in rats independently of circulating levels 3~, although not every study has shown this 32. It is difficult to dismiss the weight of these data as artefactual, as some have done27. In particular, the identification of specific mRNAs for the RAS components in tissues cannot be disregarded lightly. Nevertheless, some authors have expressed reservations because of the practical difficulties associated with distinguishing between systemic and tissue components of the system, and the failure, in some cases, to identify critical specific functions for the tissue RAS (Refs 10,1 1). However, on this point too, there is now convincing evidence (see below).
Specific roles for tissue renin-angiotensin system The t~tsk here is to identify functions that would be inappropriately met by the systemic RAS. The difficulty has been recognized, and in the case of both the adrenal and the cardiovascular tissue systems, it has been proposed that the systemic RAS is concerned with acute homeostatic functions, whereas the tissue system controls longer-term trophic events n°'26"33.It is thought this would be consistent with the likely dynamics of angiotensin availability from the two sources. Other quite different functions have been described, however. For example, angiotensin II stimulates ovarian oestrogen production 34, while m
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RAS present in both male and female reproductive tracts is that it exists to provide angiotensin II to sperm throughout their transit through the reproductive tract. Clearly, this is an angiotensin II function that requires the regulated production of angiotensin II in an entirely independent manner from the systemic source.
Uterus
Figure 4. Immunofluorescence staining of AT 1 receptors, using monoclonal antibody 6313/G2, in the secretory epithelium of human breast tissue. Scale bar = 50 Ixm.
The presence of angiotensin II receptors in the uterus has long been recognized, although only AT2 type receptors are found in humans 36. However, the role of angiotensin II has not been clearly defined. Angiotensin II receptors are present in uterine smooth muscle cells and, in rat uterus, angiotensin II induces uterine contractions 37, in addition to its vasoactive effects38. Uterine contractions are inhibited both by the specific AT1 antagonist, DuP753, and by monoclonal antibody 6313/G2. The antibody identifies the receptor in the luminal epithelium of the uterus, as well as in muscle and vascular sites, and it is possible that angiotensin II has specific actions on the functions of these cells as well.
Brain In brain, one set of actions of angiotensin lI is concerned with drinking behaviour. Intracerebrospinal injection of angiotensin lI rapidly induces increased water intake in rats, and a slower increase in NaCl intake 39, which is blocked by AT 1, but not by AT2, receptor antagonists 4°. All of these examples clearly indicate the requirement for tissue renin-angiotensin systems to be regulated independently from the systemic system.
Compartmentalization of tissue renin-angiotensin systems
Figure 5. Immunofluorescence staining of AT 1 receptors, using monoclonal antibody 6313/G2, in Leydig cells, primary spermatogonia and spermatid tails of rat testis. Scale bar = 25 }~m. Reproduced, with permission, from Ref. 9.
the angiotensin II antagonist saralasin inhibited ovulation in perfused rat ovaries35. We can add three examples of tissue differentiated angiotensin II functions that are more clearly distinguishable from those of systemic angiotensin II, and which evidently demand independent regulation.
Spermatozoa Following our development of a specific monoclonal antibody for the angiotensin II AT~ receptor (6313/G2), we identified AT 1 receptors in several components of the reproductive tract, including vascular and secretory epithelial components. In addition, however, we also identified the receptor in the tails of developing rat spermatozoa (Fig. 5), and ejaculated rat and human sperm. These receptors are indeed functional: our data show increased sperm motility in human ejaculates after five minutes exposure to angiotensin II (Ref. 9). Immunoblotting of solubilized sperm components after sodium dodecylsulphate (SDS) gel electrophoresis confirms the specificity of the immunorecognition: a single sperm component reacts with the antibody, and this component has a molecular weight that is identical with that of the angiotensin AT~ receptor from the adrenal cortex. Accordingly, an interpretation of the tissue m
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Clearly, it has to be established how the tissue systems, and the responses of the angiotensin receptors, are maintained separately from systemic events: in some cases there may be physical barriers; for example, the blood-brain or blood-testis barriers. In addition, we need to know how the delivery of angiotensin II to its receptors is regulated in the paracrine situation. There are several aspects to this. One is that the acquisition of precise data on the cellular localization of the sites of production of the components of the renin-angiotensin system, their regulation, and their relationship to the sites of angiotensin II action forms an absolute requirement for the interpretation of these systems, and this has not been achieved in most cases. As an example, it is clear that, in the adrenal cortex, the glomerulosa itself is the site of angiotensin II production. The question arises as to the relationship between the cellular production of angiotensin II and its receptors. If both occur in the same cell type, what implications does this hold for receptor occupancy, and the capacity of the adrenal gland to respond to physiological demand? One possibility is that angiotensin receptors are in some way sequestrated intracellularly so that their availability to locally generated angiotensin II can be regulated. However, the existence of non-receptor-binding proteins, as well as different receptor isoforms, their internalization, processing and recycling, and different mechanisms of intracellular signalling may all contribute to this separation of function. These questions need to be addressed.
Conclusions Our understanding of the role and functions of the RAS in the tissues needs to be widened significantly. • Angiotensin receptors are ubiquitous, and are widespread in many tissues that are not historically associated with angiotensin II action. In addition, several tissues contain all of the elements required for the
M O L E C U L A R MEDICINE T O D A Y
generation of angiotensin II in situ, and these systems are regulated separately f r o m the systemic system. It therefore follows that the locally generated angiotensin II acts in a paracrine fashion to elicit responses that m a y be c o n c e r n e d with tissue g r o w t h and repair, or with other functions that are highly specific, and which are not necessarily related to the functions of the systemic RAS. • M e c h a n i s m s must exist to ensure that the paracrine target cells respond in a specific m a n n e r to locally generated angiotensin II, and differently, if at all, to the systemic system. Such m e c h a n i s m s m a y include sequestration of the receptors f r o m systemic angiotensin II, partly by m e a n s of physical barriers, such as the b l o o d - b r a i n , or b l o o d - t e s t i s barriers, and also p e r h a p s by local regulation of angiotensin II receptor expression, and internalization, or by differential responses of different receptor subtypes. By analogy with other paracrine hormones, such as the insulin-like growth factors, it is also possible that there are angioter~sin II-binding proteins that regulate h o r m o n e delivery to the sites of action. • G i v e n the complexity of these mechanisms, it is conceivable that the system can be perturbed in disease. From the examples we give here, it is obvious that malfunction of tissue RAS could give rise to any of a spectrum of quite unrelated problems. In view of the widespread incidence of the AT~ receptor in epithelial tissue, and the potential for i n v o l v e m e n t of the tissue system in g r o w t h - p r o m o t i n g and t i s s u e - m o d e l l i n g events, one further striking possibility is that the R A S m a y be i n v o l v e d in cancer. The presence of AT~ receptors in breast tumours is consistent with this view. Future work should address these hypotheses.
The outstanding questions • Why do tissues generate the hormone angiotensin II in addition to receiving it from the bloodstream? • How do target tissues distinguish between tissue-generated and circulating angiotensin II? • What specific functions do the tissue-generated angiotensin II perform? • What factors regulate tissue generation of angiotensin 1I, and how is this different from the regulation of the circulating hormone?
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