Angiotensin II receptors and functional correlates.

AJH

1992;

5:221S-235S

Angiotensin II Receptors and Functional Correlates Pieter BM.W.M. Timmermans, Pamela Benfield, Andrew T. Chiu, William F. Herblin, Pancras G Wong, and Ronald D . Smith

t

2

2

2

A

ngiotensin II (ANG II) is the primary mediator of the renin-angiotensin system (RAS). ANG II acts on specific receptors to induce vascular contraction and aldosterone release. It is through these actions of ANG II that the RAS maintains its critical role in the long-term control of blood 1,2

Losartan, which represents the first of a new class of therapeutic agents, is currently undergoing clinical trials. A growing number of other ATj-selective ANG II-receptor antagonists are under development, including L-158,809, SKF 108566, and GR117285. Rat AT^receptor subtypes have been cloned and sequenced ( A T and A T ) . Human ANG II receptors have also been cloned and shown to have high affinity for losartan. A number of atypical angiotensin-binding sites have been identified from mycoplasma, amphibians, and mouse neuroblastoma, which are not sensitive to either losartan or PD123177. Our view of the ANG II receptor continues to grow in its complexity, as losartan and other nonpeptide antagonists are used as tools to define the functional role(s) of ANG II. Am J Hypertens 1992;5:221S-235S 1A

1B

KEY WORDS: Angiotensin II, losartan (DuP 753), angiotensin II receptor, renin-angiotensin system.

pressure, conceptualized as the vasoconstrictor-volume theory or the renal fluid volume pressure control mechanism. The major biosynthetic pathway of ANG II starts with the formation of ANG I from angiotensinogen by the action of renin. ANG II is then formed from ANG I by the action of angiotensin converting enzyme (ACE). The ANG II receptor has been classically characterized by evaluating the responses of isolated tissue. ANG II-receptor subtypes were suggested by differences in the relative potency of the peptide agonists. The use of receptor-binding assays led to the description of a distinct angiotensin receptor for des-AspÂNG II (ANG 3

4

5

6,7

From The Du Pont Merck Pharmaceutical Company, Wilmington, Delaware. Address correspondence and reprint requests to P.B.M.W.M. Timmermans, Ph.D., The Du Pont Merck Pharmaceutical Company, Experimental Station, P.O. Box 80400, Wilmington, DE 19880-0400

© 1992 by the American Journal of Hypertension, Inc.

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0895-7061/92/$5.00

Downloaded from http://ajh.oxfordjournals.org/ at New York University on April 21, 2015

Angiotensin II (ANG II) is the primary mediator of the renin-angiotensin system, which has an important functional role in cardiovascular homeostasis. The angiotensin receptor and its functional correlates have been redefined by the cloning of angiotensin receptors and the discovery and widespread study of specific nonpeptide ANG II-receptor antagonists losartan (AT selective) and PD123177 (AT selective). With these antagonists, it has been possible to extend the concept of ANG II-receptor heterogeneity to virtually every tissue and species. The losartan-sensitive sites have been shown to mediate all of the major ANG II-induced biologic effects, including vasoconstriction, aldosterone and catecholamine release, and central, ANG II-induced drinking behavior. The function of the A T site is not fully understood, but it may be involved in neuronal ion channel modulation and in fibroblast collagen metabolism. The presence of A T sites in fetal tissues and in discrete locations in the brain has encouraged continued research.

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The widespread use of losartan and PD123177 as subtype-specific receptor antagonists has generated a wealth of data about ANG II-receptor heterogeneity and the functional role of ANG II in a variety of tissues. ANG II-receptor heterogeneity has been defined by (1) the relative potency of ANG II- and peptide-analog agonists to elicit changes in second messenger systems and in tissue, organ, or whole animal responses; (2) the effects of peptide-receptor (such as saralasin) or nonpeptide-receptor (such as losartan and PD123177) antagonists; (3) observing the effects of blocking ANG II production with ACE or renin inhibitors; and (4) interference with ANG II binding to isolated "receptors" (such as semipurified membrane or microsomal preparations). It is the latter competitive-binding and autoradiographic techniques that have generated both interest and controversy concerning the functional significance of ANG II-receptor subtypes. The very discrete pattern of distribution of A T and AT -receptor subtypes in certain tissues—such as the brain, the adrenal and, in some species, the k i d n e y — and the marked changes in these patterns at birth have stimulated the search for receptor subtypes in virtually every tissue and organ system. Further, species differ markedly in the distribution of ANG II-receptor subtypes. Although increasing evidence suggests that the AT receptor can be functionally coupled (see below), its physiologic significance is not yet established. Not surprisingly, ANG II receptors in lower species may have different binding and functional characteristics. In the chicken, for example, ANG II may cause both pressor and depressor effects. As will be discussed below, receptor heterogeneity across species reflects differences in the amino acid sequence of the natural ligand. The following discussion will focus on (1) the functional correlates of ANG II-receptor subtypes; (2) the evidence for further AT!-receptor subtypes ( A T , A T ) ; (3) possible AT -receptor "functions"; and (4) atypical ANG II receptors—for example, in amphibians, fowl, Mycoplasma hyorhinis, and the mas proto-oncogene f a m i l y . 28

r

2

2

29

30

1A

1B

2

31

32

33

34,35

A T j - R E C E P T O R SUBTYPE FUNCTION The functional definition of ANG II receptors has included all of the steps in ligand-receptor-response coupling (Table 1). The response to ANG II, and therefore the "response" to antagonists, is specific to species, receptor, and cell. Losartan, which is the first nonpeptide-receptor antagonist to undergo clinical development, is being studied for its effects in virtually every setting in which ANG II elicits a biologic response. It has been concluded that virtually all of the well-known actions of ANG II are mediated by the AT receptor subtype, because losartan blocks these responses. This conclusion is supported by growing experience with losartan or its r

28


III) and supported the concept of multiple angiotensin receptors. Subsequent studies characterizing receptor subtypes in rat liver and rat kidney cortex suggested further ANG II receptor heterogeneity. ' The function of ANG II receptors has been defined by the study of the effects of peptide agonists and antagonists in isolated tissue or following injection directly into organs such as the brain. Investigations before 1989 were limited by the use of peptide-receptor antagonists that had partial agonist activity and limited duration of action. The introduction of ANG II-synthesis inhibitors (primarily the ACE inhibitors captopril and enalapril) dramatically expanded the appreciation of the physiologic roles of ANG II and its pathophysiologic role in essential hypertension, congestive heart failure, and a number of other clinical conditions, including scleroderma, proteinuria, and diabetic glomerulopathy. The ACE inhibitors, however, also inhibit kininase II, so a role for ANG II could not always be differentiated from a possible role for bradykinin. Recent developments have been instrumental in redefining the ANG II receptor and its functional consequences. First, although ANG II remains the primary effector molecule of the RAS, there is increasing evidence that angiotensin fragments are biologically active. Second, molecular biological techniques have been used to clone ANG II receptors, to produce transgenic hypertensive animals (rats and mice) by REN2 gene transfer, to explore the importance of tissue RAS, and to carry out chromosomal mapping to identify the "candidate gene" for hypertension. Finally, most important for the purposes of this review, has been the discovery and development of two series of nonpeptide ANG II-receptor antagonists represented by losartan (DuP 753) and PD123177. The discovery of losartan has been described previously. In brief, the initial lead for losartan, S - 8 3 0 7 , was chemically modified to yield a series of progressively more potent and orally active nonpeptide ANG II antagonists. Losartan has 10,000-fold greater affinity for the angiotensin receptor than S-8307 but, like all the phenyl- or biphenyl-substituted imidazoles, losartan did not totally displace radiolabeled ANG II, suggesting that there were multiple receptor subtypes. The disclosure of a series of imidazopyridines, exemplified by PD123177 and P D 1 2 3 3 1 9 , provided the necessary additional tools to complete the current concept that ANG II has two distinct binding sites. Similar findings were reported using a peptide antagonist CGP42112A. A standard nomenclature for these two ANG II-receptor subtypes has been proposed, in which the ANG II receptors inhibited by losartan (and dithiothreitol, DTT) are designated AT receptors, and those inhibited by PD123177 (or related compounds, like PD123319), as well as by CGP42112A, are designated A T receptors.

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TABLE 1. VARIABLES IN THE FUNCTIONAL DEFINITION OF ANGIOTENSIN II RECEPTORS Functional Response Coupling

ι

3

Ang II (Val -Ang II for chicken) ATj-AT (species, subtype specific)

ι

44

3

45

46

5

Natural ligand

125

3

ACE (or other proteases, eg, chymase)

Ligand synthesis pathway

binding assays in vitro and in challenge-type experi ments in vitro and in vivo. In binding assays, unlabeled losartan inhibits the binding of [ H] ANG II, [ I] ANG II, [ÎJSarMhÂNG II, or [ H]losartan while having no effect on [ H]prazosin or [ H]nitrendipine binding. Likewise, losartan did not inhibit opioid, dopamine-2, serotonin-2, phenycyclidine, neurotensin, cholinergic, or glycine receptors. In isolated vascular tissue, losar tan had no effect on the response to norepinephrine or potassium chloride, acetylcholine, bradykinin, or bombesin. In isolated guinea pig ileum, losartan had no effect on the responses of bradykinin, acetylcholine, or norepinephrine. In pithed rats, losartan had no ef fect on the responses to vasopressin, norepinephrine, or isoproterenol. Recently, losartan was shown to have no significant effect on the response of the feline hindquarter to U446619, norepinephrine, serotonin, endothelin-1, or BAY K 8 6 4 4 . The selectivity for the ANG II-ATi recep tor is further supported by losartan's lack of effect in preparations where the RAS is not functional. Losartan, for example, has no acute effect on blood pressure in normotensive or DOCA-salt hypertensive or L-NAME hypertensive animals. Losartan's lack of partial agonist (ANG II-like) activ ity is an important consideration, because the absence of this property clearly differentiates the nonpeptide-receptor antagonist losartan from the peptide-receptor an tagonists, such as saralasin. Saralasin is one of a num ber of peptide (ANG II analogs) antagonists that have been synthesized and studied biologically. Each of these compounds has significant agonist activity. Sara lasin has a potent pressor effect in rats and humans. Losartan is devoid of agonist activity in hepatocytes, in isolated vascular or nonvascular smooth muscle prepa rations, in normotensive rats, and in humans. In the normotensive rat, losartan blocked the partial ago nist effects of saralasin, confirming that this is an ANG II-receptor-mediated effect of saralasin. Whether losartan is absolutely selective or is com pletely devoid of partial agonist activity will require fur ther study. The vast majority of data support this con clusion, although several observations, primarily in cultured cells, should be mentioned. First, in rat aortic smooth muscle cells, a slight (10%) increase in the C a transient (fura-2 signal) was observed following admin istration of losartan. However, an earlier, similar study in rat aortic smooth muscle cells in our laboratory failed to detect any effect of losartan itself. In human mesangial cells, losartan and EXP3174 have been reported to increase the C a transient. These investigators found no effect of either compound on phosphoinositide metabolism, thymidine incorpora tion, or prostaglandin release. In a series of studies, lo sartan has been shown to release prostacyclin from por cine smooth muscle cells, C6-glioma cells, and human 3

Variable

223S

n

47

48

G protein (sensitivity to GTPyS)

Receptor

47

Coupling

49

C a transient, JTcAMP, ITcGMP, PI hydrolysis, DAG release, arachidonate release, Τ tyrosine phosphatase 2+

ι Second messenger

ι

Cell/tissue/organ response Intracellular response

50

Contraction (vascular, nonvascular smooth muscle), Secretion/release (AVP, aldosterone, catecholamine), Transport (Na , HCOj), Proliferation/hypertrophy Autocrine, paracrine, and hormonal actions of endogenous Ang II, Dose, duration of exposure to exogenous Ang II, Partial agonist effects of antagonists, Specificity of antagonist for Ang II receptor (or receptor subtype), Pharmacokinetics of antagonists) +

Whole organism response

ACE, angiotensin converting enzyme; Ang II, angiotensin II; AT, angio tensin II receptor subtypes; AVP, arginine vasopressin.

49,51

47

6,52,53

47

54

55

48

56,57

active metabolite EXP3174, and with other newly intro duced AT -selective agents such as SKF 1 0 8 5 6 6 , L-158,809, - S R 4 7 4 3 6 , I C I D 8 7 3 1 , G R 1 1 7 2 8 9 , and S C - 5 1 8 9 5 . The function of the ANG II-AT -receptor subtype has been defined by two kinds of experiments: "antagonism type," in vitro or in vivo, in which exogenously admin istered ANG II is given before and after selective antago nists are administered; and "pharmacodynamic type," in which a parameter, such as blood pressure, is moni tored following the administration of a selective recep tor antagonist. So far, the majority of the experience with AT!-selective nonpeptide ANG II-receptor antago nists has been with losartan. The validity of its use as a tool for defining ANG II-receptor function includes its (1) selectivity for the ANG II receptor; (2) lack of partial (ANG II-like) agonist effects; and (3) nonpeptide nature, which gives rise to its activity after oral administration and its appreciable duration of action. The selectivity of losartan has been demonstrated in 36

1

37

38

39

40

43

a

41,42

47

58

47

2 +

59

60

2 +

61


ι

Τ PLC, TPLD, adenylate cyclase

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64

TABLE 2. FUNCTIONAL RESPONSES TO ANGIOTENSIN II

65

67

69

Response

Blood vessels/ vascular smooth muscle

Contraction Hypertrophy Angiogenesis Intimal hyperplasia Proximal tubular ion transport Constriction of afferent/ efferent arterioles Aldosterone release Catecholamine release Contractility Hypertrophy Inhibition of collagenase Arginine vasopressin release Thirst/drinking Luteinizing hormone and prolactin "Behavior" Myometrial contraction (from pregnant animals)

70,71

Kidney

72

7 3

Receptor Subtype

Tissue

66

68

5, NO. 12, PART 2

Adrenal cortex Adrenal medulla Myocardium

Brain

74

75

Reproductive organs

AT, AT, AT,/AT AT, AT,/AT

2

2

AT, AT, AT, AT, AT, AT,/AT AT, AT, AT,/AT AT,/AT AT,

2

2

2

1

1,76

2 +

77,78

2 +

2 +

79

80,81

3

4 5

2 +

56,82

59,60,83

s

s

48

perfused vascular beds of the rat and the cat, losartan is a specific antagonist of ANG II-induced changes in vascular r e s i s t a n c e . Losartan, likewise, is a competitive antagonist of ANG II (and ANG III), and induced changes in blood pressure in pithed r a t s and conscious r a t s , dogs, and monkeys. The antihypertensive effects of losartan can be attributed, to specific inhibition of endogenously released ANG II. Losartan lowered pressure in rats with renovascular hypertension or partial renal infarct, or with reduced renal mass, and in rats with hypertension induced by genes, aortic banding, TGR(MREN2)27S gene, chronic ANG II infusion, and cold. In renal hypertensive monkeys, losartan administered orally lowered blood pressure from approximately 129 mm Hg to approximately 105 mm H g . In contrast, losartan did not lower blood pressure in DOCA salt-hypertensive or normotensive rats. The vascular hypertrophic effect of ANG II has been studied with losartan as the AT -selective antagonist in smooth muscle cells, in chronic hypertensive models, and in studies of response to balloon injury preparations. Losartan blocks ANG II-induced thymidine incorporation (DNA synthesis) in cultured rat aortic smooth muscle cells. Chronic treatment with losartan of spontaneously hypertensive rats (SHR) lowered blood pressure and significantly reversed the vascular (and cardiac) hypertrophy observed in the untreated S H R . " The role of ANG II in the response to balloon injury was 50,86,87

71,88

39,89

90

39

91,92

93

94

95

96

97

98

99

100

49

X

60

1 0 1

1 0 3


astrocytes, " but this has not been confirmed. Losartan, PD123177, and CGP42112A displayed agonist activity in cultured mesangial and proximal tubular epithelial cells. Losartan, when administered centrally to rats, elicited a transient increase in blood pressure, a slight (7%) acute decrease in blood pressure, and a delayed prolonged decrease, but did not affect blood pressure in other studies. In dog, rapid bolus intravenous doses of losartan, but not EXP3174, caused a significant but transient ( < 10 min) reduction of blood pressure that cannot be explained based on ANG II-receptor antagonism. Very-high-dose infusions of losartan (1000 //g/kg/ min for 2 h) have been reported to produce a "somatic disturbance. " This is 20 to 30 times the infusion rate needed to produce effective ANG II-receptor blockade. Losartan blocked the response to ANG II, but not to glutamate, in brain slices from the dorsal medulla oblongata of the rat. The same group showed that ANG II and glutamate responses were blocked in the rostral ventrolateral medulla in vivo. As losartan use increases, the significance of these observations will be determined. The general conclusion, however, is that losartan has little or no independent effect other than specifically blocking the responses to ANG II at its receptor. Losartan is AT -receptor subtype specific; thus, if it blocks the response, the response is designated as AT-mediated. ANG II binds to its specific, high-affinity receptor and exerts its characteristic cellular effects (such as smooth muscle contraction or aldosterone release from the adrenal glomerulosa) primarily through guanine triphosphate (GTP)-coupled second messenger systems. ANG II acts to release C a from intracellular s t o r e s . In vascular smooth muscle cells, the release of C a initiates formation of the C a calmodulin complex, activation of the myosin light chain kinase (MLCK) phosphorylation of the 20 kDa myosin light chain, and, ultimately, actin-myosin interaction and contraction. ANG II may also negatively couple to adenylate cyclase, leading to a decrease in cyclic adenosine monophosphate (cAMP) l e v e l s . The effect of ANG II antagonists can thus be "functionally" defined by their effects on the following: 1) the intracellular second-messenger-system response (for example, inositol 1,4,5-triphosphate [IP ], calcium release), 2) the cellular response (steroidogenesis, catecholamine release), 3) the organ/tissue response (contraction, secretion), and 4) the response of the whole animal (reduction in blood pressure. Losartan blocks the effects of ANG II at each level of ligand-receptor-response coupling (Table 2). In cultured vascular smooth muscle cells, losartan inhibits ANG II-induced C a flux, calcium transients, and increases in inositol phospholyj 6 3 , 8 4 , 8 5 Contractions of isolated vascular strips or rings of both arteries and veins are blocked by losartan. In

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1 0 5

1 0 8

109

110,111

112

113

114,115

2

116

82,117

2 /

1 1 8

119

1 2 0

2 +

119,121

66,118

122,123

124

125

126

127

128

129

130

58

1 2 2 , 1 3 1

132,133

134

135

X

+

136

3

+

3

137

The adrenal is an important target organ of ANG II and, although both subtypes are p r e s e n t , the pri mary endocrine function of both the adrenal cortex (al dosterone release) and the adrenal medulla (catechol amine) are subserved by the ATi receptor subtype. Losartan has been shown to block the ANG II (or III)-induced release of aldosterone in r a t s . This effect has also been observed with a new antagonist, L - 1 5 8 , 8 0 9 . Steroidogenesis was completely inhibited by losartan in cultured glomerulosa c e l l s . In an isolated perfused adrenal preparation, losartan blocked the ANG II-in duced release of catecholamines. Baseline aldoster one was reduced by selective AT -receptor blockade in pacing-induced failure in d o g s , whereas little effect was noted in a similar congestive heart failure model in s h e e p . In normal volunteers, losartan had little acute effect on aldosterone levels. The cardiac renin-angiotensin system involves func tional ANG II receptors on myocytes, coronary arteries and veins, sympathetic ganglia, postganglionic sympa thetic nerve terminals, vascular endothelium, and fibroblasts. " The clear understanding of the impor tance of ANG II in overall cardiac function, however, has been hampered by the differences observed among species in the distribution of angiotensin receptors, " in the responses to ANG I I , and in the direct versus indirect (for example, catecholamine releasing) effects of ANG I I . ANG II can also be formed in the heart by proteases other than ACE, such as human heart chymase. Defining the cardiac role of ANG II with ACE inhibi tors may be qualified by their potential action to poten tiate bradykinin. However, it was the effectiveness of ACE inhibitors to reduce cardiac hypertrophy that lead to the CONSENSUS trial of enalapril in patients with congestive heart failure. The clear efficacy of enala pril in that trial has stimulated interest in the cardiac role of ANG II. The use of the receptor subtype-specific an tagonists has shown that cardiac tissue of most species contains low levels of both AT! and A T binding sites. The contractile response of human atrial tis sue to ANG II is blocked by losartan even though both receptor subtypes are present. Losartan reduced the cardiac hypertrophy observed in S H R , in rats with aortic banding ligation, and in rats with coronary ar tery ligation. Losartan reduced the hypertension, but it did not block the cardiac or renal hypertrophy induced by chronic exposure to cold, which is mediated through sympathetic activation. In newborn pigs, losartan, like enalapril, reduced the rate of cardiac growth. ATj-receptor blockade has been shown to have no negative inotropic effect in iso lated cardiac tissue (P. Wong, personal communication) or in dogs at constant pressure and heart r a t e . ANG II has been shown to increase peak L-type calcium channel response in isolated canine ventricular myocytes, and 56,138

47,86

37

139,140

141

x

128

129

58

142

144

145

1 4 7

148

149

150

151

2

145,147

152

95,101

153

154

99

155

156


first shown by the use of ACE inhibitors. Losartan significantly reduced the neointimal proliferation pro duced by balloon injury in both the r a t " and rab b i t . Interestingly, the doses used were from 10 to 20 mg/kg/day given beginning 1 h to 2 weeks before the injury and continued for 12 to 14 days afterward. The extent of protection was variable, but comparable to ACE inhibitors. Losartan effects microvascular density or rarefaction in the r a t , angiogenesis in chick chorioallantoic membrane (at least in the larger microvessels), and angiotensin-induced angiogenesis in a sponge implant assay in r a t s . It can be concluded that AT! is the pre dominant receptor subtype for the various actions of ANG II in vascular smooth muscle. In the kidney, ANG II modulates renal function by its effects on vascular smooth muscle, tubular cells, glo merular (mesangial) cells, juxtaglomerular (JG) cells, and sympathetic nerves, and by its effects on endothe lial cell or smooth muscle release of vasoactive peptides or growth f a c t o r s . The ANG II-receptor subtype A T appears to be the primary mediator of these effects. The afferent and efferent arterioles in isolated perfused rat kidney are constricted by similar concentrations of ANG II, and constriction of both is inhibited by losar t a n . Losartan selectively blocks the actions of ANG II to increase production of inositol 1,4,5-triphosphate and P G E and to stimulate type I collagen synthesis, phospholipase D , hypertrophy, and intracellular C a mobilization in cultured me sangial cells of various species. The juxtaglomerular cells of the kidney play a critical role in providing the major source of renin for virtually all peripheral t i s s u e s . Elevated ANG II levels exert an inhibitory effect on renin release. EXP7711, a lo sartan precursor, blocks the inhibitory effect of ANG II on renin release from renal slices. Although not stud ied directly, losartan increases plasma renin activity in rats, guinea p i g s , dogs, sheep, monkeys, and humans, presumably by blocking the feedback inhibitory action of ANG II on renin release. These data suggest that the ΑΤι-subtype receptor is responsible. The blood pressure-lowering effect of losartan is elim inated by removing the kidneys of S H R . Sympa thetic nerves can play an important modulatory role in renal f u n c t i o n , and ANG II has a facilitates norepi nephrine release. In dogs, losartan inhibits the renal nerve stimulation-induced increase in renal vascular re sistance (RVR) while not affecting the RVR response to exogenous norepinephrine. Thus the presynaptic ANG II receptor in the sympathetic nerve terminal is the AT type. Renal tubular effects of ANG II have been described in the proximal convoluted tubule to increase N a and H C 0 reabsorption. Losartan blocks the proximal tubule (SI segment) reabsorption of both N a and H C 0 in r a t s .

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157

1 5 8

159

160

161

162

t

2

163,164

2

ί

X

163,164

2

165

166

167,168

169

41,170

171

172

173

174

175

175

2

176

3

2 +

2

Two lines of evidence suggest that there are at least structural, if not functional, differences in the ANG II ΑΤι-receptor subtypes. Data from cultured rat mesan gial and tubular epithelial cells provide the first line of evidence. In these preparations, the so-called A T 1B

2 +

2

66

1 7 7 - 1 7 9

180

181

182

177-179

X

177

1 A

179

1B

1B

1A

178

1

183

r

183

184

POSSIBLE FUNCTIONS OF THE ANG II AT -"RECEPTOR" SUBTYPE 2

The A T receptor has not been cloned, so it can only be described by its binding and functional characteristics. The AT -binding sites in human myometrium, bovine cerebral cortex, and rat fetal skin and skeletal mus c l e are not affected by GTP(y)S and are therefore not G-protein coupled to a second messenger system (Fig ure 1). Two cell lines have been shown to contain exclu2

EVIDENCE OF AT^RECEPTOR SUBTYPES

66,176

receptor subtype has a higher affinity for PD123177 and a lower affinity for losartan. These different receptor subtypes appear to be functionally linked to both IP mediated C a mobilization in mesangial cells and phospholipase A (PLA )-mediated C a mobilization intransporting renal tubular epithelial cells. Differences in the potencies of the selective antagonists provided evidence for ATj-receptor subtype heterogeneity. Direct evidence of structurally distinct AT!-receptor subtypes has been provided by the cloning of two losartan-sensitive receptors from the r a t . Previously, the ATi receptors in bovine adrenal, rat vascula ture, and rat (SHR) kidney had been cloned and sequenced. Each of these ATi receptors contained 359 amino acids and displayed very high sequence homol ogy. More recently, several laboratories have reported successful cloning of multiple ATi receptors from rat genomic DNA l i b r a r i e s . In each of these studies, two structurally distinct AT receptors were described. The differences among the resultant cloned two-recep tor subtypes involved 17 to 18 amino acids, primarily in the intracellular and extracellular d o m a i n s . " The pattern of distribution of these ATi-receptor subtypes (so-called A T and A T ) is not fully characterized, but both appear to be widely distributed. The adrenal and pituitary gland express primarily A T mRNA, whereas vascular smooth muscle and lung primarily A T mRNA. The human ANG II AJ receptor has also now been cloned from lymphocyte and hepatic cDNA libraries. Within the coding region of a clone isolated from a human liver cDNA library, the human receptor nucleo tide sequence was 86 and 9 1 % homologous to the rat and bovine sequences, respectively. This receptor was expressed in COS cells and shown to be an A T r e ceptor subtype, functionally coupled to mobilization of intracellular calcium. A virtually identical receptor from a human liver cDNA library was cloned, se quenced, and expressed in COS-7 cells. In contrast to the rat, no human AT!-receptor subtypes have been identified to date. The functional significance of the ATj-receptor subtypes is unknown. Since losartan blocks both receptor subtypes, it will be difficult to eval uate possible differences in post-receptor G-protein (guanine triphosphate-binding protein) or second mes senger-response coupling.

2

185

186


this response is blocked by losartan. In an intravascu lar two-dimensional Doppler ultrasound study in dogs, intracoronary losartan was shown to increase coronary blood flow more than enalapril (15 ± 2 % ν 4 ± 2 % ) , suggesting that ANG II has a tonic vasoconstrictor role with respect to coronary circulation. ANG II adminis tered directly into the brain can cause an increase in central blood pressure, a dipsogenic response, and a release of vasopressin and other hormones from the pituitary, such as prolactin and luteinizing hormone. These responses are blocked by losartan, suggesting that the AT is predominant functional receptor subtype. Most of these data come from rat studies, however, and there are differences in the distribution of AT! and A T receptors in human brain compared to rat b r a i n . These differences are primarily in the locus coeruleus and subthalamus nucleus where A T sites predominate in the rat, but ΑΊ sites predominate in the human brain. In both species, AT receptors predominate in areas of the brain associated with cardiovascular function (circumventricular organs), drinking behavior (subfornical organ, lamina terminalis), and neuroendocrine actions (posterior and anterior hypothalamus). The func tional correlates of the A T receptors in other brain areas associated with the auditory or visual system are un known. The "resetting" action of ANG II on the baroreceptor reflex also appears to be antagonized by losartan. The role of ANG II in learning and memory has been sug gested by studies in rats and mice treated with ACE inhibitors or l o s a r t a n . Although losartan can ac cess the brain, there is some question whether func tional blockade is achieved when losartan is adminis tered peripherally. The presence of angiotensin receptors and other com ponents of the RAS in ovaries and testes, their impor tance in regulating uterine and placental blood flow, a possible role in ovulation, and the potential mater nal/fetal toxicity of ACE inhibition have stimulated research in this area. The experience with losartan is limited, but long-term safety assessment and reproduc tive studies are in progress. Recently it has been reported that ANG II contracts the myometrium of the gravid but not the nonpregnant ewe, and that these responses are blocked by losartan. During gestation and shortly after delivery, there is a marked increase in total ANG II binding in the myometrium and the ratio of A T ^ A ^ changes significantly, and by 18 days postpartum the A T subtype predominates.

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227S

CGP42112A,

Saralasin

AT A

AT

t

AT

I AT,

2

1 B

|

Amphibian

G Protein G Protein Coupled Noncoupled

G Protein /

TcGMP

IcAMP

TIP

Tyrosine Phosphatase

IcGMP

sively AT -binding sites: Swiss 3T3 c e l l s and PC12W cells. In the 3T3 cells, ANG II had no effect on phosphoinositol turnover, cAMP, cyclic guanosine monophosphate (cGMP), tyrosine kinase, or arachidonate rel e a s e . Likewise, in PC12W cells, ANG II had no effect on basal cAMP, cGMP, arachidonate release, prostacyclin release, intracellular calcium mobilization, or H thymidine incorporation. In contrast, ANG II was reported to inhibit basal and atrial natriuretic peptide-stimulated guanylate cyclase activity in PC12W cells and to inhibit phosphotyrosine phosphatase. Orthovanadate blocked the ANG II effect in these cells. Virtually all of the major responses to ANG II are blocked by losartan and are designated as effects of AT!. A number of reports, however, have shown ANG II responses that are selectively blocked by PD123319 (or PD123177) or by both PD123319 and losartan (Table 3). Both A T and A T subtype antagonists inhibit proximal tubule sodium reabsorption in the microperfused rat kidney, scopolamine-induced amnesia in r a t s , thymidine incorporation in SHSY5Y neuroblastoma cells, and the release of luteinizing hormone and prolactin. The ANG II-induced increase in cGMP in cultured neurones from 1-day-old rat brain, increase in K current in the same 1 day old rat brain neurons, the inhibition of trypsin-activated collagenase activity in cultured rat cardiac fibroblasts, and the dilation of rat pial brain arterioles are all blocked by PD123117, but not by losartan. Whether A T receptors mediate prostaglandin release is controversial. CGP42112A has been shown to block ANG II-induced prostacyclin (PGI ) release in cultured astrocytes and porcine smooth muscle cells, while having no effect on P G I release in rat C6 glioma. In these studies, losartan had no effect on the ANG I I P G I release from human astrocytes, but it blocked, or partially blocked, the release from C6 glioma. Other inves187

2

188

187

3

189

190

128

t

191

193

194

+

196

197

198

2

2

199

62

2

2

2

65

2 +

200

112

2

2

192

195

tigators have shown that ANG II-induced P G E release from rat astrocytes and P G I release from porcine smooth muscle cells was blocked by losartan but not by P D 1 2 3 1 7 7 . In bovine medullary cells, losartan inhibited the ANG II response, but PD123177 altered the nature of the C a transient response. In chick chorioallantoic membrane preparation (note probable difference in receptor and natural chick ligand), losartan blocked the growth of first and second order vessels, but only CGP42112A blocked the third and fourth order vessels as determined by videodensitometry. The functional characterization of the A T receptor is hampered by (1) the lack of bioavailability data for the PD123177/PD123319 series of compounds; (2) the peptide nature and partial agonist effect of CGP42112A; (3) the inhibition of AT! binding by high doses/concentrations of PD123177; and (4) the possible displacement of losartan from protein-binding sites,

63

2

TABLE 3. POSSIBLE ANGIOTENSIN II AT RECEPTOR "FUNCTIONS"

2

Decrease in cGMP in neurons cultured from 1 day old rat brains Proximal tubule sodium re-absorption in microperfused rat kidney Scopolamine-induced amnesia in mice Modulation of C a transient in bovine medullary cells Increased thymidine incorporation in SHSY5Y neuroblastoma cells Prostaglandin release from cultured cells Increase in K current in neurons cultured from rat brain Dilation of rat brain arterioles Microvascular growth of chick chorioallantoic membranes Inhibition of collagenase activity in cultured cardiac fibroblasts Luteinizing hormone and prolactin secretion in rats Drinking response in rats 195

191

192

2+

200

193

62199

+

196

198

197

162

160

As defined by PD123177/PD123319

or CGP42112A.

112


/

FIGURE 1 . The multiple subtypes of the angiotensin II receptors and their intracellular responses. (IP3, inositol 1,4,5-triphosphate; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; G-protein, guanine triphospate (GTP)- binding protein.)

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2

2

141,145

141

141

160

1 4 1

2 0 3 , 2 0 4

160

2

205,206

r

2

ATYPICAL ANG II RECEPTORS The functional characterization of ANG II receptors now extends beyond the current definition of ΑΊ and A T . ANG II binding sites, functionally coupled re ceptors, or both, have been described which are not sensitive to either losartan or PD123177 (Table 4). For most mammals (humans, pigs, rats, rabbits, dogs, ί

27

2

5, NO. 12, PART 2

guinea pigs), the natural octapeptide ligand is the same. The structure of ANG II is Asp Arg Val Tyr lie His Pro Phe ; sheep, ox, bullfrogs, and avian species, such as the domestic chicken, have a Val instead of the He . Other differences also exist in the precursor peptides. In the anesthetized domestic fowl, the depressor re sponse to Val -ANG II was only partially attenuated by either PD123319 or losartan, and these effects were eliminated by reserpine and prazosin pretreatment. Binding of Val -ANG II to endothelium or smooth mus cle cells was little affected by either PD123319 or losar tan at a concentration < 10" mol, suggesting differences in the avian and mammalian receptors. ANG II signifi cantly increases microvascular density in the chicken chorioallantoic membrane preparation. In that study, losartan only blocked the increase in the larger microvessels, whereas CGP42112A blocked both the longer and smaller vessels. Mouse neuroblastoma neuro-2A cells also express a binding site that has affinity for ANG II but not for ANG III. The binding of ANG II is not inhibited by losartan, PD123319, GTP(y)S, or Gpp(NH)p. ANG II pro duced a concentration-related increase in cGMP levels in these cells which is not blocked by either losartan or P D 1 2 3 3 1 9 . An amphibian ANG II receptor has been expressed in frog oocytes that affects the release of in tracellular calcium. The calcium transient is inhibited by the nonselective peptide-receptor antagonist sarala sin, but it is not inhibited by > 400-fold-higher concen trations of either losartan or P D 1 2 3 1 7 7 . Likewise, saralasin and CGP42112A markedly inhibited binding to ANG II receptors from amphibian cardiac membrane, but losartan and PD123177 had virtually no effect at > 1 0 0 //mol. Mycoplasma hyorhinis is a common tissue culture con taminant that been shown to contain specific, highaffinity binding sites for ANG I I . These binding sites have not been functionally characterized but, although they are sensitive to DTT, they are not inhibited by either losartan or CGP42112A. Only saralasin concen trations of > 10" mol had any effect. The sensitivity of this binding site to bacitracin and aprotinin should allow investigators to differentiate culture contamination from a novel ANG II receptor. The MAS oncogene was reported to encode an angio tensin receptor. The oncogene was transiently ex pressed in Xenopus oocytes and selected responses of angiotensins on the phospholipase C-linked chloride current were observed. Although neither losartan nor PD123177 blocked this response, a tachykinin antago nist competitively blocked the MAS/angiotensin re sponse. The unique structure of this receptor was demonstrated by its affinity for [ H]ANG II or III, but not for [ I]ANG II. This has been confirmed by mono specific antipeptide antibodies chemically or photochemically cross-linked to the [ H]ANG II. The physiologic role of this "receptor" is unknown. A 1

7

2

3

4

5

6

8

5

5

30

5

32

5

5

32

112

207

208

209

209

31

210

TABLE 4. ANGIOTENSIN II "RECEPTOR" SUBTYPES Subtype

Occurrence

ATj

Throughout body in all species (cell sur face and nucleus) (subtypes AT and AT in rats) Discretely localized in brain and adrenal, species variable

Functional Response All principle responses to Ang II

1A

1B

AT

2

AT „ n

AT , n

AT „ AT ,„ AT n

n

?

Possible role in neuro nal ion channel modulation, angiogenesis, brain arteri olar dilation Mediates increase in Neuro-2A cells cGMP Avian (turkey, chicken) Aldosterone, catechol amine release Unknown Amphibian Mycoplasma hyorhinis Unknown Mas gene family (mas, Modulates intracellu mrg) lar Ang II actions

33

6

33

211

212

3

125

3

8

212


giving rise to higher free levels of losartan. These com pounds are highly protein bound and, if submaximal doses of losartan are administered to the renal hyper tensive rats, PD123177 may further lower pressure by displacing losartan from plasma p r o t e i n . Although the importance of the reported functions of the A T receptor is not known, a rapidly growing litera ture on the subject describes negative data with AT -selective antagonists. Neither PD123177 nor PD123319 (1) block ANG II (or III) pressor e f f e c t s ; (2) lower blood pressure in renin-dependent models ; (3) block aldosterone or vasopressin release ; (4) block the vasopressor response to ANG II in a variety of species; or (5) block the dipsogenic response to ANG I I . ' As was the case with losartan, a high dose of PD123177, centrally administered, was reported to block the dipso genic effect of ANG II in r a t s . The function of the A T receptor remains of interest because of its specific distribution in brain, human kid ney, fetal tissue, and reproductive organs, and its en hanced expression in wound h e a l i n g . Losartan and the other AT receptor antagonists under development significantly elevate plasma ANG II levels, raising the possibility that novel AT -receptor functions could be unmasked by such treatment. To date, no such unex pected effect of long-term losartan treatment in animals or humans has been observed.

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MAS-related gene (mrg) has been cloned from a human genomic library and, when injected into Xenopus oocytes, increases the electrophysiologic response to ANG II. The mrg amplification of the ANG II response was observed in oocytes but not in CHO or COS cells. It was concluded that the seven transmembrane proteins encoded by the MAS gene family are not "angiotensin receptors" but may serve a modulatory role to the re sponse to ANG II.

8.

Papdimitriou A, Worcel M: Dose-response curves for angiotensin II and synthetic analogues in three types of smooth muscle: existence of different forms of receptor sites for angiotensin II. Br J Pharmacol 1974;50:291297.

9.

Goodfriend TL, Peach MJ: Angiotensin III: (des-aspartic acid^-angiotensin II. Circ Res 1975;37(suppl I):I38148.

10.

Devynck MA, Pernollet MG, Matthews PG, et al: Spe cific receptors for des-Asp -angiotensin II ("angioten sin ΠΙ") in rat adrenals. Proc Natl Acad Sci USA 1977;74:4029-4032.

35

35

1

SUMMARY AND CONCLUSIONS

1

2

t

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The discovery and widespread availability of specific nonpeptide ANG II-receptor antagonists has acceler ated the search for new functional roles for ANG II. The principle actions of ANG II on the blood vessels, the adrenal gland, and the kidney are clearly mediated through ATj (losartan-sensitive) receptors. Many other potentially important roles of ANG II on the brain, on vascular response to injury, and on neuronal catechol amine release also appear to be AT -receptor mediated. A functional role of the AT -receptor subtype cannot be ruled out, however, as a number of studies have shown that either PD123177-like compounds or CGP42112A, or both, alter responses to ANG II. The cloning of the mammalian AT receptors should allow the further un derstanding of the nature of the interaction between the intracellular domain of the ANG II receptor(s) and the G-protein/second-messenger systems. Whether there are additional, as yet undiscovered, human ANG II re ceptors remains to be determined. Our current view of the ANG II receptor and its func tional correlates (Figure 1) continues to grow in its com plexity. The nonpeptide ANG II-receptor antagonists now being developed should provide additional tools for the still-finer definition of the physiologic role of ANG IL

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Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum.

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Circulating Exosomes Induced by Cardiac Pressure Overload Contain Functional Angiotensin II Type 1 Receptors.

Intranasal angiotensin II in humans reduces blood pressure when angiotensin II type 1 receptors are blocked.

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Structural basis for selectivity and diversity in angiotensin II receptors.

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Specific receptors for des-Asp1-angiotensin II (("angiotensin III") in rat adrenals.

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