Director, Harvard Center for the Study of Kidney Disease, and. Samuel A. ... I. Hyp.;rtcnsion. I. Laragh .... CV-2961 (Takeda). IC 50 15 000 ... TCV-116 (Takeda) ?
Hypertension Pathophysiology, Diagnosis, and Management
Second Edition VOLUME ONE
Editors
John H. Laragh, M.D. Director, Cardiovascular Center and Hypertension Center Chief, Cardiology Division Hilda Altschul Master Professor ofMedicine The New York HospitalCornell Medical Center New York, New York
Barry M. Brenner, M.D. Director, Renal Division Department ofMedicine Brigham and Women's Jfospital, and Director, Harvard Center for the Study ofKidney Disease, and Samuel A. Levine Professor a/Medicine Harvard Medical School Boston, Jvfassachusetts
Raven Press §
New York
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Ra1cn Pre,,, Ltd., 1185 A 1cnuc of the America,, :--;c1, York, :--;en York IOOJ6
e, 1995 by Raven Press. Ltd. :\II rights reserved. This book is protected by copyright. ~o part ofit may oc reproduced. stored in a retrieval system. or transmitted, in any form or by any means. ekctronic, mechanic:il, photocopying, recording, or othawise, \\ithout prior \Hillen permission of the publisher. ~lade in the Cnited States of ,\merica Library of Congrc" Cataloging-in-Public:1tion Data !-lyp.;rtension: pathophysiology, diagnosis. and management/editors. John 1-1. Laragh, Barry :-..t. Brenner.-2nd ed. p. cm. Includes bibliographical references and index. !SB'.\/ 0-7817-0 I 57-0 (set) I. Hyp.;rtcnsion. I. Laragh,John II., 1924II. Brenner, Barry M., 1937- . [D'.\/LM: I. Hypertension. \VG 34011996354 1995] RC685.1181-19144 1995 6 I6.1'32-dc20 D>IL:-..t;DLC for Library ofCongn:ss 94-26916 CIP
The material contained in this rnlumc was submitted as previously unpublished material, except in the instances in which some of the illustrative material was derived. Great care has ocen taken to maintain the accuracy of the information contained in the volume. However, neither Raven Press nor the editors can oc held responsible for errors or for any consequences arising from the use of the information contained herein. Materials appearing in this book prepared by individuals as part of their official duties as U.S. Government employees arc not covered by the above-mentioned copyright.
9
8
7
6
5 4
3
2
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-11
-10
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concentration (log M) 125
FIG. 4. Competition for [ 1]Ang II binding in rat vascular smooth muscle cells (AT,, upper panel) and human myometrium (AT 2 , lower panel) membrane particulates by Ang I (•). Ang II (e), Ang Ill ("'), valsartan (+), and CGP 42112 (T). The method has been described in { 19}. The concentration of radioiodinated Ang II was 0.15 nM. The binding buffer contained 2 mg/ml bovine serum albumin. Ang I and Ang II do not distinguish the AT, and AT 2 receptors, whereas the affinity of Ang Ill for AT 2 is greater than for the AT, receptor. Valsartan {17} and CGP 42112 {19} bind preferentially to the AT, and AT 2 receptor, respectively.
nalization process in smooth muscle cells is blocked by losartan, confirming that it is an event mediated by the AT 1 receptor (43). Similarly, in neuroblastoma NIE-115 cells, Ang II downregulates 85 percent of the AT1 receptors within 5 min at 37 degrees C. There is a rapid but incomplete recovery within 4 hours which involves receptor recycling and an SO-percent recovery by 18 hours that requires protein synthesis and new receptor formation (44). Subtypes ofAT1 Receptors, Evidence from Binding Studies
There appears to be subtle differences between the binding properties of central and peripheral AT 1 receptors. Indeed, AT 1 receptors in the brain appear to be less
In addition to the binding studies outlined above, molecular biology has provided strong evidence for AT I receptor heterogeneity in animals. For example, the AT 1 receptor expressed by Xenopus oocytes after inoculation with rat brain messenger ribonucleic acid (mRNA), discriminates poorly between Ang II and Ang III. This is in contrast to the AT 1 receptor expressed in this cell type after liver or pituitary mRNA inoculation which discrimates highly between Ang II and Ang III (50). In rat adrenal, two clones encoding for different AT 1 receptor subtypes have been identified from genomic libraries (51-53). These AT 1 receptor clones, also termed AT1a and A T1b, bear little resemblance to the AT ta and AT1b receptor classification proposed from binding studies in rat kidney (4 7). The two rat adrenal clones, which have a 90-percent identity, differ in their amino-acid sequence, primarily in the last intracellular loop of the receptor. The AT 1a gene, located on chromosome 17, is mainly expressed in rat vascular smooth muscle, in the rat kidney, and in rat lung. The AT lb gene, located on chromosome 2, is expressed in rat adrenal glomerulosa, pituitary, and liver (51). An additional cDNA, AT 1c, which is highly homologous to the AT 1a and AT 1h genes, has recently been identified in cultured rat vascular smooth muscle cells and in rat mesangium (54). Hybridization experiments using brain cDNA probes have also revealed the existence of additional mRNAs, suggesting differences between central and peripheral Ang II receptors (55). Interestingly, transfected COS-7 cells express both AT 1a and AT 1b receptors. When these cells are labeled with radioactive Ang II, solubilized, and the supernatant run on isoelectric focusing gels, a single peak of specific radioactivity (pl 6.8) is obtained (56). These results as well as the similar binding signature of the clones
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PHYSIOLOGIC Rou~ OF ANGIOTENSIN
(57) indicate that AT,a and AT 1b receptors may actually be isoforms rather than specific subtypes of the AT I receptor. In contrast to the rat, only one single gene encoding for the AT, receptor has been reported so far in bovine and human tissues. Recently, however, a novel type of human AT has been cloned from placental CDHA library. It has identity with the receptor cloned from human heart CDHA and appears pharmacologically distinct (4 I ,58-60). The regulation of the expression of both AT,a and AT11, receptors differs. Angiotensin II downregulates AT,a mRNA in vascular SMC but induces the upregulation of AT 11, mRNA in rat adrenal (6 I). Furthermore, bilateral nephrectomy is accompanied by a decreased expression of the AT,a receptor in liver and by an increase of the AT,h receptor in the adrenal (36). Treatment with adrenocorticotropic hormone (ACTH) or Ang II antagonists downregulates AT 1b mRNA in adrenal, whereas insulin upregulates it (33). These observations indicate that both receptor subtypes are regulated, but in opposite directions. It has been proposed that at least two pathways downregulate AT, mRNA in rat glomerular mesangial cells (62). One pathway is an Ang-II-induced, protein-kinase-C-independent but cycloheximide-sensitive pathway, indicating that it involves protein synthesis. The other is Ang-II-independent, induced by cyclic adenosine monophosphate (cAMP) but is cycloheximide-insensitive. Are the AT,a and AT,h receptor subtypes pharmacologically and functionally different? Indeed, Ang III appears more potent when acting through the AT 1h receptor subtype than through the AT,a receptor subtype. In addition, the dose-response curves for Ang-II-induced calcium mobilization differ between the two receptor subtypes (63). Moreover, the two subtypes seem differentially coupled to inhibition of adenylate cyclase, since ACTH-induced CAMP formation is inhibited by Ang II in adrenocortical tumor cells transfected with the AT,a-but not the AT 11, receptors (64). Thus, although there appear to be some pharmacological differences between the two AT I receptor subtypes, there is so far no conclusive evidence indicating a ditlerence in their functional roles. Distribution oftl,e AT1 Receptor
The anatomical distribution of the AT, receptor has received considerable attention and conventional receptor binding methodology, as well as autoradiography, have been used to identify AT, receptors in several tissues from various species, including humans. Reports that have identified AT, receptors in somatic and brain tissues are summarized in Table 2. Some tissues express a nearly homogeneous population of AT, receptors, while others are characterized by a mixture of both the AT, and AT 2 receptor subtypes (6,24,65,66). In general, AT, receptors predominate in virtually all organs and tissues
II
RECEPTORS
/
1701
involved in fluid-electrolyte balance and blood pressure regulation. Thus, the AT, receptor is found primarily in adrenals, vascular smooth muscle, kidney, and heart. AT 1 receptors are also present in specific brain areas such as the circumventricular organs, the hypothalamus, the suprachiasmatic nucleus, the supraoptic nucleus, the para ventricular nucleus, and the nucleus of the solitary tract which are implicated in the dipsogenic actions of Ang II, the release of vasopressin, and the neurogenic control of blood pressure (67,68). Major differences exist in the distribution of AT, receptors between species (6,24). For example, bovine adrenal glomerulosa and human adrenal cortical cells express only AT, receptors; whereas rat, rabbit, and dog adrenal cortex exhibit both AT I and AT 2 receptors ( 19,21,69, 70). In rat and rabbit kidney, only the AT, receptor is expressed, but both receptors are present in the kidney parenchyme of the rhesus monkey (71, 72) and in the large cortical vessels of human kidney (73). Similar variations between species have been observed in the distribution of the AT, receptor in the brain. For example, human locus coeruleus, cerebellum, and the subthalamic nucleus express the AT I subtype, whereas the same brain areas of the rat contain mainly AT 2 receptors (46,74-80). Similar species differences are found with regard to the relative proportion of AT 1 and AT 2 receptors in tissues where both receptors are expressed (24). Thus, in rabbit aorta, 90 percent of Ang II receptors are of the AT I receptor subtype, whereas in rat and monkey aorta, only 60 percent of the Ang II receptors are of the AT, subtype. Furthermore, rat heart differs from rabbit, monkey, and human heart in expressing over 90-percent AT I receptors (81-83). These observations indicate that a marked species variation exists in both the distribution and the proportion of the Ang II receptor. The AT 1 Receptor-Signaling Mechanisms
G-proteins
The AT 1 receptor has been recently cloned and is a member of the 7-transmembrane domain, G-protein coupled receptor superfamily (40,4 I). The AT I receptor has been shown to be independently coupled through Gproteins (Gi and Gq) to a variety of signal transduction pathways, including adenylate cyclase, phospholipase C, phospholipase A2 , phospholipase D, and ion channels (39, 174-176). These various mechanisms mediate a variety of tissue responses to Ang II which have been extensively reviewed (6,177). Adenylate Cyclase
Angiotensin II has been found to either inhibit or stimulate adenylate cyclase. In the majority of studies, Ang II inhibits adenylate cyclase. For example, in adrenal and
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1702 /
CHAPTER102 TABLE 2. Distribution of the angiotensin II subtypes in various tissues of different species
System Cardiovascular Heart
Aorta
Renal artery Pulmonary artery Anterior cerebral artery Portal vein Mesenteric artery Vascular SMC Neointima
Species Rabbit, pig, monkey Rat, guinea pig Human Rabbit, pig, dog Rat, monkey Rat fetus Human Rat Rat R~ R~ Rat, human Rat
AT,
AT2
+
+
+
+ + + ±
+
+ +
+ + +
+
+ +
+ + ?
+
+ Spleen Platelet Excretory Kidney Glomeruli Mesangial cells Proximal tubule Tick ascending loop LLC-PK1 cells Capsule Preglomerular vessel Urinary Bladder Endocrine Adrenal Cortex Fasciculata Medulla
Pituitary PC12Wcells Digestive system Liver PLC-PRF-5 cells Clone 9 Intestine Pancreas Reproductive system Uterus
Ovary atretic follicle corpus luteum Placenta
Vas Deferens Epididymis Adipocyte
R~ Human
+
Rat, rabbit Monkey, human Rat, human Rat R~.rabblt Rat Pig tubular epithelial cell Rabbit R~ Human Guinea pig
+
Rat, rabbit, dog, human Bovine, monkey Bovine, human Rat, human Rabbit Bovine R~ Murine anterior pituitary tumor Rat pheochromocytoma
+
Rat, rabbit Rat fetus Hepatoma cell line Rat liver cell line Guinea pig, rat Dog
+ + + + +
Rat, rabbit Monkey, human Ovine during pregnancy Ovine outside pregnancy Rat, bovine Rat Rat, human Rabbit (fetal portion) Guinea pig, rabbit (chorion) Rabbit Rat Rat epididymis
+
+ +
+ + + +
+
+
Th is mate ria I was rnpied
+ +
+
+ + ± + +
+ + ±
+
+
±
+
+ + + + + +
± ± ±
+ + +
+ + +
Reference (81 ,82,84,85) (81,86-89) (83) (81,90) (81,91-93) (94) (19) (95) (96) (97) (98) (19,27,99, 100) (101,102) (103) (104) (105) (71,73,81,87, 106-108) (71,81,109) (109-113) (47) (114,115) (115) (116) (106) (117) (118) (119)
(19,21-23,27,30,69,81, 106,108) (69,81) (70,120) (19,21,27,30,69,81, 108) (106) (69,121,122) (123,124) (125) (25,127,208) (25,27, 128,129) (94) (130) (22,131) (132-134) (135,136) (19,22,27) (19,137) (138) (138) (139-141) (139) (142-144) (145) (145) (146) (147) (148)
PHYSIOLOGIC ROLE OF ANGIOTENSIN
II
RECEPTORS
I 1703
TABLE 2. Continued.
AT 1
AT2
Rat Rat fetus
+ +
+
Rat, monkey Rabbit Rat Rat
+ + + +
Rat, rabbit
+
(46,76,77, 108, 153-155)
Rat, human Rabbit Rat, rabbit, human
+
(74,76-78) (74,153,157) (74,77, 152,153,158,159)
Rat Rat Hamster Rat, rabbit
+ +
Species
System Respiratory system Lung Nervous system Brain
Astrocytes Choroid plexus Circumventricular organ Organum vasculosum laminae terminalis Area postrema Median eminence Septum and hypothalamus Hypothalamus Suprachiasmatic nucleus Supraoptic nucleus Thalamus Paraventricular nucleus Subthalamic nucleus
Mediodorsal thalamic nucleus Amygdala Medial amygdala Dentate gyrus Midbrain and pons Superior colliculus Locus ceruleus
Substantia nigra
Medial geniculate nucleus Medulla oblongata Nucleus tractus solitarius Dorsal motor neuron of vagus Inferior olive
NG108-15 cells NIE-115 cells Glial cells Neuronal cells Miscellaneous Fetal/newborn skin Skeletal muscle Fibroblast R3T3 cells
+ ±
+ +
+
Rat, rabbit, human Rat Human Rabbit, hamster Rat Rabbit Rat Rat Rabbit
+ +
+
±
Rat, human, rabbit Rat, Human Rat, human Rabbit, hamster Human Mouse neuroblastoma X rat glioma hybrid Murine neuroblastoma Rat Rat Human, rat, monkey, Rat fetus Rat fetal skin Line derived from Swiss 3T3 fibroblasts
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+
+ +
+ ±
+
+ + +
+ +
+
+
(46,74,78,79,153,155, 156,161,162) (76,77) (74) (153,160) (76-79, 155) (153)
(46,76-79, 108,123,157) (76-78,123) (153) (74) (153) (153) (74) (108, 152, 157) (74,160)
+
± ±
(46, 75, 79,123) (46,76,78, 123) (160) (46, 78, 153-155, 161)
+ +
+ + +
(46,75,79,81,99, 150) (81) (151) (78,152)
(76, 78,152) (153) (153)
±
+
(142,149) (94)
+ +
Rat Rat Rabbit Human Rat Rabbit Human Rat Human, hamster
Reference
(76-79, 123,155) (74,76,77,79) (74,76-78) (153,160) (80) (163)
+
(44,164) (165,166) (165,166)
+ + + +
(142,167-172) (94) (171) (173)
1704 /
CHAPTER102
in kidney membrane particulate ( 177-180). However, in intact adrenal glomerulosa cells, in bovine adrenal fasciculata, in fetal fibroblasts in culture, in freshly isolated and cultured microvessel endothelial cells, as well as in cultured vascular smooth muscle cells, Ang II stimulates cAMP production ( 171, 181-184). The effect of Ang II to stimulate adenylate cyclase activity may not be via a direct interaction of the AT I receptor with adenylate cyclase through Gi but may be secondary to 1,4,5-triphosphate (IP 3 ) stimulation of a calciumcalmodulin-dependent mechanism ( 183) or may result from the activation of other pathways such as phospholipase A2 , responsible for the generation of prostanoids, which in turn can stimulate adenylate cyclase. Phospholipase C
Interaction of Ang II with AT I receptors results in the activation of phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate (PIP 2) leading to the production of inositol 1,4,5 trisphosphate (IP 3 ) and 1,2diacylglycerol (DAG). Each of these two metabolites activates a different pathway. Generation ofIP 3 results in the release of calcium from intracellular stores whereas DAG activates protein kinase C (PKC). These two pathways are the most extensively investigated signal transduction pathways of Ang II ( 180).
formation in response to hormone-receptor binding may feedback to terminate IP 3 generation and calcium mobilization ( 190). This process is involved in desensitization of the cell to Ang II. Protein kinase C also contributes to the stimulation of prostaglandin (PG) synthesis and smooth muscle cell contraction (186,191,192). Recently it has been suggested that this enzyme also modulates cardiac sodium channels and can stimulate steroidogenesis (193,194). In addition, PKC probably plays an essential role in the growth-promoting effects of Ang II by activating kinases such as MAP kinase and Raf- I kinase (195) involved in the mitogenic cascade, and by inducing the expression of growth factors such as platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), or transforming growth factor-/3 (TGF-/3) and protooncogenes like fas, myc, jun and jun-B (9, I 0, 12, 196-199). However, Ang II can also activate protein phosphorylation through PKC-independent mechanisms (195). Phospholipase A 2 and Phospholipase D
In mesangial cells, Ang II has been shown to stimulate phospholipase A 2 through a pertussis toxin-sensitive Gprotein ( 186) and phospholipase D through a G-protein as yet undetermined (200).
IP3 Generation
Phospholipase A 2
The rapid rise in intracellular calcium induced by IP 3 generation initiates a variety of cellular responses, including the synthesis and secretion of aldosterone (180), the secretion of peptide hormones such as prolactin, ACTH, vasopressin and oxytocin ( 185), vasoconstriction (186), and protein-tyrosine phosphorylation ( 187). Increased intracellular calcium has also been shown to decrease intracellular cyclic guanosine monophosphate (GMP) levels, secondary to activation of a calciumcalmodulin-dependent phosphodiesterase ( 188). Interestingly, mutation of Asp 74 in the AT I receptor to Asn or Gin decreases tenfold the affinity for losartan, increases 20-fold that of CGP 42112 and the mutants become unable to stimulate IP 3 production and Ca2+ mobilization. This indicates that Asp 74 is essential for the signaling mechanism of the AT I receptor ( 189).
Phospholipase A2 (PLA2) hydrolyses phospholipids to generate PG, prostacyclin, thromboxane A2, and other eicosanoids. These molecules probably play a role in the regulation of Ang II actions since PG formation by Ang II has been shown to attenuate vasoconstriction induced by the peptide. Furthermore, a PG metabolite , 5' 6epoxyeicosatrienoic acid, has been suggested to inhibit sodium entry in proximal tubules, probably through stimulation of voltage-gated calcium channels. Thus, stimulation of phospholipase A 2 may also contribute to modulation of the renal actions of Ang II on proximal tubular ion transport (177). Interestingly, pertussis toxin that abolishes Ang-II-induced decrement in CAMP production does not inhibit Ang II stimulation of PLA2 in proximal tubular epithelium ( 177). Phospholipase D
DAG Formation
DAG has a dual role: it activates PKC and it stimulates arachidonic acid formation which leads to the formation of eicosanoids. Protein kinase C phosphorylates various cellular proteins including hormone receptors and acts synergistically with cytosolic-free calcium to initiate a wide variety of cellular responses. For example, DAG
Stimulation of phospholipase Din glomerular mesangial cells and vascular SMC results in hydrolysis of phosphatidylcholine and generation of DAG and phosphatidic acid, two metabolites which activate PKC and lead to the synthesis of PGs (200,20 I). The physiological role of this enzyme has not yet been elucidated, but it may be involved in stimulation of cell growth and contraction.
This material wasrnpied
PHYSIOLOGIC ROLE OF ANGIOTENSIN
Regulation of Calcium, Potassium, Sodium, and Chloride Channels
II
RECEPTORS /
1705
AT.1 receptor, ~owever, .much less is known about its signaling mechamsm and Its physiological role.
Calcium Channels
Binding Properties
In adrenal glomerulosa cells, Ang II acting via the AT 1 receptor stimulates voltage-gated calcium channels and blocks potassium channels [204]. These two mechanisms are responsible for an increase in intracellular calcium concentration which ultimately leads to aldosterone release (180,202-204). The nature of the type of calcium channel activated by Ang II is still controversial and may vary according to the species and tissue. It has ?ecn suggested that in bovine glomerulosa the channels mvolved in Ang II stimulus-secretion coupling are of the T type (205), whereas in proximal tubular epithelium they are of the L type (177).
The AT2 receptor binds Ang peptides with the following order of potency: Ang III;;:: Ang II> Ang (3-8) > Ang (I- 7) = Ang I. The AT 2 receptor has a 10-times-higher affinity for Ang III (pKi 9 .5) than the AT 1 receptor (24,173,208). As described previously, the AT 2 receptor has a low affinity for losartan (Ki ;;:: 10-4 M) and related compounds but a high affinity for CGP 42112, a peptide analogue of Ang II (Ki I l nM) and nonpeptidic, spinacine derivatives, examplified by PD 123319 and analogues (Ki - 10 nM) (19,21,22). CGP 42112 and PD 123319 have at least a 2000-fold greater affinity for the AT2 than for the AT 1 receptor (28).
Potassium Channels Biochemical Properties
A low-conductance, adenosine-triphosphate (ATP)sensitive potassium channel has been found to be inhibited by Ang II via the AT 1 receptor in cultured porcine coronary artery smooth muscle cells (206). Blockade of Potassium channels in this tissue results in depolarization and sustained calcium influx through voltagedependent channels as described previously in glomerulosa cells and may be involved in Ang II induced vasoconstriction (204,206).
In contrast to their effect on the binding properties of the AT1 receptor, reducing agents such as DTT either increase or have no effect on the binding of Ang II to the AT 2 receptor (19 ,22,32,81,94, 139, 141,209,210). Sodium ions do not affect the binding of Ang II to the AT 2 receptor, except in bovine cerebellum where the affinity is lowered two- to threefold (35). Physicoclzemical Properties
Sodium Channels Angiotensin II modulates sodium channels in isolated rat ventricular cardiomyocytes ( 193). In these cells, which express only AT 1 receptors (81 ), Ang II increases the frequency ofopening of sodium channels and hence the rate of activation of single-channel sodium currents ( 193). The phorbol ester TPA mimics the effect of Ang II, suggesting the involvement of PKC as the second messenger.
Chloride Channels Angiotensin II has been reported to increase calciumactivated chloride conductance in mesangial cells ~ 190,207). This effect is thought to be mediated by an Increase in the concentration of intracellular calcium secondary to IP 3 generation and to the activation of PKC (207). The AT2 Receptor Subtype
The binding characteristics and the distribution of the AT2 receptor have been well described. In contrast to the
The molecular weight of the AT2 receptor has been characterized by covalent cross-linking and photoaffinity labeling (137,139,173). In R3T3 cells, a mouse fibroblast cell line derived from Swiss 3T3 cells, a specifically labeled protein corresponding to the AT 2 receptor has been detected with a molecular weight of 100 kDa. In ovarian granulosa cells, a molecular weight of 79 kDa has been reported ( 139). Similar experiments performed using membranes prepared from NG108-15 hybridoma cells and human myometrium indicate that glycosylated AT 2 receptors in these tissues have molecular weights of 92 and 68 kDa, respectively. After deglycosylation, the molecular weight of the AT 2 receptor in NG 108-15 cells falls to 56 kDa and to 49 kDa in human uterus [(137); Bonnafous et al., personal communication]. The difference in the molecular weights of the AT 2 receptor in different cell lines or tissues, together with the observed differences in glycosylation, raises the possibility that the AT 2 receptor may be a heterogeneous protein. The AT 2 receptor in rat adrenal tissue has an isoelectric point of 6.3 (56,211). Using expression cloning from fetal and pheochromocytoma cell line (PC 12W) libraries, two groups of investigators have recently reported the successful isolation of a full-length cDNA encoding the AT 2
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1706 / CHAPTER102 receptor based on its identical binding specificities, DTT enhancement of binding, and developmental and tissuespecific expression. Surprisingly, this cloned AT 2 receptor has a 7-transmembrane topology, but ligand binding was unaffected by guanosine triphosphate (GTP) analogues. The novel receptor is a protein of 363 aminoacid residues with an estimated molecular weight of 40.9 kDa. It has 32% homology with the AT 1 receptor (356358). This finding will certainly catapult research on the AT 2 receptor into a new era.
Internalization Unlike the AT 1 receptor, the AT 2 receptor expressed in ovarian atretic follicles and in R3T3 cells does not undergo agonist-induced endocytosis (139,173). Upregulation of the A T 2 receptor has been noted in R3T3 cells treated with AT2 ligands (212). In contrast, in neuroblastoma NIE-115 cells, which express both AT 1 and AT2 receptors, there is a late downregulation of the AT 2 receptor occurring 30 minutes after exposure to Ang II (44). These observations may suggest that AT2 receptors in neural and peripheral tissues are heterogeneous.
Relative Proportion and Distribution Proportion The proportion of Ang receptors that are of the AT 2 subtype varies between tissues and species (see Table 2). As an example of interspecies variations, nonpregnant sheep, marmosets, and human myometrium express only the AT 2 receptor, whereas non pregnant rabbit and rat myometrium express both receptors (19,137,138). An example of intertissue variations has been reported in the monkey where the relative proportion of the AT2 receptor varies between less than 10 percent to a maximum of 58 percent in adrenal and kidney cortex respectively (81 ).
Distribution A number of studies have been published describing the distribution of Ang II receptors in brain (see Table 2). The AT2 receptor is located mainly in areas related to the control and learning of motor activity, as well as in sensory and visual areas and in the limbic system (213). Centers involved in the regulation of blood pressure, drinking, salt appetite, and vasopressin release do not express AT 2 receptors. AT 2 receptors are particularly abundant in the fetus where they show a transient pattern of expression (142,209,214). They will be described later in relation to their hypothesized role in growth and development.
Signaling Mechanisms Cells such as atretic follicular cells, NG I 08-15 cells, R3T3 cells, and PC 12W pheochromocytoma cells express only the AT 2 receptor and have been extensively utilized to study the intracellular mechanisms linked to this receptor. Despite extensive studies in these cell lines, the exact nature of the intracellular mechanisms coupled to the AT 2 receptor have not been clearly determined (139,173,208,215). Thus, the AT 2 receptor does not signal through G-proteins and does not affect inositol phosphate production, phosphatidylcholine turnover, intracellular calcium mobilization, or arachidonate metabolism. Neither does Ang II binding to the AT 2 receptor affect CAMP or tyrosine kinase activity (25,35,37, 127,139,141,173,208). Working with cultured neurones isolated from neonatal rat brain, which express predominantly AT 2 receptors, Sumners et al. ( 166) recently demonstrated that Ang II decreased intracellular cyclic guanosine monophosphate (cGMP). This effect of Ang II in neuronal cultures was found to be blocked by high doses of PD 123177 and CGP 42112, suggesting the involvement of AT 2 receptors. However, this effect appears to be linked 2 to a Ca +-calmodulin-dependent phosphodiesterase mechanism. Recent studies by Bottari et al. (6,216) indicate that in PC 12W cells stimulation of AT 2 receptors leads to a 40-percent decrease in both basal and atrialnatriuretic-peptide (ANP)-stimulated cGMP levels regardless of the presence ofa phosphodiesterase inhibitor. There is no effect on nitroprusside-stimulated cGMP levels, ruling out interactions between the AT 2 receptor and soluble guanylate cyclase (217). Particulate guanylate cyclase activity in both membrane particulates as well as in intact PC12W cells maintained in culture was inhibited by Ang II. These effects of Ang II were not affected by losartan but were mimicked by CGP 42112 (6,216,218220). Using the same experimental model, such an effect was not observed in smooth muscle cells, excluding suggestion that the effects of Ang II in PC 12W cells are AT 1linked. These data suggest that particulate guanylate cyclase and especially the ANP-receptor are targets of the AT2 receptor. Bottari et al. (6,216) have also demonstrated that the effect of Ang II on intracellular cGMP levels in PC12W cells can be abolished by 100-µM sodium orthovanadate, an inhibitor of protein tyrosine phosphatase, but not by okadaic acid which is a serine/threonine phosphatase inhibitor. Immunoblotting experiments using an antiphosphotyrosine monoclonal antibody have shown that Ang II induces a rapid dephosphorylation of tyrosine-phosphorylated proteins in PC 12W cells and that this effect is inhibited by orthovanadate. Furthermore (Fig. 5), Ang II dose-dependently stimulates the protein tyrosine phosphatase activity of PC 12W cell membranes using paranitrophenyl-phosphate as a sub-
PHYSIOLOGIC ROLE OF ANGIOTENSIN
150
....>. ·s:
===(.) ltl ltl U/ Q) ltl U/ .0 ltl~
0.~
140 130 120
I-
0.
110 100 0
1
5
10
50
100
Ang II (nM) FIG. 5. Effect of Ang II on phosphotyrosine phosphatase (PTPase) activity in PC12W plasma membrane particulate (mean ± SEM; n = 7). Membranes were preincubated for 20 mins at room temperature in the presence of increasing concentrations of Ang II. PTPase activity was determined after 60 min incubation using para-nitrophenylphosphate as a substrate. The results are expressed as the percentage of basal PTPase activity. (Florio et al. [222] and Pan et al. [223]).
II
RECEPTORS /
1707
high affinity for CGP 42112 and PD I 231 77 but which differ from "classical" AT 2 receptors in their sensitivity to OTT, GTP-')'-S, and pertussis toxin (224,225). These newly discovered binding sites which are found in the ventral thalamic nuclei, the medial geniculate nucleus, and the locus ceruleus have been designated AT23 while the "classic" AT 2 receptor found in the inferior olive is designated as AT 2b in this classification. Fluharty et al. (226,227) have also demonstrated the existence of two populations of AT2 receptors in cells of the NIE-115 neuroblastoma line, both of which exhibit a high affinity for CGP 42112 but no affinity for losartan. They differ in their sensitivity to PD 123319 and to DTT (Fluharty, personal communication). Moreover, using a specific anti-PLC-a antiserum, these investigators suggest that one of the CGP-42112-sensitive receptor subpopulations is coupled to PLC-a through a Gq-like protein. Classification of putative subpopulations will be postitive when more receptors will have been cloned. Angiotensin Binding Sites Other Than AT1 and AT2
The Cytoso/ic Binding Protein strate (221 ). It is therefore suggested that AT2 receptors signal by stimulating protein tyrosine phosphatase activity which modulates a series of cellular effector systems. A similar pathway has been recently reported for two members of the 7-transmembrane domain receptor superfamily, somatostatin and dopamine receptors (222,223). Decreased levels of cyclic GMP in response to Ang II is not a conclusion shared by all investigators who have utilized this cellular system (127,208,356). Moreover, in stably transfected cells, Ang II mediated on inhibition of protein cytosine phosphatase or was devoid of effect (357,358). Thus, the observations of Bottari et al. in PC 12W cells need to be confirmed and extended to other tissue systems before this putative signaling system of the AT2 receptor can be accepted. Another cellular target for the AT 2 receptor has recently been described in NG 108-15 hybridoma cells Which express only the AT 2 receptor (I 63). Using the technique of whole cell patch-clamping, Ang II, as well as CGP 42112, have been shown to inhibit T-type calcium currents at membrane potentials higher than -40 m V and to shift the current voltage curve to lower potentials. !hese effects are not affected by losartan but are inhibited by orthovanadate. The intracellular mechanisms mediating these effects of Ang II have not yet been determined.
Heterogeneity Tsutsumi and Saavedra (224) have recently shown the Presence of Ang II binding sites in rat brain that have a
A cytosolic protein with high affinity for Ang II and particularly Ang III has been detected in many tissues including liver, neonatal rat cardiomyocytes, myometrium, and in the brain (228-231). This Ang-II-binding protein of 80.8 kDa which has been isolated, purified, and cloned (230), shares no sequence homology with the AT I receptor. Neither losartan nor PD 123177 have any reasonable affinity for this protein (228). CGP 42112, however, shows a low affinity for this binding site ( 1 µM), similar to its affinity for the AT I receptor (24 ).
The Nuclear Binding Site Early studies demonstrated that injection of tritiated Ang II into the left ventricle of adult rats resulted in radioactivity appearing in the nucleus of endothelial and smooth muscle cells. These observations, which have since been confirmed in rat liver and spleen, suggest that Ang II or a fragment binds to a nuclear protein and may exert an action on nuclear function (232,233). The nuclear Ang II receptor binds losartan with high affinity but has a very low affinity for PD 1231 77 (234,235). Thus, the nuclear receptor appears to have binding properties similar to AT I receptors present in the plasma membrane and has a similar molecular weight. Another similarity between the nuclear binding protein and the plasma membrane AT I receptor is its coupling to G-proteins in a manner similar to that recently reported for the somatostatin nuclear receptor. The nature of the nuclear second messengers linked to the nuclear-binding-receptor-G-protein complex has not
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yet been reported, but possible candidates for signal transduction such as PKC, calcium-ATPase, and inositol trisphosphate-sensitive calcium channels are known to be localized in the nucleus (236,237). Slight differences between their binding properties and binding requirement suggest that the nuclear receptor, the cytosolic binding protein, and the plasma membrane AT, receptor are independent entities (24). Binding Sites for the Angiotensin II Fragments Ang (J-7) and Ang (3-8)
Angiotensin II is rapidly degraded both in blood and in tissues (238-240). Among the various metabolites of Ang II, a heptapetide (Ang 1-7) and a hexapeptide (Ang 3-8) have been detected (241 ).
itself exhibits very little affinity for either AT 1 or AT2 receptors (24). The density of this putative Ang (3-8) binding site is generally higher than that of AT I and AT 2 receptors. GTP--y-S has no effect on the binding of Ang II (3-8) in bovine aorta smooth muscle cells, indicating that this receptor is probably not coupled to a G-protein (254). Possible intracellular signaling mechanisms of this "receptor" have not been defined. However, calcium metabolism may be involved, since in rat aorta smooth muscle cells, Ang (3-8) produces a sustained increase in intracellular calcium and inositol trisphosphate, which are not altered by treatment with pertussis toxin (255). It has been proposed to call this receptor AT4 • Nonmammalian Angiotensin II Receptors
Ang II receptors expressed in amphibian and avian tissues, as well as in mycoplasma, appear to differ from mammalian receptors (24).
Ang (1-7)
Ang ( 1-7) has been shown to stimulate vasopressin release and to decrease blood pressure after injection into the area postrema, the nucleus of the tractus solitarius, or into the ventrolateral medulla (242,243). Ang ( 1-7) enhances prostaglandin synthesis in endothelial cells, vascular smooth muscle cells, astrocytes, glioma cells, and in perfused rat hearts (244-247). Furthermore, in the rat kidney, Ang ( 1-7) produces a substantial natriuresis and diuresis and an increase in glomerular filtration rate without affecting renal vascular resistance (248). In contrast to Ang II, Ang ( 1-7) does not produce peripheral vasoconstriction or dypsogenesis and does not stimulate aldosterone secretion (242). Since Ang ( 1-7) has actions different from those of Ang II and has only a weak affinity for either AT, and AT 2 receptors (24 ), it has been proposed that this heptapeptide acts via a separate receptor subtype. However, Ang (1-7) has a high affinity for the AT 1b receptor subtype present in proximal tubular epithelium (47). Thus in the kidney, Ang (1-7) may affect renal sodium and water handling by interacting with the AT,b receptor (248).
Amphibian Receptors
In amphibians, Ang II receptors are functionally similar to the AT, subtype since they mediate phosphoinositide hydrolysis and calcium mobilization. However, they bind peptide and nonpeptide ligands with different affinities than either mammalian AT, or AT 2 receptors. Thus, amphibian Ang II receptors have virtually no affinity for either losartan or PD 123177. CGP 42112, which selectively binds to the mammalian AT 2 receptor, also has high (nM) affinity for the amphibian Ang II receptor (256,257). Functionally, CGP 42 l 12, in contrast to losartan or PD 123177, inhibits Ang-II-induced increased calcium flux in frog oocytes, a pathway typical of the AT, receptor (257,258). Molecular cloning and sequencing of an amphibian Ang II receptor have pointed to a 60-percent amino acid identity and 65-percent nucleotide homology with the coding region of the mammalian AT, receptor (258). Avian Receptors
Ang (3-8)
Although Ang (3-8) has been termed Ang JV, a physiological role for this peptide has not yet been precisely defined. It has, however, been proposed that Ang (3-8) may be involved in endothelial cell-dependent vasodilation, renal cortical blood flow, and learning acquisition (249-251). Definite and distinct binding sites for Ang (3-8) have been described in sensitive tissues from a variety of species (250,252,253). Ang (3-8) binding in these tissues is saturable, reversible, and specific. Its affinity for its binding site is high, in the nanomolar range. Neither losartan nor CGP 42112 or PD 123177 are able to compete with radiolabeled Ang (3-8) for binding. Ang (3-8)
Losartan and PD 123319 bind poorly to Ang II receptors present in vascular smooth muscle and endothelial cells obtained from chicken tissues, as well as from the chicken egg chorio-allantoic membrane (259). CGP 42112, however, binds with high affinity to the Ang II receptor present in the chorio-allantoic membrane (260) and in this respect resembles the Ang II receptor found in amphibian membranes. In contrast to the mammalian AT, receptor, nonhydrolyzable GTP analogues do not alter the dissociation rate of Ang II bound to the avian receptor (261 ). Functionally, CGP 42112 is able to inhibit Ang-II-induced angiogenesis of pre- and postcapillary vessels, whereas PD 123319 and losartan are in-
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PHYSIOLOGIC ROLE OF ANGIOTENSIN
effective (260). In contrast, the initial blood pressure reduction induced by the natural ligand, Val 5-Ang II, in the anesthetized chicken was attenuated by both losartan and PD 123319 (259). These conflicting reports suggest that the avian receptor may not be identical to mammalian AT I or AT 2 receptors. Recently, a receptor has been cloned from turkey adrenocortical cells. It shows 78-percent amino acid identity and 85-percent nucleotide homology with the rat aortic smooth muscle AT1 receptor (262). lvficroorganisms
Microorganisms of the class Mollicutes such as lvfycop/asma and Ac/10/eplasma are common cell culture contaminants which also express an Ang II binding site. This binding site, which differs markedly from mammalian AT1 and AT 2 receptors (263,264), is characterized by a very low affinity for Ang III and a high affinity for both Ang ( 1-7) and Ang I. Losartan, PD 123319, and CGP 42112 do not bind to this site. Low concentrations of DTT increase the affinity of Ang II for this binding site by 35-50 percent, whereas bacitracin inhibits binding (ICso 30 µM). It is unknown at the present time whether the Ang II binding site in Aclw!eplasma is coupled to intracellular second messengers or whether Ang II medi~tes a physiological function through this site. Therefore, it cannot yet be considered a true receptor. Miscel/a11eous A11giote11si11 II Bi11di11g Sites
II
RECEPTORS /
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Iv!as-Oncogene
The mas-oncogene has been reported to code for a novel neuronal Ang II receptor. This receptor has been cloned and is reported to possess a molecular weight of 37 .5 kDa (268). When transfected into Xenopus oocytes, binding sites appear which mediate increased membrane chloride conductance and intracellular calcium mobilization in response to high concentrations (~ 1o- 6 M) of Ang II and Ang III. These findings suggest that the binding site encoded by the mas-oncogene in Xenopus oocytes is coupled to the endogenous inositol lipid/calcium mobilizing pathway in these cells. The changes in intracellular biochemistry produced by Ang II in this cell line are not blocked either by losartan or PD 123177 (269), suggesting that the mas-oncogene does not code for a classical, functional AT I or AT2 receptor (270). Indeed, the recent cloning of AT1 receptor cDNA has revealed that mas bears only a minor structural resemblance with less than 10 percent overall identity to this receptor. Several groups have cloned mas-related gene [mrg, rat thoracic aorta (RTA)] (271,272). Neither mrg nor RTA have been detected in tissues which display Ang II binding. Thus the 7-fransmembrane proteins encoded by the mas gene family are not authentic Ang II receptors. They appear, however, to enhance the expression or signaling sensitivity of endogenous Ang II receptors and their signaling pathways. Additional Angiotensin II Binding Sites
Other poorly characterized Ang II binding sites have been described (24). Mouse Neuroblastoma (Neuro-2a)
This cell line expresses a binding site that has a high affinity for Ang II but not for Ang III. Ligand binding in neuro-2a cells is not blocked by losartan or PD 123319 ~nd is not affected by GTP analogues. Disulfide bridge integrity does not play an essential role in ligand binding since DTT has only minor effects on receptor affinity. !his site has been tentatively named AT3 since its binding properties differ from the AT 1and AT2 receptors that are present in mammalian cells (265). In neuro-2a cells, Ang II at concentrations 1000-fold higher than its Ki for binding, causes a concentration-dependent increase in soluble guanylate cyclase activity which is not blocked by losartan or PD 123319 (266). This stimulatory effect on guanylate cyclase appears to be secondary to an increase in nitric oxide synthesis. Increased calcium influx via an ion channel distinct from the L type or N type may be the initiatory event in this response (267). In terms of receptor binding, the "AT3 receptor" in neuro-2a cells closely resembles the Ang-II-binding site in Acho!ep/asma and Mycop!asma.
Additional Ang II binding sites have been described in fetal mesenchyme (142) and after nephrectomy in the adrenal cortex (273). These are not typical AT 1 or AT2 receptors as they have poor affinity, if any, for losartan, PD 123177, or CGP 42112. More binding sites will be most probably discovered in the near future as our tools become more sophisticated. The Physiological Role of AT 1 and AT2 Receptors
Since the development of subtype-specific ligands, it has been possible to determine which tissues express Ang II receptor subtypes. In many tissues the intracellular processes and the ultimate response or responses mediated through the AT I receptor have been clearly established. It appears that the majority, if not all, of the known effects of Ang II are mediated through the AT 1 receptor (274). A large number of selective antagonists have been synthesized (see Fig. 3) (275,276) and all studies have demonstrated that, following activation of the renin-angiotensin system, blockade of the AT I receptor decreases blood pressure (BP) and aldosterone release affects drinking behavior, and modulates many of th~
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effects of Ang II on renal function (277). Thus, AT 1 antagonists, like ACE inhibitors, may have utility in the clinical settings of hypertension and congestive heart failure. In addition to effects on BP and fluid balance, Ang II acting through the AT 1 receptor stimulates the growth of various cells in culture and can reverse cardiac hypertrophy and neointima proliferation after experimental balloon angioplasty (278-282). Finally, AT I receptor blockade may also influence neural transmission and, at least in rodents, affect general behavior (283,284). In view of the numerous actions of AT 1 antagonists on systems other than hemodynamics and fluid balance, future studies with this class of compounds are anticipated to reveal potential therapeutic indications beyond hyper-
valsartan losartan
/
6d t AT
1
aldosterone production contraction release of hormones fluid regulation growth others
tension and heart failure. A description of the tissue responses linked to the AT 1 receptor has recently been the subject of several excellent journal reviews and are discussed in detail in other chapters of this book (6,66,277,285). The interested reader is therefore referred to these and other sources for further detailed information concerning the physiological processes linked to the AT 1 receptor. Blockade of the AT I receptor in animal and in man is accompanied by a compensatory increase in circulating Ang II (219,275,276,286-292). This may result in Ang II exerting some effects via the unblocked, i.e., non-AT 1 receptors (Fig. 6). The functional importance and clinical relevance of receptors other than AT I are not known
Ang II Ang Ill
.
Ang 1-7, Ang 3-8 other fragments
[VJ
[VJ
AT
AT
2
1
X
1
unknown
growth? proliferation ? differentiation ? neuronal channel modulation ? angiogenesis ? brain art. dilation ? others? FIG. 6. Blockade of the AT1 receptor on the juxtaglo_merular cells leads to an increase in renin and Ang
11 plasma levels. The unblocked receptors are accessible to Ang II and its various metabolites. This may lead to additional effects, the clinical relevance of which is still unknown, as the function of the AT recep~or and ot~er binding sites is still poorly understood. So far, the tolerance of the AT1 antagonists i: good 1n both animals and humans.
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PHYSIOLOGIC ROLE OF ANGIOTENSIN
and further investigation is therefore required to understand the possible therapeutic application of Ang II receptor blockade in cardiovascular and other diseases.
Is There a Physiological Role/or the AT1 Receptor? In contrast to our knowledge of the AT 1 receptor, the intracellular mechanisms and physiological processes mediated through the AT 2 receptor are still poorly, if at all, understood. Nonetheless, many reports have appeared in the recent literature demonstrating that AT 2 selective ligands have actions in several organ systems. Whether these observations can be considered evidence of functional AT rmediated responses is a matter of debate. Part of the confusion concerning the interpretation of studies using AT 2 specific ligands is related to our poor understanding of their mechanism of action. For example, it is often assumed that CGP 42112, PD 123319, and PD 123177 are antagonists of Ang II acting at AT2 receptors. However, this assumption has not been convincingly demonstrated. Indeed, it appears, at least in two cell lines, that CGP 42112 is a full agonist at AT 2 receptors (163,218); whereas both PD 123319 and PD 123177 display antagonistic properties (218,358). Furthermore, none of the selective ligands available for study are completely specific for the AT 2 receptors (29). Therefore, many studies performed with AT2 selective ligands are virtually impossible to interpret because of the very high concentrations of these compounds used. Indeed, it has recently been very clearly shown that at high doses CGP 42112 acts as a partial agonist at AT 1 receptors (293) and that PD 123319 may have some agonistic properties (48). In view of the confusion concerning the use of ATr specific ligands and the difficulty in correctly interpreting data from experiments where they have been used in Whole animal studies, this section is confined to a critical review of the current literature concerning the functional role of the AT2 receptor.
II
RECEPTORS /
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Angiotensin II-Induced Pressor Response Selective administration of Ang II into the cerebral ventricles or into certain brain structures, such as the paraventricular nucleus, leads to peripheral vasoconstriction and to an increase in systemic BP. There appears to be almost universal agreement that the pressor response to centrally administered Ang II is mediated by AT1 receptors (277). Thus, in both conscious and anesthetized rats and in the spontaneous hypertensive rat, intracerebroventricular (ICY) injection of 40-350 ng Ang II directly into the paraventricular nucleus produces an increase in BP which is completely blocked by losartan (0.7-10.0 µg ICY or 3 mg/kg ICY), but is unchanged by PD 123177 or PD 123319 (7 .0 µg ICY or 10 mg/kg IV) (295-30 I). The observations suggesting that the central cardiovascular effects of Ang II are mediated by AT 1 receptors are in agreement with the distribution of this receptor subtype in areas of the brain where Ang II produces its pressor action. In contrast, AT 2 receptors are present only outside areas involved in cardiovascular regulation (76). Interestingly, ICY injection of PD 123319 (80 µg) has recently been shown to produce a long-lasting antagonism (>48 hours) of the pressor effects of centrally administered Ang II ( 100 ng) (300). However, in this study PD 1233 I 9 was used at a high dose and the possibility exists that the AT2 antagonist may be blocking the central effects of Ang II at the AT 1 receptor or be exhibiting nonspecific properties (300).
Angiotensin-Il-Induced Thirst Injection of Ang II (40-100 ng) into the brain ventricles causes drinking in animals. This response to Ang II appears to be mediated by AT 1 receptors since it is blocked by hr.1· Res Co1111111111 1992: J 88:298-303. 62. Makita N. lwai N, lnagami T, Badr KF. /Jioche111 /JiofJh.rs Res ('111111111111 1992: 185: 142-146. 63. Sandberg K. Ji II, Clark AJ, Shapira II. Catt KJ. J Biol Chem 1992: 26 7:9455-9458. 64. Sandberg K. Tian Y. Ji II. 75th A111111al .\leering Tire Endocrine Sociefl' 1993; 355-l 220B. 65. Hodg~s JC. Hamby J M. Blankley CJ. Drugs F11111rc I 992; I 7: 575-593. 66. Smith RD, Chiu AT. Wong PC. llerhlin WF, Timmermans PB. A111111 Rel' l'lwrmacol Toxicol 1992; 32: 135-165. 67. Steckelings UM, Obermuller N, Bottari SP, Qadri F. Vcltmar A. Unger T. l'lwr111acol '/inicol 1992: 70:S23-S27. 68. Bunncrnann B. Fuxc K, Gantcn D. Reg11I l'efJl I 993:46:487-509. 69. Balla T, Baukal AJ, Eng S. Catt KJ. ,\fol l'lwr111acol 1991;40: 401-406. 70. Na ville D, Lcbrcthon MC. Kcrmabon A Y, Roucr E. Bcnarous R. Sac, J. FUJS l.
