Regulatory Peptides, 40 (1992) 409-419 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00
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REGPEP 01204
Rapid communication
Discovery of a distinct binding site for angiotensin II (3-8), a putative angiotensin IV receptor Geoffrey N. Swanson a, Jodie M. Hanesworth a, Michael F. Sardinia a, John K.M. Coleman b, John W. Wright a'b, Keith L. Hall ~, Allison V. Miller-Wing ~, Jeffrey W. Stobb ~, Victoria I. Cook a, Erin C. Harding ~ and Joseph W. Harding a'b aDepartments of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA (USA) and bDepartment of Psychology, Washington State University, Pullman, WA
(USA) (Received 14 April 1992; revised version received and accepted 7 May 1992)
Key words: Radioligand binding; Receptor autoradiography; Bovine adrenal; Angiotensin; Renal blood flow
Summary We report here the discovery of a unique and novel angiotensin binding site and peptide system based upgn the C-terminal 3-8 hexapeptide fragment of angiotensin II (NH 3 +-Val-Tyr-Ile-His-Pro-Phe-COO-) (AII(3-8) (AIV)). This fragment binds saturably, reversibly, specifically, and with high affinity to membrane-binding sites in a variety of tissues and from many species. The binding site is pharmacologically distinct from the classic angiotensin receptors (AT 1 or AT2) displaying low affinity for the known agonists (AII and AIII) and antagonist (SarX,IleS-AII). Although a definitive function has not been assigned to this system in many of the tissues in which it resides, AIV's interaction with endothelial cells may involve a role in endothelial cell-dependent vasodilation. Consequent to this action, AIV is a potent stimulator of renal cortical blood flow.
Correspondence to: J.W. Harding, Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University Pullman, WA 99164-6520, USA.
410
Introduction
The renin-angiotensin system has wide-ranging actions on numerous tissues, playing a role primarily directed at cardiovascular and electrolyte homeostasis [2,12,16,20]. The current view suggests that angiotensin II (AII) and angiotensin III (AIII) are the biologically active forms of angiotensin and that they are synthesized via a cascade of enzymatic cleavages beginning with the action of renin on angiotensinogen to produce angiotensin I (AI), a biologically inactive decapeptide. Angiotensin II, the classical bioactive peptide, is formed by the action of angiotensin converting enzyme on circulating AI. AIII, which is derived from All by aminopeptidase action, is also active and may have important functions in the brain [ 11 ]. Angiotensin II and AIII activity is thought to be terminated by enzymatic degradation to a series of fragments. Until now, fragments smaller than AIII were thought to be biologically inactive and of little physiological significance. This assumption has been based on the general inability of angiotensin metabolites to activate clas sic angiotensin-dependent processes [ 5,8,19,20 ]. Additionally, N-terminal deleted fragments smaller than AIII exhibit low binding affinities to angiotensin receptors as determined by radioligand binding studies [4,9]. Despite the lack of interaction between angiotensin fragments and All receptors, we report here the existence of an entirely new class of angiotensin binding site highly specific for one such fragment, the 3-8 fragment of AII, which we have called AIV [22]. This new site does not bind All or AIII unless they undergo prior metabolic conversion to AIV. This delineation of a separate AIV binding site is not without prior physiological support. In cultured chick cardiocytes, AIV has been shown to antagonize All induced increases in RNA synthesis and protein synthesis, despite its inability to compete for 125I-AII binding [3]. This observation is consistent with two distinct receptors. Also, intracerebroventricularly applied AIV has been shown to enhance recall of passive avoidance conditioning in rats [6]. Given the low affinity of AIV for AT t and AT 2 receptors [4,9], the receptor system documented here could explain this finding. Further, AII and AIV appear to employ different mechanisms of calcium mobilization in rat vascular smooth muscle cells [7]. The results reported here extend these diverse observations and definitively demonstrate a high affinity binding site for AIV that is distinct and separate from All receptors. This demonstration provides a biochemical basis for many previously unexplained physiological observations.
Materials and Methods
Radioligand binding studies Initial experiments were conducted to examine the kinetic parameters of ~25I-AIV binding sites present in membranes of bovine adrenal cortical cells, and to compare this binding to the classic AII binding site as defined by lzSI-Sarl,IleS-AII. Binding was carded out in a buffer system designed to minimize metabolism of both receptor and ligand. 1 g of adrenal cortex was first homogenized in 10 ml hypotonic buffer (50 mM Tris, 5 mM EDTA, pH 7.4 at 4 ° C). Homogenized tissue was then centrifuged at 1000 rpm for 10 min (500 g). Membranes located in the supernatant were decanted off the
411
pellet and saved. The pellet was homogenized a second time in 10 ml hypotonic buffer, and centrifuged again at 1000 rpm for 10 min. The supernatants were combined and then pelleted at 20,000 rpm for 20 min (40,000 g). The pellet was washed with 10 ml hypotonic buffer and spun at 20,000 rpm for 20 rain. This pellet was resuspended in 2 ml hypotonic buffer and a Lowry assay [ 14] was performed to determine protein content. After adjusting tissue to a concentration of 2.5 mg/ml, 10 #l/tube of this tissue suspension was then used in binding assays. Assays were carried out in triplicate in an incubation volume of 250/~1 for 2 h at 37°C, filtered through glass fiber filters (Schleicher and Schuell, No. 32), and washed four times with 5 ml of phosphatebuffered saline. The incubation buffer contained 50 mM Tris, 150 mM NaC1, 5 mM EDTA, 20 #M bestatin, 100 #M PMSF, 50 #M 2-mercaptomethyl-3-guanidoethylthioproprionic acid and 0.1 ~o heat-treated bovine serum albumin (BSA).
Solubilized receptor studies/ligand metabolism Membranes were prepared as described above for the membrane-binding assay. The membranes were then loaded onto a discontinuous sucrose gradient (0.65 M, 1.3 M) and centrifuged at 38,000 rpm for 90 min (100,000 g). The tissue interface was removed in 8 ml and incubated 10 rain with 20 mM MgC12. The tissue was then heated for 20 min at 60°C. After the heat step, the tissue was diluted 1:5 with hypotonic buffer and centrifuged at 20,000 rpm for 30 min (40,000 g) to remove most of the sucrose. Pellets were rehomogenized in 1 ml hypotonic buffer, combined, a Lowry [ 14] protein assay performed and the tissue suspension adjusted to a protein concentration of 10 mg/ml. Tissue was then solubilized in 1~o CHAPS for 2 h at 4°C. At the end of the solubilization, tissue was spun for 60 rain at 38,000 rpm (100,000 g) and filtered through a 0.22 #M filter. Supernatant was collected, diluted 1:4, and 10 #1 of this was used in the assay. Metabolism of the labeled ligand was assessed directly in the binding assays themselves. At the conclusion of incubation, the binding assay suspensions were filtered through 1 ml gel filtration columns (Biogel P6, Bio-Rad) to separate bound from free. The bound ligand passed through the column, leaving the free labeled ligand trapped in the column. This bound receptor-ligand complex was collected directly into 20~o TCA to denature any proteins present. Afterwards, the denatured protein was centrifuged out and the samples were analyzed by HPLC. The HPLC method employed is described in detail in Abhold and Harding [1 ].
Receptor autoradiography Tissue was frozen sectioned at 20 micron thickness and sections were thaw mounted on subbed slides and stored at -20 ° C. Prior to incubation, sections were thawed and preincubated in Coplin jars containing isotonic buffer with the same composition and concentration of inhibitors used in the homogenate binding assays for 30 rain at room temperature. The sections were then placed in incubation jars containing radiolabeled ligands (0.6 nM) and displacers (100 nM to define nonspecific binding) for 2 h at room temperature. After incubation was complete, sections were washed in a series of three 2-min washes and dried under a stream of air. Sections were then exposed to film (Kodak 5B-5) for 2 days.
412
Renal cortical blood flow A final set of experiments were carried out to investigate the relationship of AIV and renal blood flow. Superficial blood flow in the rat kidney cortex was assessed using a laser Doppler flowmeter in anesthetized rats during direct infusion of test substances via the renal artery. All rats were anesthetized with Ketamine-Rompun (100 and 2 mg/ml respectively: 1 ml/kg). The animals were prepared with carotid catheters which allowed for the measurement of blood pressure. Continuous monitoring of blood pressure was accomplished via Statham transducer and polygraph (Grass Instruments, Model 5B). The left kidney was exposed using a ventrolateral approach, the renal artery was isolated from the surrounding fascia, and an approx. 1 cm section was exposed. Experimental and control infusions were delivered via a 33 g needle inserted into the surgically exposed renal artery. The 33 g needle was attached to tubing (PE 10, Clay Adams) which in turn was connected to the infusion pump (Sage Instruments). The subjects were infused with the following compounds: 0.15 M NaC1, 100 pmol angiotensin II (AII), 100 pmol D-Val I AIV or 100 pmol AIV for 10 min at 25/~l/min. The experimental infusions were counterbalanced with the saline infusions to control for order of effects. Blood flow was measured in the kidney cortex, using laser Doppler flowmetry [ 15]. The laser Doppler probe was placed on the posterior lateral aspect of the renal capsule, thus allowing for the assessment of renal cortical blood flow.
Results Both 125I-Sar1,Ile8-AII and 125I-AIV binding were characterized by slow association rates (K 1 --- 1.01 + 0.12.10 -2 and 5.58 + 0.64.10-2 (nM-1 min-1), respectively), very slow dissociation rates ( K 1 = 2.36 + 0.49.10- 2 and 2.57 + 0.05.10- 2 (min- 1), respectively), and high affinity binding (calculated K d = 2.25 + 0.26.10-- l0 and 4.42 + 0.46.10- 10 (M), respectively) (n = 4). Equilibrium dissociation constants from saturation isotherms also demonstrated high affinity binding for both ligands Kd (~25Isarl,IleS-AII: 0.54 + 0.14" 10- 9M; x25I-AIV: 0.74 + 0.14-10- 9M; mean ___S.D.; n = 4) and large differences in Bmax (125I-Sarl,Ile8-AII: 1.03 + 0.26 pmol/mg protein; 1251AIV: 3.82 + 1.12 pmol/mg protein; mean + S.D.; n = 4) which suggested distinct binding sites. An example saturation isotherm for 125I-AIV binding to bovine adrenal cortical membranes is shown in Fig. 1. Both ligands' binding was characterized by Hill Coefficients of 1.00 + 0.03 (mean + S.D.; n = 4) indicating no cooperativity and suggestive of a single class of binding site for each ligand. Competition analysis of bovine adrenal binding (Table I) highlights the different structural requirements for the two binding sites and indicates their distinct nature. The site defined by 125I-Sarl,IleS-AII was effectively bound by sarl,IleS-AII, AII, AIII and DuP 753, but AIV, AII(3-7), D-Val I AIV and CGP 42112A exhibited very little affinity for the AII binding site. This pattern of binding is consistent with an AT1 AII binding site [ 18]. The site defined by ~25I-AIV bound AIV and, to a lesser extent, AIII, AII(3-7), and AII(3-6). Further N-terminal shortening to AII(3-5) drastically lowered the affinity for this highly specific site as did the substitution of D-Val for L-Val at position number 1. The AIV site bound neither DuP 753 nor CGP 42112A demon-
413 30
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0.1,
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0.o
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.
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.
•
30
o 0
i
i
i
1
2
3
4
Total(nM)
Fig. 1. Saturation isotherm of ~251-AIV binding in membranes from bovine adrenal cortex. Nonspecific binding was defined as binding in the presence of 100 nM AIV. Assays were carried at 37°C for 2 h in a cocktail of peptidase inhibitors (see Materials and Methods for details) using 25 ~g of membrane protein. Ligand metabolism at the end of the incubation period was typically less than 10 ~o. The above example curve was developed from 12 data points run in duplicate. Binding constants calculated from these data were as follows: KI9 = 6.33' 10 - 10 M; B~a x = 2.89 pmol/mg protein; and, Hill coefficient = 1.01. The rfor the Rosenthal plot was 0.975.
strating that it did not possess the pharmacological characteristics of either A T 1 o r A T 2 All binding sites [18,21]. Angiotensin III binding to the AIV site appears to be an artifact of N-terminal metabolism of AIII to AIV because bound ligand recovered from
TABLE I Competition curves for ]25I-Sar~,IleS-AnglI and ~25I-AIV(3-8) binding to bovine adrenal cortical membranes Competitor
125I-Sarl,IleS-Angll binding (Ki, M)
125I-AIV binding (Ki, M)
Sarq,IleS-AII AII AIII AIV [ A I I ( 3 - 8 ) ] AII(4-8) DuP753 C G P 42112A PD123177 D-Val I AIV
0.22 + 0.10' 10- 9, 2.01 + 0 . 6 7 ' 1 0 - 9 1.15 + 0.34.10- 9 > 10-6 > 10 -6 3.10 + 0.67' 10- s > 10 - 4 > 10 -4 > 10 -6
> 10- 6 > 10 -6 14.50 + 2.3' 10- 9 0.58 + 0.15' 10- 9 > 10 -6 > 10- 4
AII(3-7) AII(3-6) AII(3-5) AII(3-4) AII(1-7)
ND ND ND ND ND
1.65 _+ 1.18' 10 - 8 8.31 _+3.79' 10 - 8 > 10- 6 > 10 - 6 > 10 - 6
* Mean _+S.D., n = 2-4. N D = not determined.
>
10 - 4
> 10 - 4 > 10 -6
414 solubilized bovine adrenal receptors, subsequent to incubation with 125I-AIII, was 100~o ~25I-AIV. Furthermore, it was shown that the binding achieved with ~25I-AIII in different solubilized receptor preparations was directly proportional to the metabolic formation of 125I-AIV (data not shown). The inability of A I I and Sar~,IleS-AII to bind to the AIV site suggests that N-terminal elongation of AIV beyond L-Val is detrimental to binding. In addition, many other N-terminal extended analogs of AIV which have been synthesized in our laboratory also exhibited low affinity for the AIV site ( K i s > 10 - 6 M, data not shown). In addition, the loss of the N-terminal L-Val or its substitution by D-Val resulted in a massive decrease in binding affinity. A I I ( 3 - 7 ) still binds with reasonable affinity while smaller C-terminal deleted fragments bind poorly. It has yet to be determined what effect elongation of the C-terminal beyond position 8 will have on binding affinity. Together, these structure-binding studies directed at the N- and C-terminals of the bound ligand indicate strict structural requirements for the Nterminal, clearly different for AT~ or AT 2 receptors [4,9,18,21], while the C-terminal requirements of the receptor may be much less stringent. A second major approach employed to define separate and distinct binding sites was to examine their relative species (Table II) and tissue (Table III) distribution. Since guinea-pig adrenal also exhibited high levels of AIV binding, we utilized this species to conduct a general tissue distribution study of 125I-AIV binding. As presented in Table III, AIV binding sites were prevalent in all tissues of cardiovascular interest including brain, heart, kidney, aorta, liver and lung. Although large variations in the levels of binding can be seen among the guinea-pig tissues, no tissue is devoid of the AIV binding site. This is not surprising given the high concentration of AIV sites on vascular endothelial cells (Table II) and the fact that all tissues are vascularized. In comparison to 125I-Sar1,IleS-AII binding, the binding of 125I-AIV ranged from nearly equivalent in the liver to more than 20-times greater in the uterus.
TABLE II Distribution of 125I-SI-AIIand 125I-AIVbinding to mammalian tissuesa Species
Tissue
125I-Sarl,IleS-AIIbindingh
125I-AIVbinding
Bovine Pig Horse Dog Cat Rabbit Guinea-pig Bovine
adrenal medula whole adrenal whole adrenal whole adrenal whole adrenal whole adrenal whole adrenal coronary venular endothelial cells heart
218.8 + 56.2 < 1.0 1.8 + 1.0 4.6 + 0.7 3.3 _+2.3 79.6 _+21.6 45.6 + 9.2
397.3 + 53.6 70.8 + 6.7 72.7 +_13.5 36.5 + 5.4 199.6 + 19.7 105.3 _+15.6 101.2 + 26.3
2.9 _+0.3 10.6 + 3.6
85.1 + 3.3 249.9 + 36.3
Rabbit
a 25 #g of membrane protein was incubated with 500,000 cpm of label (0.6 nM). Specific binding was defined as total binding-nonspecificbinding at 100 nM unlabeled peptide. b fmol/mg protein; n = 2-6; mean _+S.D.
415 TABLE III Distribution of 125I-Sarl,IleS-AnglIand '25I-AIVbinding sites in the membranesof guinea-pig tissuesa Tissue
125I-Sara,IleS-AnglIbindingb
125I-AIV
binding
Aorta Brain Heart Kidney Liver Lung Uterus
3.17 + 2.2 17.7 + 9.5 5.7 + 0.6 8.1 + 1.9 22.4 + 5.3 12.2_+0.4 4.0 + 1.5
45.4 + 11.0 60.8 + 13.5 83.3 + 20.8 22.7 + 12.1 28.9 + 6.4 56.1 + 10.1 87.0 _+6.3
n = 4; mean + S.D.
a Binding was carried out as described in Table II. b fmol/mgprotein.
Not only is the AIV receptor system widely distributed in guinea-pig tissues, but the AIV receptor system was found to be present in a wide range of mammalian species. Table II provides examples of the location of binding sites among several species and highlights the universality of this system. In those cases where the AIV receptor system has been examined in detail, the binding characteristics have been nearly identical. These detailed analyses have been carried out in bovine coronary venular endothelial cells (K d = 0.70 + 0.10 nM, Bmax = 476 + 57 fmol/mg protein, n = 4, mean + S.D.), rabbit heart (K d = 1.7 + 0.5 nM, B m a x = 731 + 163 fmol/mg protein, n = 4, mean + S.D.), and guinea-pig brain (K d = 0.42 _+0.09 nM, Bmax -- 118.9 + 20.1 fmol/mg protein, n = 4, mean + S.D.). In all cases, the binding sites exhibited a structure-binding relationship identical to that observed for bovine adrenal cortex (Table I). In order to clearly illustrate and underscore the distinct nature of the AIV and S ar~,Ile8-AII binding sites, receptor autoradiographs of 20 micron thick serial sections of guinea-pig brain have been included. The sample sections (Fig. 2) reveal distinct 125I-AIV binding in the hippocampus, thalamic nuclei and cerebral cortex (Fig. 2A) with ~zSI-Sarl,IleS-AII binding primarily localized to fiber systems (Fig. 2D). While 125I-AIV binding was totally displaced by 100 nM AIV (Fig. 2B), it was unaffected by 100 nM Sarl,IleS-AII (Fig. 2C). Conversely, 125I-Sarl,IleS-AII was displaced by 100 nM Sarl,Ile8-AII (Fig. 2F), but not 100 nM AIV (Fig. 2E). In agreement with the competition curves, the autoradiographic data support the existence of two separate receptors. Together these distribution studies establish the conservation of the 12SI-AIV binding site across species and tissues. The results obtained from infusion of AII, AIV and D-Val 1 AIV are presented in Fig. 3 and demonstrate that AIV infused at 100 pmol/min stimulated a profound and sustained increase in blood flow, while AII at 100 pmol/min, produced a dramatic decrease in flow. The infusion of the nonbinding analog D-vall-AIV or 0.15 M NaC1 had no effect on renal blood flow. Consistent with the involvement of different receptors in the mediation of AII and AIV effects, the specific AII antagonist Sar~,IleS-AII (1 nmol/min - 10 rain pretreatment) completely blocked the AII effect while having no effect on AIV (data not shown). The increase in blood flow witnessed with AIV was
416
A
B
(_~
Fig. 2. Receptor autoradiography of coronal sections of guinea-pig brain. (A) Total t251-AIV binding. (B) ~25I-AIV binding displaced with 100 nM AIV. (C) 12~I-AIV binding displaced with 100 nM Sarl,IleS-AII. (D) Total 125I-SarX,IleS-AII binding. (E) 125I-Sarl,IleS-All binding displaced with 100 nM AIV. (F) 125ISarJ,IleS-AII displaced with 100 nM Sar~,IleS-AII. 60
40 20
"~o
I~
0 -20 -40 0
2
4
6
8
10
Time (rain) Fig. 3. Renal cortical blood flow in rats following infusion of angiotensins into the renal artery. The above figure depicts the percent change in renal blood flow due to infusions of 100 pmol AIV (n = 13) (O), 0.15 M saline (0.15 M NaC1) (n = 9) (O), 100 pmol D-Val 1 AIV (n = 9) (m) and 100 pmol AII (n = 8) ([~) into the renal artery at 25/A/min. See text for details of the method.
not accompanied by alterations in mean arterial pressure suggesting that AIV's effects may be limited to selective vascular beds, may induce compensatory changes in cardiac output, or that AIV is rapidly degraded in the kidney.
417
Discussion The universal nature of the AIV system encourages speculation that this system must play an important physiological role. In an initial attempt to demonstrate a physiological function for the AIV system, we have focused on its role in the control of renal blood flow. The rationale for initially choosing to examine the kidney was four-fold. First, the AIV binding site is found in significant concentrations in kidney and endothelial cells. Second, vascular endothelial cells are known to regulate vascular tone by several mechanisms and play a key role in the control of renal blood flow. Third, AIV has been reported to decrease renin release in humans following systemic infusion [ 131. Since renin release is inversely proportional to renal blood flow, it follows that if AIV is vasodilatory via its action on renal endothelial cells, it should be expected to produce the observed decrease in renin release. And finally, AII(3-8) has been implicated as a vasodilator in rat cerebral arteries where it appears to act via an endothelial-derived relaxing factor (EDRF) mechanism [lo]. Given the evidence that AIV can mediate vasodilation in the kidney and cerebral arterioles [ lo], that AIV receptors are highly concentrated on endothelial cells, and that endothelial cells regulate the level of vasodilation by release of endogenous regulators like EDRF, it is tempting to speculate that AIV’s interaction with its endothelial cell receptor stimulates EDRF synthesis and release. This possible link of AIV with EDRF may, in fact, extend beyond the vascular system to the brain. The enrichment of AIV receptors in the hippocampus, its known ability to enhance cognitive function [6], the hippocampus’ known involvement in memory and learning, and the recent linkage of EDRF to memory-related processes in the hippocampus [ 171 further suggest a plausible link between AIV and EDRF. In addition to its role in the regulation of vasodilation and cognitive function, AIV may mediate other actions. The distribution of AIV binding sites in a number of tissues including brain, adrenals, heart, pituitary, and blood vessels where they are not exclusively localized on endothelial cells is consistent with various functions. Autoradiographic data and studies on cells in culture demonstrate that AIV receptors found in the above mentioned tissues are not exclusively endothelial in nature, but are located directly on cardiocytes, adrenal cortical and adrenal chromafIin cells, and smooth muscle cells (Harding, Hall, Hainesworth, unpublished data). It is presently unknown whether the action of AIV on various tissues will be characterized by a common physiological function (e.g., growth control) or whether the role of AIV will be as diverse as the tissues themselves. In summary, this study demonstrates the existence of an entirely new angiotensin receptor with unique specificity, distribution and functional characteristics. Additionally, the present investigation should stimulate an examination of: (a) the physiological role of NH, + -Val-Tyr-Ile-His-Pro-related peptides; (b) the synthetic process responsible for their production; (c) the biochemical properties of their receptor(s) and the genes coding these proteins; (d) the regulation of tissue responsiveness to these nentides: and (e) alterations in the svstem that might accomnanv certain nathol-
418
Acknowledgements This study was supported by NIH grant HL 32063 and an American Heart Association Established Investigator Award to Joseph W. Harding. Animals employed in this study were housed in an AAALAC-approved facility. All surgical procedures were carried out in accordance with institutional guidelines. We would like to thank Paul Barron-Bates, Bea O'Neill, and Ruth Day's efforts in preparing this manuscript and Dr. Robert Speth's assistance with the autoradiography. In addition we would like to thank Dr. Ron Smith of Dupont-Merck for the gift of DuP 753 and Marc de Gasparo of Ciba-Geigy for the gift of CGP 42112A, and David Taylor of Warner Lambert-Parke Davis for the gift of PD 123177.
References 1 Abhold, R.H. and Harding, J.W., Metabolism of angiotensin II and III by membrane peptidases from rat brain, J. Pharmacol. Exp. Ther., 245 (1988) 171-177. 2 Antonaccio, M.J. and Wright, J.J., Renin-angiotensin system, converting enzyme, and renin inhibitors. In M. Antonaccio (Ed.), Cardiovascular Pharmacology, Raven Press, New York, 1990, pp. 201-228. 3 Baker, K.M. and Aceto, J.F., Angiotensin II stimulation of protein synthesis and cell growth in chick heart cells, Am. J. Physiol., 259 (1990) H610-H618. 4 Bennett, J. P., Jr. and Snyder, S.H., Angiotensin II binding to mammalian brain membranes, J. Biol. Chem., 251 (1976) 7423-?430. 5 Blair-West, J. R., Coghlan, J.P., Denton, D.A., Funder, J.W., Scoggins, B.A. and Wright, R.D., The effect of the heptapeptide (2-8) and hexapeptide (3-8) fragments of angiotensin II on aldosterone secretion, J. Clin. Endocrinol. Metab., 32 (1971) 575-578. 6 Braszko, J.J., Kupryszewski, G., Witczuk, B. and Wisniewski, K., Angiotensin II(3-8)-hexapeptide affects motor activity, performance of passive avoidance and conditioned avoidance response in rats, Neuroscience 27 (1988) 777-783. 7 Dostal, D. E., Murahashi, T. and Peach, M.J., Regulation of cytosolic calcium by angiotensins in vascular smooth muscle, Hypertension, 15 (1990) 815-822. 8 Fitzsimons, J.T., The effect on drinking of peptide precursors and of shorter chain peptide fragments of angiotensin II injected into the rats' diencephalon, J. Physiol. Lond., 214 (1971) 295-303. 9 Glossman, H., Baukal, A. and Catt, K.J., Properties of angiotensin II receptors in the bovine and rat adrenal cortex, J. Biol. Chem., 249 (1974) 825-834. 10 Haberl, R.L., Decker, P.J. and Einh~tupl, K.M., Angiotensin degradation products mediate endothelium-dependent dilation of rabbit brain arterioles, Circ. Res., 68 (1991) 1621-1627. 11 Harding, J.W., Jensen, L.L., Quirk, W. S., Dewey, A.L. and Wright, J.W., Brain angiotensin: Critical role in the ongoing regulation of body fluid homeostasis and cardiovascular function, Peptides, 10 (l 989) 261-264. 12 Johnston, C.I., Biochemistry and pharmacology of the renin-angiotensin system, Drugs, 39 (Suppl. 1) (1990) 21-31. 13 Kono, T., Ikeda, F., Tamiguchi, A., Imura, H., Oseko, F., Yoshioka, M. and Khosla, M., Responses of patents with Barter's syndrome to angiotensin III and angiotensin II-(3-8)-hexapeptide, Acta Endocrinol., 109 (1985) 249-253. 14 Lowry, O., Rosebrough, N., Farr, A. and Randall, R., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 15 Miller, J.M., Marks, N.J. and Goodwin, P.C., Laser Doppler measurements of cochlear blood flow, Hear. Res., 11 (1983) 385-394. 16 Phillips, M.I., Functions of angiotensin in the central nervous system, Annu. Rev. Physiol., 49 (1987) 413-435.
419 17 Schumann, E.M. and Madison, D.V., A requirement for the intercellular messenger nitric oxide in long-term potentiation, Science, 254 (1991) 1503-1506. 18 Timmermans, P.B., Wong, P.C., Chui, A.T. and Harblin, W.F., Nonpeptide angiotensin II receptor antagonists, Trends Pharmacol. Sci., 12 (1991) 55-62. 19 Tonnaer, J.A., Weigant, V.M., DeJong, W. and DeWied, D., Central effects of angiotensins on drinking and blood pressure: Structure-activity relationships, Brain Res., 236 (1982) 417-428. 20 Unger, T., Badoer, E., Ganten, D., Lang, R.E. and Rettig, R., Brain angiotensin: Pathways in pharmacology, Circulation, 77 (Suppl. I) (1988) 1-40-1-54. 21 Wong, P.C., Tam, S.W., Herblin, W.F. and Timmermans, P.B., Further studies on the selectivity of DuP 753, a nonpeptide angiotensin II receptor antagonist, Eur. J. Pharmacol., 196 (1991) 201-203. 22 Wright, J. W., Roberts, K.A. and Harding, J. W., Drinking to intracerebroventricularly infused angiotensin II, III and IV in the SHR, Peptides, 9 (1988) 979-984.
409
REGPEP 01204
Rapid communication
Discovery of a distinct binding site for angiotensin II (3-8), a putative angiotensin IV receptor Geoffrey N. Swanson a, Jodie M. Hanesworth a, Michael F. Sardinia a, John K.M. Coleman b, John W. Wright a'b, Keith L. Hall ~, Allison V. Miller-Wing ~, Jeffrey W. Stobb ~, Victoria I. Cook a, Erin C. Harding ~ and Joseph W. Harding a'b aDepartments of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA (USA) and bDepartment of Psychology, Washington State University, Pullman, WA
(USA) (Received 14 April 1992; revised version received and accepted 7 May 1992)
Key words: Radioligand binding; Receptor autoradiography; Bovine adrenal; Angiotensin; Renal blood flow
Summary We report here the discovery of a unique and novel angiotensin binding site and peptide system based upgn the C-terminal 3-8 hexapeptide fragment of angiotensin II (NH 3 +-Val-Tyr-Ile-His-Pro-Phe-COO-) (AII(3-8) (AIV)). This fragment binds saturably, reversibly, specifically, and with high affinity to membrane-binding sites in a variety of tissues and from many species. The binding site is pharmacologically distinct from the classic angiotensin receptors (AT 1 or AT2) displaying low affinity for the known agonists (AII and AIII) and antagonist (SarX,IleS-AII). Although a definitive function has not been assigned to this system in many of the tissues in which it resides, AIV's interaction with endothelial cells may involve a role in endothelial cell-dependent vasodilation. Consequent to this action, AIV is a potent stimulator of renal cortical blood flow.
Correspondence to: J.W. Harding, Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University Pullman, WA 99164-6520, USA.
410
Introduction
The renin-angiotensin system has wide-ranging actions on numerous tissues, playing a role primarily directed at cardiovascular and electrolyte homeostasis [2,12,16,20]. The current view suggests that angiotensin II (AII) and angiotensin III (AIII) are the biologically active forms of angiotensin and that they are synthesized via a cascade of enzymatic cleavages beginning with the action of renin on angiotensinogen to produce angiotensin I (AI), a biologically inactive decapeptide. Angiotensin II, the classical bioactive peptide, is formed by the action of angiotensin converting enzyme on circulating AI. AIII, which is derived from All by aminopeptidase action, is also active and may have important functions in the brain [ 11 ]. Angiotensin II and AIII activity is thought to be terminated by enzymatic degradation to a series of fragments. Until now, fragments smaller than AIII were thought to be biologically inactive and of little physiological significance. This assumption has been based on the general inability of angiotensin metabolites to activate clas sic angiotensin-dependent processes [ 5,8,19,20 ]. Additionally, N-terminal deleted fragments smaller than AIII exhibit low binding affinities to angiotensin receptors as determined by radioligand binding studies [4,9]. Despite the lack of interaction between angiotensin fragments and All receptors, we report here the existence of an entirely new class of angiotensin binding site highly specific for one such fragment, the 3-8 fragment of AII, which we have called AIV [22]. This new site does not bind All or AIII unless they undergo prior metabolic conversion to AIV. This delineation of a separate AIV binding site is not without prior physiological support. In cultured chick cardiocytes, AIV has been shown to antagonize All induced increases in RNA synthesis and protein synthesis, despite its inability to compete for 125I-AII binding [3]. This observation is consistent with two distinct receptors. Also, intracerebroventricularly applied AIV has been shown to enhance recall of passive avoidance conditioning in rats [6]. Given the low affinity of AIV for AT t and AT 2 receptors [4,9], the receptor system documented here could explain this finding. Further, AII and AIV appear to employ different mechanisms of calcium mobilization in rat vascular smooth muscle cells [7]. The results reported here extend these diverse observations and definitively demonstrate a high affinity binding site for AIV that is distinct and separate from All receptors. This demonstration provides a biochemical basis for many previously unexplained physiological observations.
Materials and Methods
Radioligand binding studies Initial experiments were conducted to examine the kinetic parameters of ~25I-AIV binding sites present in membranes of bovine adrenal cortical cells, and to compare this binding to the classic AII binding site as defined by lzSI-Sarl,IleS-AII. Binding was carded out in a buffer system designed to minimize metabolism of both receptor and ligand. 1 g of adrenal cortex was first homogenized in 10 ml hypotonic buffer (50 mM Tris, 5 mM EDTA, pH 7.4 at 4 ° C). Homogenized tissue was then centrifuged at 1000 rpm for 10 min (500 g). Membranes located in the supernatant were decanted off the
411
pellet and saved. The pellet was homogenized a second time in 10 ml hypotonic buffer, and centrifuged again at 1000 rpm for 10 min. The supernatants were combined and then pelleted at 20,000 rpm for 20 min (40,000 g). The pellet was washed with 10 ml hypotonic buffer and spun at 20,000 rpm for 20 rain. This pellet was resuspended in 2 ml hypotonic buffer and a Lowry assay [ 14] was performed to determine protein content. After adjusting tissue to a concentration of 2.5 mg/ml, 10 #l/tube of this tissue suspension was then used in binding assays. Assays were carried out in triplicate in an incubation volume of 250/~1 for 2 h at 37°C, filtered through glass fiber filters (Schleicher and Schuell, No. 32), and washed four times with 5 ml of phosphatebuffered saline. The incubation buffer contained 50 mM Tris, 150 mM NaC1, 5 mM EDTA, 20 #M bestatin, 100 #M PMSF, 50 #M 2-mercaptomethyl-3-guanidoethylthioproprionic acid and 0.1 ~o heat-treated bovine serum albumin (BSA).
Solubilized receptor studies/ligand metabolism Membranes were prepared as described above for the membrane-binding assay. The membranes were then loaded onto a discontinuous sucrose gradient (0.65 M, 1.3 M) and centrifuged at 38,000 rpm for 90 min (100,000 g). The tissue interface was removed in 8 ml and incubated 10 rain with 20 mM MgC12. The tissue was then heated for 20 min at 60°C. After the heat step, the tissue was diluted 1:5 with hypotonic buffer and centrifuged at 20,000 rpm for 30 min (40,000 g) to remove most of the sucrose. Pellets were rehomogenized in 1 ml hypotonic buffer, combined, a Lowry [ 14] protein assay performed and the tissue suspension adjusted to a protein concentration of 10 mg/ml. Tissue was then solubilized in 1~o CHAPS for 2 h at 4°C. At the end of the solubilization, tissue was spun for 60 rain at 38,000 rpm (100,000 g) and filtered through a 0.22 #M filter. Supernatant was collected, diluted 1:4, and 10 #1 of this was used in the assay. Metabolism of the labeled ligand was assessed directly in the binding assays themselves. At the conclusion of incubation, the binding assay suspensions were filtered through 1 ml gel filtration columns (Biogel P6, Bio-Rad) to separate bound from free. The bound ligand passed through the column, leaving the free labeled ligand trapped in the column. This bound receptor-ligand complex was collected directly into 20~o TCA to denature any proteins present. Afterwards, the denatured protein was centrifuged out and the samples were analyzed by HPLC. The HPLC method employed is described in detail in Abhold and Harding [1 ].
Receptor autoradiography Tissue was frozen sectioned at 20 micron thickness and sections were thaw mounted on subbed slides and stored at -20 ° C. Prior to incubation, sections were thawed and preincubated in Coplin jars containing isotonic buffer with the same composition and concentration of inhibitors used in the homogenate binding assays for 30 rain at room temperature. The sections were then placed in incubation jars containing radiolabeled ligands (0.6 nM) and displacers (100 nM to define nonspecific binding) for 2 h at room temperature. After incubation was complete, sections were washed in a series of three 2-min washes and dried under a stream of air. Sections were then exposed to film (Kodak 5B-5) for 2 days.
412
Renal cortical blood flow A final set of experiments were carried out to investigate the relationship of AIV and renal blood flow. Superficial blood flow in the rat kidney cortex was assessed using a laser Doppler flowmeter in anesthetized rats during direct infusion of test substances via the renal artery. All rats were anesthetized with Ketamine-Rompun (100 and 2 mg/ml respectively: 1 ml/kg). The animals were prepared with carotid catheters which allowed for the measurement of blood pressure. Continuous monitoring of blood pressure was accomplished via Statham transducer and polygraph (Grass Instruments, Model 5B). The left kidney was exposed using a ventrolateral approach, the renal artery was isolated from the surrounding fascia, and an approx. 1 cm section was exposed. Experimental and control infusions were delivered via a 33 g needle inserted into the surgically exposed renal artery. The 33 g needle was attached to tubing (PE 10, Clay Adams) which in turn was connected to the infusion pump (Sage Instruments). The subjects were infused with the following compounds: 0.15 M NaC1, 100 pmol angiotensin II (AII), 100 pmol D-Val I AIV or 100 pmol AIV for 10 min at 25/~l/min. The experimental infusions were counterbalanced with the saline infusions to control for order of effects. Blood flow was measured in the kidney cortex, using laser Doppler flowmetry [ 15]. The laser Doppler probe was placed on the posterior lateral aspect of the renal capsule, thus allowing for the assessment of renal cortical blood flow.
Results Both 125I-Sar1,Ile8-AII and 125I-AIV binding were characterized by slow association rates (K 1 --- 1.01 + 0.12.10 -2 and 5.58 + 0.64.10-2 (nM-1 min-1), respectively), very slow dissociation rates ( K 1 = 2.36 + 0.49.10- 2 and 2.57 + 0.05.10- 2 (min- 1), respectively), and high affinity binding (calculated K d = 2.25 + 0.26.10-- l0 and 4.42 + 0.46.10- 10 (M), respectively) (n = 4). Equilibrium dissociation constants from saturation isotherms also demonstrated high affinity binding for both ligands Kd (~25Isarl,IleS-AII: 0.54 + 0.14" 10- 9M; x25I-AIV: 0.74 + 0.14-10- 9M; mean ___S.D.; n = 4) and large differences in Bmax (125I-Sarl,Ile8-AII: 1.03 + 0.26 pmol/mg protein; 1251AIV: 3.82 + 1.12 pmol/mg protein; mean + S.D.; n = 4) which suggested distinct binding sites. An example saturation isotherm for 125I-AIV binding to bovine adrenal cortical membranes is shown in Fig. 1. Both ligands' binding was characterized by Hill Coefficients of 1.00 + 0.03 (mean + S.D.; n = 4) indicating no cooperativity and suggestive of a single class of binding site for each ligand. Competition analysis of bovine adrenal binding (Table I) highlights the different structural requirements for the two binding sites and indicates their distinct nature. The site defined by 125I-Sarl,IleS-AII was effectively bound by sarl,IleS-AII, AII, AIII and DuP 753, but AIV, AII(3-7), D-Val I AIV and CGP 42112A exhibited very little affinity for the AII binding site. This pattern of binding is consistent with an AT1 AII binding site [ 18]. The site defined by ~25I-AIV bound AIV and, to a lesser extent, AIII, AII(3-7), and AII(3-6). Further N-terminal shortening to AII(3-5) drastically lowered the affinity for this highly specific site as did the substitution of D-Val for L-Val at position number 1. The AIV site bound neither DuP 753 nor CGP 42112A demon-
413 30
~"
0.3
20'
O O
E e,,,,i
o rn
0.1,
lO. [
0.o
t
~
.
,
.
•
30
o 0
i
i
i
1
2
3
4
Total(nM)
Fig. 1. Saturation isotherm of ~251-AIV binding in membranes from bovine adrenal cortex. Nonspecific binding was defined as binding in the presence of 100 nM AIV. Assays were carried at 37°C for 2 h in a cocktail of peptidase inhibitors (see Materials and Methods for details) using 25 ~g of membrane protein. Ligand metabolism at the end of the incubation period was typically less than 10 ~o. The above example curve was developed from 12 data points run in duplicate. Binding constants calculated from these data were as follows: KI9 = 6.33' 10 - 10 M; B~a x = 2.89 pmol/mg protein; and, Hill coefficient = 1.01. The rfor the Rosenthal plot was 0.975.
strating that it did not possess the pharmacological characteristics of either A T 1 o r A T 2 All binding sites [18,21]. Angiotensin III binding to the AIV site appears to be an artifact of N-terminal metabolism of AIII to AIV because bound ligand recovered from
TABLE I Competition curves for ]25I-Sar~,IleS-AnglI and ~25I-AIV(3-8) binding to bovine adrenal cortical membranes Competitor
125I-Sarl,IleS-Angll binding (Ki, M)
125I-AIV binding (Ki, M)
Sarq,IleS-AII AII AIII AIV [ A I I ( 3 - 8 ) ] AII(4-8) DuP753 C G P 42112A PD123177 D-Val I AIV
0.22 + 0.10' 10- 9, 2.01 + 0 . 6 7 ' 1 0 - 9 1.15 + 0.34.10- 9 > 10-6 > 10 -6 3.10 + 0.67' 10- s > 10 - 4 > 10 -4 > 10 -6
> 10- 6 > 10 -6 14.50 + 2.3' 10- 9 0.58 + 0.15' 10- 9 > 10 -6 > 10- 4
AII(3-7) AII(3-6) AII(3-5) AII(3-4) AII(1-7)
ND ND ND ND ND
1.65 _+ 1.18' 10 - 8 8.31 _+3.79' 10 - 8 > 10- 6 > 10 - 6 > 10 - 6
* Mean _+S.D., n = 2-4. N D = not determined.
>
10 - 4
> 10 - 4 > 10 -6
414 solubilized bovine adrenal receptors, subsequent to incubation with 125I-AIII, was 100~o ~25I-AIV. Furthermore, it was shown that the binding achieved with ~25I-AIII in different solubilized receptor preparations was directly proportional to the metabolic formation of 125I-AIV (data not shown). The inability of A I I and Sar~,IleS-AII to bind to the AIV site suggests that N-terminal elongation of AIV beyond L-Val is detrimental to binding. In addition, many other N-terminal extended analogs of AIV which have been synthesized in our laboratory also exhibited low affinity for the AIV site ( K i s > 10 - 6 M, data not shown). In addition, the loss of the N-terminal L-Val or its substitution by D-Val resulted in a massive decrease in binding affinity. A I I ( 3 - 7 ) still binds with reasonable affinity while smaller C-terminal deleted fragments bind poorly. It has yet to be determined what effect elongation of the C-terminal beyond position 8 will have on binding affinity. Together, these structure-binding studies directed at the N- and C-terminals of the bound ligand indicate strict structural requirements for the Nterminal, clearly different for AT~ or AT 2 receptors [4,9,18,21], while the C-terminal requirements of the receptor may be much less stringent. A second major approach employed to define separate and distinct binding sites was to examine their relative species (Table II) and tissue (Table III) distribution. Since guinea-pig adrenal also exhibited high levels of AIV binding, we utilized this species to conduct a general tissue distribution study of 125I-AIV binding. As presented in Table III, AIV binding sites were prevalent in all tissues of cardiovascular interest including brain, heart, kidney, aorta, liver and lung. Although large variations in the levels of binding can be seen among the guinea-pig tissues, no tissue is devoid of the AIV binding site. This is not surprising given the high concentration of AIV sites on vascular endothelial cells (Table II) and the fact that all tissues are vascularized. In comparison to 125I-Sar1,IleS-AII binding, the binding of 125I-AIV ranged from nearly equivalent in the liver to more than 20-times greater in the uterus.
TABLE II Distribution of 125I-SI-AIIand 125I-AIVbinding to mammalian tissuesa Species
Tissue
125I-Sarl,IleS-AIIbindingh
125I-AIVbinding
Bovine Pig Horse Dog Cat Rabbit Guinea-pig Bovine
adrenal medula whole adrenal whole adrenal whole adrenal whole adrenal whole adrenal whole adrenal coronary venular endothelial cells heart
218.8 + 56.2 < 1.0 1.8 + 1.0 4.6 + 0.7 3.3 _+2.3 79.6 _+21.6 45.6 + 9.2
397.3 + 53.6 70.8 + 6.7 72.7 +_13.5 36.5 + 5.4 199.6 + 19.7 105.3 _+15.6 101.2 + 26.3
2.9 _+0.3 10.6 + 3.6
85.1 + 3.3 249.9 + 36.3
Rabbit
a 25 #g of membrane protein was incubated with 500,000 cpm of label (0.6 nM). Specific binding was defined as total binding-nonspecificbinding at 100 nM unlabeled peptide. b fmol/mg protein; n = 2-6; mean _+S.D.
415 TABLE III Distribution of 125I-Sarl,IleS-AnglIand '25I-AIVbinding sites in the membranesof guinea-pig tissuesa Tissue
125I-Sara,IleS-AnglIbindingb
125I-AIV
binding
Aorta Brain Heart Kidney Liver Lung Uterus
3.17 + 2.2 17.7 + 9.5 5.7 + 0.6 8.1 + 1.9 22.4 + 5.3 12.2_+0.4 4.0 + 1.5
45.4 + 11.0 60.8 + 13.5 83.3 + 20.8 22.7 + 12.1 28.9 + 6.4 56.1 + 10.1 87.0 _+6.3
n = 4; mean + S.D.
a Binding was carried out as described in Table II. b fmol/mgprotein.
Not only is the AIV receptor system widely distributed in guinea-pig tissues, but the AIV receptor system was found to be present in a wide range of mammalian species. Table II provides examples of the location of binding sites among several species and highlights the universality of this system. In those cases where the AIV receptor system has been examined in detail, the binding characteristics have been nearly identical. These detailed analyses have been carried out in bovine coronary venular endothelial cells (K d = 0.70 + 0.10 nM, Bmax = 476 + 57 fmol/mg protein, n = 4, mean + S.D.), rabbit heart (K d = 1.7 + 0.5 nM, B m a x = 731 + 163 fmol/mg protein, n = 4, mean + S.D.), and guinea-pig brain (K d = 0.42 _+0.09 nM, Bmax -- 118.9 + 20.1 fmol/mg protein, n = 4, mean + S.D.). In all cases, the binding sites exhibited a structure-binding relationship identical to that observed for bovine adrenal cortex (Table I). In order to clearly illustrate and underscore the distinct nature of the AIV and S ar~,Ile8-AII binding sites, receptor autoradiographs of 20 micron thick serial sections of guinea-pig brain have been included. The sample sections (Fig. 2) reveal distinct 125I-AIV binding in the hippocampus, thalamic nuclei and cerebral cortex (Fig. 2A) with ~zSI-Sarl,IleS-AII binding primarily localized to fiber systems (Fig. 2D). While 125I-AIV binding was totally displaced by 100 nM AIV (Fig. 2B), it was unaffected by 100 nM Sarl,IleS-AII (Fig. 2C). Conversely, 125I-Sarl,IleS-AII was displaced by 100 nM Sarl,Ile8-AII (Fig. 2F), but not 100 nM AIV (Fig. 2E). In agreement with the competition curves, the autoradiographic data support the existence of two separate receptors. Together these distribution studies establish the conservation of the 12SI-AIV binding site across species and tissues. The results obtained from infusion of AII, AIV and D-Val 1 AIV are presented in Fig. 3 and demonstrate that AIV infused at 100 pmol/min stimulated a profound and sustained increase in blood flow, while AII at 100 pmol/min, produced a dramatic decrease in flow. The infusion of the nonbinding analog D-vall-AIV or 0.15 M NaC1 had no effect on renal blood flow. Consistent with the involvement of different receptors in the mediation of AII and AIV effects, the specific AII antagonist Sar~,IleS-AII (1 nmol/min - 10 rain pretreatment) completely blocked the AII effect while having no effect on AIV (data not shown). The increase in blood flow witnessed with AIV was
416
A
B
(_~
Fig. 2. Receptor autoradiography of coronal sections of guinea-pig brain. (A) Total t251-AIV binding. (B) ~25I-AIV binding displaced with 100 nM AIV. (C) 12~I-AIV binding displaced with 100 nM Sarl,IleS-AII. (D) Total 125I-SarX,IleS-AII binding. (E) 125I-Sarl,IleS-All binding displaced with 100 nM AIV. (F) 125ISarJ,IleS-AII displaced with 100 nM Sar~,IleS-AII. 60
40 20
"~o
I~
0 -20 -40 0
2
4
6
8
10
Time (rain) Fig. 3. Renal cortical blood flow in rats following infusion of angiotensins into the renal artery. The above figure depicts the percent change in renal blood flow due to infusions of 100 pmol AIV (n = 13) (O), 0.15 M saline (0.15 M NaC1) (n = 9) (O), 100 pmol D-Val 1 AIV (n = 9) (m) and 100 pmol AII (n = 8) ([~) into the renal artery at 25/A/min. See text for details of the method.
not accompanied by alterations in mean arterial pressure suggesting that AIV's effects may be limited to selective vascular beds, may induce compensatory changes in cardiac output, or that AIV is rapidly degraded in the kidney.
417
Discussion The universal nature of the AIV system encourages speculation that this system must play an important physiological role. In an initial attempt to demonstrate a physiological function for the AIV system, we have focused on its role in the control of renal blood flow. The rationale for initially choosing to examine the kidney was four-fold. First, the AIV binding site is found in significant concentrations in kidney and endothelial cells. Second, vascular endothelial cells are known to regulate vascular tone by several mechanisms and play a key role in the control of renal blood flow. Third, AIV has been reported to decrease renin release in humans following systemic infusion [ 131. Since renin release is inversely proportional to renal blood flow, it follows that if AIV is vasodilatory via its action on renal endothelial cells, it should be expected to produce the observed decrease in renin release. And finally, AII(3-8) has been implicated as a vasodilator in rat cerebral arteries where it appears to act via an endothelial-derived relaxing factor (EDRF) mechanism [lo]. Given the evidence that AIV can mediate vasodilation in the kidney and cerebral arterioles [ lo], that AIV receptors are highly concentrated on endothelial cells, and that endothelial cells regulate the level of vasodilation by release of endogenous regulators like EDRF, it is tempting to speculate that AIV’s interaction with its endothelial cell receptor stimulates EDRF synthesis and release. This possible link of AIV with EDRF may, in fact, extend beyond the vascular system to the brain. The enrichment of AIV receptors in the hippocampus, its known ability to enhance cognitive function [6], the hippocampus’ known involvement in memory and learning, and the recent linkage of EDRF to memory-related processes in the hippocampus [ 171 further suggest a plausible link between AIV and EDRF. In addition to its role in the regulation of vasodilation and cognitive function, AIV may mediate other actions. The distribution of AIV binding sites in a number of tissues including brain, adrenals, heart, pituitary, and blood vessels where they are not exclusively localized on endothelial cells is consistent with various functions. Autoradiographic data and studies on cells in culture demonstrate that AIV receptors found in the above mentioned tissues are not exclusively endothelial in nature, but are located directly on cardiocytes, adrenal cortical and adrenal chromafIin cells, and smooth muscle cells (Harding, Hall, Hainesworth, unpublished data). It is presently unknown whether the action of AIV on various tissues will be characterized by a common physiological function (e.g., growth control) or whether the role of AIV will be as diverse as the tissues themselves. In summary, this study demonstrates the existence of an entirely new angiotensin receptor with unique specificity, distribution and functional characteristics. Additionally, the present investigation should stimulate an examination of: (a) the physiological role of NH, + -Val-Tyr-Ile-His-Pro-related peptides; (b) the synthetic process responsible for their production; (c) the biochemical properties of their receptor(s) and the genes coding these proteins; (d) the regulation of tissue responsiveness to these nentides: and (e) alterations in the svstem that might accomnanv certain nathol-
418
Acknowledgements This study was supported by NIH grant HL 32063 and an American Heart Association Established Investigator Award to Joseph W. Harding. Animals employed in this study were housed in an AAALAC-approved facility. All surgical procedures were carried out in accordance with institutional guidelines. We would like to thank Paul Barron-Bates, Bea O'Neill, and Ruth Day's efforts in preparing this manuscript and Dr. Robert Speth's assistance with the autoradiography. In addition we would like to thank Dr. Ron Smith of Dupont-Merck for the gift of DuP 753 and Marc de Gasparo of Ciba-Geigy for the gift of CGP 42112A, and David Taylor of Warner Lambert-Parke Davis for the gift of PD 123177.
References 1 Abhold, R.H. and Harding, J.W., Metabolism of angiotensin II and III by membrane peptidases from rat brain, J. Pharmacol. Exp. Ther., 245 (1988) 171-177. 2 Antonaccio, M.J. and Wright, J.J., Renin-angiotensin system, converting enzyme, and renin inhibitors. In M. Antonaccio (Ed.), Cardiovascular Pharmacology, Raven Press, New York, 1990, pp. 201-228. 3 Baker, K.M. and Aceto, J.F., Angiotensin II stimulation of protein synthesis and cell growth in chick heart cells, Am. J. Physiol., 259 (1990) H610-H618. 4 Bennett, J. P., Jr. and Snyder, S.H., Angiotensin II binding to mammalian brain membranes, J. Biol. Chem., 251 (1976) 7423-?430. 5 Blair-West, J. R., Coghlan, J.P., Denton, D.A., Funder, J.W., Scoggins, B.A. and Wright, R.D., The effect of the heptapeptide (2-8) and hexapeptide (3-8) fragments of angiotensin II on aldosterone secretion, J. Clin. Endocrinol. Metab., 32 (1971) 575-578. 6 Braszko, J.J., Kupryszewski, G., Witczuk, B. and Wisniewski, K., Angiotensin II(3-8)-hexapeptide affects motor activity, performance of passive avoidance and conditioned avoidance response in rats, Neuroscience 27 (1988) 777-783. 7 Dostal, D. E., Murahashi, T. and Peach, M.J., Regulation of cytosolic calcium by angiotensins in vascular smooth muscle, Hypertension, 15 (1990) 815-822. 8 Fitzsimons, J.T., The effect on drinking of peptide precursors and of shorter chain peptide fragments of angiotensin II injected into the rats' diencephalon, J. Physiol. Lond., 214 (1971) 295-303. 9 Glossman, H., Baukal, A. and Catt, K.J., Properties of angiotensin II receptors in the bovine and rat adrenal cortex, J. Biol. Chem., 249 (1974) 825-834. 10 Haberl, R.L., Decker, P.J. and Einh~tupl, K.M., Angiotensin degradation products mediate endothelium-dependent dilation of rabbit brain arterioles, Circ. Res., 68 (1991) 1621-1627. 11 Harding, J.W., Jensen, L.L., Quirk, W. S., Dewey, A.L. and Wright, J.W., Brain angiotensin: Critical role in the ongoing regulation of body fluid homeostasis and cardiovascular function, Peptides, 10 (l 989) 261-264. 12 Johnston, C.I., Biochemistry and pharmacology of the renin-angiotensin system, Drugs, 39 (Suppl. 1) (1990) 21-31. 13 Kono, T., Ikeda, F., Tamiguchi, A., Imura, H., Oseko, F., Yoshioka, M. and Khosla, M., Responses of patents with Barter's syndrome to angiotensin III and angiotensin II-(3-8)-hexapeptide, Acta Endocrinol., 109 (1985) 249-253. 14 Lowry, O., Rosebrough, N., Farr, A. and Randall, R., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 15 Miller, J.M., Marks, N.J. and Goodwin, P.C., Laser Doppler measurements of cochlear blood flow, Hear. Res., 11 (1983) 385-394. 16 Phillips, M.I., Functions of angiotensin in the central nervous system, Annu. Rev. Physiol., 49 (1987) 413-435.
419 17 Schumann, E.M. and Madison, D.V., A requirement for the intercellular messenger nitric oxide in long-term potentiation, Science, 254 (1991) 1503-1506. 18 Timmermans, P.B., Wong, P.C., Chui, A.T. and Harblin, W.F., Nonpeptide angiotensin II receptor antagonists, Trends Pharmacol. Sci., 12 (1991) 55-62. 19 Tonnaer, J.A., Weigant, V.M., DeJong, W. and DeWied, D., Central effects of angiotensins on drinking and blood pressure: Structure-activity relationships, Brain Res., 236 (1982) 417-428. 20 Unger, T., Badoer, E., Ganten, D., Lang, R.E. and Rettig, R., Brain angiotensin: Pathways in pharmacology, Circulation, 77 (Suppl. I) (1988) 1-40-1-54. 21 Wong, P.C., Tam, S.W., Herblin, W.F. and Timmermans, P.B., Further studies on the selectivity of DuP 753, a nonpeptide angiotensin II receptor antagonist, Eur. J. Pharmacol., 196 (1991) 201-203. 22 Wright, J. W., Roberts, K.A. and Harding, J. W., Drinking to intracerebroventricularly infused angiotensin II, III and IV in the SHR, Peptides, 9 (1988) 979-984.
