CHARACTERIZATION OF THE RABBIT VENTRICULAR MYOCARDIAL RECEPTOR FOR ANGIOTENSIN-II - EVIDENCE FOR 2 SITES OF DIFFERENT AFFINITIES AND SPECIFICITIES

Abstract

Angiotensin II binding sites in a rabbit ventricular myocardial particulate fraction were identified and characterized with the radioligand 125I-angiotensin II. The order of potency in competing with 125I-angiotensin II for these sites was similar to that observed in physiological studies. Computer-assisted analysis of the competition of binding sites for 0.3 nM 125I-angiotensin II by unlabeled angiotensin II (3 .times. 10-11 M to 1 .times. 10-5 M) demonstrated that optimal fitting of the competition curves was attained with a 2-site model having 1 site of high affinity (KA1 [angiotensin II binding site constant] = 2.4 .+-. 0.6 .times. 109 M-1), low capacity (N1 = 7.8 .+-. 0.8 fmol/mg of protein) and a 2nd site low affinity (KA2 = 9.6 .+-. 0.6 .times. 106 M-1) and high capacity (N2 = 219 .+-. 128 fmol/mg of protein). Analysis of competition by Sar1-Ile8 angiotensin II for 125I-angiotensin II binding sites indicated that the antagonist interacted with the 1st site with high affinity (KA1 = 8 .times. 109 M-1), but interacted minimally with the second site (KA2 = 105 M-1). Monovalent cations (Na+, K+, Li+, NH4+) were roughly equipotent in decreasing 125I-angiotensin II binding by reducing the number of high-affinity sites (N1 = 2.6 .+-. 0.7 fmol/mg of protein with 100 mM Na+) without changing the affinity of either site or the number of low-affinity sites. The number of high-affinity sites was increased to 14.4 .+-. 1.5 fmol/mg of protein by 5 mM Mg2+. In the presence of divalent cations, nucleotides reduced binding of 125I-angiotensin II with the potency order guanosyl-5''-yl-imidodiphosphate > GTP > GDP > ATP > GMP. Guanosyl-5''yl-imido-diphosphate significantly reduced the affinity of the high-affinity site (KA1 = 1.0 .+-. 0.2 .times. 109 M-1) and perhaps of the low-affinity site (KA2 = 1.0 .+-. 2.2 .times. 106 M-1). Computer-assisted assessment of dissociation of 0.3 nM 125I-angiotensin II from rabbit myocardial membranes corroborated the equilibrium data: dissociation was biphasic (k-1 = 0.19 .+-. 0.2 min-1 for a rapidly dissociating site, k-1 = 2.5 .+-. 2.1 .times. 10-3 min-1 for a slowly dissociating site); 5 mM Mg2+ did not significantly change either dissociation rate; but guanosyl-5''-yl-imidodiphosphate significantly increased dissociation rates from both sites. Despite the indirect evidence that these angiotensin II receptors interact with guanine nucleotide regulatory proteins, angiotensin II (10-6 M) failed to influence adenylate cyclase activity. The physiological implications of the presence in ventricular myocardium of 2 distinct angiotensin II receptors and in particular the implications of a receptor-associated guanine nucleotide regulatory protein which does not couple to adenylate cyclase, require further investigation.