Expression of endothelin receptor subtypes in rabbit saphenous vein

ML Webb, EC Liu, H Monshizadegan, CC Chao, J Lynch, SM Fisher and PM Rose

Department of Cardiovascular Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.

Recent investigations have revealed the presence of vasoconstrictory endothelin (ET)-B receptors in several tissues, including the rabbit saphenous vein (RSV). To determine the nature of the ET binding sites in RSV, radioligand-receptor binding studies with selective ligands and Northern analyses with probes from the ET-A and ET-B receptor cDNAs were conducted. ET-1 inhibited 125I-ET-1 binding to RSV in a monophasic manner, with an inhibition constant (Ki) of 0.08 +/- 0.02 nM and a slope factor of 0.9 +/- 0.1. ET-3 inhibition of 125I-ET-1 binding was biphasic, with 68% of the 125I-ET-1 binding sites being displaceable with a Ki value of 31 +/- 4 nM. The remaining 32% of the sites displayed high affinity for ET-3 (Ki = 0.2 +/- 0.1 nM). The ET-A- selective peptide BQ-123 inhibited 125I-ET-1 binding in a biphasic manner, with Ki values of 10.4 +/- 1.9 nM and 3.2 +/- 0.9 microM. The high affinity BQ-123 site comprised 70% of the binding sites, whereas the low affinity site comprised 30%. The correspondence of high affinity binding sites for BQ-123 and low affinity binding sites for ET- 3 is consistent with the suggestion that 70% of the 125I-ET-1 binding sites in this tissue are ET-A receptors. To further investigate the nature of the ET-B binding sites in RSV, 125I-ET-3 competition binding experiments were conducted. ET-1 and BQ-123 inhibited 125I-ET-3 binding in RSV with Ki values of 40 +/- 7 pM and 7.2 microM, respectively, whereas inhibition curves for ET-3 and the ET-B receptor-selective agonist sarafotoxin S6c (S6c) were best fit to two-site models. Resultant Ki values for ET-3 and S6c were 50 pM (71%)/4 pM (29%) and 0.7 nM (71%)/95 nM (29%), respectively. Binding in RSV differed from that in rat cerebellum, where ET-3 and S6c inhibition of 125I-ET-3 binding was monophasic (Ki values of 70 pM and 1.1 nM for ET-3 and S6c, respectively). The presence of the nonhydrolyzable guanine nucleotide analog guanosine-5'-O-(3-thio)triphosphate (200 microM) did not affect 125I-ET-3 binding. Low stringency Northern analysis of RSV RNA with [alpha-32P]dCTP-labeled fragments from the ET-A or ET-B receptor cDNAs revealed similar hybridization patterns with both probes, with two resolved RNA species migrating at 4.7 and 1.8 kilobases.(ABSTRACT TRUNCATED AT 400 WORDS)

Volume 44, Issue 5, pp. 959-965, 11/01/1993
Copyright © 1993 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				D. Eguchi, J. Nishimura, S. Kobayashi, K. Komori, K. Sugimachi, and H. Kanaide Down-regulation of endothelin B receptors in autogenous saphenous veins grafted into the arterial circulation Cardiovasc Res, August 1, 1997; 35(2): 360 - 367. [Abstract] [Full Text] [PDF]