Expression of endothelin receptor subtypes in rabbit saphenous vein
Webb, M.L.; Liu, E.C.; Monshizadegan, H.; Chao, C.C.; Lynch, J.; Fisher, S.M.; Rose, P.M.
Molecular Pharmacology 44(5): 959-965
1993
ISSN/ISBN: 0026-895X PMID: 8246919 Document Number: 410561
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-8 receptor cDNAs were conducted. ET-1 inhibited 125I-ET-1 binding to RSV in a monophasic manner, with an inhibition constant (K-i) 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 K-i value of 31 +- 4 nM. The remaining 32% of the sites displayed high affinity for ET-3 (K-i = 0.2 +- 0.1 nM). The ET-A-selective peptide BQ-123 inhibited 125I-ET-1 binding in a biphasic manner, with K-i values of 10.4 1.9 nm and 3.2 0.9 mu-M. 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-1 23 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 BQ123 inhibited 125I-ET-3 binding in RSV with K-i values of 40 +- 7 pM and 7.2 mu-M, 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 K-i 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 (K-i 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 mu-M) 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. These data indicate that the ET receptor population in RSV is composed of approximately 70% ET-A receptors and 30% ET-B receptors, with a component of the ET-B population displaying very high affinity for ET-3 and moderate affinity for S6c.