Expression of endothelin receptor subtypes in rabbit saphenous vein.
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)
Webb
M L
ML
Department of Cardiovascular Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.
Liu
E C
EC
Monshizadegan
H
H
Chao
C C
CC
Lynch
J
J
Fisher
S M
SM
Rose
P M
PM
D000818:Animals; D001667:Binding, Competitive; D018076:DNA, Complementary; D016232:Endothelins / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D011817:Rabbits; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000378:metabolism*; D012501:Saphenous Vein / Q000378:metabolism*
D018076:DNA, Complementary; D016232:Endothelins; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin