title: Characterization of the endothelin receptor in primary cultures of human aortic smooth muscle cells.
abstract: We characterized the endothelin receptor subtypes in primary cultures of human aortic smooth muscle cells (HASMCs) by binding studies. [125I]-Endothelin (ET)-1 saturation experiments showed the existence of a homogeneous population of binding sites with the high affinity (KD value) of 97 +/- 37 pM and maximum number of binding sites (Bmax) of 54 +/- 10 fmol/mg protein. However, almost no specific [125I]-ET-3 binding was observed. Inhibition of [125I]-ET-1 binding in the HASMCs membrane by nonlabeled compounds showed the following order of effectiveness: ET-1 = ET-2 = FR139317 >> ET-3. These results suggest that the endothelin receptor of HASMCs is of the ETA type. We also studied the effect of ET-1 on the cytosolic [Ca2+]i in HASMCs loaded with fura-2/AM. In 1.3 mM Ca2+, ET-1 produced a dose-dependent, biphasic increase in signal with a maximal effect at 10 nM. At this concentration, ET-1 produced a transient increase in [Ca2+]i that reached a peak at 1 min, which was followed by a slow but sustained increase in [Ca2+]i. This second phase was attenuated in Ca(2+)-deficient medium. Furthermore, ET-1 increased inositol 1,4,5-triphosphate in a time- and dose-dependent manner. These results suggest that the endothelin receptors of HASMCs are of the ETA type, which couple with Ca2+ channels.
authors.affiliation: Drug Serendipity Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Yazawa
authors.forename: E
authors.initials: E
authors.lastname: Iida-Kubota
authors.forename: Y
authors.initials: Y
authors.lastname: Honma
authors.forename: K
authors.initials: K
authors.lastname: Honda
mesh_terms: D001011:Aorta / Q000378:metabolism*; D001381:Azepines / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D017466:Receptors, Endothelin / Q000378:metabolism*; D012044:Regression Analysis
chemical_list: D001381:Azepines; D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D007211:Indoles; D017466:Receptors, Endothelin; C079574:FR 139317; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium