Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells.
Specific binding sites for synthetic porcine endothelin (pET), a novel potent vasoconstrictor peptide isolated from the supernatant of cultured porcine endothelial cells, and its effects on cytosolic free Ca2+ concentrations ([Ca2+]i) and phosphatidylinositol (PI) response were studied in cultured rat aortic vascular smooth muscle cells (VSMC). Binding of 125I-labeled-pET to rat VSMC was time- and temperature-dependent and the cell-bound 125I-labeled-pET was resistant to dissociate. Scatchard analysis of binding studies indicated the presence of a single class of high-affinity binding sites: the apparent Kd was 2-4 X 10(-10) M and the maximal binding capacity was 11,000-13,000 sites/cell. The binding was highly specific for pET because neither well-recognized vasoconstrictors, peptide neurotoxins, nor Ca2+-channel blockers affected the binding. pET dose-dependently (10(-9)-10(-7) M) induced a transient and sustained increase in [Ca2+]i in fura-2-loaded cells of which effect was largely dependent on extracellular Ca2+, whereas it had no significant effect on PI response in 3H-myoinositol-prelabeled cells. The present data clearly demonstrates the presence of specific receptors for pET distinct from those of the well-recognized vasoconstrictors and voltage-dependent Ca2+-channels in cultured rat VSMC, and suggest that pET-induced increase in [Ca2+]i is involved in the mechanism of its vasoconstriction.
D000818:Animals; D001572:Benzofurans; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D016232:Endothelins; D016257:Fura-2; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D013552:Swine; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D001572:Benzofurans; D016232:Endothelins; D010455:Peptides; D010716:Phosphatidylinositols; D014662:Vasoconstrictor Agents; D002118:Calcium; D016257:Fura-2