Dependence of endothelin-1 secretion on Ca2+.
The role of Ca2+ and protein kinase C (PKC) activity in the release of immunoreactive endothelin-1 (ET-1) from cultured porcine aortic endothelial cells of first or second passage has been studied. ET-1 accumulation within cells and secretion into cell-conditioned medium over 3 and/or 5 hr was measured. Confluent cells incubated in medium containing 1.8 mM Ca2+ (control condition) accumulated and released ET-1 in a time-dependent way. Reducing intracellular free Ca2+ concentration ([Ca2+]i) by adding the Ca2+ entry blockers NiCl2 (0.5 mM) and amiloride (1 mM) or the Ca2+ chelator EGTA (5 mM) to the incubation medium reduced ET-1 secretion to between 50 and 30% of controls, respectively (P < 0.01). To determine the effect of high [Ca2+]i on ET-1 release, cells were incubated with thapsigargin (10-1000 nM) or Ca2+ ionophore A23187 (1 microM) which raised [Ca2+]i progressively from 190 nM (control) to > 1 microM. Both agents reduced ET-1 secretion in a concentration-dependent manner to between 50 and 20% of controls (P < 0.01). Intracellular levels of ET-1 were also reduced at both low and high [Ca2+]i (P < 0.01). In the presence of the PKC inhibitors chelerythrine (50 microM) and H-7 (60 microM), basal ET-1 secretion was reduced to below 20% of controls (P < 0.01). The PKC activator phorbol 12-myristate 13-acetate (0.4 microM) stimulated ET-1 release 1.4-fold (P < 0.01) and its effect was abolished by EGTA (5 mM). Increased [Ca2+]i stimulated the production and release of cyclic guanosine-3',5'-monophosphate, but basal ET-1 secretion rates correlated poorly with nucleotide levels. These data indicate that: (i) at resting [Ca2+]i concentrations, ET-1 release is close to maximal and is reduced at lower and higher concentrations, resulting in a bell-shaped relationship between [Ca2+]i and ET-1 release; and (ii) basal ET-1 release is largely determined by Ca(2+)-dependent PKC activity.
Brunner
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Institut für Pharmakologie und Toxikologie, Universität Graz, Austria.
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D016232:Endothelins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D053119:Benzophenanthridines; D016232:Endothelins; D007546:Isoquinolines; D010617:Phenanthridines; D010879:Piperazines; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C016299:chelerythrine; D011493:Protein Kinase C; D006152:Cyclic GMP; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium