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 mu M) which raised [Ca2+](i) progressively from 190 nM (control) to > mu M 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 mu M) and H-7 (60 mu M), basal ET-1 secretion was reduced to below 20% of controls (P < 0.01). The PKC activator phorbol 12-myristate 13-acetate (0.4 mu M) 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 Ca2+-dependent PKC activity.