ROLES OF CALCIUM AND KINASES IN REGULATION OF THROMBIN-STIMULATED PREPROENDOTHELIN-1 TRANSCRIPTION

The release of the vasoactive peptide endothelin-l (ET-1) is Ca2+ depe ndent after thrombin stimulation; however, little is known about the p athways involved. We studied the importance of Ca2+-dependent signal t ransduction pathways on preproET-1 mRNA induction in human endothelial cells. Thrombin-mediated preproET-1 mRNA induction was inhibited afte r clamping of cytosolic free Ca2+ concentration ([Ca2+](i)) with 1,2-b is(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid. Chelation of ext racellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N' ,N'-tetraacetic acid also had a significant inhibitory effect on the i nduction of preproET-1 mRNA. The Ca2+ ionophore A23187 induced constit utive as well as thrombin-stimulated preproET-1 mRNA expression. Mobil ization of Ca2+ stores into the cytosol by inhibition of endoplasmic r eticulum Ca2+-adenosinetriphosphatase with thapsigargin was effective also in inducing preproET-1 mRNA. Calmodulin antagonists W-7 and calmi dazolium, as well as Ca2+/calmodulin-dependent kinase II inhibitor KN- 62, significantly reduced thrombin-induced preproET-1 mRNA. Inhibition by cyclosporin A of the Ca2+ calmodulin-dependent phosphatase calcine urin potentiated constitutive preproET-1 mRNA, These data suggest that , in human endothelial cells, thrombin-mediated preproET-1 gene induct ion is regulated by a stimulatory Ca2+/calmodulin kinase II-dependent pathway.