Release mechanism of endothelin-1 and big endothelin-1 after stimulation with thrombin in cultured porcine endothelial cells.
Cultured porcine endothelial cells (EC) released immunoreactive endothelin-1 (ir-endothelin-1) and big endothelin-1 (ir-big endothelin-1) into the medium in a time-dependent way. Reverse-phase high-pressure liquid chromatography coupled with radioimmunoassay showed that the major component of ir-endothelin-1 corresponded to standard endothelin-1 (1-21) and that the major component of ir-big endothelin-1 corresponded to standard big endothelin-1 (porcine 1-39). This release was strongly inhibited by cycloheximide and was, therefore, related to de novo protein synthesis. The release of greater amounts was stimulated by thrombin. The protein kinase C (PKC) inhibitors from two chemical classes, H7 and staurosporine, inhibited release following such stimulation in a relatively dose-dependent way. Neither H7 nor staurosporine affected the basal release of both endothelin-1 and big endothelin-1. Phorbol myristate acetate, which activates PKC and the Ca2+ ionophore A23187, stimulated the release of ir-endothelin-1 and ir-big endothelin-1 in a dose-dependent way, respectively. In addition, the combination of both compounds had a synergistic effect. An inactive enantiomer of phorbol ester, 4 alpha-phorbol-12,13-didecanoate had no effect on the release of ir-endothelin-1 and ir-big endothelin-1. These results suggest that cultured EC release endothelin-1 and big endothelin-1 simultaneously, and that thrombin stimulates this release by a mechanism that probably involves intracellular Ca2+ mobilization and the activation of PKC.
Kohno
M
M
Department of Internal Medicine, Osaka City University Medical School, Japan.
Yokokawa
K
K
Horio
T
T
Yasunari
K
K
Murakawa
K
K
Ikeda
M
M
Takeda
T
T
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003513:Cycloheximide / Q000494:pharmacology; D019332:Endothelin-1; D016232:Endothelins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011498:Protein Precursors / Q000378:metabolism*; D019311:Staurosporine; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D000470:Alkaloids; D019332:Endothelin-1; D016232:Endothelins; D007546:Isoquinolines; D010879:Piperazines; D011498:Protein Precursors; D000001:Calcimycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D003513:Cycloheximide; D011493:Protein Kinase C; D013917:Thrombin; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate