Role of Ca2+ and protein kinase C in shear stress-induced actin depolymerization and endothelin 1 gene expression.
Vascular endothelial cells adapt to changes in blood flow by altering the cell architecture and by producing various substances. We have previously reported that low shear stress induces endothelin 1 (ET-1) expression in endothelial cells and that this induction is mediated by depolymerization of actin fiber. In the present study, we examined the role of Ca2+ and protein kinase C (PKC) in shear stress-induced actin depolymerization and subsequent ET-1 gene expression. Exposure of cultured porcine aortic endothelial cells to low shear stress (5 dyne/cm2) for 3 hours increased the ratio of G-actin to total actin from 54 +/- 0.8% to 80 +/- 1.0%. This shear stress-induced actin depolymerization was completely blocked by chelation of extracellular Ca2+ with EGTA and partially inhibited by intracellular Ca2+ chelation with the tetraacetoxymethyl ester of BAPTA (BAPTA/AM). Pretreatment with staurosporine, a PKC inhibitor, or desensitization of PKC by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 hours also resulted in partial inhibition of shear stress-induced actin depolymerization. Although PKC activation by TPA mildly increased G-actin content, the effect of TPA and shear stress on actin depolymerization was not additive. Moreover, shear stress-induced ET-1 gene expression was inhibited by EGTA, BAPTA/AM, and staurosporine to a degree similar to the inhibition of actin depolymerization. In contrast, ET-1 gene expression induced by cytochalasin B, an actin-disrupting agent, was not affected by staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)
Morita
T
T
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Kurihara
H
H
Maemura
K
K
Yoshizumi
M
M
Nagai
R
R
Yazaki
Y
Y
D000199:Actins / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D016232:Endothelins / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D007202:Indicators and Reagents; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D019311:Staurosporine; D013314:Stress, Mechanical; D013552:Swine
D000199:Actins; D000470:Alkaloids; D002614:Chelating Agents; D016232:Endothelins; D007202:Indicators and Reagents; D010703:Phorbol Esters; D012333:RNA, Messenger; D004533:Egtazic Acid; D011493:Protein Kinase C; D019311:Staurosporine; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium