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-I gene expression. Exposure of cultured porcine aortic endothelial cells to low shear stress (5 dyne/cm(2)) 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. These results suggest that shear stress can induce actin fiber depolymerization through, at least in part, Ca2+- and PKC-dependent pathways and that the resultant actin depolymerization leads to induction of ET-1 gene expression in a PKC-independent manner.