PROTEIN-KINASE C-BETA REGULATES HETEROLOGOUS DESENSITIZATION OF THROMBIN RECEPTOR (PAR-1) IN ENDOTHELIAL-CELLS

We studied the effects of protein kinase C (PKC) activation on endothe lial cell surface expression and function of the proteolytically activ ated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was as sessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), a nd receptor activation was assessed by measuring increases in cytosoli c Ca2+ concentration in human dermal microvascular endothelial cells ( HMEC) exposed to alpha-thrombin or phorbol ester, 12-O-tetradecanoylph orbol-13-acetate (TPA). Immunofluorescence showed that thrombin and TP A reduced the cell surface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1 desensitization. In contrast, prior activation of PKC with TPA produced desensitization to thrombin and histamine, indicati ng heterologous PAR-1 desensitization. Treatment of cells with stauros porine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Deplet ion of PKC beta isozymes (PKC beta(I) and PKC beta(II)) by transducing cells with antisense cDNA of PKC beta(I) prevented the TPA-induced de crease in cell surface PAR-1 expression and restored similar to 60% of the cytosolic Ca2+ signal in response to thrombin. In contrast, deple tion of PKC beta isozymes did not affect the loss of cell surface PAR- 1 and induction of homologous PAR-1 desensitization by thrombin. There fore, homologous PAR-1 desensitization by thrombin occurs independentl y of PKC beta isozymes, whereas the PKC beta-activated pathway is impo rtant in signaling heterologous PAR-1 desensitization in endothelial c ells.