REGULATION OF ENDOTHELIN-INDUCED CA2-MUSCLE CELLS BY PROTEIN-KINASE-C( MOBILIZATION IN SMOOTH)

We have investigated the role of protein kinase C (PKC) in regulating vascular smooth muscle cell responses to endothelin (ET). During the i nitial phase of the response, ET stimulated rapid formation of diacylg lycerol due to rapid and transient activation of phosphatidyl inositol -specific phospholipase C and to rapid and prolonged activation of pho spholipase D. Concurrently, ET stimulated translocation of PKC activit y that reached a peak at 1 min and remained elevated for at least 20 m in. Activation of PKC produced early inhibitory effects. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) 5 min before stimulat ion with ET inhibited total inositol phosphate formation by >50%. Beca use each inositol phosphate isomer was equally affected, the target ap pears to be either phospholipase C or some upstream component of the r eceptor coupling mechanism. Activation of PKC was important for sustai ned response to ET. Treatment of cells with staurosporine significantl y reduced sustained elevation of cytosolic free Ca2+ concentration ([C a2+](i)) normally seen with ET. We had previously shown that sustained elevation of [Ca2+](i) initiated by ET was due to continued activity of L-type Ca2+ channels. Our current data suggest that PKC is importan t in this response. For example, staurosporine inhibited both ET-induc ed Ca-45(2+) and Mn2+ entry occurring 10 min after stimulation of infl ux mechanisms by the agonist. Similarly, pretreatment of cells for 18 h with phorbol dibutyrate depleted the cells of PKC and blocked the su stained activity of Ca2+ entry mechanisms stimulated by ET. Finally, P MA initiated a slowly developing, sustained elevation of [Ca2+](i). Th e time required to reach peak [Ca2+](i) was dependent on the concentra tion of PMA. The rise in [Ca2+](i) and the entry of Mn2+ induced by PM A was inhibitable by nicardipine, suggesting that L-type Ca2+ channels are involved. Thus it appears that the activation of PKC during the i nitial phase of the cellular response to ET produces inhibitory signal s on initial coupling mechanisms and plays an essential role in contin ued Ca2+ entry and sustained elevation of [Ca2+](i).