Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C
Xuan, Y.T.; Wang, O.L.; Whorton, A.R.
American Journal of Physiology 266(6 Pt 1): C1560-C1567
1994
ISSN/ISBN: 0002-9513 PMID: 8023888 Document Number: 424590
We have investigated the role of protein kinase C (PKC) in regulating vascular smooth muscle cell responses to endothelin (ET). During the initial phase of the response, ET stimulated rapid formation of diacylglycerol due to rapid and transient activation of phosphatidyl inositol-specific phospholipase C and to rapid and prolonged activation of phospholipase D. Concurrently, ET stimulated translocation of PKC activity that reached a peak at 1 min and remained elevated for at least 20 min. Activation of PKC produced early inhibitory effects. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) 5 min before stimulation with ET inhibited total inositol phosphate formation by gt 50%. Because each inositol phosphate isomer was equally affected, the target appears to be either phospholipase C or some upstream component of the receptor coupling mechanism. Activation of PKC was important for sustained response to ET. Treatment of cells with staurosporine significantly reduced sustained elevation of cytosolic free Ca-2+ concentration ((Ca-2+)-i) normally seen with ET. We had previously shown that sustained elevation of (Ca-2+)-i initiated by ET was due to continued activity of L-type Ca-2+ channels. Our current data suggest that PKC is important in this response. For example, staurosporine inhibited both ET-induced 45Ca-2+ and Mn-2+ entry occurring 10 min after stimulation of influx mechanisms by the agonist. Similarly, pretreatment of cells for 18 h with phorbol dibutyrate depleted the cells of PKC and blocked the sustained activity of Ca-2+ entry mechanisms stimulated by ET. Finally, PMA initiated a slowly developing, sustained elevation of (Ca-2+)-i. The time required to reach peak (Ca-2+)-i was dependent on the concentration of PMA. The rise in (Ca-2+)-i and the entry of Mn-2+ induced by PMA was inhibitable by nicardipine, suggesting that L-type Ca-2+ channels are involved. Thus it appears that the activation of PKC during the initial phase of the cellular response to ET produces inhibitory signals on initial coupling mechanisms and plays an essential role in continued Ca-2+ entry and sustained elevation of (Ca-2+)-i.