CALCIUM-ENTRY ACTIVATED BY STORE DEPLETION IN CORONARY ENDOTHELIUM ISPROMOTED BY TYROSINE PHOSPHORYLATION

Application of substance P (SP), a potent endothelium-dependent vasodi lator, to porcine coronary artery endothelial cells (PCAECs) results i n release of Ca2+ from intracellular stores followed by extracellular Ca2+ influx. We tested the hypothesis that intracellular store depleti on results in tyrosine phosphorylation, which promotes Ca2+ influx. PC AECs labeled with antiphosphotyrosine antibody conjugated to fluoresce in isothiocyanate showed a 3.3- to 3.4-fold increase in fluorescence i n response to SP or 2,5-di-tert-butylhydroquinone (BHQ), an agent that depletes intracellular stores by inhibiting the endoplasmic reticulum Ca2+-adenosinetriphosphatase. In both cases, the tyrosine kinase inhi bitor, genistein, reduced the fluorescence intensity to near-basal lev els. Pretreatment of PCAECs with the tyrosine kinase inhibitors, genis tein or tyrphostin, induced a significant reduction in the plateau pha se of SP-induced Ca2+ elevation with no effect on the release of Ca2from stores. Neither daidzein, a structurally similar but inactive ana logue of genistein, nor H-7, a serine-threonine kinase inhibitor, affe cted SP-induced Ca2+ influx. Voltage-clamp recordings using the perfor ated patch technique with simultaneous Ca2+ measurements showed that i ntracellular Ca2+ elevation and inward current activated by SP and BHQ were reduced by 60-70% in response to genistein. These data indicate that the link between store depletion and Ca2+ influx in endothelial c ells requires tyrosine phosphorylation.