AJP - Heart and Circulatory Physiology, Vol 270, Issue 1 267-H274, Copyright © 1996 by American Physiological Society

ARTICLES

Calcium entry activated by store depletion in coronary endothelium is promoted by tyrosine phosphorylation

N. R. Sharma and M. J. Davis
Department of Medical Physiology, Texas A&M University Health Science Center, College Station 77843, USA.

Application of substance P (SP), a potent endothelium-dependent vasodilator, to porcine coronary artery endothelial cells (PCAECs) results in release of Ca2+ from intracellular stores followed by extracellular Ca2+ influx. We tested the hypothesis that intracellular store depletion results in tyrosine phosphorylation, which promotes Ca2+ influx. PCAECs labeled with antiphosphotyrosine antibody conjugated to fluorescein isothiocyanate showed a 3.3- to 3.4-fold increase in fluorescence in response to SP or 2,5-di-tert-butylhydroquinone (BHQ), an agent that depletes intracellular stores by inhibiting the endoplasmic reticulum Ca(2+)-adenosinetriphosphatase. In both cases, the tyrosine kinase inhibitor, genistein, reduced the fluorescence intensity to near-basal levels. Pretreatment of PCAECs with the tyrosine kinase inhibitors, genistein or tyrphostin, induced a significant reduction in the plateau phase of SP-induced Ca2+ elevation with no effect on the release of Ca2+ from stores. Neither daidzein, a structurally similar but inactive analogue of genistein, nor H-7, a serine-threonine kinase inhibitor, affected SP-induced Ca2+ influx. Voltage-clamp recordings using the perforated patch technique with simultaneous Ca2+ measurements showed that intracellular 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 cells requires tyrosine phosphorylation.

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