Calcium entry activated by store depletion in coronary endothelium is promoted by tyrosine phosphorylation.
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.
Sharma
N R
NR
Department of Medical Physiology, Texas A&M University Health Science Center, College Station 77843, USA.
Davis
M J
MJ
D000818:Animals; D002118:Calcium / Q000172:deficiency* / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D004553:Electric Conductivity; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D019833:Genistein; D006873:Hydroquinones / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D010766:Phosphorylation; D013373:Substance P / Q000494:pharmacology; D013552:Swine; D014443:Tyrosine / Q000378:metabolism*
D002121:Calcium Channel Blockers; D006873:Hydroquinones; D007529:Isoflavones; C019359:2,5-di-tert-butylhydroquinone; D013373:Substance P; D014443:Tyrosine; D019833:Genistein; D002118:Calcium