Calcium entry activated by store depletion in coronary endothelium
is promoted by tyrosine phosphorylation.
Sharma, Neeraj R., Michael J. Davis.
Department of Medical Physiology, Texas A&M University Health
Sciences Center, College Station, TX 77843
APStracts 2:0301H, 1995.
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 fluorescene isothiocyanate showed a 3.3 - 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 Ca2+-ATPase. 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 analog of genistein,
nor H7, 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.
Received 1 May 1995; accepted in final form 10 July 1995.
APS Manuscript Number H416-5.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 30 July 1995.