Pregnancy-specific enhancement of agonist-stimulated ERK-1/2 signaling in uterine artery endothelial cells increases Ca2+ sensitivity of endothelial nitric oxide synthase as well as cytosolic phospholipase A(2)

Uterine artery endothelial cells (UAEC) from pregnant ewes (P-UAEC) demonst rate generally enhanced ability to couple growth factor and G protein-coupl ed receptors to the ERK-1/2 signaling pathway and stimulate NO production i ndependently of elevated [Ca2+]. Herein we investigate the signaling and va sodilator responses to ATP, an agonist that also elevates [Ca2+](i) in both NP and P-UAEC, to determine the relative importance of Ca2+ vs. ERK-1/2 in the activation of eNOS. We observed in both NP-UAEC and P-UAEC that ATP ac ts through G protein-coupled P-2Y receptors to activate phospholipase C and dose-dependently elevate [Ca2+](i) independently of extracellular Ca2+. Th e small reduction in the [Ca2+](i) response in NP us. P-UAEC did not, howev er, account for the difference in NO production by P-UAEC much greater than NP-UAEC. ATP had no stimulatory effect on Akt phosphorylation but rapidly stimulated ERK-1/2 phosphorylation in P-UAEC much greater than NP-UAEC in a manner that correlated with NO production. In both NP- and P-UAEC, both ER K-1/2 and Ca2+ were absolutely required for eNOS as well as cPLA(2) activat ion and the Ca2+ sensitivity of eNOS was enhanced through the cytosolic [Ca 2+](i) range in P-UAEC much greater than NP-UAEC. Thus ERK-1/2 may regulate the Ca2+ sensitivity of eNOS to an even greater extent than is known to oc cur for cPLA(2).