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 A2

Uterine artery endothelial cells (UAEC) from pregnant ewes (P-UAEC) demonstrate generally enhanced ability to couple growth factor and G protein-coupled receptors to the ERK-1/2 signaling pathway and stimulate NO production independently of elevated [Ca2+]. Herein we investigate the signaling and vasodilator 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 acts through G protein-coupled P-2Y receptors to activate phospholipase C and dose-dependently elevate [Ca2+](i) independently of extracellular Ca2+. The small reduction in the [Ca2+](i) response in NP us. P-UAEC did not, however, 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 ERK-1/2 and Ca2+ were absolutely required for eNOS as well as cPLA(2) activation and the Ca2+ sensitivity of eNOS was enhanced through the cytosolic [Ca2+](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 occur for cPLA(2).