Pregnancy-specific enhancement of agonist-stimulated ERK-1/2 signaling in uterine artery endothelial cells increases Ca(2+) sensitivity of endothelial nitric oxide synthase as well as cytosolic phospholipase A(2).
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 [Ca(2+)]. Herein we investigate the signaling and vasodilator responses to ATP, an agonist that also elevates [Ca(2+)](i) in both NP and P-UAEC, to determine the relative importance of Ca(2+) 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 [Ca(2+)](i) independently of extracellular Ca(2+). The small reduction in the [Ca(2+)](i) response in NP vs. P-UAEC did not, however, account for the difference in NO production by P-UAEC>>NP-UAEC. ATP had no stimulatory effect on Akt phosphorylation but rapidly stimulated ERK-1/2 phosphorylation in P-UAEC>>NP-UAEC in a manner that correlated with NO production. In both NP- and P-UAEC, both ERK-1/2 and Ca(2+) were absolutely required for eNOS as well as cPLA(2) activation and the Ca(2+) sensitivity of eNOS was enhanced through the cytosolic [Ca(2+)](i) range in P-UAEC>>NP-UAEC. Thus ERK-1/2 may regulate the Ca(2+) sensitivity of eNOS to an even greater extent than is known to occur for cPLA(2).
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Perinatal Research Laboratories, Department of Obstetrics/Gynecology, University of Wisconsin-Madison, Madison, Wisconsin 53715, USA.
Sullivan
J A
JA
Magness
R R
RR
Zhang
L
L
Bird
I M
IM
D000818:Animals; D001158:Arteries / Q000166:cytology / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004730:Endothelium, Vascular / Q000737:chemistry / Q000502:physiology*; D005260:Female; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010741:Phospholipases A / Q000378:metabolism*; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000502:physiology*; D012756:Sheep; D015398:Signal Transduction / Q000502:physiology; D014599:Uterus / Q000098:blood supply*
D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D020928:Mitogen-Activated Protein Kinases; D010741:Phospholipases A; D002118:Calcium