Pregnancy-enhanced endothelial nitric oxide synthase (eNOS) activation in uterine artery endothelial cells shows altered sensitivity to Ca2+, U0126, and wortmannin but not LY294002--evidence that pregnancy adaptation of eNOS activation occurs at multiple levels of cell signaling.
During pregnancy, vascular remodeling and vasoactive agents such as nitric oxide (NO) increase blood flow to the uteroplacental unit. Using our uterine artery endothelial cell (UAEC) culture model, based on cells from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes, we investigate the relative physiological roles of Ca(2+) vs. kinase in the regulation of endothelial NO synthase (eNOS) activity. When Ca(2+) mobilization is fully inhibited using inhibitors of phospholipase C (PLC) (U73122) and the inositol triphosphate (IP3) receptor (IP3-R) (2-APB), significant residual eNOS activity remains in both P- and NP-UAEC. No change in ATP-stimulated ERK2, Akt, or eNOS phosphorylation is observed with U73122 (0.01-1 microM) or 2-APB (1-50 microM). The MAPK kinase (MEK) 1/2 inhibitor U0126 (10 microM) did not alter ATP-stimulated eNOS activity in P-UAEC, but potentiated the ATP response in NP-UAEC. Using two phosphatidylinositol 3-kinase (PI3-K) inhibitors, we observed no effect with LY294002 (10 microM) on eNOS activity in P- and NP-UAEC, but wortmannin (10 microM) inhibited both P- and NP-UAEC eNOS activation. Expression of constitutively active Akt (ca-Akt) in UAEC resulted in slight elevation of basal eNOS activity, but relative ATP-stimulated eNOS activation was not altered by ca-Akt. Wortmannin continued to inhibit eNOS activation by ATP in the presence of ca-Akt; LY294002 still had no inhibitory effect. Our data indicate both [Ca(2+)](i) and multiple kinases are involved in the regulation of eNOS activity in our model. We report that pregnancy adaptation of eNOS activation includes the reduced sensitivity to ERK-mediated attenuation of eNOS activity and enhanced stimulation of eNOS activity through a wortmannin-sensitive, LY294002-insensitive, Akt-independent mechanism.
Sullivan
Jeremy A
JA
Department of Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, Madison, 53715, USA.
Grummer
Mary A
MA
Yi
Fu-Xian
FX
Bird
Ian M
IM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002867:Chromones / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes / Q000494:pharmacology; D016257:Fura-2 / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D015233:Models, Statistical; D009025:Morpholines / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology*; D010766:Phosphorylation; D011247:Pregnancy; D011270:Pregnancy, Animal; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction; D013997:Time Factors; D014599:Uterus / Q000166:cytology* / Q000201:enzymology*; D000077191:Wortmannin
D000730:Androstadienes; D002070:Butadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009025:Morpholines; D009570:Nitriles; C113580:U 0126; D049452:Green Fluorescent Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000255:Adenosine Triphosphate; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D002118:Calcium; D016257:Fura-2; D000077191:Wortmannin