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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

Department of Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, Madison, Wisconsin 53715

Address all correspondence and requests for reprints to: Ian M. Bird, Ph.D., University of Wisconsin-Madison, Department of Obstetrics and Gynecology, Perinatal Research Laboratories, 7E Meriter Hospital/Park, 202 South Park Street, Madison, Wisconsin 53715. E-mail: Imbird{at}wisc.edu.

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 Ca²⁺ vs. kinase in the regulation of endothelial NO synthase (eNOS) activity. When Ca²⁺ 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 µM) or 2-APB (1–50 µM). The MAPK kinase (MEK) 1/2 inhibitor U0126 (10 µM) 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 µM) on eNOS activity in P- and NP-UAEC, but wortmannin (10 µM) 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 [Ca²⁺]_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.

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