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Sullivan JA, Grummer MA,
Yi FX, Bird IM.
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.
Endocrinology. 2006 May;147(5):2442-57
Department of Obstetrics and Gynecology, Perinatal Research
Laboratories, University of Wisconsin, Madison, 53715, USA.
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.
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