Investigation of mechanisms regulating nitric oxide and prostacyclin production in an ovine uterine artery endothelial cell culture model

Pregnancy is associated with a dramatic increase in uteroplacental blood flow. Coincident with this, during pregnancy there is an increase of circulating factors, including endothelial derived Nitric Oxide (NO) and Prostacyclin (PGI2) that function to facilitate the increase in flow. We have reported that while pregnancy is associated with an increase in expression of the limiting proteins involved in the production of NO (eNOS) and PGI2 (cPLA2, COX-1, PGI2). Nonetheless, in our uterine artery endothelial cell primary culture model (UAEC) where the levels of such proteins are normalized, we still see differences in NO and PGI2 production. We also detected changes in cell signaling at three fundamental levels, namely Ca2+, MEK/ERK-1/2 pathway, and what we term a 'wortmannin sensitive factor' that has yet to be elucidated.; Chemical inhibition of agonist stimulated intracellular Ca2+ levels only partially inhibits ATP stimulated eNOS activity and PGI2 production. We examined if ERK2 phosphorylation could account for the residual eNOS activity or PGI2 production by investigating phosphoryaltion sites on both eNOS and cPLA2 reported to be targets for active ERK2. ATP did not stimulate an increase in cPLA2 S505 phosphorylation, an ERK2 associated site. We also observed that while ATP was able to stimulate an increase in eNOS phosphorylation at S1179, 5617 and S635, the regulatory nature of these phosphorylation events is questioned since Ca2+ mobilization antagonists that block eNOS activity fail to alter ATP stimulated phosphorylation, indicating a dissociation between eNOS phosphorylation and eNOS activity. Another kinase implicated in eNOS phosphorylation is Akt. We found that ATP did not couple to Akt phosphorylation and treatment of UAEC with the general PI3-K inhibitor, LY294002 did not affect ATP stimulated eNOS activity. With many published studies implicating Akt as an eNOS regulator, we introduced a constitutively active Akt protein in our cells using adenoviral delivery and observed no increase in hormone sensitive eNOS activity and furthermore, the previously mentioned wortmannin sensitivity was not affected by the expression of this Akt protein. We conclude that mechanisms underlying the pregnancy-induced enhancement of NO and PGI2 in UAEC are Ca2+ related, but cannot be described by Ca 2+ mobilization alone.