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This Article Full Text Full Text (PDF) All Versions of this Article: 290/4/H1433    most recent 00942.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Cale, J. M. Articles by Bird, I. M. Search for Related Content PubMed PubMed Citation Articles by Cale, J. M. Articles by Bird, I. M.

Dissociation of endothelial nitric oxide synthase phosphorylation and activity in uterine artery endothelial cells

Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, Wisconsin

Submitted 6 September 2005 ; accepted in final form 1 November 2005

Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca²⁺ and kinase signaling pathways. Using UAEC derived from pregnant ewes (P-UAEC), as well as COS-7 cells transiently expressing ovine endothelial nitric oxide synthase (eNOS), we investigated the role of phosphorylation of five known amino acids following treatment with physiological calcium-mobilizing agent ATP and compared with the effects of PMA (also known as TPA) alone or in combination with ATP. In P-UAEC, ATP stimulated eNOS activity and phosphorylation of eNOS S617, S635, and S1179. PMA promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in phospho (p)-T497 and potentiation of p-S1179. In COS-7 cells, PMA inhibition of ATP-stimulated eNOS activity was associated with p-T497 phosphorylation. Although T497D eNOS activity was reduced to 19% of wild-type eNOS with ATP and 44% with A23187 [GenBank] , we nonetheless observed more p-S1179 with ATP than with A23187 [GenBank] (3.4-fold and 1.8-fold of control, respectively). Furthermore, the S1179A eNOS mutation partly attenuated ATP- but not A23187 [GenBank] -stimulated activity, but when combined with T497D, no further reduction of eNOS activity was observed. In conclusion, although phosphorylation of eNOS is associated with activation in P-UAEC, no single or combination of phosphorylation events predict activity changes. In COS-7 cells, phosphorylation of T497 can attenuate activity but also influences S1179 phosphorylation. We conclude that in both cell types, observed changes in phosphorylation of key residues may influence eNOS activation but are not sufficient alone to describe eNOS activation.

ovine; pregnancy; calmodulin; bovine aortic endothelial cells