Submitted on September 6, 2005
Accepted on November 1, 2005
DISSOCIATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE PHOSPHORYLATION AND ACTIVITY IN UTERINE ARTERY ENDOTHELIAL CELLS
Jacqueline M Cale1 and Ian M Bird1*
1 OB/Gyn, University of Wisconsin, Madison, Wi, USA
* To whom correspondence should be addressed. E-mail: imbird{at}wisc.edu.
Pregnancy is known to induce programming of maternal uterine artery endothelial cells, manifested in part as increased agonist responsive cell signaling and associated nitric oxide production. Endothelial nitric oxide synthase (eNOS) is in turn regulated by numerous posttranslational mechanisms including both Ca2+ and phosphorylation. Using uterine artery endothelial cells derived from pregnant ewes (P-UAEC) and COS-7 cells transiently expressing ovine eNOS, we measured activity and phosphorylation of five known amino acids following treatment with physiologic calcium mobilizing agent ATP alone and compared the effects to those of phorbol 12-myristate 13-acetate (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 alone promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in p-T497 and potentiation of p-S1179. Studies of phosphorylation and mutagenesis of eNOS in COS-7 cells indicated that PMA inhibition of ATP stimulated eNOS activity was associated with p-T497 phosphorylation. T497D-eNOS has been shown by others to decrease A23187-stimulated activity and p-S1179. In the current study, T497D-eNOS activity was reduced to 19% of wt-eNOS with ATP and 44% with A23187. Nonetheless, despite these relative changes in activity, we observed more p-S1179 with treatment with ATP than A23187 (3.4-fold and 1.8 fold of control, respectively). Furthermore, the S1179A-eNOS mutation partly attenuated ATP- but not A23187-stimulated activity, but when this mutation was combined with T497D, no further reduction of eNOS activity was observed. In conclusion, in P-UAEC our data suggest that while phosphorylation events are associated with eNOS activity changes, no single or combination of phosphorylation events, especially of S1179 or T497, is sufficient alone to predict or reflect activity changes. Furthermore, our findings in COS-7 cells confirm that given sufficient phosphorylation of T497, attenuation of activity can occur but this event also results in alteration of S1179 phosphorylation. We conclude that in both cell types, changes in phosphorylation of key residues could be observed and may influence eNOS activation but are not sufficient alone to describe eNOS activation.
