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

Abstract

Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca(2+) 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, we nonetheless observed more p-S1179 with ATP than with 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 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.

Pregnancy Enhances Sustained Ca2+ Bursts and Endothelial Nitric Oxide Synthase Activation in Ovine Uterine Artery Endothelial Cells Through Increased Connexin 43 Function1

“…Such studies showed that the sustained phase of capacitative entry in UAECs is, in fact, not a constant elevation of [Ca 2þ ] i but a repeated periodic increase in [Ca 2þ ] i ([Ca 2þ ] i bursts) [11]. In this study, we further report novel findings from extended simultaneous recordings of individual cells from both NP-UAECs and P-UAECs.…”

“…Prior to stimulation, cells were washed twice and then incubated for 1 h in Krebs buffer, which was then removed and replaced with fresh buffer or with 300 lM (43,37)GAP27 in Krebs buffer. Serum starvation continued for three more hours prior to addition of 1.5 lCi per well of L-[2,3,4,5-3 H]arginine monohydrochloride (61 Ci/mmol; GE Healthcare Bio-Sciences), followed by addition of vehicle or ATP (30 min, final concentration, 100 lM) to the appropriate wells; the assay was then continued as described previously [10,11].…”

“…Nonetheless, pregnancy enhancement of capacitative entry and NOS3 activation occur in spite of the fact that the P2Y2 receptor, its associated G proteins, PLCB, as well as TRPC3 and ITPR2 themselves are expressed equally in nonpregnant or pregnant uterine artery endothelial cell (UAEC) preparations [8,9]. We also reported recently that when individual pregnant UAECs (P-UAECs) are observed for prolonged periods (30 min), the greater sustained phase of capacitative entry is observed as a series of periodic [Ca 2þ ] i bursts [11]; no comparative data from individual nonpregnant UAECs (NP-UAECs) have been published. The questions that remain, and the focus of the current study, are: What is the comparative nature of the [Ca 2þ ] i response in individual NP-UAECs to that observed in P-UAECs, and how can pregnancy enhance sustained-phase Ca 2þ signaling without altered expression of these key signaling molecules that otherwise collectively mediate cyclic capacitative entry?…”

Transcriptome analysis of porcine PBMCs after in vitro stimulation by LPS or PMA/ionomycin using an expression array targeting the pig immune response

“…PMA, also known as 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent tumor promoter often used in biomedical research [27]. Ionomycin is an ionophore produced by Streptomyces conglobatus .…”

Role of Nitric Oxide in Isoproterenol-Induced Myocardial Infarction

“…Cardiac myocytes express the three isoforms although eNOS is mainly expressed in the endothelial cells. eNOS is constitutively expressed and can be stimulated with the increase of cytosolic calcium and phosphorylation [Cale & Bird, 2006]. In contrast, iNOS is a cytokine-inducible isoenzyme.…”