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

2006 ◽  
Vol 290 (4) ◽  
pp. H1433-H1445 ◽  
Author(s):  
Jacqueline M. Cale ◽  
Ian M. Bird
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Uterine Artery ◽  
Cell Types ◽  
Enos Activity ◽  
Key Residues ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

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, 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.

Abstract 3635: Inhibition of Inhibitor Kappa B Kinase Beta Augments Endothelial Nitric Oxide Synthase Activity and Nitric Oxide Production in Aortic Endothelial Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Sumathy Mohan ◽  
Ryzard Konopinski ◽  
Mohan Natarajan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Vascular Endothelial Cells ◽  
No Production ◽  
Enos Activity ◽  
Hsp 90 ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hall-mark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases (inhibitor kappa B kinases α, β and γ) in non-vascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90 and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (4.02 ± 0.81-folds) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.04 ± 0.06-folds). Another important and novel finding is that both IKKβ and eNOS could be co-immunoprecipitated with Hsp-90 (Figures A & B ) thus indicating the possible existence of a complex of IKKβ and eNOS interacting with single pool of Hsp-90. Inhibition of Hsp-90 with geldanamycin (2μM) or Radicicol (20μM) mitigated (0.45 ± 0.04 -fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.416-fold). Blocking of IKKβ expression by IKK inhibitor II (15μM wedelolactone) or siRNA improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.

Alkalosis stimulates endothelial nitric oxide synthase in cultured human pulmonary arterial endothelial cells

2002 ◽  
Vol 283 (1) ◽  
pp. L113-L119 ◽  
Author(s):  
Shiro Mizuno ◽  
Yoshiki Demura ◽  
Shingo Ameshima ◽  
Seitaro Okamura ◽  
Isamu Miyamori ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Extracellular Ph ◽  
Enos Activity ◽  
Pulmonary Arterial ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Arterial Endothelial Cells

To investigate the effect of extracellular pH on endothelial nitric oxide synthase (eNOS) in human pulmonary arteries, we measured eNOS activity and expression as well as some ion channels in human pulmonary arterial endothelial cells (HPAEC) exposed to various pH levels (6.6–8.0). eNOS activity was found to increase with alkalization and decrease with acidification, while Ca2+ uptake into HPAEC increased with alkalization. The addition of 3′,4′-dichlorobenzamil hydrochloride, an inhibitor of the Na+/Ca2+ exchanger (NCX), prevented the increase of eNOS activity with alkalosis. Exposure to alkalosis and acidosis increased eNOS and NCX mRNA levels. These results suggest that an elevation of extracellular pH activates eNOS via the influx of extracellular Ca2+ and that NCX also regulates eNOS activity during alkalosis. Furthermore, NCX may have a tight interaction with eNOS at the level of transcription and might affect pulmonary circulation during alkalosis and acidosis.

scholarly journals Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells

2009 ◽  
Vol 297 (5) ◽  
pp. G930-G939 ◽  
Author(s):  
Willson Kwok ◽  
Sang Ho Lee ◽  
Cathy Culberson ◽  
Katarzyna Korneszczuk ◽  
Mark G. Clemens
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Molecular Mechanism ◽  
Endothelial Nitric Oxide Synthase ◽  
Liver Sinusoidal Endothelial Cells ◽  
Caveolin 1 ◽  
Enos Activity ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-β-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 μg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs.

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