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eNOS Phosphorylation (eno + phosphorylation)

Distribution by Scientific Domains
Medical Sciences83%

Selected Abstracts

Activation of eNOS in rat portal hypertensive gastric mucosa is mediated by TNF-, via the PI 3-kinase,Akt signaling pathway

HEPATOLOGY, Issue 2 2002
Hirofumi Kawanaka
Activation of endothelial nitric oxide synthase (eNOS) in portal hypertensive (PHT) gastric mucosa leads to hyperdynamic circulation and increased susceptibility to injury. However, the signaling mechanisms for eNOS activation in PHT gastric mucosa and the role of TNF-, in this signaling remain unknown. In PHT gastric mucosa we studied (1) eNOS phosphorylation (at serine 1177) required for its activation; (2) association of the phosphatidylinositol 3-kinase (PI 3-kinase), and its downstream effector Akt, with eNOS; and, (3) whether TNF-, neutralization affects eNOS phosphorylation and PI 3-kinase,Akt activation. To determine human relevance, we used human microvascular endothelial cells to examine directly whether TNF-, stimulates eNOS phosphorylation via PI 3-kinase. PHT gastric mucosa has significantly increased (1) eNOS phosphorylation at serine 1177 by 90% (P < .01); (2) membrane translocation (P < .05) and phosphorylation (P < .05) of p85 (regulatory subunit of PI 3-kinase) by 61% and 85%, respectively; (3) phosphorylation (P < .01) and activity (P < .01) of Akt by 40% and 52%, respectively; and (4) binding of Akt to eNOS by as much as 410% (P < .001). Neutralizing anti,TNF-, antibody significantly reduced p85 phosphorylation, phosphorylation and activity of Akt, and eNOS phosphorylation in PHT gastric mucosa to normal levels. Furthermore, TNF-, stimulated eNOS phosphorylation in human microvascular endothelial cells. In conclusion, these findings show that in PHT gastric mucosa, TNF-, stimulates eNOS phosphorylation at serine 1177 (required for its activation) via the PI 3-kinase,Akt signal transduction pathway. [source]

Role of LOX-1 in monocyte adhesion-triggered redox, Akt/eNOS and Ca2+ signaling pathways in endothelial cells

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009
Nobuo Sakamoto
This study was conducted to examine the role of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in monocyte adhesion-induced redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in endothelial cells (ECs). LOX-1 was blocked by an antibody-neutralizing LOX-1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47phox, and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30,min and NF-,B phosphorylation within 1,h, resulting in redox-sensitive gene expression. Akt and eNOS phosphorylation was induced 15,min after adding monocytes and returned to control level after 30,min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX-1 blunted the monocyte adhesion-triggered redox-sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca2+ mobilization and Ca2+ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX-1 is involved in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of oxidized low-density lipoprotein (ox-LDL). Furthermore, blockade of Ca2+ inhibited monocyte adhesion-triggered Rac1 and p47phox activation and ROS generation in ECs, whereas Ca2+ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor-, or ox-LDL. We provide evidence that LOX-1 plays a role in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of the ox-LDL,LOX-1 axis. J. Cell. Physiol. 220: 706,715, 2009. © 2009 Wiley-Liss, Inc. [source]

Post-translational Regulation of Endothelial Nitric Oxide Synthase (eNOS) by Estrogens in the Rat Vagina

THE JOURNAL OF SEXUAL MEDICINE, Issue 5 2010
Biljana Musicki PhD
ABSTRACT Introduction., Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown. Aims., Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression. Methods., We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17, (15 µg). Main Outcome Measures., Molecular mechanisms of eNOS regulation by estrogen in the rat vagina. Results., We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina. Conclusions., These results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS,caveolin-1 interaction. Musicki B, Liu T, Strong TD, Lagoda GA, Bivalacqua TJ, and Burnett AL. Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina. J Sex Med 2010;7:1768,1777. [source]

Age-Related Changes in Phosphorylation of Endothelial Nitric Oxide Synthase in the Rat Penis

THE JOURNAL OF SEXUAL MEDICINE, Issue 3 2005
Biljana Musicki PhD
ABSTRACT Aim., Aging is associated with erectile dysfunction (ED) attributed to reduced nitric oxide synthase (NOS) activity and nitric oxide bioavailability. However, the mechanism for this effect has not been fully investigated. We evaluated (i) whether age-related ED involves dysregulation of endothelial NOS (eNOS) phosphorylation; and (ii) whether vascular endothelial growth factor (VEGF) exerts erectile effects and operates via eNOS phosphorylation in aged rats. Methods., Male Fischer 344 "young" (4-month-old) and "aged" (19-month-old) rats were used. Electrical stimulation of the cavernous nerve (CNS) was performed to generate penile erection. Erectile response in the presence of rhVEGF165 was evaluated by intracavernosal pressure monitoring 25 minutes after intracavernosal injection of VEGF. Penes were excised at baseline, with or without rhVEGF treatment, and after CNS for Western immunoblot of phospho-eNOS (Ser-1177 and Thr-495), phospho-Akt, and eNOS. Results., Erectile response was significantly reduced in aged rats compared with young rats. Phospho-eNOS (Ser-1177) and phospho-Akt were significantly reduced, while phospho-eNOS (Thr-495) was significantly increased, in the aged penis at baseline and after CNS. rhVEGF significantly improved erection and reversed downregulated Ser-1177, but not upregulated Thr-495 phosphorylation, on eNOS in aged penes. eNOS protein was significantly increased in aged penes. Conclusions., Age-related ED is associated with eNOS inactivation through a decrease in phosphorylation of its positive regulatory site (Ser-1177) and an increase in phosphorylation of its negative regulatory site (Thr-495) in the penis. Altered phosphorylation/constitutive activation of eNOS by fluid shear stress may be a major determinant of compromised vascular homeostasis of the aged penis. The finding that VEGF rapidly induces erection and partly corrects alterations in eNOS phosphorylation in the aged rat penis suggests impaired eNOS activation by deficient endogenous VEGF and supports the potential for growth factor therapy in the treatment of age-related ED. [source]

Tamoxifen dilates porcine coronary arteries: roles for nitric oxide and ouabain-sensitive mechanisms

BRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2006
H S Leung
Background and purpose: Experiments were designed to determine the mechanism of the relaxation induced by tamoxifen in porcine coronary arteries at the tissue, cellular and molecular levels. Experimental approach: Porcine left circumflex coronary arteries were isolated and isometric tension was measured. [Ca2+]i in native endothelial cells of intact arteries was determined by a calcium fluorescence imaging technique and eNOS ser1177 phosphorylation was assayed by Western blotting. Key results: Tamoxifen induced an endothelium-dependent relaxation that was antagonized by ICI 182,780 and abolished by NG -nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). L-Arginine reversed the effect of L-NAME while indomethacin was without effect. Tamoxifen-induced relaxation was attenuated by charybdotoxin (CTX) plus apamin, ouabain or by incubation in a K+ -free solution. Moreover, tamoxifen triggered extracellular Ca2+ -dependent increases in endothelial [Ca2+]i and this effect was abolished by ICI 182,780. Endothelium-independent relaxation to sodium nitroprusside was also inhibited by ouabain or in a K+ -free solution. Furthermore, tamoxifen increased endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177 and ICI 182,780 prevented this effect. Conclusions and Implications: The present results suggest that tamoxifen mainly induces endothelium-dependent relaxation and that endothelial nitric oxide (NO) is the primary mediator of this effect. NO-dependent responses may result from elevated [Ca2+]i in endothelial cells; an effect abolished by ICI 182,780. NO activates Na+/K+ -ATPase in vascular smooth muscle, leading to relaxation. These results suggest that tamoxifen is able to modulate eNOS phosphorylation directly. British Journal of Pharmacology (2006) 149, 703,711. doi:10.1038/sj.bjp.0706921 [source]