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Alpha(1)-adrenergic-mediated eNOS phosphorylation in intact arteries.

ABSTRACT: Activation of arterial smooth muscle alpha(1)-adrenergic receptors results in vasoconstriction, as well as a secondary release of nitric oxide and slow vasodilation, presumably through gap junction communication from smooth muscle to endothelium. We hypothesized that this slow vasodilation is due to activation of eNOS through phosphorylation at Ser1179 and dephosphorylation at Thr495. Phosphorylation was measured by western blot using mouse mesenteric arteries that were cannulated and pressurized (75 mm Hg) and treated either by 1) 5 min of phenylephrine superfusion (10(-5)M) (PE5), 2) 15 min of phenylephrine (PE15), 3) 15 min phenylephrine followed by acetylcholine (10(-4)M) (PE+ACh), or 4) 20 min time control with no treatment (NT) [4-5 arteries pooled per treatment per blot; 5 blots performed]. These treatments allowed correlation between vasomotor changes, namely maximal constriction (PE5), slow vasodilation (PE15), and maximal dilation (PE+ACh), and relative phosphorylation changes. Phosphorylation of eNOS at Ser1179 was increased relative to NT by more than 2-fold at PE5 and remained similarly increased at PE15 and PE+ACh. Phosphorylation of eNOS at Thr495 was less in all treatments relative to NT, but not significantly. Treatment with L-NAME (10(-4)M) or endothelial denudation indicated that the slow dilation in response to phenylephrine was completely due to nitric oxide synthase and was endothelial dependent. These results indicate that eNOS phosphorylation at Ser1179 occurs before the slow dilation and is not actively involved in this vasodilation or dilation to acetylcholine, but may play a permissive role in eNOS activation by other mechanisms. It is not yet known what mechanism is responsible for Ser1179 phosphorylation with phenylephrine stimulation.

SUBMITTER: Looft-Wilson RC

PROVIDER: S-EPMC3534816 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Alpha(1)-adrenergic-mediated eNOS phosphorylation in intact arteries.

Looft-Wilson Robin C RC Todd Sarah E SE Araj Christina A CA Mutchler Stephanie M SM Goodell Cara A Raphael CA

Vascular pharmacology 20120913 1-2

Activation of arterial smooth muscle alpha(1)-adrenergic receptors results in vasoconstriction, as well as a secondary release of nitric oxide and slow vasodilation, presumably through gap junction communication from smooth muscle to endothelium. We hypothesized that this slow vasodilation is due to activation of eNOS through phosphorylation at Ser1179 and dephosphorylation at Thr495. Phosphorylation was measured by western blot using mouse mesenteric arteries that were cannulated and pressurize ...[more]

PMID: 22982055

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Project description:Hyperhomocysteinemia (HHcy) impairs endothelium-dependent vasodilation by increasing reactive oxygen species, thereby reducing nitric oxide (NO.) bioavailability. It is unclear whether reduced expression or function of the enzyme that produces NO., endothelial nitric oxide synthase (eNOS), also contributes. It is also unclear whether resistance vessels that utilize both NO.and non-NO.vasodilatory mechanisms, undergo alteration of non-NO.mechanisms in this condition. We tested these hypotheses in male C57BL/6 mice with chronic HHcy induced by 6-wk high methionine/low-B vitamin feeding (Hcy: 89.2 +/- 49.0 microM) compared with age-matched controls (Hcy: 6.6 +/- 1.9 microM), using first-order mesenteric arteries. Dilation to ACh (10(-9)-10(-4) M) was measured in isolated, cannulated, and pressurized (75 mmHg) arteries with and without N(G)-nitro-l-arginine methyl ester (l-NAME) (10(-4) M) and/or indomethacin (10(-5) M) to test endothelium-dependent dilation and non-NO.-dependent dilation, respectively. The time course of dilation to ACh (10(-4) M) was examined to compare the initial transient dilation due to non-NO., non-prostacyclin mechanism and the sustained dilation due to NO.. These experiments indicated that endothelium-dependent dilation was attenuated (P < 0.05) in HHcy arteries due to downregulation of only NO.-dependent dilation. Western blot analysis indicated significantly less (P < 0.05) basal eNOS and phospho-S1179-eNOS/eNOS in mesenteric arteries from HHcy mice but no difference in phospho-T495-eNOS/eNOS. S1179 eNOS phosphorylation was also significantly less in these arteries when stimulated with ACh ex vivo or in situ. Real-time PCR indicated no difference in eNOS mRNA levels. In conclusion, chronic diet-induced HHcy in mice impairs eNOS protein expression and phosphorylation at S1179, coincident with impaired NO.-dependent dilation, which implicates dysfunction in eNOS post-transcriptional regulation in the impaired endothelium-dependent vasodilation and microvascular disease that is common with HHcy.

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Alpha(1)-adrenergic-mediated eNOS phosphorylation in intact arteries.

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Alpha(1)-adrenergic-mediated eNOS phosphorylation in intact arteries.

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