Flow does not alter eNOS phosphoryation at Ser1179 or Thr495 in preconstricted mouse mesenteric arteries.

In arteries, endothelium-dependent vasodilatory agonists and flow-induced shear stress cause vasodilation largely by activation of the endothelial enzyme eNOS, which generates nitric oxide that relaxes vascular smooth muscle. Agonists activate eNOS in part through increased phosphorylation at Ser1179 and decreased phosphorylation at Thr495. We previously found that preconstriction of intact, isolated mouse mesenteric arteries with phenylephrine also caused increased Ser1179 and decreased Thr495 eNOS phosphorylation, and sequential treatment with the vasodilatory agonist acetylcholine did not cause any further change in phosphorylation at these sites, despite producing vasodilation. The present study tests the hypothesis that luminal flow in these arteries preconstricted with phenylephrine also produces vasodilation without phosphorylation changes at these sites. First-order mesenteric arteries, isolated from male C57/BL6 mice (7-20 weeks of age) anesthetized with pentobarbital (50 mg/kg, i.p.), were cannulated, pressurized, and treated with stepped increases in luminal flow (15-120 µL/min). Flow resulted in dilation that plateaued at ~60 µL/min (31.3 ± 3.0% dilation) and was significantly (P < 0.001) NOS-dependent at all flow rates (determined by 10-4 mol/L L-NAME treatment). In separate arteries, preconstriction with phenylephrine (10-5 mol/L) resulted in increased eNOS phosphorylation at Ser1179 (P < 0.05) and decreased phosphorylation at Thr495, but subsequent flow at 60 µL/min for 5 or 15 min did not cause further changes in phosphorylation, despite causing dilation. Thus, flow-induced dilation does not require changes in these eNOS phosphorylation sites beyond those induced by alpha1-adrenergic stimulation with phenylephrine, indicating that eNOS is activated by other mechanisms during acute flow-induced dilation of preconstricted arteries.

Chronic diet-induced hyperhomocysteinemia impairs eNOS regulation in mouse mesenteric arteries.

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.