Compartmentalized Connexin 43 S-Nitrosylation/Denitrosylation Regulates Heterocellular Communication in the Vessel Wall

Objective-To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. Methods and Results-Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NOx concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release. Conclusion-This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication. (Arterioscler Thromb Vasc Biol. 2011;31:399-407.

The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo

NO plays critical roles in vascular function. We show that modulation of the eNOS serine 1179 (S1179) phosphorylation site affects vascular reactivity and determines stroke size in vivo. Transgenic mice expressing only a phosphomimetic (S1179D) form of eNOS show greater vascular reactivity, develop less severe strokes, and have improved cerebral blood flow in a middle cerebral artery occlusion model than mice expressing an unphosphorylatable (S1179A) form. These results provide a molecular mechanism by which multiple diverse cardiovascular risks, such as diabetes and obesity, may be centrally integrated by eNOS phosphorylation in vivo to influence blood flow and cardiovascular disease. They also demonstrate the in vivo relevance of posttranslational modification of eNOS in vascular function

Plasminogen Activator Inhibitor-1 Regulates Myoendothelial Junction Formation

Rationale: Plasminogen activator inhibitor-1 (PAI-1) is a biomarker for several vascular disease states; however, its target of action within the vessel wall is undefined. Objective: Determine the ability of PAI-1 to regulate myoendothelial junction (MEJ) formation. Methods and Results: MEJs are found throughout the vasculature linking endothelial cells (ECs) and vascular smooth muscle cells. Using a vascular cell coculture we isolated MEJ fractions and performed two-dimensional differential gel electrophoresis. Mass spectrometry identified PAI-1 as being enriched within MEJ fractions, which we confirmed in vivo. In the vascular cell coculture, recombinant PAI-1 added to the EC monolayer significantly increased MEJs. Conversely, addition of a PAI-1 monoclonal antibody to the EC monolayer reduced the number of MEJs. This was also observed in vivo where mice fed a high fat diet had increased PAI-1 and MEJs and the number of MEJs in coronary arterioles of PAI-1(-/-) mice was significantly reduced when compared to C57Bl/6 mice. The presence of MEJs in PAI-1(-/-) coronary arterioles was restored when their hearts were transplanted into and exposed to the circulation of C57Bl/6 mice. Application of biotin-conjugated PAI-1 to the EC monolayer in vitro confirmed the ability of luminal PAI-1 to translocate to the MEJ. Functionally, phenylephrine-induced heterocellular calcium communication in the vascular cell coculture was temporally enhanced when recombinant PAI-1 was present, and prolonged when PAI-1 was absent. Conclusion: Our data implicate circulating PAI-1 as a key regulator of MEJ formation and a potential target for pharmacological intervention in diseases with vascular abnormalities (eg, diabetes mellitus). (Circ Res. 2010; 106: 1092-1102.

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

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 mm 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. Phosphotylation 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. (C) 2012 Elsevier Inc. All rights reserved

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 μM) compared with age-matched controls (Hcy: 6.6 ± 1.9 μM), 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 NG-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