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

Looft‐Wilson, Robin; Ashley, Blair; Billig, Janelle; Wolfert, Madeline; Ambrecht, Lindsay; Bearden, Shawn E.

doi:10.1152/ajpregu.00833.2007

Cited by 21 publications

(31 citation statements)

References 34 publications

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“…In four of the groups the baseline diameter after 30 min of equilibration at 37° C was either not changed ( PE5 : 229 ± 8 μm; PE15 : 235 ± 7 μm), or increased slightly but significantly (p<0,05, paired t-test) ( NT : 235 ± 5 μm; PE+ACh : 245 ± 8 μm), indicating an absence of basal tone, which is consistent with our previous results (Looft-Wilson et al, 2008). However, the L-NAME and Denuded groups had significantly decreased (p<0.05, paired t-test) diameters after the 30 min equilibration ( L-NAME : 195 ± 11 μm; Denuded: 198 ± 12 μm), indicating basal nitric oxide release from the endothelium in this artery type.…”

Section: Methodssupporting

confidence: 92%

Alpha1-adrenergic-mediated eNOS phosphorylation in intact arteries

Looft‐Wilson

Todd

Araj

et al. 2013

Vascular Pharmacology

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Activation of arterial smooth muscle alpha1-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 mmHg) and treated either by 1) 5 min of phenylephrine superfusion (10−5 M) (PE5), 2) 15 minutes 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.

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Section: Methodssupporting

confidence: 92%

Alpha1-adrenergic-mediated eNOS phosphorylation in intact arteries

Looft‐Wilson

Todd

Araj

et al. 2013

Vascular Pharmacology

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“…Elevated cGMP levels in response to ACh stimulation are an indirect measure of the activation of eNOS. In resistance microvessels, we have previously reported significantly lower basal expression of eNOS and the pS1179eNOS-to-eNOS (activation site) ratio in mice with diet-induced HHcy with no difference in the pT495eNOS-to-eNOS (deactivation site) ratio (101). S1179 eNOS phosphorylation was also significantly less in these vessels when they were stimulated with ACh, both ex vivo and in situ.…”

Section: Endothelial Dysfunction In Cbs Deficiencymentioning

confidence: 77%

Vascular complications of cystathionine β-synthase deficiency: future directions for homocysteine-to-hydrogen sulfide research

Beard

Bearden

2011

American Journal of Physiology-Heart and Circulatory Physiology

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104

110

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Homocysteine (Hcy), a cardiovascular and neurovascular disease risk factor, is converted to hydrogen sulfide (H(2)S) through the transsulfuration pathway. H(2)S has attracted considerable attention in recent years for many positive effects on vascular health and homeostasis. Cystathionine β-synthase (CBS) is the first, and rate-limiting, enzyme in the transsulfuration pathway. Mutations in the CBS gene decrease enzymatic activity, which increases the plasma Hcy concentration, a condition called hyperhomocysteinemia (HHcy). Animal models of CBS deficiency have provided invaluable insights into the pathological effects of transsulfuration impairment and of both mild and severe HHcy. However, studies have also highlighted the complexity of HHcy and the need to explore the specific details of Hcy metabolism in addition to Hcy levels per se. There has been a relative paucity of work addressing the dysfunctional H(2)S production in CBS deficiency that may contribute to, or even create, HHcy-associated pathologies. Experiments using CBS knockout mice, both homozygous (-/-) and heterozygous (+/-), have provided 15 years of new knowledge and are the focus of this review. These murine models present the opportunity to study a specific mechanism for HHcy that matches one of the etiologies in many human patients. Therefore, the goal of this review was to integrate and highlight the critical information gained thus far from models of CBS deficiency and draw attention to critical gaps in knowledge, with particular emphasis on the modulation of H(2)S metabolism. We include findings from human and animal studies to identify important opportunities for future investigation that should be aimed at generating new basic and clinical understanding of the role of CBS and transsulfuration in cardiovascular and neurovascular disease.

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“…The antieNOS antibodies recognized a specific immunolabeled band around 130 kDa (Fig. 3A) corresponding to the reported molecular mass of murine eNOS (19,34). Densitometric analysis revealed that eNOS expression levels were reduced by ϳ40% in the aortic endothelium of Cx40 Ϫ/Ϫ mice compared with Cx40 ϩ/ϩ mice (Fig.…”

Section: Expression Of Enos Is Decreased In the Aortic Endothelium Ofmentioning

confidence: 87%

Loss of connexin40 is associated with decreased endothelium-dependent relaxations and eNOS levels in the mouse aorta

Alonso

Boittin

Bény

et al. 2010

American Journal of Physiology-Heart and Circulatory Physiology

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Alonso F, Boittin FX, Bény JL, Haefliger JA. Loss of connexin40 is associated with decreased endothelium-dependent relaxations and eNOS levels in the mouse aorta. Am J Physiol Heart Circ Physiol 299: H1365-H1373, 2010. First published August 27, 2010; doi:10.1152/ajpheart.00029.2010.-Upon agonist stimulation, endothelial cells trigger smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). Endothelial cells of mouse aorta are interconnected by gap junctions made of connexin40 (Cx40) and connexin37 (Cx37), allowing the exchange of signaling molecules to coordinate their activity. Wild-type (Cx40 ϩ/ϩ ) and hypertensive Cx40-deficient mice (Cx40 Ϫ/Ϫ ), which also exhibit a marked decrease of Cx37 in the endothelium, were used to investigate the link between the expression of endothelial connexins (Cx40 and Cx37) and endothelial nitric oxide synthase (eNOS) expression and function in the mouse aorta. With the use of isometric tension measurements in aortic rings precontracted with U-46619, a stable thromboxane A2 mimetic, we first demonstrate that ACh-and ATPinduced endothelium-dependent relaxations solely depend on NO release in both Cx40 ϩ/ϩ and Cx40 Ϫ/Ϫ mice, but are markedly weaker in Cx40 Ϫ/Ϫ mice. Consistently, both basal and ACh-or ATP-induced NO production were decreased in the aorta of Cx40 Ϫ/Ϫ mice. Altered relaxations and NO release from aorta of Cx40 Ϫ/Ϫ mice were associated with lower expression levels of eNOS in the aortic endothelium of Cx40 Ϫ/Ϫ mice. Using immunoprecipitation and in situ ligation assay, we further demonstrate that eNOS, Cx40, and Cx37 tightly interact with each other at intercellular junctions in the aortic endothelium of Cx40 ϩ/ϩ mice, suggesting that the absence of Cx40 in association with altered Cx37 levels in endothelial cells from Cx40 Ϫ/Ϫ mice participate to the decreased levels of eNOS. Altogether, our data suggest that the endothelial connexins may participate in the control of eNOS expression levels and function. connexin40; connexin37; endothelium-dependent relaxation; endothelial nitric oxide synthase

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

Cited by 21 publications

References 34 publications

Alpha1-adrenergic-mediated eNOS phosphorylation in intact arteries

Alpha1-adrenergic-mediated eNOS phosphorylation in intact arteries

Vascular complications of cystathionine β-synthase deficiency: future directions for homocysteine-to-hydrogen sulfide research

Loss of connexin40 is associated with decreased endothelium-dependent relaxations and eNOS levels in the mouse aorta

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