Differences in eNOS Activity Because of Subcellular Localization Are Dictated by Phosphorylation State Rather than the Local Calcium Environment

Church, Jarrod E; Fulton, David

doi:10.1074/jbc.m505968200

Cited by 68 publications

(81 citation statements)

References 43 publications

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“…Although the mechanism for this translocation has not been resolved, it requires the presence of the mitochondrial targeting sequence previously identified in eNOS (17). However, it has been proposed that eNOS redistribution from the plasma membrane to other sub-cellular compartments may be a mechanism for regulating the enzymatic activity of eNOS (16), and plasma membrane-bound eNOS and Golgi-bound eNOS generate more NO than cytosolic eNOS (10,22,60). Alternatively, since NO has been shown to regulate the electron transport chain (8) in the mitochondria, it may be involved in regulating ATP generation.…”

Section: Discussionmentioning

confidence: 99%

Disruption of Endothelial Cell Mitochondrial Bioenergetics in Lambs with Increased Pulmonary Blood Flow

Sun

Sharma

Fratz

et al. 2013

Antioxidants & Redox Signaling

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Aims: The mitochondrial dysfunction in our lamb model of congenital heart disease with increased pulmonary blood flow (PBF) (Shunt) is associated with disrupted carnitine metabolism. Our recent studies have also shown that asymmetric dimethylarginine (ADMA) levels are increased in Shunt lambs and ADMA increases the nitration of mitochondrial proteins in lamb pulmonary arterial endothelial cells (PAEC) in a nitric oxide synthase (NOS)-dependent manner. Thus, we determined whether there was a mechanistic link between endothelial nitric oxide synthase (eNOS), ADMA, and the disruption of carnitine homeostasis in PAEC. Results: Exposure of PAEC to ADMA induced the redistribution of eNOS to the mitochondria, resulting in an increase in carnitine acetyl transferase (CrAT) nitration and decreased CrAT activity. The resulting increase in acyl-carnitine levels resulted in mitochondrial dysfunction and the disruption of mitochondrial bioenergetics. Since the addition of l-arginine prevented these pathologic changes, we examined the effect of l-arginine supplementation on carnitine homeostasis, mitochondrial function, and nitric oxide (NO) signaling in Shunt lambs. We found that the treatment of Shunt lambs with l-arginine prevented the ADMA-mediated mitochondrial redistribution of eNOS, the nitration-mediated inhibition of CrAT, and maintained carnitine homeostasis. In turn, adenosine-5¢-triphosphate levels and eNOS/heat shock protein 90 interactions were preserved, and this decreased NOS uncoupling and enhanced NO generation. Innovation: Our data link alterations in cellular l-arginine metabolism with the disruption of mitochondrial bioenergetics and implicate altered carnitine homeostasis as a key player in this process. Conclusion: l-arginine supplementation may be a useful therapy to prevent the mitochondrial dysfunction involved in the pulmonary vascular alterations secondary to increased PBF.

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Section: Discussionmentioning

confidence: 99%

Disruption of Endothelial Cell Mitochondrial Bioenergetics in Lambs with Increased Pulmonary Blood Flow

Sun

Sharma

Fratz

et al. 2013

Antioxidants & Redox Signaling

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“…To determine if the effects of NO on vWF secretion were dependent on the location of synthesis or the amount, we next expressed calcium-insensitive forms of eNOS that produce equal amounts of NO regardless of their intracellular location in eNOS knockdown HAEC (3,23). These constructs are derived from the deletion of two key autoinhibitory domains of 45 and 14 amino acids in length and thus are labeled ⌬45/⌬14 eNOS.…”

Section: Resultsmentioning

confidence: 99%

“…These constructs produce equal amounts of NO regardless of intracellular location (3,23), and, when the amount of NO produced is equalized between the Golgi and PM, the ehanced ability of PM eNOS to elicit cGMP signaling or inhibit vWF secretion is lost. Although these results provide evidence against the importance of location in NO signaling, it is possible that the greater amounts of NO generated by PM eNOS vs. Golgi eNOS in our study result in a larger sphere of influence and less compartmentalization of protein S-nitrosylation.…”

Section: Discussionmentioning

confidence: 99%

Role of local production of endothelium-derived nitric oxide on cGMP signaling and S-nitrosylation

Qian

Zhang

Church³

et al. 2010

American Journal of Physiology-Heart and Circulatory Physiology

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Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), exerts control over vascular function via two distinct mechanisms, the activation of soluble guanylate cyclase (sGC)/cGMP-dependent signaling or through S-nitrosylation of proteins with reactive thiols (S-nitrosylation). Previous studies in cultured endothelial cells revealed that eNOS targeted to the plasma membrane (PM) releases greater amounts of NO compared with Golgi tethered eNOS. However, the significance of eNOS localization to sGC-dependent or -independent signaling is not known. Here we show that PM-targeted eNOS, when expressed in human aortic endothelial cells (HAEC) and isolated blood vessels, increases sGC/cGMP signaling to a greater extent than Golgi-localized eNOS. The ability of local NO production to influence sGC-independent mechanisms was also tested by monitoring the secretion of Von Willebrand factor (vWF), which is tonically inhibited by the Snitrosylation of N-ethylmaleimide sensitive factor (NSF). In eNOS "knockdown" HAECs, vWF secretion was attenuated to a greater degree by PM eNOS compared with a Golgi-restricted eNOS. Moreover, the PM-targeted eNOS induced greater S-nitrosylation of NSF vs. Golgi eNOS. To distinguish between the amount of NO generated and the intracellular location of synthesis, we expressed Golgi and PM-targeted calcium-insensitive forms of eNOS in HAEC. These constructs, which generate equal amounts of NO regardless of location, produced equivalent increases in cGMP in bioassays and equal inhibition of vWF secretion. We conclude that the greater functional effects of PM eNOS are due to the increased amount of NO produced rather than effects derived from the local synthesis of NO. nitric oxide; endothelial nitric oxide synthase; endothelium; nitrosylation; intracellular location; Von Willebrand factor CARDIOVASCULAR DISEASE (CVD) remains the principal cause of death in both developed and developing countries, and almost 800,000 die annually in the United States from CVDs that include atherosclerosis, hypertension, congestive heart failure, and stroke (18). Endothelium-derived nitric oxide (NO) as synthesized by endothelial nitric oxide synthase (eNOS) plays a vital role in maintaining cardiovascular homeostasis and has potent effects on vascular tone, smooth muscle cell proliferation and migration, leukocyte adhesion, and platelet aggregation (9). Numerous studies have shown that eNOS is protective against pathological vascular remodeling, hypertension, and atherosclerosis (25,34,40). Moreover, reduced expression and dysregulation of eNOS, which results in the decreased synthesis of NO and the increased production of superoxide instead of NO, increases the severity of CVD (30, 31). However, the mechanisms by which eNOS affords vascular protection are incompletely understood.Endothelial-derived NO controls vascular function via at least two distinct mechanisms. The first is the well-characterized activation of the soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signaling ...

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“…After 60 min of incubation, culture medium was collected and ethanol-precipitated to remove proteins. 50 l of the reaction mix were loaded to the SIEVERS machine for NO x (NO 2 and NO 3 ) measurement according to standard manufacturer's instruction as previously described (24). Protein contents in the cell lysates were determined by Lowry's method.…”

Section: Methodsmentioning

confidence: 99%

eNOS-β-Actin Interaction Contributes to Increased Peroxynitrite Formation during Hyperoxia in Pulmonary Artery Endothelial Cells and Mouse Lungs

Kondrikov

Elms

Fulton

et al. 2010

Journal of Biological Chemistry

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Oxygen toxicity is the most severe side effect of oxygen therapy in neonates and adults. Pulmonary damage of oxygen toxicity is related to the overproduction of reactive oxygen species (ROS). In the present study, we investigated the effect of hyperoxia on the production of peroxynitrite in pulmonary artery endothelial cells (PAEC) and mouse lungs. Incubation of PAEC under hyperoxia (95% O 2 ) for 24 h resulted in an increase in peroxynitrite formation. Uric acid, a peroxynitrite scavenger, prevented hyperoxia-induced increase in peroxynitrite. The increase in peroxynitrite formation is accompanied by increases in nitric oxide (NO) release and endothelial NO synthase (eNOS) activity. We have previously reported that association of eNOS with ␤-actin increases eNOS activity and NO production in lung endothelial cells. To study whether eNOS-␤-actin association contributes to increased peroxynitrite production, eNOS-␤-actin interaction were inhibited by reducing ␤-actin availability or by using a synthetic peptide (P326TAT) containing a sequence corresponding to the actin binding site on eNOS. We found that disruption of eNOS-␤-actin interaction prevented hyperoxia-induced increases in eNOS-␤-actin association, eNOS activity, NO and peroxynitrite production, and protein tyrosine nitration. Hyperoxia failed to induce the increases in eNOS activity, NO and peroxynitrite formation in COS-7 cells transfected with plasmids containing eNOS mutant cDNA in which amino acids leucine and tryptophan were replaced with alanine in the actin binding site on eNOS. Exposure of mice to hyperoxia resulted in significant increases in eNOS-␤-actin association, eNOS activity, and protein tyrosine nitration in the lungs. Our data indicate that increased association of eNOS with ␤-actin in PAEC contributes to hyperoxia-induced increase in the production of peroxynitrite which may cause nitrosative stress in pulmonary vasculature.Oxygen therapy is an important element of the management of various conditions such as adult or neonatal respiratory distress syndrome, circulatory shock, infection, multiple-organ failure syndrome, and pulmonary hypertension (1-6). However, prolonged exposure to increased concentrations of oxygen induces diffuse pulmonary injuries, excessive inflammation, and lung fibrosis. The hyperoxia-induced damages to lung cells have been attributed to the generation of reactive oxygen species (ROS) 2 and subsequent formation of more potent oxidants such as peroxynitrite (ONOO Ϫ ) (7,8). Several studies have suggested that endothelial nitric-oxide synthase (eNOS) plays an important role in the pathogenesis of oxygen toxicity (8, 9). It has been reported that hyperoxia increases eNOS activity and nitric oxide (NO) release from endothelial cells (9, 10). Inhibition of eNOS using L-NAME or knock-out of eNOS reduces peroxynitrite-mediated cytotoxicity in hyperoxic cellular damage in retina (9, 11). However, the mechanism for hyperoxia-induced increases in eNOS activity and NO release has not been clarified. In the present study,...

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Differences in eNOS Activity Because of Subcellular Localization Are Dictated by Phosphorylation State Rather than the Local Calcium Environment

Cited by 68 publications

References 43 publications

Disruption of Endothelial Cell Mitochondrial Bioenergetics in Lambs with Increased Pulmonary Blood Flow

Disruption of Endothelial Cell Mitochondrial Bioenergetics in Lambs with Increased Pulmonary Blood Flow

Role of local production of endothelium-derived nitric oxide on cGMP signaling and S-nitrosylation

eNOS-β-Actin Interaction Contributes to Increased Peroxynitrite Formation during Hyperoxia in Pulmonary Artery Endothelial Cells and Mouse Lungs

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