Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation

doi:10.1016/j.freeradbiomed.2012.02.029

Free Radical Biology and Medicine

Volume 52, Issue 9, 1 May 2012, Pages 1806-1819

https://doi.org/10.1016/j.freeradbiomed.2012.02.029 Get rights and content

Abstract

The NADPH oxidases (Noxs) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of this study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and this inhibition was reversible with chronic, but not acute, exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, and eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation, or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO sensitivity, and biotin labeling. Furthermore, coexpression of the denitrosylation enzymes thioredoxin 1 and GSNO reductase prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was in the order Nox1 ≥ Nox3 > Nox5 > Nox2, whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5.

Highlights

► Nitric oxide dose-dependently inhibits NADPH oxidase activity. ► Reduced Nox5 activity was not due to nitration, glutathiolation or phosphorylation. ► Nox5 was S-nitrosylated on at least 4 sites and SNO-Nox5 reduced by C694S mutation. ► Sensitivity to NO inhibition varied across Nox isoforms, Nox1≥Nox3>Nox5>Nox2>Nox4.

Section snippets

Cell culture and transfection

COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% fetal calf serum. For transfection, COS-7 cells were seeded and transfected the next day using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Human aortic endothelial cells (HAECs) and human aortic smooth muscle cells (HASMCs) were purchased from Cascade Biologics and grown in endothelial cell basal medium-2 (Clonetics) or smooth

Results

To investigate whether NO can influence Nox5 activity, we coexpressed iNOS with Nox5. We employed iNOS because it produces relatively large amounts of NO within the cell, yet much smaller and more biologically relevant amounts than the typical concentration of NO donor used. In COS-7 cells cotransfected with Nox5 and iNOS, there was substantially less superoxide produced in response to the calcium ionophore ionomycin, compared to that produced from cells expressing Nox5 and a control plasmid,

Discussion

In this study, we show that both exogenous and endogenously produced NO can reduce the activity of Nox5 in a dose-dependent manner. Furthermore, blockade of endogenously produced NO in human endothelial and smooth muscle cells increases Nox5-dependent ROS production. The inhibitory effects of NO are seen under basal conditions and after stimulation with both calcium-dependent and independent agonists. Reduced activity is also observed in an isolated enzyme activity assay and is independent of

Conclusions

Thus the ability of NO to suppress Nox2 activity is clearly only part of the story. Nox5, Nox1, and Nox3 must be added to the list of NO-sensitive ROS-generating oxidoreductases. Nox5 is directly modified by reversible S-nitrosylation and future studies are needed to identify whether the other Nox enzymes are similarly S-nitrosylated and to identify the mechanism by which Nox4 resists NO inhibition. Collectively, these mechanisms may contribute to the redox balance observed in blood vessels and

Acknowledgments

This work was supported by National Institutes of Health Grants R01 HL085827 (D.J.R.F., R.C.V.), R01HL092446 (D.J.R.F., D.W.S.), and P01 HL101902-01A1 (D.J.R.F., S.M.B.) and an Established Investigator Award from the American Heart Association (D.J.R.F.).

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Original ContributionNitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation

Abstract

Highlights

Section snippets

Cell culture and transfection

Results

Discussion

Conclusions

Acknowledgments

J. Mol. Cell. Cardiol.

Free Radic. Biol. Med.

J. Biol. Chem.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

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J. Biol. Chem.

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J. Biol. Chem.

J. Biol. Chem.

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Free Radic. Biol. Med.

J. Biol. Chem.

Cell. Signal.

Cell

Cell

Chem. Biol.

Biochimie

Cell

J. Biol. Chem.

J. Biol. Chem.

Nitric oxide synthases: structure, function and inhibition

Biochem. J.

Deletion of protein tyrosine phosphatase 1b improves peripheral insulin resistance and vascular function in obese, leptin-resistant mice via reduced oxidant tone

Circ. Res.

Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly

Am. J. Physiol.

Identification of S-nitrosylated targets of thioredoxin using a quantitative proteomic approach

Biochemistry

Arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels

Circulation

Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress

Circ. Res.

Hsp90 regulates NADPH oxidase activity and is necessary for superoxide but not hydrogen peroxide production

Antioxid. Redox Signal.

Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase

J. Clin. Invest.

Measurement of reactive oxygen species in cardiovascular studies

Hypertension

Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide

Proc. Natl. Acad. Sci. U. S. A.

Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay

Am. J. Physiol. Cell Physiol.

Endothelial nitric oxide synthase in vascular disease: from marvel to menace

Circulation

A protein microarray-based analysis of S-nitrosylation

Proc. Natl. Acad. Sci. U. S. A.

Regulation of nitric oxide-sensitive guanylyl cyclase

Circ. Res.

Nox5 and the regulation of cellular function

Antioxid. Redox Signal.

NAD(P)H oxidase: role in cardiovascular biology and disease

Circ. Res.

Original Contribution
Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation