S-Nitrosothiols signal hypoxia-mimetic vascular pathology
Lisa A. Palmer, … , Timothy Macdonald, Benjamin Gaston
Lisa A. Palmer, … , Timothy Macdonald, Benjamin Gaston
Published September 4, 2007
Citation Information: J Clin Invest. 2007;117(9):2592-2601. https://doi.org/10.1172/JCI29444.
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Research Article

S-Nitrosothiols signal hypoxia-mimetic vascular pathology

Abstract

NO transfer reactions between protein and peptide cysteines have been proposed to represent regulated signaling processes. We used the pharmaceutical antioxidant N-acetylcysteine (NAC) as a bait reactant to measure NO transfer reactions in blood and to study the vascular effects of these reactions in vivo. NAC was converted to S-nitroso-N-acetylcysteine (SNOAC), decreasing erythrocytic S-nitrosothiol content, both during whole-blood deoxygenation ex vivo and during a 3-week protocol in which mice received high-dose NAC in vivo. Strikingly, the NAC-treated mice developed pulmonary arterial hypertension (PAH) that mimicked the effects of chronic hypoxia. Moreover, systemic SNOAC administration recapitulated effects of both NAC and hypoxia. eNOS-deficient mice were protected from the effects of NAC but not SNOAC, suggesting that conversion of NAC to SNOAC was necessary for the development of PAH. These data reveal an unanticipated adverse effect of chronic NAC administration and introduce a new animal model of PAH. Moreover, evidence that conversion of NAC to SNOAC during blood deoxygenation is necessary for the development of PAH in this model challenges conventional views of oxygen sensing and of NO signaling.

Authors

Lisa A. Palmer, Allan Doctor, Preeti Chhabra, Mary Lynn Sheram, Victor E. Laubach, Molly Z. Karlinsey, Michael S. Forbes, Timothy Macdonald, Benjamin Gaston

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Figure 5

S-Nitrosothiols prevent normoxic ubiquitination and degradation of HIF 1α.

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S-Nitrosothiols prevent normoxic ubiquitination and degradation of HIF ...
(A) NAC treatment (10 mg/ml; 3 weeks) increased whole-lung HIF 1–DNA binding activity. Complexes were supershifted (ss) with anti–HIF 1β and eliminated with excess cold probe (P). (B) SNOAC (1 μM), like GSNO (5, 13, 38), increased normoxic HIF 1α expression in nuclear extracts isolated from primary murine pulmonary endothelial cells. NAC alone (50 μM) did not affect HIF 1α expression. β-Actin was used as a protein load control. (C) In BPAECs transfected with HA-tagged HIF 1α and dominant-negative His-6-Myc–tagged ubiquitin (DN-Ub), ubiquitinated proteins were isolated using a nickel column and immunoblotted for HIF 1α. Both hypoxia and GSNO (G; 10 μM) inhibited HIF 1α ubiquitination relative to normoxia. (D) In COS cells cotransfected with HA-tagged HIF 1α and FLAG-tagged pVHL, GSNO (10 μM) prevented the coimmunoprecipitation of HIF 1α with pVHL. (E) S-nitrosylation of pVHL by SNOAC (5 μM) in equal protein aliquots isolated from HeLa cells was identified by biotin substitution (49); in the absence of ascorbate, S-nitrosylated pVHL was not detected. (F) Similarly, SNOAC and GSNO (5 μM) increased pVHL S-nitrosylation in pVHL-overexpressing 786-O cells. (G) C162, but not C77, was identified by biotin substitution to be S-nitrosylated in BPAECs transfected with wild-type cysteine 77 to serine mutant (C77S), C162S, or combined C77S/C162S pVHL exposed to SNOAC (1 μM). Native pVHL and MAPK immunoblots represented the pVHL expression and protein load controls, respectively. All in vitro treatments were for 4 hours.