Abstract
Dysfunction of vascular nitric oxide (NO)/cGMP signaling is believed to contribute essentially to various cardiovascular disorders. Besides synthesis and/or bioavailability of endothelial NO, impaired function of soluble guanylate cyclase (sGC) may play a key role in vascular dysfunction. Based on the proposal that desensitization of sGC through S-nitrosation contributes to vascular NO resistance (
Proc Natl Acad Sci U S A:-12317, 2007), we exposed purified sGC to dinitrosyl iron complexes (DNICs), known as potent nitrosating agents. In the presence of 2 mM GSH, DNICs stimulated cGMP formation with EC50 values of 0.1 to 0.5 μM and with an efficacy of 70 to 80% of maximal activity measured with 10 μM 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO). In the absence of GSH, the efficacy of DNICs was markedly reduced, and sGC stimulation was counteracted by the inhibition of both basal and DEA/NO-stimulated cGMP formation at higher DNIC concentrations. Inactivation of sGC was slowly reversed in the presence of 2 mM GSH and associated with stoichiometric S-nitrosation of the protein (2.05 ± 0.18 mol S-nitrosothiol per mol of 143-kDa heterodimer). S-Nitrosoglutathione and sodium nitroprusside caused partial inhibition of DEA/NO-stimulated sGC that was prevented by GSH, whereas nitroglycerin (0.3 mM) had no effect. Our findings indicate that nitrosation of two cysteine residues in sGC heterodimers results in enzyme inactivation. Protection by physiologically relevant concentrations of GSH (10 μM to 3 mM) suggests that S-nitrosation of sGC may contribute to vascular dysfunction in inflammatory disorders associated with nitrosative and oxidative stress and GSH depletion.
Footnotes
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This work was supported by the Fonds zur Föderung der Wissenschaftlichen Forschung in Austria [Grant P20669]; the Deutsche Forschungsgemeinschaft [Grant KO1157/4-1]; the European Vascular Genomic Network [Grant LSHM-CT-2003-503254]; and the Forschungsvereinigungen “Otto von Guericke” [Grant PRO INNO II: KF0193301RK5].
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ABBREVIATIONS: sGC, soluble guanylate cyclase; BSA, bovine serum albumin; DEA/NO, 2,2-diethyl-1-nitroso-oxyhydrazine; DNIC, dinitrosyl iron complex; GSNO, S-nitrosoglutathione; GTN, glyceroltrinitrate (nitroglycerin); PBS, phosphate-buffered saline; SNP, sodium nitroprusside; TEA, triethanolamine; YC-1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole; RSNO, S-nitrosothiol.
- Received September 18, 2008.
- Accepted December 23, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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