Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets
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Nitric oxide (NO), S-nitrosoglutathione (GSNO) and S-nitrosocysteine are highly potent signaling molecules, acting both by cGMP-dependent and cGMP-independent mechanisms. The NO metabolite nitrite (NO2 −) is a major NO reservoir. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to reduce/convert nitrite to NO. We evaluated the role and the physiological importance of CA for an extra-platelet CA/nitrite/NO/cGMP pathway in human platelets. Authentic NO was analyzed by an NO-sensitive electrode. GSNO and GS15NO were measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS). cGMP was determined by LC–MS/MS or RIA. In reduced glutathione (GSH) containing aqueous buffer (pH 7.4), human and bovine erythrocytic CAII-mediated formation of GSNO from nitrite and GS15NO from 15N-nitrite. In the presence of l-cysteine and GSH, this reaction was accompanied by NO release. Incubation of nitrite with bovine erythrocytic CAII and recombinant soluble guanylyl cyclase resulted in cGMP formation. Upon incubation of nitrite with bovine erythrocytic CAII and washed human platelets, cGMP and P-VASPS239 were formed in the platelets. This study provides the first evidence that extra-platelet nitrite and erythrocytic CAII may modulate platelet function in a cGMP-dependent manner. The new nitrite-dependent CA activity may be a general principle and explain the cardioprotective effects of inorganic nitrite in the vasculature. We propose that nitrous acid (ONOH) is the primary CA-catalyzed reaction product of nitrite.
KeywordsCarbonic anhydrase cGMP Nitric oxide Nitrite S-Nitrosothiols Platelets
Bovine serum albumin
Cyclic guanosine monophosphate
Liquid chromatography–tandem mass spectrometry
VASP phosphorylated at Ser239
Soluble guanylyl cyclase
Stable-isotope dilution mass spectrometry
We are grateful to Prof. R. Seifert from the Institute of Pharmacology and Prof. V. Kaever from the Core Unit Metabolomics, both Hannover Medical School, for providing us with the recombinant soluble guanylyl cyclase and for the LC–MS/MS analysis of cGMP. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grant TS60/4-1 (to D.T.).
Compliance with ethical standards
Conflicts of interest
The authors declare that they have no conflict of interest.
The study on human platelets and human erythrocytes was approved by the Ethics Committee of the Hannover Medical School.
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