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Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets

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Abstract

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.

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Abbreviations

AlbSNO:

S-Nitrosoalbumin

BSA:

Bovine serum albumin

CA:

Carbonic anhydrase

cGMP:

Cyclic guanosine monophosphate

CVD:

Cardiovascular disease

CysSH:

Reduced l-cysteine

CysSNO:

S-Nitrosocysteine

GC:

Gas chromatography

GSH:

Glutathione

GSNO:

S-Nitrosoglutathione

GTP:

Guanosine triphosphate

HbSNO:

S-Nitrosohemoglobin

HMM:

High-molecular-mass

IS:

Internal standard

LC:

Liquid chromatography

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

LMM:

Low-molecular-mass

NO:

Nitric oxide

NR:

Nitrate reductase

PKG:

Protein kinase

P-VASPS239 :

VASP phosphorylated at Ser239

RSNO:

S-Nitrosothiol

sGC:

Soluble guanylyl cyclase

SIDM:

Stable-isotope dilution mass spectrometry

SNP:

Sodium nitroprusside

VASP:

Vasodilator-stimulated phosphoprotein

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Acknowledgments

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.).

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Authors and Affiliations

  1. Centre of Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Erik Hanff, Anke Böhmer, Maximilian Zinke, Alexandra Schwarz & Dimitrios Tsikas

  2. Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia

    Stepan Gambaryan

  3. Department of Cytology and Histology, S. Petersburg State University, Universitetskaya Nab 7/9, 199034, S. Petersburg, Russia

    Stepan Gambaryan

  4. Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019, Sesto Fiorentino, Florence, Italy

    Claudiu T. Supuran

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  1. Erik Hanff
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  2. Anke Böhmer
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  3. Maximilian Zinke
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  4. Stepan Gambaryan
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  6. Claudiu T. Supuran
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  7. Dimitrios Tsikas
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Correspondence to Dimitrios Tsikas.

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The authors declare that they have no conflict of interest.

Ethical statement

The study on human platelets and human erythrocytes was approved by the Ethics Committee of the Hannover Medical School.

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E. Hanff and A. Böhmer have contributed equally to this article and are both first authors.

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Hanff, E., Böhmer, A., Zinke, M. et al. Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets. Amino Acids 48, 1695–1706 (2016). https://doi.org/10.1007/s00726-016-2234-z

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