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

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 GS(15)NO 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 GS(15)NO from (15)N-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-VASP(S239) 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.

Carbonic anhydrase II does not regulate nitrite‐dependent nitric oxide formation and vasodilation

“…Previously, CAII inhibitors, such as dorzolamide, were shown to paradoxically increase NO production from nitrite and bovine CAII using a NO‐specific electrode by Aamand et al (), although no clear mechanism for this was established. However, we found no effect of dorzolamide on NO‐production using NO chemiluminescence (data not included), in line with recent other reports (Andring et al, ; Hanff et al, ). The mechanism of CAII inhibition by dorzolamide has been well characterized, and structural data illustrate that dorzolamide and related inhibitors bind directly to the zinc in the active site of CAII (Pinard, Boone, Rife, Supuran, & McKenna, ).…”

“…Despite the fact that CAII is expressed abundantly in red blood cells, we found that CAII KO red blood cells exhibited similar nitrite‐dependent platelet inhibition compared with wild‐type red blood cells (Figure ). Recently, Hanff et al () reported that nitrite and CAII can generate S‐nitrosothiols (RSNOs) in the presence of cysteine, but we did not observe in vivo or in vitro vasodilation with CAII KO (Figures and ). While RSNOs are potent vasodilators, our observation suggests that this chemistry is not occurring in vivo.…”

Discovery of potent carbonic anhydrase, acetylcholinesterase, and butyrylcholinesterase enzymes inhibitors: The new amides and thiazolidine-4-ones synthesized on an acetophenone base

“…The CA I isoform is available in cerebral and retinal illness, and we pursued the supposition that proteins of the CA I isoform could demonstrate treatment purposes for inhibiting CA I‐induced vascular permeance . The HCA I isoform is significantly expressed in the human body, and it is available in great concentrations in the gastrointestinal tract and blood .…”

“…4.2.1.1), as isoforms I and II, are extremely available in the gastrointestinal tract and in red blood cells of vertebrates, arriving at concentrations as high as 0.2 mM and having a significant role in blood pH homeostasis as well as in carrying carbon dioxide (CO 2 ) to the lungs. [ Both of these cytosolic isoenzymes are the predominant ones among the 16 CAs recorded throughout history in the tissues of vertebrates/different cells . Twelve of them are described by a common Zn 2+ ion at the active site, while they differ in tissue and organ expression, molecular features, catalytic activity cellular localization, and how they respond to various groups of inhibitors …”

Evaluation of acetylcholinesterase and carbonic anhydrase inhibition profiles of 1,2,3,4,6-pentasubstituted-4-hydroxy-cyclohexanes

“…In this test, variations in absorbance were obtained during 3 min at 30°C. p ‐Nitrophenyl acetate (NPA) was used as a substrate and transformed by both isoforms to p ‐nitrophenolate ion . Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was examined for registration of both isoforms purity .…”