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

Abstract

Background and purpose Although it has been reported that bovine carbonic anhydrase CAII is capable of generating NO from nitrite, the function and mechanism of CAII in nitrite‐dependent NO formation and vascular responses remain controversial. We tested the hypothesis that CAII catalyses NO formation from nitrite and contributes to nitrite‐dependent inhibition of platelet activation and vasodilation. Experiment approach The role of CAII in enzymatic NO generation was investigated by measuring NO formation from the reaction of isolated human and bovine CAII with nitrite using NO photolysis‐chemiluminescence. A CAII‐deficient mouse model was used to determine the role of CAII in red blood cell mediated nitrite reduction and vasodilation. Key results We found that the commercially available purified bovine CAII exhibited limited and non‐enzymatic NO‐generating reactivity in the presence of nitrite with or without addition of the CA inhibitor dorzolamide; the NO formation was eliminated with purification of the enzyme. There was no significant detectable NO production from the reaction of nitrite with recombinant human CAII. Using a CAII‐deficient mouse model, there were no measurable changes in nitrite‐dependent vasodilation in isolated aorta rings and in vivo in CAII−/−, CAII+/−, and wild‐type mice. Moreover, deletion of the CAII gene in mice did not block nitrite reduction by red blood cells and the nitrite‐NO‐dependent inhibition of platelet activation. Conclusion and implications These studies suggest that human, bovine and mouse CAII are not responsible for nitrite‐dependent NO formation in red blood cells, aorta, or the systemic circulation.

Novel benzoic acid derivatives: Synthesis and biological evaluation as multitarget acetylcholinesterase and carbonic anhydrase inhibitors

“…[ 51,52 ] The MW of these hCA isoenzymes was computed by this method as well as with the SynGene imaging tool, which was approximately 29 kDa. [ 53 ] The esterase activity of the hCA isoforms was determined by following the change in absorbance at 348 nm over 3 min [ 54 ] according to the method described by Verpoorte et al [ 55 ] One enzyme unit (EU) was defined as the hydrolysis of 1.0 µmol 4‐nitrophenyl acetate to 4‐nitrophenyl per minute at pH 7.4 and temperature 25°C. AAZ (C 4 H 6 N 4 O 3 S 2 , N ‐(5‐sulfamoyl‐1,3,4‐thiadiazol‐2‐yl)acetamide, PHR1908) was used as the reference drug.…”

The Role of NO/sGC/cGMP/PKG Signaling Pathway in Regulation of Platelet Function

“…Both CA1 and CA2 are expressed in platelets in a relatively high level (2500 and 36,100 copies/platelet respectively) [ 84 ]; however, it is not clear whether platelet CA can reduce nitrite to NO. sGC is strongly activated in washed human platelets incubated with recombinant bovine CA2 together with nitrite [ 83 ], whereas others showed that human, bovine, or mouse CA2 does not regulate nitrite-dependent NO formation in blood [ 85 ]. Recently in experiments with washed human platelets, we showed that cysteine is required for nitrite-dependent sGC activation, which was independent from CA inhibitor [ 86 ], but this assumption needs future experimental confirmations.…”

Nitrous anhydrase activity of carbonic anhydrase II: cysteine is required for nitric oxide (NO) dependent phosphorylation of VASP in human platelets

“…A sophisticated animal study was performed by Wang et al 14 . In CA II þ/þ , CA II ± and CA II -/mice (26 À 32 g body weight), infusion of NaNO 2 (30, 50, 100, 500, 2500 nmol over 5 min corresponding to doses of up to 3.8 mg nitrite/kg body weight) lowered mean arterial blood pressure equally from about 70 mmHg down to 40 mmHg in CA II þ/þ , CA II ± and CA II -/-, suggesting a mechanism independent of CA II 14 . Unfortunately, the authors did not report on the possible formation of S-nitrosothiols, which could have been an indication of hCA II involvement.…”

“…There are contradictory reports in the literature regarding the above mentioned newest CA II activities and even a disputation of a physiological role of CA II in the bioactivation of nitrite [11][12][13][14] . Here, we provide new experimental data in support of the nitrous anhydrase activity of CA II.…”