Nitrite Is Reduced to Nitric Oxide by Xanthine Oxidoreductase and Nitric Oxide Synthase in the Erythrocyte Membrane in Hypoxemia.

Abstract Evidence is emerging that inorganic nitrite, previously thought to be an inactive metabolite of nitric oxide (NO.), provides an alternative source of functionally active NO. under ischemic conditions when normal NO. synthesis, via nitric oxide synthase (NOS), is suppressed. Our previous studies have demonstrated that the reduction of nitrite to NO. in the ischemic heart is cardioprotective and dependent on xanthine oxidoreductase (XOR) activity (Webb et al., PNAS, 2004; 101:13683–13688). Recent evidence in erythrocytes suggests that nitrite is converted to NO. by a different mechanism involving deoxyhemoglobin (Cosby et al., Nat Med, 2003; 9:1498–1505). We hypothesized that erythrocytes may also contain XOR with nitrite reductase activity, and we investigated this possibility using suspensions of washed erythrocytes prepared from blood taken from healthy volunteers. Whole erythrocytes were incubated with nitrite (100 μM) in anaerobic conditions at pH 7.4 and at pH 6.8, mimicking normal physiology and severe acidosis respectively, and NO. production measured in parts per billion (ppb) using ozone chemiluminescence. Addition of erythrocytes to nitrite caused an elevation of NO. generation at pH 7.4 (0.28±0.06; p<0.05; n=6) and pH 6.8 (0.50±0.09; p<0.05; n=6) significantly above that produced by simple acidification (0.07±0.02 at pH 7.4, n=6; 0.17±0.06 at pH 6.8). NO. generation at pH 6.8 was enhanced by ~43% (P<0.01, n=6) in the presence of XOR substrate xanthine (10 μM) and attenuated by ~65% (P<0.05, n=6) by the XOR inhibitor allopurinol (100 μM). In contrast, xanthine and allopurinol did not alter nitrite-derived NO. generation at pH 7.4 (n=6). Treatment of erythrocytes with the NOS inhibitor L-NAME (300 μM) abolished nitrite-derived NO. generation at pH 7.4 but inhibited the response by only ~57% at pH 6.8 (n=6, P<0.05). Western blot analysis of erythrocyte membranes confirmed the presence of endothelial NOS (eNOS), as has been demonstrated recently (Kleinbongard et al., Blood, 2006; 107(7):2943–2951). Furthermore the presence of XOR in whole purified erythrocytes was confirmed both immunohistochemically and using immunoflorescence. In summary we have demonstrated that erythrocytes reduce nitrite to NO. through the enzymatic actions of XOR and eNOS. These mechanisms of NO. production complement that of deoxyhemoglobin and are likely to contribute to the protective effects of nitrite attributed to NO. generation in hypoxemic and ischaemic conditions.