Red blood cell induced conversion of nitrite to NO.: mechanisms of synthesis

Krishnaraj S Rathod, Andrew J Webb, Matthew J Lovell, Florence Lecomte, Amrita Ahluwalia. Clinical Pharmacology, William Harvey Research Institute, Queen Mary University London, UK, EC1M 6BQ.

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 ischaemic conditions when normal NO. synthesis, via NO. synthase (NOS), is suppressed. Our previous studies have demonstrated that the reduction of nitrite to NO. in the ischaemic heart is cardioprotective and dependent on xanthine oxidoreductase (XOR) activity (Webb et al., 2004). Recent evidence in erythrocytes suggests that nitrite is converted to NO. by a different mechanism involving deoxyhemoglobin (Cosby et al., 2003). 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 (100μM) 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 (100 µM) caused an elevation of NO. generation at pH 7.4 (from 0.070±02 to 0.280±0.06 ppb.μl-1 . s -1; P<0.05; n=6) and at pH 6.8 (from 0.170±06 to 0.500±0.09 ppb.μl-1.s-1; P<0.05; n=6) significantly above that produced by chemical acidification alone (0.070±02 at pH 7.4, n=6; 0.170±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 (10μM) and attenuated by ~65% (P<0.05, n=6) by the XOR inhibitor allopurinol (100μM). 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 (300μM) completely abolished nitrite-derived NO. generation at pH 7.4 and inhibited the response by ~57% at pH 6.8 (n=6, P<0.05). Incubation of erythrocytes with L-arginine (300μM; the substrate for NOS and conventional NO. synthesis) at pH 7.4 inhibited nitrite reductase activity by ~37% (P<0.05, n=6) whilst the inactive isomer D-arginine (300μM) had no effect. Western blot analysis of erythrocyte membranes collected from the blood of 5 individuals confirmed the presence of endothelial NOS (eNOS), as has been demonstrated recently (Kleinbongard et al., 2006). 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.

Cosby et al., 2003; Nat Med, 9:1498-1505
Kleinbongard et al., 2006 ; Blood, 107(7):2943-2951
Webb et al., PNAS, 2004; 101:13683-13688