Erythroid anion Exchanger-1 (band 3) transports nitrite for nitric oxide metabolism

Nitrite (NO₂^-) interacts with hemoglobin (Hb) in various ways to regulate blood flow. During hypoxic vasodilation, nitrite is reduced by deoxyHb to yield nitric oxide (NO). While NO, a hydrophobic gas, could freely diffuse across the cell membrane, how the reactant nitrite anion could permeate through the red blood cell (RBC) membrane remains unclear. We hypothesized that Cl^-/HCO₃^- anion exchanger-1 (AE1; band 3) abundantly embedded in the RBC membrane could transport NO₂^-, as HCO₃^- and NO₂^- exhibit similar hydrated radii. Here, we monitored NO/N₂O₃ generated from NO₂^- inside human RBCs by DAF-FM fluorophore. NO₂^-, not NO₃^-, increased intraerythrocytic DAF-FM fluorescence. To test the involvement of AE1-mediated transport in intraerythrocytic NO/N₂O₃ production from nitrite, we lowered Cl^- or HCO₃^- in the RBC-incubating buffer by 20 % and indeed observed slower rise of the DAF-FM fluorescence. Anti-extracellular AE1, but not anti-intracellular AE1 antibodies, reduced the rates of NO formation from nitrite. The AE1 blocker DIDS similarly reduced the rates of NO/N₂O₃ production from nitrite in a dose-dependent fashion, confirming that nitrite entered RBCs through AE1. Nitrite inside the RBCs reacted with both deoxyHb and oxyHb, as evidenced by 6.1 % decrease in deoxyHb, 14.7 % decrease in oxyHb, and 20.7 % increase in methemoglobin (metHb). Lowering Cl^- in the milieu equally delayed metHb production from nitrite-oxyHb and nitrite-deoxyHb reactions. Thus, AE1-mediated NO₂^- transport facilitates NO₂^--Hb reactions inside the red cells, supporting NOx metabolism in circulation.