Comparative analysis of nitrite uptake and hemoglobin-nitrite reactions in erythrocytes: sorting out uptake mechanisms and oxygenation dependencies

Abstract

Nitrite uptake into red blood cells (RBCs) precedes its intracellular reactions with hemoglobin (Hb) that forms nitric oxide (NO) during hypoxia. We investigated the uptake of nitrite and its reactions with Hb at different oxygen saturations (So2), using RBCs with (carp and rabbit) and without (hagfish and lamprey) anion exchanger-1 (AE1) in the membrane, with the aim to unravel the mechanisms and oxygenation dependencies of nitrite transport. Added nitrite rapidly diffused into the RBCs until equilibrium. The distribution ratio of nitrite across the membrane agreed with that expected from HNO2diffusion and AE1-mediated facilitated NO2−diffusion. Participation of HNO2diffusion was emphasized by rapid transmembrane nitrite equilibration also in the natural AE1 knockouts. Following the equilibration, nitrite was consumed by reacting with Hb, which created a continued inward diffusion controlled by intracellular reaction rates. Changes in nitrite uptake with So2, pH, or species were accordingly explained by corresponding changes in reaction rates. In carp, nitrite uptake rates increased linearly with decreasing So2over the entire So2range. In rabbit, nitrite uptake rates were highest at intermediate So2, producing a bell-shaped relationship with So2. Nitrite consumption increased ∼10-fold with a 1 unit decrease in pH, as expected from the involvement of protons in the reactions with Hb. The reaction of nitrite with deoxyhemoglobin was favored over that with oxyhemoglobin at intermediate So2. We propose a model for RBC nitrite uptake that involves both HNO2diffusion and AE1-mediated transport and that explains both the present and previous (sometimes puzzling) results.