Diffusion of Peroxynitrite in the Presence of Carbon Dioxide

Peroxynitrite, the reactive species formed in vivo by the reaction of nitric oxide with superoxide anion, is capable of diffusing across erythrocyte membranes via anion channels and passive diffusion (A. Denicola, J. M. Souza, and R. Radi, Proc. Natl. Acad. Sci. USA 95, 3566–3571, 1998). However, peroxynitrite diffusion could be limited by extracellular targets, with the reaction with CO2 (k2 = 4.6 × 104 at 37°C and pH 7.4) the most relevant. Herein, we studied the influence of physiological concentrations of CO2 on peroxynitrite diffusion across intact red blood cells. The presence of CO2 inhibited the oxidation of intracellular oxyhemoglobin by externally added peroxynitrite. However, the inhibition by CO2 decreased at increasing red blood cell densities. At 45% hematocrit, 1.3 mM CO2 (in equilibrium with 24 mM bicarbonate, at pH 7.4 and 25°C) only inhibited 30% of intracellular oxyhemoglobin oxidation. This partial inhibition was also observed in red blood cells pretreated with the anion exchanger inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, ruling out a competition between peroxynitrite and bicarbonate for the transport through the anion channel. A theoretical model was developed to estimate the diffusion distance and half-life of extracellular peroxynitrite before reacting with intracellular oxyhemoglobin, at different red blood cell densities, and in the presence or absence of CO2. The theoretical model correlated well with the experimental data. Our results indicate that, even in the presence of CO2, peroxynitrite is able to diffuse and reach the inside of the erythrocyte.