Diffusion of peroxynitrite in the presence of carbon dioxide

Peroxynitrite, the reactive species formed in vivo by the reaction of nitri c oxide with superoxide anion, is capable of diffusing across erythrocyte m embranes via anion channels and passive diffusion (A. Denicola, J. M. Souza , and R. Radi, Proc. Natl. Acad. Sci. USA 95, 3566-3571, 1998). However, pe roxynitrite diffusion could be limited by extracellular targets, with the r eaction with CO2 (k(2) = 4.6 x 10(4) at 37 degrees C and pH 7.4) the most r elevant. Herein, we studied the influence of physiological concentrations o f CO2 on peroxynitrite diffusion across intact red blood cells. The presenc e of CO2 inhibited the oxidation of intracellular oxyhemoglobin by external ly added peroxynitrite. However, the inhibition by CO2 decreased at increas ing red blood cell densities. At 45% hematocrit, 1.3 mM CO2 (in equilibrium with 24 mM bicarbonate, at pH 7.4 and 25 degrees C) only inhibited 30% of intracellular oxyhemoglobin oxidation, This partial inhibition was also obs erved in red blood cells pretreated with the anion exchanger inhibitor 4,4' -diisothiocyanatostilbene-2,2'-disulfonic acid, ruling out a competition be tween peroxynitrite and bicarbonate for the transport through the anion cha nnel. A theoretical model was developed to estimate the diffusion distance and half-life of extracellular peroxynitrite before reacting with intracell ular oxyhemoglobin, at different red blood cell densities, and in the prese nce or absence of CO2. The theoretical model correlated well with the exper imental data. Our results indicate that, even in the presence of CO2, perox ynitrite is able to diffuse and reach the inside of the erythrocyte. (C) 19 99 Academic Press.