Hematocrit and Oxygen Dependence of Nitric Oxide Scavenging by Red Blood Cells.

Human, animal and in vitro studies suggest that reactions involving nitric oxide (NO) and cell-free hemoglobin (Hb) occur up to 1000 times faster than those with red blood cell (RBC) encapsulated hemoglobin. Thus, cell-free Hb resulting from hemolysis or infusion upsets vascular homeostasis and contributes to pathological conditions in hemolytic anemias such as sickle cell disease and paroxysmal nocturnal hemoglobinuria. The slow NO uptake by RBCs results either from internal factors such as a physical barrier set up by the RBC membrane or external factors such as the time required for NO to diffuse to the cell. We have conducted experiments in which both external and internal factors play a role in limiting NO uptake wherein excess NO is rapidly mixed with RBCs and NO uptake is monitored by absorption spectroscopy. In addition, we have measured NO uptake under conditions where the role of the external factors are reduced by having cell-free and RBC encapsulated Hb compete for limited amounts of slowly released NO, measuring the reaction products using electron paramagnetic resonance spectroscopy. We are the first to have conducted these types of measurements at physiologically relevant hematocrits. Our major findings are that (1) the relative rate that deoxygenated red blood cells scavenge nitric oxide is 3–8 times faster than oxygenated red cells; surprisingly, this effect appears to be partially attributable to a physical membrane barrier, and (2) the rate of NO scavenging by oxygenated RBCs increases significantly (about 3 fold) when the hematocrit is raised from 15% to 50%. Our biophysical data support the thesis that cell-free Hb is capable of greatly reducing the bioavailability of NO and thereby negatively effect hemodynamics and endothelial function. The hematocrit dependence of NO scavenging by intact erythrocytes provides a potential pathogenic mechanism for hypertension during erythropoietin therapy, mortality associated with eNOS inhibition in polycythemic mice, and endothelial dysfunction in acquired polycythemic conditions. The observed oxygen dependence of NO uptake rate supports models in which the submembrane red cell protein scaffolding contributes to NO homeostasis, but not to the extent that has been previously proposed by others. Modulation of the effects of the scaffolding on scavenging and export of NO activity by RBCs under normoxic and hypoxic conditions requires further investigation.