Effects of hydroxyurea and L-arginine on the production of nitric oxide metabolites in cultures of normal and sickle erythrocytes

Abstract

Previous in vitro studies suggest that erythrocytes may be a source of nitric oxide (NO) produced by nitric oxide synthase (NOS) or by oxyhemoglobin-mediated oxidation of hydroxyurea (HU). This study was performed to determine the roles of HU and NOS in the production of NO by normal and sickle erythrocytes. Red blood cells (RBCs) from normal adult hemoglobin (HbAA) and homozygous sickle cell subjects (HbSS) were incubated with PBS containing 0.2 mM hydrogen peroxide (control) for 2 h at 37 degrees C in the presence and absence of l-arginine, the substrate for NOS, and with l-arginine plus HU in the presence and absence of l-NMMA, a specific inhibitor of NOS. The nitrate and nitrite metabolites of NO, expressed as [NOx], were measured. [NOx] in the HbAA and HbSS RBC cultures was not significantly different in the presence and absence of 1.0 mM l-arginine (p>0.1). [NOx] in the HbAA and HbSS cultures treated with a clinically relevant dose of HU (1.0 mM) plus 1.0 mM l-arginine was significantly greater than that in controls incubated with PBS and with l-arginine p < 0.01. However, [NOx] in the HbAA and HbSS cultures treated with 50 microg/ml l-NMMA was not significantly different than that in the cultures treated with HU plus l-arginine in the absence of l-NMMA. These findings suggest that NOx production by erythrocytes may be increased by treatment with HU and may not be decreased by inhibiting NOS. Therefore, we conclude that a therapeutic dose of HU may increase the plasma concentration of NO by a mechanism that does not require erythrocytes NOS activity.