Normal circulating adult human red blood cells contain inactive NOS proteins

Human red blood cells (RBCs) are considered to play a significant role in b oth blood pressure (BP) regulation and tissue oxygenation, because they can bind as well as release previously bound nitric oxide (NO) from hemoglobin (Hb) and other intracellular components. Two reports indicate that the hum an RBC possesses nitric oxide synthase (NOS) activity-by the accumulation o f nitrite across a membraned chamber in one and by the hydrolysis of labele d L-arginine, presumably to labeled L-citrulline, in the other. Furthermore , NOS proteins have been identified by immunoblot In RBCs, If true, the pre sence of NOS activity would convert the human RBC to a donor with limited a bility to bind exogenously generated NO. In considering the importance of t he question of the presence or not of NO synthetic capacity of this cell in BP regulation and tissue perfusion, whether human RBCs are, indeed, able t o hydrolyze L-arginine to L-citrulline, the coproduct of NO was explored, R BC samples collected from control subjects were assayed for NOS activity by incubation of homogenized cellular fractions with labeled tritiated L-argi nine in the presence of 0.5 mmol/L NADPH. By this method, the amino acid co product of NO, tritiated L-citrulline, would be recovered in the supernatan t after removal of unused substrate by cationic resin treatment. At first, activity appeared to be present in the RBC supernatant but not In the pelle t. However, activity was not suppressed by known inhibitors of NOS, whereas activity was suppressed by norvaline, an inhibitor of arginase activity wi th no known effect on NOS. By contrast, RBC arginase activity was not inhib ited by N-omega-nitro-L-arginine, N-G-mgthyl-L-arginine, or aminoguanidine, known inhibitors of NOS, but was inhibited by norvaline. The label recover ed by thin-layer chromatography was determined not to be tritiated L-citrul line but was instead tritiated L-ornithine, the product of arginase activit y. Thus the enzymatic hydrolysis of L-arginine was not caused by NOS but wa s a result of the action of the enzyme arginase, which abounds in this cell . However, proteins interacting with antibodies to the endothelial and indu cible isoforms of NOS were detected in human RBCs by immunoblot. Together, these findings indicate that human RBCs collected from normal adult individ uals possess proteins that react with monoclonal antibodies to the inducibl e and endothelial isoforms of NOS, but the proteins are without catalytic a ctivity.