EPR EVIDENCE FOR NITRIC-OXIDE PRODUCTION FROM GUANIDINO NITROGENS OF L-ARGININE IN ANIMAL-TISSUES INVIVO

Administration of Fe2+-citrate complex (50 mg/kg of FeSO4 or FeCl2 plus 250 mg/kg of sodium citrate) subcutaneously in the thigh or Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) intraperitoneally, (i.p.) to mice induced NO formation in the livers in vivo at the rate of 0.2-0.3-mu-g/g wet tissue per 0.5 h. The NO synthesized was specifically trapped with Fe2+-diethyldithiocarbamate complex (FeDETC2), formed from endogenous iron and diethyldithiocarbamate (DETC) administered i.p. 0.5 h before decapitation of the animals. NO bound with this trap resulted in the formation of a paramagnetic mononitrosyl iron complex with DETC (NO-FeDETC2), characterized by an EPR signal at g perpendicular-to = 2.035, g parallel-to = 2.02 with triplet hyperfine structure (HFS) at g perpendicular-to. This allowed quantification of the amount of NO formed in the livers. An inhibitor of enzymatic NO synthesis from L-arginine, N(G)-nitro-L-arginine (NNLA, 50 mg/kg) attenuated the NO synthesis in vivo. L-Arginine (500 mg/kg) reversed this effect. Injection of L-[guanidineimino-N-15(2)]arginine combined with Fe2+-citrate or LPS led to the formation of the EPR signal of NO-FeDETC2 characterized by a doublet HFS at g perpendicular-to, demonstrating that the NO originates from the guanidino nitrogens of L-arginine in vivo.