Inactivation of nitric oxide synthase isoforms by diaminoguanidine and N-G-amino-L-arginine

Diaminoguanidine (DAG) and N-G-amino-L-arginine each produced a time- and concentration-dependent inactivation of the citrulline-forming activity of all three NOS isoforms. DAG inactivates both the NADPH-oxidase and the citrulline-forming activities of GH(3) pituitary nNOS while N-G-amino-L-arginine inactivates only its citrulline-forming activity. The inactivation by DAG of GH(3) nNOS NADPH-oxidase and citrullineforming activities is stimulated by (6R)-5,6,7,8-tetrahydrobiopterin (BH4) cofactor, follows pseudo-first-order kinetics and is not substrate saturable. DAG-induced inactivation of the citrulline-forming activity for the iNOS and eNOS isoforms displayed maximal inactivation rates of 0.37 and 0.14 min(-1) and K-i values of 385 and 670 mu M, respectively. At 1 mM DAG and saturating BH4, half-times of inactivation of 0.7, 8, and 2 min were observed for the nNOS, eNOS, and iNOS isoforms, respectively. N-G-Amino-L-arginine-induced inactivation of the citrulline-forming activity of the nNOS, iNOS, and eNOS isoforms displayed maximal inactivation rates of 0.35, 0.26, and 0.53 min(-1) and K-i values of 0.3, 3, and 2.5 mu M, respectively. The inactivation of the NOS activities by both DAG and N-G-amino-L-arginine in preincubations required the presence of oxygen and Ca2+, consistent with an inactivation mechanism that requires active metabolism by NOS. Methylguanidine and 1,1-dimethylguanidine exhibited a reversible inhibition pattern in contrast to all three NOS isoforms. Neither agent exhibited significant isoform selectivity. (C) 1996 Academic Press, Inc.