INHIBITION OF NITRIC-OXIDE SYNTHESIS BY N-G-NITRO-L-ARGININE METHYL-ESTER (L-NAME) - REQUIREMENT FOR BIOACTIVATION TO THE FREE ACID, N-G-NITRO-L-ARGININE

1 The L-arginine derivatives N-G-nitro-L-arginine (L-NOARG) and NG-nit ro-L-arginine methyl ester (L-NAME) have been widely used to inhibit c onstitutive NO synthase (NOS) in different biological systems. This wo rk was carried out to investigate whether L-NAME is a direct inhibitor of NOS or requires preceding hydrolytic bioactivation to L-NOARG for inhibition of the enzyme. 2 A bolus of L-NAME and L-NOARG (0.25 mu mol ) increased coronary perfusion pressure of rat isolated hearts to the same extent (21+/-0.8 mmHg; n=5), but the effect developed more rapidl y following addition of L-NOARG than L-NAME (mean half-time: 0.7 vs. 4 .2 min). The time-dependent onset of the inhibitory effect of L-NAME w as paralleled by the appearance of L-NOARG in the coronary effluent. 3 Freshly dissolved L-NAME was a 50 fold less potent inhibitor of purif ied brain NOS (mean IC50 = 70 mu M) than L-NOARG (IC50 = 1.4 mu M), bu t the apparent inhibitory potency of L-NAME approached that of L-NOARG upon prolonged incubation at neutral or alkaline pH. H.p.l.c. analyse s revealed that NOS inhibition by L-NAME closely correlated with hydro lysis of the drug to L-NOARG. 4 Freshly dissolved L-NAME contained 2% of L-NOARG and was hydrolyzed with a half-life of 365+/-11.2 min in bu ffer (pH 7.4), 207+/-1.7 min in human plasma, and 29+/-2.2 min in whol e blood (n = 3 in each case). When L-NAME was preincubated in plasma o r buffer, inhibition of NOS was proportional to formation of L-NOARG, but in blood the inhibition was much less than expected from the rates of L-NAME hydrolysis. This was explained by accumulation of L-NOARG i n blood cells. 5 These results suggest that L-NAME represents a prodru g lacking NOS inhibitory activity unless it is hydrolyzed to L-NOARG. Bioactivation of L-NAME proceeds at moderate rates in physiological bu ffers, but is markedly accelerated in tissues such as blood or vascula r endothelium.