Inhibition of nitric oxide synthesis by NG‐nitro‐L‐arginine methyl ester (L‐NAME): requirement for bioactivation to the free acid, NG‐nitro‐L‐arginine

1 The L‐arginine derivatives NG‐nitro‐L‐arginine (L‐NOARG) and NG‐nitro‐L‐arginine methyl ester (L‐NAME) have been widely used to inhibit constitutive NO synthase (NOS) in different biological systems. This work 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 μmol) increased coronary perfusion pressure of rat isolated hearts to the same extent (21 ± 0.8 mmHg; n = 5), but the effect developed more rapidly 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 was paralleled by the appearance of L‐NOARG in the coronary effluent. 3 Freshly dissolved L‐NAME was a 50 fold less potent inhibitor of purified brain NOS (mean IC50 = 70 μm) than L‐NOARG (IC50 = 1.4 μm), but the apparent inhibitory potency of L‐NAME approached that of L‐NOARG upon prolonged incubation at neutral or alkaline pH. H.p.l.c. analyses revealed that NOS inhibition by L‐NAME closely correlated with hydrolysis 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 buffer (pH 7.4), 207 ± 1.7 min in human plasma, and 29 ± 2.2 min in whole blood (n = 3 in each case). When L‐NAME was preincubated in plasma or 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 in blood cells. 5 These results suggest that L‐NAME represents a prodrug lacking NOS inhibitory activity unless it is hydrolyzed to L‐NOARG. Bioactivation of L‐NAME proceeds at moderate rates in physiological buffers, but is markedly accelerated in tissues such as blood or vascular endothelium.