Determination of brain nitric oxide synthase inhibition in vivo: ex vivo assays of nitric oxide synthase can give incorrect results.
The in vivo potencies of N omega-nitro-L-arginine (L-NA), N omega-monomethyl-L-arginine (L-NMMA) and N omega-iminoethyl-L-ornithine (L-NIO) against brain nitric oxide synthase (NOS) were determined by assessing their ability to inhibit harmaline-induced increases in rat cerebellar cGMP. L-NA, L-NIO and L-NMMA were all able to completely prevent the harmaline-induced increase in cGMP with ID50s of 0.5, 30 and 55 mg/kg, respectively, and with the same order of potency as that seen for inhibition of cerebellar NOS in vitro. The inhibitory effects of low doses of L-NA on cerebellar cGMP were maintained for at least 8 hr. The ID50 of L-NA for inhibition of cerebellar cGMP in vivo was similar to its ID50 for inhibition of cerebellar NOS ex vivo but only when NOS activity was assayed as an initial rate. However, doses of L-NMMA and L-NIO that inhibited harmaline-induced increases in cerebellar cGMP in vivo by 50% failed to inhibit NOS ex vivo. The methyl ester of L-NA, L-NAME, produced substantial inhibition of cerebellar NOS ex vivo when given either orally, intraperitoneally or intravenously but with a slower onset of action than L-NA. These results demonstrate that measurement of NOS activity ex vivo can accurately reflect the degree of inhibition of NOS in vivo with inhibitors that dissociate slowly from the enzyme such as L-NA, but only when the initial rate of NOS activity is measured.