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-mono methyl-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 cerebella r cGMP. L-NA, L-NIO and L-NMMA were all able to completely prevent the harmaline-induced increase in cGMP with ID(50)s 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 d oses 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 a ctivity 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 slo wer onset of action than L-NA. These results demonstrate that measurem ent of NOS activity ex vivo can accurately reflect the degree of inhib ition of NOS in vivo with inhibitors that dissociate slowly from the e nzyme such as L-NA, but only when the initial rate of NOS activity is measured.