Mechanism of the inhibition of neuronal nitric oxide synthase by 1-(2-trifluoromethylphenyl) imidazole (TRIM).

We have previously reported that 1-(2-trifluoromethylphenyl) imidazole (TRIM) is a potent inhibitor of mouse cerebellar neuronal NOS (nNOS) in vitro with very much reduced activity against bovine aortic endothelial NOS (eNOS). Using purified rat brain nNOS as enzyme source we have now probed the mechanism of action of TRIM. nNOS activity was linear over the first 5 min incubation. Optimal enzyme activity occurred in the presence of NADPH (0.5 mM), calcium chloride (75 microM), tetrahydrobiopterin (12 microM) and calmodulin (10 microg/ml) as cofactors. TRIM was a poor inhibitor of nNOS (IC50, 462.0 microM) compared with L-N(G) nitro arginine (L-NOARG, IC50, 0.32 microM). Removal of tetrahydrobiopterin (but not calmodulin) from the incubation medium greatly enhanced the nNOS inhibitory activity of TRIM (IC50, 32.0 microM) but not L-NOARG (IC50, 0.34 microM). In the absence of added tetrahydrobiopterin, TRIM competed with L-arginine for the substrate binding site on the nNOS enzyme with a Ki value of 47.3 microM. The present experiments suggest that TRIM interferes with the binding of both L-arginine and tetrahydrobiopterin to their respective sites on the nNOS enzyme.