Table 1.

In vitro inhibition of NOS activities by BN 80933

Inhibitor	Ki, μM
	nNOS	eNOS	iNOS
BN 80933	0.92	111	>300
l-NA	0.21	1.15	12.6

For NOS activity assay, the standard reaction mixture contained 100 mM Hepes (pH 7.4), 2.5 mM CaCl₂, 10 μg/ml calmodulin, 2 mM dithiothreitol, 2 mM NADPH, 10 μM FAD, 10 μM FMN, and 10 μM tetrahydrobiopterin in a final volume of 200 μl, l-[³H]arginine (62.5 nM for nNOS or 100 nM for iNOS and eNOS, specific activity 40–50 Ci/mmol; Amersham), l-arginine as indicated, l-NA or BN 80933 at various concentrations, and 50 μl of the enzyme. Reaction mixtures for iNOS were similar, but CaCl₂ and calmodulin were omitted. Reaction was initiated by addition of the enzyme. After a 15-min incubation at 37°C the reaction was stopped by 2 ml of 20 mM Hepes (pH 5.5)/2 mM EDTA. The reaction mixture was applied to a 1-ml column of Dowex AG50WX-8 (Na⁺ form). l-[³H]citrulline was quantified by liquid scintillation counting of the 2-ml flow-through. K_i values shown are averages of two or three experiments made in duplicate. Duplicate measurements agreed within ±5%.