The Inhibition of the Constitutive Bovine Endothelial Nitric Oxide Synthase by Imidazole and Indazole Agents

Citrulline formation by the Ca2+ CaM-dependent nitric oxide synthase of bovine endothelium is inhibited reversibly by 7-nitroindazole, 1-phenylimidazole, and imidazole. As measured at 0.67 μM (6R)-5,6,7,8-tetrahydrobiopterin (BH4), IC50 values of 0.8, 200, and 50 μM were determined for 7-nitroindazole, 1-phenylimidazole, and imidazole, respectively. Increasing concentrations of added BH4 cofactor increased the IC50 values for 7-nitroindazole and 1-phenylimidazole but did not alter the IC50 value for imidazole. 7-nitroindazole inhibited citrulline formation by the endothelial cNOS noncompetitively versus arginine substrate but competitively versus BH4 with a Ki value of 0.8 μM. 1-Phenylimidazole inhibited citrulline formation by the endothelial cNOS competitively versus both arginine substrate and BH4 with a Ki value of 50 μM. Imidazole inhibited citrulline formation competitively versus arginine substrate but noncompetitively versus BH4 with a Ki value of 50 μM. Neither 7-nitroindazole, 1-phenylimidazole, nor imidazole inhibited the cytochrome c reductase activity of endothelial cNOS at concentrations up to 5000-fold higher than their Ki values for inhibition of citrulline formation. By comparison with the previously determined kinetic properties of the other nitric oxide synthase isoforms, these observations establish that 1-phenylimidazole displays marked specificity for inhibiting the inducible nitric oxide synthase isoform and, since 7-nitroindazole has been reported not to elevate blood pressure (McCall et al., 1991, Br. J. Pharmacol. 102, 234-238), fails to confirm the expected insensitivity of the constitutive endothelial nitric oxide synthase to inhibition by 7-nitroindazole.