Interferon-γ-Inducible Murine Macrophage Nitric-Oxide Synthase: Studies on the Mechanism of Inhibition by Imidazole Agents

Abstract

Citrulline formation by the interferon-γ/lipopolysaccharide-inducible murine macrophage nitric oxide synthase is inhibited reversibly by imidazole, 1-phenylimidazole, 4-phenylimidazole, and 2-phenylimidazole with IC50 values of 40 μM, 6 μM, 225 μM and >1 mM, respectively. 1-Phenylimidazole inhibited the maximal velocity of citrulline formation but did not alter the concentration of arginine providing half-maximal activity. 1-Phenylimidazole inhibited citrulline formation by the murine macrophage nitric oxide synthase competitively versus (6R)-5,6,7,8-tetrahydro-L-biopterin (THB) with a Ki value of 0.7 μM, but inhibited citrulline formation by Ca2+-calmodulin-dependent nitric oxide synthase from GH3 pituitary cells noncompetitively versus THB with a Ki value of 40 μM. Imidazole inhibited citrulline formation by the murine macrophage nitric oxide synthase noncompetitively versus THB with a Ki value of 48 μM. Neither imidazole nor 1-phenylimidazoIe inhibited the cytochrome c reductase activity of murine macrophage nitric oxide synthase at concentrations 100-fold higher than their IC50 values for inhibiting citrulline formation. The antifungal imidazoles miconazole, ketoconazole, and clotrimazole did not inhibit either citrulline formation or cytochrome c reduction by murine macrophage nitric oxide synthase at concentration as high as 200 μM. Ca2+-calmodulin-dependent nitric oxide synthase from GH3 pituitary cells exhibited a Kact for THB of 80 nM, while the inducible murine macrophage nitric oxide synthase exhibited a Kact of 8 μM.