Cytochrome P-450 cholesterol 7α-hydroxylase: Inhibition of enzyme deactivation by structurally diverse calmodulin antagonists and phosphatase inhibitors

Abstract

Cytochrome P-450 cholesterol 7α-hydroxylase ( P-450 ch7 α ) catalyzes the first and ratelimiting step in the conversion of cholesterol to bile acids. Incubation of rat liver microsomes in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer resulted in a timedependent deactivation of P-450 ch7 α which was markedly accelerated by the nonionic detergent Tween 80. Microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450-dependent 7-ethoxycoumarin 0-deethylase activities were unaffected under these conditions, evidencing the selectivity of the deactivation process for P-450 ch7 α . The rate ( t 1 2 = 15–19 min at 37 °C ) and maximal extent of P-450 ch7 α deactivation (⩾-90%) were both unaffected by the presence of cytosolic proteins and were also not dependent on the initial enzyme level, as shown using liver microsomes isolated from untreated, cholestyramine-fed, and xenobiotic-induced rats exhibiting an eight-fold range in P-450 ch7 α activity. Scavengers for reduced oxygen species were also without effect. P-450 ch7 α was stabilized some six- to sevenfold ( t 1 2 = 94–143 min ) by the phosphatase inhibitor NaF. of a series of other phosphatase inhibitors examined, including, among others, EDTA, vanadate, and molybdate, only phosphate-containing compounds and the calmodulin antagonist trifluoperazine, an inhibitor of the Ca 2+-calmodulin-dependent phosphatase calcineurin, effectively stabilized P-450 ch7 α . Modulation of P-450 ch7 α deactivation by these inhibitors generally paralleled their effects on isolated calcineurin. A variety of structurally diverse calmodulin antagonists examined were also found to effectively protect P-450 ch7 α from deactivation; these include calmidazolium and tamoxifen (IC 50 = 25 to 50 μ m), chlorpromazine, thioridazine, amitriptyline, imipramine, and the naphthalene sulfonamide compound W-7 (IC 50 = 50 to 300 μ m). Structure-activity analysis of several phenothiazines and their derivatives indicated that although little activity was exhibited by the sulfoxides, some protection was provided by the corresponding sulfones. On the basis of these observations, various models for the molecular basis of enzyme deactivation are considered, including the hypothesis that a calcineurin-like microsomal phosphatase mediates deactivation of this cytochrome P-450 enzyme.