Nitric Oxide 유래된 mRNA 분해와 S-nitrisylation에 의한 FMO효소계의 억제현상

초록

Objectives: The present work is to investigate the effect of nitric oxide (NO)-donors treated exogenously on the metabolic activities of flavin-containing monooxygenase in cultured primary rat hepatocytes. Background: Overproduction of NO by inducible NO synthase (iNOS) is a common phenomenon in inflammation. At this time, the NO has been implicated as the mediator of decreased catalytic activity and expression of drug-metabolizing enzymes such as cytochrome P450s (CYPs) and flavin-containing monooxygenases (FMOs). Previous work in our laboratory suggests that FMO activities in rat liver treated with LPS are decreased by down-regulation of FMO1 mRNA expression in vivo (Park et al., 1999, Mol Pharmacol). Methods: Rat hepatocytes were isolated from rat livers perfused with collagenase. The cells were then treated for 4 h with NO-donors like SNAP or SIN-1. The FMO1 mRNA expression was compared after pretreatment of actinomycin-D to examine the effect of NO on the mRNA stability. The metabolic FMO activities were determined by ranitidine N-oxidation and thiobenzamide S-oxidation estimated with HPLC and spectrophotometer, respectively, in the absence or presence of DTT, a sulfhydryl-reducing agent. Results: FMO activities determined in hepatocytes treated with NO-donors were significantly suppressed to 20-40% of those in untreated control hepatocytes. However, the expression of FMO1 mRNA was not decreased or slightly decreased. Interestingly, stability of FMO1 mRNA after pretreated with actinomycin-D was decreased significantly by exposure to NO donors. Furthermore, the inhibition of ex vivo and in vitro FMO activities estimated with microsomes exposured to NO donors was reversed completely by addition of DTT. Conclusions: These results suggest that FMO activities in hepatocytes can be decreased both by enhanced unstability of FMO1 mRNA leading to decreased FMO1 expression and by reversible S-nitrosylation of the existing FMO1.