SELECTIVE-INHIBITION OF MITOCHONDRIAL 27-HYDROXYLATION OF BILE-ACID INTERMEDIATES AND 25-HYDROXYLATION OF VITAMIN-D3 BY CYCLOSPORINE-A

It was demonstrated recently that cyclosporin A blocks bile acid synthesis in cultured rat and human hepatocytes by specific inhibition of chenodeoxycholic acid formation. The site of inhibition was found to be the 27-hydroxylation of cholesterol catalysed by the liver mitochondrial 27-hydroxylase [Princen, Meijer, Wolthers, Vonk and Kuipers (1991) Biochem J. 275, 501-505]. In this paper the mechanism by which cyclosporin A blocks mitochondrial 27-hydroxylation was further investigated. It is shown that cyclosporin A inhibited 27-hydroxylation of bile acid intermediates, depending on their polarity. In isolated rat liver mitochondria, 27-hydroxylation of cholesterol was dose-dependently blocked by the drug, giving half-maximal inhibition at 4 muM, whereas 27-hydroxylation of 5 beta-cholestane-3alpha,7alpha,12alpha-triol was not affected. A similar observation was made using electrophoretically homogeneous cytochrome P-450(27) isolated from rabbit liver mitochondria, excluding the possibility that cyclosporin A interfered with transport of substrates into the mitochondrion. Kinetic studies showed that inhibition of the 27-hydroxylation of cholesterol by cyclosporin A was of a noncompetitive type. The drug also inhibited the 25-hydroxylase activity towards vitamin D3, catalysed by the same enzyme preparation, to the same extent as 27-hydroxylation of cholesterol. These results suggest that cyclosporin A may interfere with binding of cholesterol, but not of 5beta-cholestane-3alpha,7alpha,12alpha-triol, to the active site of the enzyme. These data provide an explanation for the selective inhibition of chenodeoxycholic acid synthesis.