RT Journal Article
SR Electronic
T1 Role of the steroid 17 alpha-hydroxylase in spironolactone-mediated destruction of adrenal cytochrome P-450.
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 321
OP 325
VO 40
IS 2
A1 D C Kossor
A1 S Kominami
A1 S Takemori
A1 H D Colby
YR 1991
UL http://molpharm.aspetjournals.org/content/40/2/321.abstract
AB Previous investigations have established that spironolactone (SL) is converted to a reactive metabolite by adrenal microsomal enzymes, resulting in the degradation of cytochrome P-450 (P-450). Deacetylation of SL to 7 alpha-thiospironolactone (7 alpha-thio-SL) is the first step in the activation pathway, but further NADPH-dependent metabolism of 7 alpha-thio-SL is required for P-450 destruction. Studies were done to evaluate the role of the steroid 17 alpha-hydroxylase in the activation of 7 alpha-thio-SL by adrenal microsomes. Incubation of guinea pig adrenal microsomes with 7 alpha-thio-SL in the presence of NADPH effected greater than 50% declines in P-450 content and in 17 alpha-hydroxylase activity but no change in the rate of 21-hydroxylation. Preincubation of the microsomes with antisera to the 17 alpha-hydroxylase P-450 isozyme (P-450(17 alpha,lyase)) decreased 17 alpha-hydroxylase but not 21-hydroxylase activity and prevented the degradation of P-450 by 7 alpha-thio-SL. Control IgG had no effect on 17 alpha-hydroxylase activity or on the 7 alpha-thio-SL-mediated destruction of P-450. When added to a purified P-450(17 alpha,lyase) preparation, 7 alpha-thio-SL and the endogenous substrate progesterone caused typical type I spectral changes, but SL did not. Incubation of a purified and reconstituted 17 alpha-hydroxylase system, consisting of P-450(17 alpha,lyase), NADPH-P-450 reductase, cytochrome b5, and dilauroylphosphatidylcholine, with 7 alpha-thio-SL plus NADPH effected the complete degradation of the P-450(17 alpha,lyase). Neither progesterone nor SL caused P-450 destruction with the reconstituted enzyme preparation. The results provide direct evidence for the activation of 7 alpha-thio-SL by the 17 alpha-hydroxylase and support the hypothesis that a mechanism-based inhibition of the enzyme occurs. The data also provide additional evidence that 7 alpha-thio-SL is an obligatory intermediate in the degradation of P-450 by SL.