Role of the steroid 17 alpha-hydroxylase in spironolactone-mediated destruction of adrenal cytochrome P-450.
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.
Kossor
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DC
Department of Pharmacology and Toxicology, Philadelphia College of Pharmacy and Science, Pennsylvania 19104.
Kominami
S
S
Takemori
S
S
Colby
H D
HD
D000311:Adrenal Glands / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006168:Guinea Pigs; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009249:NADP / Q000494:pharmacology; D013148:Spironolactone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D013254:Steroid 17-alpha-Hydroxylase / Q000502:physiology*
D013148:Spironolactone; C033554:7 alpha-thiospironolactone; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D013254:Steroid 17-alpha-Hydroxylase