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Regulation of de Novo Synthesis of Cytochrome P‐450 17α in Mouse LeydigCell Cultures a
Author(s) -
HALES DALE B.,
PAYNE ANITA H.
Publication year - 1987
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.1987.tb25049.x
Subject(s) - library science , obstetrics and gynaecology , chemistry , medicine , political science , sociology , biology , computer science , pregnancy , genetics
Studies from this laboratory have shown that chronic (long term) treatment of mouse Leydig cell cultures with LH, or its intracellular second messenger, CAMP, causes a time-dependent increase in l7a-hydroxylase activity.l This CAMP-induced increase in P-45017, enzyme activity is enhanced by aminoglutethimide (AG), an inhibitor of cholesterol metabolism. The AG enhancement of CAMP-induced l7a-hydroxylase activity can be reversed by supplying exogenous testosterone (T) to the cAMP + AG-treated cultures.2 This finding suggests that testosterone produced during the cAMP induction of P-45017, activity negatively regulates the extent of this induction. The present study was designed to examine whether the CAMP-induced increase and testosterone-mediated decrease in P-45OI7, activity are due to changes in the total amount of specific enzyme protein, changes in the rate of de nouo synthesis of P-45OI7,, or activation or inactivation of preexisting enzyme protein. Purified Leydig cells were maintained in culture for 7 days prior to the initiation of treatment with 0.05 mM 8-Br-cAMP, 0.5 mM AG, cAMP + AG, or cAMP + AG + 5 p M T. To determine de nouo synthesis of P-45017,, cultures were incubated for 3 h in medium containing [35S]methionine. Immunoprecipitable P-45OI7, was separated by SDS-gel electrophoresis and visualized by fluorography. [35Slmethionine P-45OI7, was quantitated by laser densitometry. The total amount of P-45017, was determined by immunoblotting (Western blotting); SDS-gel resolved Leydig cell proteins were transferred to nitrocellulose paper, incubated with anti-P-45017, antibody, and bound antibody was detected with iodinated protein A. The total amount of P-45OI7, was quantitated by laser densitometry. 17a-Hydroxylase activity was determined after a 1 h wash, by measuring the conversion of [3H]progesterone to [3H]steroid products during a 1 h incubation. To determine whether the 8-Br-CAMP-stimulated induction of l7a-hydroxylase activity was mediated by an increase in the rate of de nouo synthesis of P450170, the rate of incorporation of ["Slmethionine into newly synthesized enzyme protein was measured over a 4-day period. The results shown in FIGURE 1 demonstrate that treatment of Leydig cells with 8-Br-CAMP resulted in the induction of de nouo synthesis of P-45OI7,. The rate of synthesis approximately doubled when cells were treated with 8-Br-CAMP + AG. To test if the AG-enhancement of