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Induction of cytochrome P-450 by pregnenolone-16 alpha-carbonitrile in primary monolayer cultures of adult rat hepatocytes and in a cell-free translation system.

N. Elshourbagy,J. Barwick,P. Guzelian

Published 1981 in Journal of Biological Chemistry

ABSTRACT

To investigate whether primary cultures of adult rat hepatocytes retain the capacity to respond to inducers of cytochrome P-450 other than polycyclic aromatic compounds, we tested the effects of pregnenolone-16acarbonitrile (PCN) which induces a novel form of the hemoprotein (PCN cytochrome P-450) i n rat liver (Elshourbagy, N. A., and Guzelian, P. S. (1980) J. EioL Chem, 255,1279-1286). Cultured hepatocytes incubated for 72 h in serum-free medium containing IO-‘ M PCN exhibited increases in the concentration of cytochrome P-450 (1.5-fold) and in the activities of ethylmorphine (4-fold) and aminopyrine (2-fold) N-demethylation as compared to values in incubated control cultures. The cytochrome P-450 induced in culture by PCN matched the PCN cytochrome P-450 purified from PCN-treated rats in the sensitivity of its catalytic activity to formspecific anti-PCN cytochrome P-450 I& in being imm~oprecipitable with this antibody and in its mobility on polyacrylamide gels (51,000 daltons). Pulse-labeling cells with C3H]leucine and isolation of PCN cytochrome P-450 from solublized cell lysates by immunoprecipitation revealed a striking increase in the relative rate of synthesis of the cytochrome protein 48 (9-fold) and 72 h (30-fold) after PCN was added to the medium of 24-h-old cultures. Moreover, RNA extracted from such PCN-treated cultures and translated in a cell-free system contained at least 6 to 8 times more mRNA activity directing synthesis of immunoprecipitable PCN cytochrome P-450 than did RNA extracted from control cultures. As judged from pulse-chase experiments, PCN cytochrome P-450 protein is degraded rapidly, at a rate 5 times faster than that of total cellular protein. In summary, our results demonstrate unequivoca~y that mature hepatocytes in maintenance culture retain the capacity to carry out inducible, de m u 0 synthesis of a form of cytochrome P-450 found in adult liver.

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