Evidence that ligand formation is a mechanism underlying the maintenance of cytochrome <i>P</i>-450 in rat liver cell culture. Potent maintenance by metyrapone

Paine, Alan J.; Villa, Pia; Hockin, Lesley J.

doi:10.1042/bj1880937

Cited by 48 publications

(5 citation statements)

References 9 publications

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“…Rat hepatocytes cultured for 24 h lose 70% of their cytochrome P450 [31,32], while in bovine adrenal zona glomerulosa cells the loss of capacity to synthesize aldosterone accompanies the decline in cytochrome P450 following isolation [33,34]. This decrease can be prevented by supplementing the culture medium with a variety of nutrients, growth factors and hormones [35,36], or by the incorporation of hyperphysiological concentrations of nicotinamide or pyridine derivatives (metapyrone) in the culture medium [37,38].…”

Section: Cell Isolationmentioning

confidence: 99%

Primary culture, cellular stress and differentiated function

Wolffe

Tata

1984

FEBS Letters

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Isolation of specialized cell types for the analysis of tissue‐specific gene function often results in loss of the differentiated phenotype. Examples of this type of phenotypic change following tissue disaggregation are reviewed together with possible explanations. Close similarities between the effects of cell isolation with those of other cellular stresses such as heat or anoxia point to common biochemical mechanisms being involved. This suggests that the study of freshly isolated cells will contribute significantly to out understanding of the nature of cellular stress and its consequences for the maintenance of phenotype and induction of tissue specific gene expression.

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Section: Cell Isolationmentioning

confidence: 99%

Primary culture, cellular stress and differentiated function

Wolffe

Tata

1984

FEBS Letters

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“…However, when rat hepatocytes were cultured with metyrapone, not only was the spontaneous loss of cytochrome P-450 prevented, but the activity of drug-metabolizing enzymes generally considered to be associated with the PBinducible cytochrome P45011B subfamily was maintained for 72 h at the same level as found in the intact liver from which the cultures were prepared. In contrast, those activities associated with the polycyclic-hydrocarbon-inducible P4501A subfamily were mark-edly induced [3,4]. In order to rationalize these observations and to document further the response of cultured hepatocytes to inducers, the present work employing polyclonal antibodies to quantify the effect of treatment with metyrapone and the archetypal inducers upon their content of P450IA and P45011B subfamilies was undertaken.…”

Section: Introductionmentioning

confidence: 99%

Immunochemical quantification of cytochrome P450IA and IIB subfamilies in the livers of metyrapone-treated rats. Relevance to the ability of metyrapone to prevent the loss of cytochrome P-450 in rat hepatocyte culture

Shean

Paine

1990

Biochemical Journal

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Polyclonal antibodies to the major beta-naphthoflavone (BNF)-inducible form of cytochrome P-450 (P450IA) and to the major phenobarbitone (PB)-inducible form (P450IIB) have been used to quantify the contribution of these subfamilies to the total amount of cytochrome P-450 in rat livers and rat hepatocyte cultures treated with PB, BNF and metyrapone for 24 and 72 h. The P450IA and IIB subfamilies were not detectable (less than 5 pmol/mg of microsomal protein) in the livers of control rats, but administration of BNF resulted in the P450IA subfamily comprising more than 80% of the total hepatic cytochrome P-450. Administration of PB and metyrapone to rats did not elevate the level of this subfamily but elevated the levels of the P450IIB subfamily to 60% and 30% respectively of the total. Thus metyrapone is a 'PB-like' inducer. However, in contrast with their effects in vivo, treatment with PB and metyrapone of rat hepatocytes did not elevate the proportion of the P450IIB subfamily relative to that in untreated cells but rather, like BNF, increased the P450IA subfamily. This would account for the ability of metyrapone to produce in hepatocyte culture, like BNF, a pronounced induction of ethoxyresorufin O-de-ethylase activity, but it does not account for why of all inducers studied only metyrapone can maintain the total cytochrome P-450 content of cultured hepatocytes, or the activity of ethylmorphine N-demethylase. This activity is generally considered to be associated with the P450IIB subfamily, but the lack of effect of metyrapone on this subfamily in hepatocyte culture must suggest that metyrapone is able to prevent the loss of the total amount of the cytochrome by increasing the expression of other cytochromes P-450.

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“…However, since most cultured cells quickly lose their in vivo levels of cytochrome P-450 and monooxygenase activities (27), studies of xenobiotic metabolism in lung at present are limited to the freshly isolated cells. The spontaneous loss of cytochrome P-450 in cultured hepatocytes can be prevented in part by the presence of nicotinamide (28), metyrapone (29) or other substances in the culture medium. These factors are now being investigated in pulmonary cell culture.…”

Section: Cell Isolation Techniquementioning

confidence: 99%

Alveolar Type II and Clara Cells: Isolation and Xenobiotic Metabolism

Devereux¹

1984

Environmental Health Perspectives

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This paper describes one isolation procedure for two pulmonary cell types and discusses how these cells are being used for toxicological studies. Alveolar Type II cells and Clara cells have been isolated from rabbits and rats and separated into highly enriched fractions. The lungs were digested with protease, and the pulmonary cell digests were separated into discrete fractions on the basis of cellular size and density differences. Several studies have been conducted to compare the metabolism of xenobiotics in these cell types and three examples are discussed. The metabolic activation and covalent binding of the pulmonary toxin, 4-ipomeanol was found to occur in both Clara and Type II cells in vitro, although to a much greater extent in the Clara cells. Also, the metabolism of several substrates, including 7-ethoxycoumarin, coumarin and benzo(a)pyrene, was compared in the isolated cell fractions and found to be much greater in the Clara cells than in the Type II cells. Immunohistochemical analysis and gel electrophoresis have also been used to demonstrate the presence of the two major rabbit pulmonary cytochrome P-450 isozymes in both the isolated Clara cells and alveolar Type II cells.

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Evidence that ligand formation is a mechanism underlying the maintenance of cytochrome P-450 in rat liver cell culture. Potent maintenance by metyrapone

Cited by 48 publications

References 9 publications

Primary culture, cellular stress and differentiated function

Primary culture, cellular stress and differentiated function

Immunochemical quantification of cytochrome P450IA and IIB subfamilies in the livers of metyrapone-treated rats. Relevance to the ability of metyrapone to prevent the loss of cytochrome P-450 in rat hepatocyte culture

Alveolar Type II and Clara Cells: Isolation and Xenobiotic Metabolism

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