Characterization of the cytochrome P-450 monooxygenase system in nonciliated bronchiolar epithelial (Clara) cells isolated from mouse lung.

C. H. Chichester, R. M. Philpot, A. J. Weir, Alan R Buckpitt, Charles Plopper

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Abstract

The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Original languageEnglish (US)
Pages (from-to)179-186
Number of pages8
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume4
Issue number2
StatePublished - Feb 1991

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Cytochrome P-450 Enzyme System
Epithelial Cells
Lung
Isoenzymes
Antibodies
Benzphetamine
Chemical activation
Quinones
NADPH-Ferrihemoprotein Reductase
Mitochondria
Epoxidation
Immunohistochemistry
Benzo(a)pyrene
High performance liquid chromatography
Xenobiotics
Rabbits
Metabolites
Electrophoresis
Smooth Endoplasmic Reticulum
Metabolism

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Pulmonary and Respiratory Medicine

Cite this

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title = "Characterization of the cytochrome P-450 monooxygenase system in nonciliated bronchiolar epithelial (Clara) cells isolated from mouse lung.",
abstract = "The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80{\%} purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)",
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T1 - Characterization of the cytochrome P-450 monooxygenase system in nonciliated bronchiolar epithelial (Clara) cells isolated from mouse lung.

AU - Chichester, C. H.

AU - Philpot, R. M.

AU - Weir, A. J.

AU - Buckpitt, Alan R

AU - Plopper, Charles

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