Postnatal changes in the expression and distribution of pulmonary cytochrome P450 monooxygenases during Clara cell differentiation in rabbits

Charles Plopper, A. J. Weir, D. Morin, A. Chang, R. M. Philpot, Alan R Buckpitt

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Abstract

Previous studies have indicated that both cytodifferentiation of Clara cells and the onset of pulmonary cytochrome P450 activity are postnatal events. However, the relationship between these two events during lung development remains poorly understood. To determine how these events interrelate, we examined rabbit Clara cells during postnatal differentiation, with the following goals in mind: 1) to identify the patterns of intracellular expression of cytochrome P450 monooxygenase isozymes 2B and 4B and cytochrome P450 reductase, 2) to describe the biogenesis of the organelles with which these isozymes are associated, namely smooth and rough endoplasmic reticulum, and 3) to compare the patterns of expression with cytochrome P450 activity in the whole lung over the same period. Lungs of rabbits ranging in age from 24 days gestational age (DGA) to 25 weeks postnatally were studied. Ultrastructural morphometry showed that smooth endoplasmic reticulum averaged <5% of the Clara cell volume in late gestational (24-30 DGA) and neonatal rabbits [0-7 days postnatally (DPN)], grew to 20-30% of the cell volume in 14-21-DPN animals, and approximated adult levels (>40%) in 28-DPN rabbits. In contrast, rough endoplasmic reticulum decreased from >10% of the cell volume at 27 DGA to <5% in adults. All postnatal animals showed considerable heterogeneity in the abundance of smooth endoplasmic reticulum among individual cells, Immunohistochemistry revealed that cytochrome P450 reductase appeared in Clara cells earlier (28 DGA) than did either isozyme 2B or 4B (1 DPN). Each antigen was detected first in the apical borders of the cells, then throughout the cytoplasm in a few cells by 7 DPN, and finally in adult abundance by 28 DPN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that cytochrome P450 protein concentrations increased postnatally. Cytochrome P450 heme protein was not detected spectrophotometrically in the lungs of animals younger than 3 DPN but increased to approximately 70% of adult levels by 28 DPN. Likewise, cytochrome P450 activity (measured as ethoxy- and pentoxyresorufin O-dealkylation) was not detected in animals younger than 2 DPN but increased to approximately 75% of adult levels by 28 DPN. We conclude that, in rabbits, 1) pulmonary cytochrome P450 monooxygenase activity begins in the perinatal period and attains adult levels after 28 DPN, 2) cytochrome P450 protein expression appears and differentiates postnatally in Clara cells, 3) the appearance of smooth endoplasmic reticulum and the expression of cytochrome P450 protein do not occur uniformly in differentiating Clara cells even in the same bronchiole, and 4) the biogenesis of endoplasmic reticulum precedes the expression of cytochrome P450 protein in differentiating Clara cells.

Original languageEnglish (US)
Pages (from-to)51-61
Number of pages11
JournalMolecular Pharmacology
Volume44
Issue number1
StatePublished - Jul 1993

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Mixed Function Oxygenases
Cytochrome P-450 Enzyme System
Cell Differentiation
NAD
Rabbits
Lung
Smooth Endoplasmic Reticulum
Isoenzymes
Gestational Age
NADPH-Ferrihemoprotein Reductase
Rough Endoplasmic Reticulum
Proteins
Dealkylation
Hemeproteins
Bronchioles
Organelle Biogenesis
Cell Size
Sodium Dodecyl Sulfate
Endoplasmic Reticulum
Polyacrylamide Gel Electrophoresis

ASJC Scopus subject areas

  • Pharmacology

Cite this

Postnatal changes in the expression and distribution of pulmonary cytochrome P450 monooxygenases during Clara cell differentiation in rabbits. / Plopper, Charles; Weir, A. J.; Morin, D.; Chang, A.; Philpot, R. M.; Buckpitt, Alan R.

In: Molecular Pharmacology, Vol. 44, No. 1, 07.1993, p. 51-61.

Research output: Contribution to journalArticle

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abstract = "Previous studies have indicated that both cytodifferentiation of Clara cells and the onset of pulmonary cytochrome P450 activity are postnatal events. However, the relationship between these two events during lung development remains poorly understood. To determine how these events interrelate, we examined rabbit Clara cells during postnatal differentiation, with the following goals in mind: 1) to identify the patterns of intracellular expression of cytochrome P450 monooxygenase isozymes 2B and 4B and cytochrome P450 reductase, 2) to describe the biogenesis of the organelles with which these isozymes are associated, namely smooth and rough endoplasmic reticulum, and 3) to compare the patterns of expression with cytochrome P450 activity in the whole lung over the same period. Lungs of rabbits ranging in age from 24 days gestational age (DGA) to 25 weeks postnatally were studied. Ultrastructural morphometry showed that smooth endoplasmic reticulum averaged <5{\%} of the Clara cell volume in late gestational (24-30 DGA) and neonatal rabbits [0-7 days postnatally (DPN)], grew to 20-30{\%} of the cell volume in 14-21-DPN animals, and approximated adult levels (>40{\%}) in 28-DPN rabbits. In contrast, rough endoplasmic reticulum decreased from >10{\%} of the cell volume at 27 DGA to <5{\%} in adults. All postnatal animals showed considerable heterogeneity in the abundance of smooth endoplasmic reticulum among individual cells, Immunohistochemistry revealed that cytochrome P450 reductase appeared in Clara cells earlier (28 DGA) than did either isozyme 2B or 4B (1 DPN). Each antigen was detected first in the apical borders of the cells, then throughout the cytoplasm in a few cells by 7 DPN, and finally in adult abundance by 28 DPN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that cytochrome P450 protein concentrations increased postnatally. Cytochrome P450 heme protein was not detected spectrophotometrically in the lungs of animals younger than 3 DPN but increased to approximately 70{\%} of adult levels by 28 DPN. Likewise, cytochrome P450 activity (measured as ethoxy- and pentoxyresorufin O-dealkylation) was not detected in animals younger than 2 DPN but increased to approximately 75{\%} of adult levels by 28 DPN. We conclude that, in rabbits, 1) pulmonary cytochrome P450 monooxygenase activity begins in the perinatal period and attains adult levels after 28 DPN, 2) cytochrome P450 protein expression appears and differentiates postnatally in Clara cells, 3) the appearance of smooth endoplasmic reticulum and the expression of cytochrome P450 protein do not occur uniformly in differentiating Clara cells even in the same bronchiole, and 4) the biogenesis of endoplasmic reticulum precedes the expression of cytochrome P450 protein in differentiating Clara cells.",
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N2 - Previous studies have indicated that both cytodifferentiation of Clara cells and the onset of pulmonary cytochrome P450 activity are postnatal events. However, the relationship between these two events during lung development remains poorly understood. To determine how these events interrelate, we examined rabbit Clara cells during postnatal differentiation, with the following goals in mind: 1) to identify the patterns of intracellular expression of cytochrome P450 monooxygenase isozymes 2B and 4B and cytochrome P450 reductase, 2) to describe the biogenesis of the organelles with which these isozymes are associated, namely smooth and rough endoplasmic reticulum, and 3) to compare the patterns of expression with cytochrome P450 activity in the whole lung over the same period. Lungs of rabbits ranging in age from 24 days gestational age (DGA) to 25 weeks postnatally were studied. Ultrastructural morphometry showed that smooth endoplasmic reticulum averaged <5% of the Clara cell volume in late gestational (24-30 DGA) and neonatal rabbits [0-7 days postnatally (DPN)], grew to 20-30% of the cell volume in 14-21-DPN animals, and approximated adult levels (>40%) in 28-DPN rabbits. In contrast, rough endoplasmic reticulum decreased from >10% of the cell volume at 27 DGA to <5% in adults. All postnatal animals showed considerable heterogeneity in the abundance of smooth endoplasmic reticulum among individual cells, Immunohistochemistry revealed that cytochrome P450 reductase appeared in Clara cells earlier (28 DGA) than did either isozyme 2B or 4B (1 DPN). Each antigen was detected first in the apical borders of the cells, then throughout the cytoplasm in a few cells by 7 DPN, and finally in adult abundance by 28 DPN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that cytochrome P450 protein concentrations increased postnatally. Cytochrome P450 heme protein was not detected spectrophotometrically in the lungs of animals younger than 3 DPN but increased to approximately 70% of adult levels by 28 DPN. Likewise, cytochrome P450 activity (measured as ethoxy- and pentoxyresorufin O-dealkylation) was not detected in animals younger than 2 DPN but increased to approximately 75% of adult levels by 28 DPN. We conclude that, in rabbits, 1) pulmonary cytochrome P450 monooxygenase activity begins in the perinatal period and attains adult levels after 28 DPN, 2) cytochrome P450 protein expression appears and differentiates postnatally in Clara cells, 3) the appearance of smooth endoplasmic reticulum and the expression of cytochrome P450 protein do not occur uniformly in differentiating Clara cells even in the same bronchiole, and 4) the biogenesis of endoplasmic reticulum precedes the expression of cytochrome P450 protein in differentiating Clara cells.

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