scholarly journals Characterization of Clara and type II cells isolated from rat lung by fluorescence-activated flow cytometry

1993 ◽  
Vol 295 (1) ◽  
pp. 73-80 ◽  
Author(s):  
J Martin ◽  
D Dinsdale ◽  
I N H White
Keyword(s):  
Flow Cytometry ◽  
Type Ii Cells ◽  
Type Ii ◽  
Clara Cells ◽  
Rat Lung ◽  
Trypan Blue Exclusion ◽  
Potent Inducer ◽  
Oxygenase Activity ◽  
Cytochrome P 450

A novel procedure for the isolation of Clara cells from rat lung is described. Single-cell suspensions from male F344/TOX rat lungs, prepared by subtilisin digestion, were treated with monochlorobimane (10 microM) and anlaysed by fluorescence-activated flow cytometry. A sub-population of about 2.8% of the total, showing the highest blue fluorescence and by inference the highest GSH concentration, was isolated as Clara cells of > 95% purity. Type II cells of similar purity were sorted after treatment with Phosphine 3R. Both sub-populations were > 90% viable, as judged by Trypan Blue exclusion. Comparison of CYP (cytochrome P-450) isoenzymes between these subpopulations, using Western blotting, showed CYP1A1 to be barely detectable. In Clara cells, CYP2B1 was 10-fold higher than in Type II cells. Mono-oxygenase activity towards the O-deethylation of 3-cyano-7-ethoxycoumarin was 2-fold higher in Clara cells. No activity was detected in macrophages. Pretreating rats with the mono-oxygenase inducers phenobarbitone, 3-methylcholanthrene or Aroclor 1254 showed the last-named to be the most potent inducer of CYP1A1. In Clara cells, CYP1A1 concentration and mono-oxygenase activities were induced > 2000- and 50-fold respectively, whereas in Type II cells increases of 300- and 3.6-fold were seen. Clara cells isolated from the lungs of control rats had a concentration of GSH (2.7 nmol/10(6) cells) that was 9- and 2-fold higher than that in Type II cells or macrophages respectively. GSH depleted by monochlorobimane treatment was restored after 2-3 h incubation with 0.2 M N-acetylcysteine. gamma-Glutamyltranspeptidase activity in Clara cells was 6- and 50-fold higher than in Type II cells or macrophages respectively.

Localization of a candidate surfactant convertase to type II cells, macrophages, and surfactant subfractions

1999 ◽  
Vol 276 (3) ◽  
pp. L452-L458 ◽  
Author(s):  
Howard Clark ◽  
Lennell Allen ◽  
Erin Collins ◽  
Frederick Barr ◽  
Leland Dobbs ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Alveolar Macrophages ◽  
Pulmonary Surfactant ◽  
Type Ii Cells ◽  
Type Ii ◽  
Clara Cells ◽  
Rat Lung ◽  
Protein Distribution ◽  
Serine Hydrolase ◽  
Surfactant Metabolism

Pulmonary surfactant exists in the alveolus in several distinct subtypes that differ in their morphology, composition, and surface activity. Experiments by others have implicated a serine hydrolase in the production of the inactive small vesicular subtype of surfactant (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222–230, 1990). Our laboratory recently identified this enzyme in the rat as the serine carboxylesterase ES-2 [F. Barr, H. Clark, and S. Hawgood. Am. J. Physiol. 274 ( Lung Cell. Mol. Physiol. 18): L404–L410, 1998]. In the present study, we determined the cellular sites of expression of ES-2 in rat lung using a digoxygenin-labeled ES-2 riboprobe. ES-2 mRNA was localized to type II cells and alveolar macrophages but not to Clara cells. Using a specific ES-2 antibody, we determined the protein distribution of ES-2 in the lung by immunohistochemistry, and it was found to be consistent with the sites of mRNA expression. Most of the ES-2 in rat bronchoalveolar lavage is in the surfactant-depleted supernatant, but ES-2 was also consistently localized to the small vesicular surfactant subfraction presumed to form as a consequence of conversion activity. These results are consistent with a role for endogenous lung ES-2 in surfactant metabolism.

scholarly journals Immunocytochemical localization of the major surfactant apoproteins in type II cells, Clara cells, and alveolar macrophages of rat lung.

1986 ◽  
Vol 34 (9) ◽  
pp. 1137-1148 ◽  
Author(s):  
S R Walker ◽  
M C Williams ◽  
B Benson
Keyword(s):  
Alveolar Macrophages ◽  
Lamellar Body ◽  
Alveolar Type ◽  
Multivesicular Bodies ◽  
Type Ii Cells ◽  
Type Ii ◽  
Clara Cells ◽  
Rat Lung ◽  
Lamellar Bodies ◽  
Thin Sections

The adsorptive properties of phospholipids of pulmonary surfactant are markedly influenced by the presence of three related proteins (26-38 KD, reduced) found in purified surfactant. Whether these proteins are pre-assembled with lipids before secretion is uncertain but would be expected for a lipoprotein secretion. We performed indirect immunocytochemistry on frozen thin sections of rat lung to identify cells and intracellular organelles that contain these proteins. The three proteins, purified from lavaged surfactant, were used to generate antisera in rabbits. Immunoblotting of rat surfactant showed that the IgG reacted with the three proteins and a 55-60 KD band which may be a polymer of the lower MW species. Specific gold labeling occurred over alveolar type II cells, bronchiolar Clara cells, alveolar macrophages, and tubular myelin. In type II cells labeling occurred in synthetic organelles and lamellar bodies, which contain surfactant lipids. Lamellar body labeling was increased fivefold by pre-treating tissue sections with a detergent. Multivesicular bodies and some small apical vesicles in type II cells were also labeled. Secondary lysosomes of alveolar macrophages were immunoreactive. Labeling in Clara cells exceeded that of type II cells, with prominent labeling in secretory granules, Golgi apparatus, and endoplasmic reticulum. These observations clarify the organelles and pathways utilized in the elaboration of surfactant. After synthesis, the proteins move, probably via multivesicular bodies, to lamellar bodies. Both lipids and proteins are present in tubular myelin. Immunologically identical or closely similar proteins are synthesized by Clara cells and secreted from granules which appear not to contain lipid. The role of these proteins in bronchiolar function is unknown.

Incorporation of biotinylated SP-A into rat lung surfactant layer, type II cells, and Clara cells

2000 ◽  
Vol 279 (1) ◽  
pp. L118-L126 ◽  
Author(s):  
Jordan Savov ◽  
J. R. Wright ◽  
S. L. Young
Keyword(s):  
Secretory Granules ◽  
Intratracheal Instillation ◽  
Time Dependent ◽  
Clara Cell ◽  
Type Ii Cells ◽  
Secretory Product ◽  
Type Ii ◽  
Clara Cells ◽  
Rat Lung ◽  
Electron Microscopic

The goal of this study was to compare the functions of Clara and type II cells during alveolar clearance and recycling of surfactant protein (SP) A, a secretory product of both cell types. We examined the incorporation of instilled biotinylated SP-A (bSP-A) into rat lung type II and Clara cells as a measure of clearance and recycling of the protein. Ultrastructural localization of bSP-A was accomplished by an electron-microscopic immunogold technique at 7, 30, and 120 min after intratracheal instillation. Localization of bSP-A was quantitatively evaluated within extracellular surfactant components (lipid-rich forms: myelin figures, vesicles, and tubular myelin; and lipid-poor hypophase) and in compartments of type II and Clara cells. bSP-A was incorporated into myelinic and vesicular forms of extracellular surfactant, but tubular myelin and hypophase had little bSP-A. Lamellar bodies of type II cells demonstrated a significant time-dependent increase in their incorporation of bSP-A. There was a concentration of bSP-A in the secretory granules and mitochondria of Clara cells, but no Clara cell compartment showed a pattern of time-dependent change in immunolabeling. Our immunolabeling data demonstrated a time-dependent movement of exogenous SP-A from extracellular components into type II cells and their secretory granules. Clara cells did not demonstrate a time-dependent incorporation of bSP-A into their secretory granules during the period of this study. If Clara cells recycle SP-A, they must reach a steady state very quickly or very slowly.

Isolation of nonciliated bronchiolar epithelial (Clara) cells and alveolar type II cells from mouse lungs

1987 ◽  
Vol 65 (12) ◽  
pp. 2368-2372 ◽  
Author(s):  
Thomas E. Massey ◽  
Barbara A. Geddes ◽  
Poh-Gek Forkert
Keyword(s):  
Viable Cell ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Clara Cells ◽  
Isolated Cells ◽  
Alveolar Type Ii Cells ◽  
Trypan Blue Exclusion ◽  
Mouse Lungs

A method is described for the isolation of alveolar type II cells and nonciliated bronchiolar epithelial (Clara) cells from mouse lungs. Following digestion of lung tissue with Sigma type I protease, viable cells were isolated to 65% purity for type II cells (6.4 ± 1.5 × 105 cells/mouse) and 55–60% purity for Clara cells (2.6 ± 0.9 × 105 cells/mouse). Viability, as assessed by trypan blue exclusion, was routinely greater than 90% in all enriched cell fractions. Although minor mitochondrial changes occurred during isolation, the morphology of the cells showed good preservation, as revealed by electron microscopy. The isolated cells were found to be metabolically active, as indicated by the presence of 7-ethoxycoumarin deethylase (a cytochrome p-450-mediated activity). The highest activity of this enzyme (278 ± 116 pmol∙min−1∙mg protein−1) was found in the fraction enriched in Clara cells. The results indicate that this method produces viable cell populations that can be of value in investigations of the cellular distribution of lung metabolism activities.

Cell-specific posttranslational processing of the surfactant-associated protein SP-B

1993 ◽  
Vol 264 (3) ◽  
pp. L290-L299 ◽  
Author(s):  
S. Hawgood ◽  
D. Latham ◽  
J. Borchelt ◽  
D. Damm ◽  
T. White ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Specific Protein ◽  
Precursor Protein ◽  
Type Ii Cells ◽  
Posttranslational Processing ◽  
Type Ii ◽  
Clara Cells ◽  
Ovary Cells

Pulmonary surfactant-associated protein B (SP-B) is a 9-kDa lung-specific protein expressed in alveolar epithelial type II cells and Clara cells. The protein markedly increases the surface activity of phospholipids and is an active component in some surfactants in clinical use. SP-B is produced from a 43-kDa precursor protein by proteolytic cleavage of flanking regions from both the NH2- and COOH-terminal ends of the active protein. In this study we have compared the nature of the posttranslational processing of the SP-B precursor in type II cells and in a heterologous cell line transfected with the SP-B precursor. We found that isolated type II cells produce the 9-kDa form of SP-B from the precursor through a series of intermediates detectable in the cell lysates. In contrast Chinese hamster ovary cells stably transfected with the full-length human SP-B precursor produce the precursor and a 26-kDa intermediate but not the 9-kDa protein. The precursor protein in both cell types is glycosylated with NH2-linked sugars. Our results suggest there is cell specificity in the posttranslational processing of the SP-B precursor.

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