Characterization of Clara and type II cells isolated from rat lung by fluorescence-activated flow cytometry.
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.
Martin
J
J
MRC Toxicology Unit, Carshalton, Surrey, U.K.
Dinsdale
D
D
White
I N
IN
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015415:Biomarkers; D001980:Bronchi / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D002469:Cell Separation / Q000379:methods*; D003374:Coumarins / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005978:Glutathione / Q000031:analogs & derivatives / Q000032:analysis; D019803:Glutathione Disulfide; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D008297:Male; D009570:Nitriles / Q000378:metabolism; D010088:Oxidoreductases / Q000096:biosynthesis; D010720:Phosphines; D011720:Pyrazoles; D051381:Rats; D011916:Rats, Inbred F344; D013194:Staining and Labeling; D013250:Steroid Hydroxylases / Q000096:biosynthesis; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis*
D015415:Biomarkers; D003374:Coumarins; D009570:Nitriles; D010720:Phosphines; D011720:Pyrazoles; C057714:3-cyano-7-ethoxycoumarin; C059597:monochlorobimane; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; C025619:steroid 16-beta-hydroxylase; D005723:gamma-Glutamyltransferase; C044646:phosphine; D005978:Glutathione; D019803:Glutathione Disulfide