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 muM) and analysed by fluorescence-activated flow cytometry. A sub-population of about 2.8 0. 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 P450) 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 0-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.