Mixed-function oxidase activity in enriched populations of ciliated cells freshly isolated from rabbit tracheas

A method is described for the preparation of enriched populations of ciliated cells from rabbit tracheas. Following protease digestion of tracheal lumen tissue, cells were subjected to centrifugal elutriation. This produced two cell fractions of interest: an 8 micron diameter fraction believed to be composed largely of basal cells, and a 15 micron diameter fraction containing a mixture of ciliated cells and Clara cells. Further treatment of the 15 micron cells with a dextran/polyethylene glycol/phosphate buffer system resulted in separation of a highly enriched ciliated cell fraction (84.3 +/- 2.7% ciliated cells with 6.5 +/- 1.5% Clara cells) from a fraction containing both ciliated cells (42.0 +/- 2.1%) and Clara cells (27.0 +/- 3.5%). The yield of cells in the enriched ciliated cell fraction was 0.68 +/- 0.09 X 10(6) cells/trachea. Analysis of mixed-function oxidase activity in tracheal cells showed 7-ethoxycoumarin deethylase and coumarin hydroxylase activities to be present in the 8 micron cells as well as in ciliated cells and Clara cells. Enzyme activities measured in the ciliated cells (152 +/- 66 pmol/min/mg protein or 51.2 +/- 20.5 pmol/min/10(6) cells for 7-ethoxycoumarin deethylase and 31.7 +/- 15.4 pmol/min/mg protein or 10.5 +/- 4.8 pmol/min/10(6) cells for coumarin hydroxylase) were not attributable to contamination with Clara cells.