Cortisol directly stimulates differentiation of chloride cells in tilapia opercular membrane

Opercular membranes from freshwater tilapia (Oreochromis mossambicus) were maintained in vitro for 4 days and exposed to several concentrations of cortisol (0, 0.01, 0.1, 1, and 10 micrograms/ml). Chloride cell size, number, and Na(+)-K(+)-ATPase content were examined using a fluorescent mitochondrial dye (dimethylaminostyrylethylpyridiniumiodine), a fluorescent analogue of ouabain (anthroylouabain) that binds specifically to Na(+)-K(+)-ATPase, and a cytological stain specific for plasma and tubular membranes. In the absence of cortisol, chloride cell density of the freshwater tilapia opercular membrane decreased (from initial levels of 6,114 +/- 451 to 18 +/- 9 cells/cm2) and was restored by cortisol in a dose-dependent manner. Chloride cell height (5.5 +/- 0.3 microns initially and 7.8 +/- 0.5 microns after 4 days in vitro) increased twofold (13.1 +/- 0.7 microns) after exposure to 1 microgram/ml cortisol. Initially and after 4 days in control medium, there was no detectable staining with anthroylouabain; exposure to 1 microgram/ml cortisol resulted in the appearance of numerous anthroylouabain-positive chloride cells. Without cortisol, Na(+)-K(+)-ATPase activity of the opercular membrane remained constant through 4 days of culture (0.4-0.6 mumol ADP.mg protein-1.h-1); addition of cortisol caused a dose-dependent increase to a maximum of 1.2 +/- 0.1 mumol Pi.mg protein-1.h-1. In vitro cortisol also maintained the size, density, and appearance of chloride cells from opercular membrane of seawater-adapted tilapia. The results indicate that in vitro cortisol exposure causes morphological and biochemical differentiation of the seawater form of the chloride cell.