Adrenaline-regulated Cl- transport in cultured single rat epididymal cells measured by an entrapped Cl-(-)sensitive fluorophore.

1. Isolated cells from primary cultures of rat epididymal epithelial cells were employed for the study of adrenaline-stimulated Cl- transport using a Cl-(-)sensitive fluorophore 6-methoxy-N-(3-sulphopropyl) quinolinium (SPQ). SPQ was loaded into the cells by the hypotonic shock method. 2. The resting intracellular Cl- concentration, estimated in the presence of nigericin and tributyltin in high-K+ solution, was 62.3 +/- 0.2 mM. This value was not altered in the presence of 1 microM adrenaline. When extracellular Cl- was replaced by NO3-, an increase in fluorescence corresponding to a decrease in intracellular Cl- was observed. The initial outward Cl- movement was estimated to be 0.54 +/- 0.08 mM s-1. This value was increased by incubating the cells with adrenaline. The stimulatory effect of adrenaline was reduced by 1 mM DPC. 3. Addition of Cl- to cells previously depleted of Cl- caused an instantaneous decrease in fluorescence due to the entry of Cl-. The initial rate of Cl- entry was -0.62 +/- 0.13 mM s-1. Adrenaline increased the rate of entry to -2.13 +/- 0.08 mM s-1. The adrenaline-stimulated rate of entry was reduced by DPC or frusemide (0.5 mM) and was completely blocked in the presence of both agents. 4. In Na(+)-free solution, the adrenaline-stimulated rise of rate of Cl- entry was reduced in the presence of DPC. Frusemide had no effect on the entry rate. 5. The stimulatory effect of adrenaline were abolished by propranolol (5 microM) but not by phentolamine (5 microM). Conversely, isoprenaline (1 microM) and forskolin (1 microM) mimicked the effects of adrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)