Cl- transport in frog retinal pigment epithelium

Cl- transport across the retinal membrane of the frog retinal pigment epithelium was studied by means of double-barrelled Cl- selective microelectrodes. Three types of experiments were performed. In the first group of experiments, the ionic dependence of Cl- influx across the retinal membrane was studied. The intracellular Cl- activity was first decreased by perfusing the retinal side of the epithelium with low Cl- solutions (3.6 mM Cl-); then the perfusate was changed to high Cl- solutions (90.1 mM), and the resulting Cl- influx was studied. In these experiments, the combined presence of extracellular Na+ and K+ was a necessary condition for Cl- influx across the retinal membrane. This supports the hypothesis of Na+,K+,Cl- co-transport across this membrane. In a second group of experiments, the effect of furosemide was studied. Furosemide (100 microM) inhibited Cl- influx when the retinal extracellular Cl- concentration was increased from 3.6 to 90.1 mM. When administered to cells in steady state, furosemide in concentrations between 5 and 1000 microM decreased the intracellular Cl- activity. Michaelis-Menten analysis yielded a Ki for furosemide of 7 +/- 2 microM. The effect of furosemide on the intracellular Cl- activity required the combined presence of extracellular Na+ and K+. When the retinal extracellular K+ concentration was increased to between 0 and 10 mM, the furosemide-sensitive Cl- influx across the retinal membrane increased. Michaelis-Menten analysis yielded a half maximal stimulation at an extracellular K+ concentration of 0.5 mM. Stimulation of the epithelium with 1 mM cAMP and 0.5 mM IBMX reduced the effect of furosemide on the intracellular Cl- activity by 26%. In a third group of experiments, the effect of transepithelial currents on the intracellular Cl- activity was investigated. Currents that depolarized the choroidal membrane potential increased the intracellular Cl- activity; currents that hyperpolarized this membrane potential decreased the intracellular Cl- activity. These findings are compatible with conductive Cl- transport across the choroidal membrane. The apparent Cl- conductance of this membrane was estimated to be 0.59 mS cm-2. This represents 27% of the total conductance in the choroidal membrane. Administration of 1 mM cAMP and 0.5 mM IBMX caused a 21% increase in the apparent Cl- conductance of the choroidal membrane.