Cl − transport in frog retinal pigment epithelium

Abstract

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 m m Cl −); then the perfusate was changed to high Cl − solutions (90·1 m m), 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 μ m) inhibited Cl − influx when the retinal extracellular Cl − concentration was increased from 3·6 to 90·1 m m. When administered to cells in steady state, furosemide in concentrations between 5 and 1000 μ m decreased the intracellular Cl − activity. Michaelis-Menten analysis yielded a K i for furosemide of 7 ± 2 μ m. 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 m m, 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 m m. Stimulation of the epithelium with 1 m m cAMP and 0·5 m m 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 m m cAMP and 0·5 m m IBMX caused a 21% increase in the apparent Cl − conductance of the choroidal membrane.