Chloride currents in freshly isolated rat retinal pigment epithelial cells

Cells of the retinal pigment epithelium (RPE) were isolated from neonatal rats. The perforated-patch clamp technique, using amphotericin-B, revealed a chloride current, which was detected as a 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)-sensitive component. A variety of chloride-channel inhibitors, other than DIDS, also blocked the chloride current, including 9-anthracenecarboxylic acid (9-AC), niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and N-phenylanthranilic acid (DPC). DIDS at 1 mmol l-1 and DPC at 2 mmol l-1 were both more potent than niflumic acid at 200 mumol l-1. The hyperosmotic condition (+50 mosmol) did not affect the chloride current. The hypo-osmotic condition (-80 mosmol), however, led to an increase of total membrane conductance, during which a chloride conductance increase occurred. The membrane-conductance increase, evoked by the hyposmotic condition, ran up and then ran down by itself. As with the steady-state chloride conductance, 1 mmol l-1 DIDS and 2 mmol l-1 DPC were more potent blockers than 200 mumol l-1 niflumic acid. DIDS at 1 mmol l-1 was more effective in blocking the outward component than 2 mmol l-1 DPC, while the inward component was blocked oppositely. When the intracellular calcium concentration was presumed to have increased, using the calcium ionophore, ionomycin, membrane conductance abruptly increased, quickly ran up and then ran down by itself. This included an elevation of a DIDS-sensitive current, and was largely carried by chloride. Chloride currents of rat RPE cells did not appear to be regulated by cyclic AMP.