Abstract
1. The whole-cell patch clamp technique was used to investigate the swelling-activated currents in bovine non-pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The I-V relationship was shifted in the direction expected for a Cl- current when the external Cl- was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), also inhibited this current. 5. The volume-activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time-dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl- current. 8. An antibody (C219) to the MDR1 gene product, P-glycoprotein, caused a functional block of the swelling-activated Cl- current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB-1 demonstrated the presence of P-glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non-pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)-independent, ATP-dependent Cl- current and that the activity of this current is associated with P-glycoprotein. 11. It is suggested that this Cl- current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.