The role of NaKCl cotransport in blood-to-aqueous chloride fluxes across rabbit ciliary epithelium

PURPOSE. TO evaluate the role of NaKCl cotransport in short-circuit current (Isc) and chloride fluxes across rabbit ciliary epithelium mounted in a Us sing-type chamber. METHODS. Bilayered intact ciliary epithelium free of stroma was obtained af ter perfusion and dissection of rabbit eyes and mounted in an Ussing-type c hamber. The effects of bumetanide and other drugs on Isc and transepithelia l Cl-36 fluxes in bicarbonate-containing Ringer's mere determined. Immunobl ot analysis was performed by standard techniques. RESULTS. Bumetanide (100 mu M) applied to the blood (pigmented epithelium [ PE]) side of the ciliary bila) er caused a dose-dependent decrease in Isc f rom 18.2 +/- 2.2 to 10.4 +/- 1.4 mu A/cm(2) (43%). Bumetanide applied to th e aqueous (nonpigmented epithelium [NPE]) side of the tissue inhibited Ise by only 12%. Immunoblots of dissected NPE and PE tissue probed with an anti body to mammalian NaKCl cotransporter detected approximately 10 times more NaKCl, cotransporter protein in PE than in NPE. Cl-36 flux studies revealed a PE-to-NPE chloride flux; of 180.3 +/- 37.2 mu Eq/cm(2) per hour and an N PE-to-PE flux of 72.3 +/- 22.9 mu Eq/cm(2) per hour, indicating a net PE-to -NPE flux of 108.0 +/- 31.3 mu Eq/cm(2) per hour across rabbit ciliary epit helium. Bumetanide inhibited the PE-to-NPE chloride flux by 52% but did not inhibit the NPE-to-PE flux. Isoproterenol (10 mu M) added to the PE side o f the bilayer increased Isc by a dose-dependent 53%. Prior addition of bume tanide to the PE side blocked the increase due to isoproterenol by 37%. Iso proterenol (10 mu M) stimulated the PE-to-NPE chloride flux by 75% but had no stimulatory effect on the NPE-to-PE chloride flux. 4,4' Diisothiocyanato stilbene-2,2' disulfonic acid (DIDS) inhibited Isc when added to either sid e of the bila) er but was more potent at low concentrations (< 100 mu M) wh en added to the NPE side and more potent at higher concentrations (>100 mu M) when added to the PE side. Prior addition of 1 mM DIDS to the NPE side d ecreased isoproterenol stimulation of Isc by 56%. CONCLUSIONS. NaKCl cotransporters located primarily on the blood side of ra bbit ciliary epithelium contribute to aqueous-negative Isc and to blood-to- aqueous chloride transport across the tissue in bicarbonate-containing medi um. DIDS-inhibitable mechanisms, possibly including HCO3-Cl exchange and Cl channels, also pla) a role. Isoproterenol stimulation of Isc involves coor dinate upregulation of PE-side NaKCl cotransport and an NPE-side DIDS-inhib itable mechanism(s).