Purpose: To evaluate the role of NaKCl cotransport in short-circuit current (Isc) and chloride fluxes across rabbit ciliary epithelium mounted in a Ussing-type chamber.
Methods: Bilayered intact ciliary epithelium free of stroma was obtained after perfusion and dissection of rabbit eyes and mounted in an Ussing-type chamber. The effects of bumetanide and other drugs on Isc and transepithelial 36Cl fluxes in bicarbonate-containing Ringer's were determined. Immunoblot analysis was performed by standard techniques.
Results: Bumetanide (100 microM) applied to the blood (pigmented epithelium [PE]) side of the ciliary bilayer caused a dose-dependent decrease in Isc from 18.2 +/- 2.2 to 10.4 +/- 1.4 microA/cm2 (43%). Bumetanide applied to the aqueous (nonpigmented epithelium [NPE]) side of the tissue inhibited Isc by only 12%. Immunoblots of dissected NPE and PE tissue probed with an antibody to mammalian NaKCl cotransporter detected approximately 10 times more NaKCl cotransporter protein in PE than in NPE. 36Cl flux studies revealed a PE-to-NPE chloride flux of 180.3 +/- 37.2 microEq/cm2 per hour and an NPE-to-PE flux of 72.3 +/- 22.9 microEq/cm2 per hour, indicating a net PE-to-NPE flux of 108.0 +/- 31.3 microEq/cm2 per hour across rabbit ciliary epithelium. Bumetanide inhibited the PE-to-NPE chloride flux by 52% but did not inhibit the NPE-to-PE flux. Isoproterenol (10 microM) added to the PE side of the bilayer increased Isc by a dose-dependent 53%. Prior addition of bumetanide to the PE side blocked the increase due to isoproterenol by 37%. Isoproterenol (10 microM) stimulated the PE-to-NPE chloride flux by 75% but had no stimulatory effect on the NPE-to-PE chloride flux. 4,4'Diisothiocyanatostilbene-2,2'disulfonic acid (DIDS) inhibited Isc when added to either side of the bilayer but was more potent at low concentrations (<100 microM) when added to the NPE side and more potent at higher concentrations (>100 microM) when added to the PE side. Prior addition of 1 mM DIDS to the NPE side decreased isoproterenol stimulation of Isc by 56%.
Conclusions: NaKCl cotransporters located primarily on the blood side of rabbit ciliary epithelium contribute to aqueous-negative Isc and to blood-to-aqueous chloride transport across the tissue in bicarbonate-containing medium. DIDS-inhibitable mechanisms, possibly including HCO3-Cl exchange and Cl channels, also play a role. Isoproterenol stimulation of Isc involves coordinate upregulation of PE-side NaKCl cotransport and an NPE-side DIDS-inhibitable mechanism(s).