Primary cultured RCrECL were grown at an air-interface on Clear–Snapwells™ precoated with collagen/fibronectin/laminin and mounted in a modified Ussing-type chamber for the evaluation of their active ion transport processes under short-circuited conditions. Contribution of active Na+ and Cl− transport to overall short-circuit current (Isc) was evaluated by removing Na+ and Cl−, respectively, from bathing fluids of RCrECL and measurements of net fluxes of Na+ and Cl− using 22Na and 36Cl, respectively. Amiloride and benzamil were used to determine the role of apical Na+-channel activities to net Na+ fluxes. N-phenylanthranilic acid (NPAA), ouabain, BaCl2 and bumetanide were used to determine the role of basolateral Na,K-ATPase, apical Cl−-channel, and basolateral K+-channel and Na+(K+)2Cl−-cotransporter activities, respectively, in active ion transport across RCrECL.
Isc of RCrECL derived from pigmented rabbits was comprised of 64±2%and 44±5%for active Na+ and Cl− transport, respectively, consistent with net Na+ absorption and Cl− secretion of 0.062±0.006 and 0.046±0.008 μEq/cm2/hr estimated from radionuclide fluxes. Apical amiloride and benzamil inhibited Isc by up to 50%with an IC50 of 1 and 0.1 μm, respectively, consistent with participation of apical epithelial Na+-channels to net Na+ absorption across RCrECL cultured from pigmented rabbits. Addition of ouabain to the basolateral, NPAA to the apical, BaCl2 to the basolateral and bumetanide to basolateral fluid decreased Isc by 86±1.5%, 53±3%, 18±1.8%and 13±1.9%in RCrECL cultured from pigmented rabbits, while 85±0.7%, 36±1.6%, 38±1.8%and 15±3.5%decrea ses are observed for RCrECL from albino rabbits, respectively.
Air-interface cultured RCrECL from either pigmented or albino rabbits exhibited active ion transport properties similar to those present in excised tissues. This primary culture system may be a reliable in-vitro model for mechanistic characterization of corneal epithelial function and regulation of transport properties.