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University of California, School of Optometry, Berkeley 94720.
1. Radioactive tracer flux measurements demonstrate that active ion transport across the isolated bovine retinal pigment epithelium (RPE)-choroid preparation can be maintained for hours after the eye is enucleated and the tissue removed from the eye. 2. It has been shown that 86Rb tracer fluxes can be used to monitor potassium (K+) transport across bull-frog RPE. In bovine RPE, net 86Rb (K+) absorption is zero. Apical barium (Ba2+) elevated active K+ absorption from zero to approximately 0.3 mu equiv cm-2 h-1. This Ba2(+)-induced increase in active K+ absorption was inhibited either by ouabain or bumetanide in the apical bath. 3. In control Ringer solution, buffered with bicarbonate and CO2, the RPE-choroid actively absorbs chloride (Cl-) at a rate of approximately 0.5 mu equiv cm-2 h-1. In contrast, sodium (Na+) is secreted at a rate of approximately 0.5 mu equiv cm-2 h-1. Chloride absorption was inhibited by apical bumetanide, and Na+ secretion was inhibited by apical ouabain. These drugs were only effective when placed in the solution bathing the apical or retinal side of the tissue. 4. Net Cl- absorption requires an exit mechanism at the basolateral membrane. DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) in the basal bath completely inhibited net Cl- absorption in bicarbonate-free Ringer solution. 5. These experiments show that the chloride transport pathway contains at least two components: (1) a bumetanide-sensitive uptake mechanism at the apical membrane; and (2) an efflux mechanism at the basolateral membrane that is blocked by DIDS. 6. Three apical membrane mechanisms were identified that could help modulate [K+]o in the subretinal or extracellular space that separates the distal retina and the RPE apical membrane. They are: (1) an ouabain-sensitive Na(+)-K+ pump; (2) a bumetanide-sensitive mechanism, the putative Na(+)-K(+)-Cl- co-transporter; (3) a barium-sensitive K+ channel that recycles, to the apical bath, most or all of the potassium that is actively taken up by the Na(+)-K+ pump and the co-transporter. 7. These data suggest that light-induced alterations in subretinal potassium that occur in vivo can activate the chloride transport pathway and help modulate RPE intracellular Cl- during transitions between the light and dark.
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