INHIBITION OF NA+-K+-ATPASE ACTIVATES NA+-K+-2CL- COTRANSPORTER ACTIVITY IN CULTURED CILIARY EPITHELIUM

Rb-86 uptake experiments were conducted to measure Na+-K+-ATPase activity and Na+-K+-2Cl(-) cotransporter activity in a cell line derived from rabbit nonpigmented ciliary epithelium. The presence of a Na+-K+-2Cl(-) cotransporter was supported by the observation of a bumetanide-sensitive Rb-86 uptake component that was dependent on the extracellular concentration of both sodium and chloride. Potassium influx mediated by the Na+-K+-2Cl(-) cotransporter and Na+-K+-ATPase accounted for similar to 46 and 33% of total potassium uptake, respectively, whereas both ouabain- and bumetanide-resistent uptake accounted for 9%. Inhibition of the Na+-K+-ATPase had a stimulatory effect on Na+-K+-2Cl(-) cotransporter activity, which was dependent on the extent and duration of Na+-K+-ATPase inhibition. Ouabain treatment stimulated the potassium (Rb-86) efffux rate and reduced intracellular potassium ([K](i)). Potassium channel blockers suppressed the ouabain-activated potassium efflux and inhibited the ouabain-induced activation of the Na+-K+-2Cl(-) cotransporter. We conclude that Na+-K+-ATPase inhibition leads to the opening of potassium channels, which exacerbates the depletion of cellular potassium; Na+-K+-2Cl(-) cotransporter stimulation caused by the fall of [K](i) overrides the tendency of increased cellular sodium to inhibit the cotransporter.