Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na,K,Cl cotransport

Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (approximately 30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K+] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K+] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, Cl reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),Cl cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and Cl, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and Cl in the ratio of 1:1:2.