Na +-Cl --K + Cotransport Activity in Cultured Bovine Lens Epithelial Cells and its Absence in Intact Bovine Lenses

Abstract

Using 86RbCl, experiments were directed to identify the pathways for K + entry in bovine lens cells. Experiments were performed on cultured bovine lens epithelial cells, which are known to transform spontaneously in vitro, and on whole bovine lenses. In cultured epithelial monolayers, K + inflow occurred nearly equally via the Na +-K + pump and a bumetanide-sensitive, Na +- and Cl --dependent process. In the simultaneous presence of ouabain and bumetanide, uptake was inhibited by 98%. Significant passive movement of label through K + channels could not be detected as indicated by the lack of an effect by 5 mM Ba 2+. In the presence of ouabain, the concentration of bumetanide producing half-maximal inhibition of K + uptake was 1·2 × 10 -7 M, a value similar to those of other cell systems in which Na +-Cl --K + cotransport has been demonstrated by various methods. The present system was not affected by attempts to independently perturb cell Ca 2+ and cAMP, but K + uptake was inhibited upon exposure to phorbol esters and to a cell permeable analog of the protein kinase C (PKC) activator diacylglycerol. From experiments with phorbol 12-myristate-13-acetate (PMA), the reduced inflow was attributed to an inhibition of the bumetanide-sensitive pathway. In contrast, treatment with a phorbol ester known not to activate PKC, 4α-phorbol 12,13-didecanoate (4α-PDD), was ineffective; in toto suggesting that the inositol signaling system regulates K + fluxes in these cells primarily by affecting the rate via a Na +-Cl --K + cotransport mechanism. These results contrasted with those garnered with the intact lens. Lenses were isolated in a bicameral Ussing-type arrangement so that their anterior and posterior aspects could be bathed independently, K + inflow across the epithelial surface was predominately mediated by the Na +-K + pump, i.e. ouabain inhibited uptake by 87%. Addition of bumetanide to the anterior bath resulted in an uptake rate not different from that found under control conditions. The present results are consistent with findings in other cell systems which indicate that the Na +-Cl --K + cotransport activity is associated with highly proliferative cells, such as the cultured cells used. It remains to be determined whether activation, or expression, of the cotransporter has a role in the intact lens.