A mechanism for regulatory volume decrease in cultured lens epithelial cells

Purpose. To identify mechanisms contributing to regulatory volume decrease in lens epithelial cells. Methods. Cells of the lens epithelial cell line alpha TN4 were cultured in four-well culture dishes in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. After confluence cell water space was determined by measuring the equilibrium distribution of 3-O-methylglucose. Potassium influx and efflux in isotonic and hypotonic solutions were measured using (86)rubidium (Rb-86) as tracer. Total cell potassium and sodium content were determined with atomic absorption spectroscopy. Protein content per well was assayed with a modified Lowry assay and flux data and ion concentrations were normalized per mg of protein. Results. Lens epithelial cells responded to hypotonic solutions with rapid swelling followed by regulatory volume decrease (RVD). During swelling and subsequent volume decrease the unidirectional Rb efflux was increased proportionaly to the osmotic challenge. Rubidium efflux was highly sensitive to changes in extracellular osmolarity and responded with a measurable activation to changes of 12.5 mOsm. No changes in Rb-86 influx were observed with small changes (<20%) in osmolarity and only relatively small changes occurred with larger changes in osmolarity. The resulting net loss of Rb-86 and potassium (K+) was demonstrated by measuring the change of intracellular [K+] in hypotonic solutions using atomic absorption spectroscopy. The K+-channel blockers quinine-HCl and BaCl2 and the Cl--channel blockers diphenyl-2-carboxylate (DPC) and 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB) did not significantly affect the Rb-86 efflux induced by hypotonic solutions. However, [(dihydroindenyl)oxy]alkanoic acid (DIOA), reported to be a specific inhibitor of the K-Cl cotransporter, inhibited the activation of Rb-86 efflux. Rb-86 efflux could be activated in isosmotic solutions by the addition of 1 mM N-ethylmaleimide (NEM). This activation of Rb efflux could be prevented by the addition of 1 mM dithiothreitol and could be 90% blocked by DIOA. The activation of rubidium efflux by NEM led to a significant decrease of the intracellular water content. The volume regulatory changes in NEM and in hypotonic solutions could be inhibited in DIOA. Conclusions. The observations are consistent with the presence in lens epithelial cells of a K-Cl cotransporter serving as a mechanism for regulatory volume decrease.