Effect of long-term hyperosmolality on the Na+/H+ exchanger isoform NHE-3 in LLC-PK1 cells

Abstract

The effects of long-term exposure to hyperosmotic medium on the Na+/H+ exchanger isoform NHE-3 were examined in cultured renal epithelial cells (LLC-PK1). LLC-PK1 cells were grown to confluence in control medium (310 mOsm/kg H2O) and then either switched to a hyperosmotic medium (510 mOsm/kg H2O; addition of NaCl or mannitol) or maintained in the control medium for 48 hours. The Na+/H+ exchanger activity was then assessed in isosmotic solutions by measurement of amiloride-sensitive acid-stimulated 22Na+ influx or Na+-dependent acid extrusion. Acid-stimulated 22Na+ influx was decreased significantly in cells incubated in hyperosmotic medium (10.5 +/- 0.9 nmol/mg protein, control vs. 5.8 +/- 0.6, hyperosmotic; P < 0.01). Incubation in hyperosmotic medium also decreased the initial rate of Na+-dependent acid extrusion by approximately 60% over the intracellular pH range 6.9 to 7.3. Intracellular buffering power did not differ in the control and hyperosmotic groups. The Na+/H+ exchanger isoform NHE-3 mRNA and protein, assessed by Northern hybridization and immunoblot analysis, respectively, were unchanged in LLC-PK1 cells incubated in hyperosmotic medium compared with controls, suggesting post-translational regulation by high osmolality. These results demonstrate that long-term exposure to hyperosmotic medium causes an adaptive decrease in Na+/H+ exchange (NHE-3) activity in LLC-PK1 cells, and that this effect is unlikely to involve antiporter gene regulation or a change in protein abundance.