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

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 ~~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.