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 ce lls (LLC-PK1). LLC-PK1 cells were grown to confluence in control mediu m (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 c ontrol medium for 48 hours. The Na+/H+ exchanger activity was then ass essed in isosmotic solutions by measurement of amiloride-sensitive aci d-stimulated Na-22(+) influx or Na+-dependent acid extrusion. Acid-sti mulated Na-22(+) 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 a lso decreased the initial rate of Na+-dependent acid extrusion by simi lar to similar to 60% over the intracellular pH range 6.9 to 7.3. Intr acellular buffering power did not differ in the control and hyperosmot ic groups. The Na+/H+ exchanger isoform NHE-3 mRNA and protein, assess ed by Northern hybridization and immunoblot analysis, respectively, we re unchanged in LLC-PK, cells incubated in hyperosmotic medium compare d with controls, suggesting post-translational regulation by high osmo lality. These results demonstrate that long-term exposure to hyperosmo tic medium causes an adaptive decrease in Na+/H+ exchange (NHE-3) acti vity in LLC-PK, cells, and that this effect is unlikely to involve ant iporter gene regulation or a change in protein abundance.