Na⁺-H⁺ exchanger in cultured rat hepatocytes

Utilization of Laser cytometer

Abstract

The role of Na⁺-H⁺ exchanger in the maintenance of intracellular pH (pH_i) was examined with 24 hour-cultured couplet cells isolated from rat liver. The pH_i was measured with bis (carboxyethyl)carboxyfluorescein (BCECF) and Laser cytometer, and the recovery rate of the pH_i was mesured by the period of time for the acidified cellular pH with NH₄Cl to return to pH of the cellular original set point after removal of NH4Cl solution. The recovery was found to be dependent on the presence of extracellular Na⁺ and it was blocked by amiloride, and Na⁺-H⁺ exchanger exhibited a set point. The pH_i recovery rate (ΔpH/min) varied with the pH_i. The phorbol ester, 12-0-tetradecanoyl-phorbol-13-acet-ate (TPA), increased the recovery rate and shifted pH of set point. The observations indicate that the Na⁺-H⁺ exchanger regulates pHi in cultured couplet cells of rat hepatocytes, and the exchanger may act through protein kinase C. Laser cytometer is very useful for determination of pumping function of cell membrane by observation of pH_i changes.