Intracellular pH of hepatocytes in primary monolayer culture

Nonproliferating rat hepatocytes in primary monolayer culture were used for determining liver cell intracellular pH and the degree of intracellular pH homeostasis. The dimethyloxazolidinedione weak acid distribution method was adapted for use in monolayer culture. Intracellular pH of cultured hepatocytes in bicarbonate:CO2 medium was relatively constant at 6.85-7.05 over the external pH range of 7.0-8.0. Below an external pH of 7.0, intracellular pH fell below 6.8. Varying PCO2 between 15 and 40 mmHg did not alter the extracellular versus intracellular pH curve. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid medium, in the absence of bicarbonate, intracellular pH homeostasis was less well defended. In this setting, the intracellular versus extracellular pH relationship curve could be described by a straight line with slope of 0.59 +/- 0.04. The system responded to the addition of the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone with an increase in the transmembrane pH gradient. Addition of nigericin in 5 mM K+ medium resulted in intracellular acidification to pH 5.5 +/- 0.2. Metabolism of 20 mM added fructose resulted in intracellular acidification. Incubation in sodium-free media at extracellular pH of 7.6 reduced intracellular pH to 6.67 +/- 0.02 compared with an intracellular pH of 6.99 +/- 0.04 in cultures exposed to medium sodium concentrations of 20-80 meq/liter.