Ionic determinants of pH of acidic compartments under hypertonic conditions in trout hepatocytes

Exposure of trout hepatocytes to hypertonicity induced a decrease in acridine orange (AO) fluorescence, indicating a corresponding decrease in pH inside the lumen of acidic compartments (pH(L)). Pre-exposure of cells to the specific V-ATPase inhibitor bafilomycin A1 (0.3 micromol l(-1)) increased AO fluorescence - unmasking H(+) leaks under steady-state conditions - and partially removed the hypertonicity-induced pH(L) decrease. The sustainability of the luminal acidification, but not the acidification itself, appeared to depend on a low K(+) and a high Cl(-) conductance under hypertonic conditions. Increasing K(+) conductance using the specific ionophore valinomycin (10 micromol l(-1)) or removal of extracellular Cl(-) after an instant drop in AO fluorescence resulted in a reversal of luminal acidity. The alkalinization measured under hypertonic conditions in the absence of Cl(-) was largely attenuated when cells were bathed in HCO(3)(-)-free medium, signifying the possible presence of Cl(-)/HCO(3)(-) exchange. Under steady-state conditions, while a slight and brief pH(L) increase was measured upon exposure of cells to valinomycin, Cl(-) removal, unexpectedly, induced a decrease in pH(L), indicating a role for extracellular Cl(-) in limiting luminal acidification. This was confirmed by the substantial pH(L) decrease measured upon exposure of cells to the anion exchanger inhibitor SITS (0.5 mmol l(-1)). Furthermore, hypertonicity-induced acidification was still noticeable in the presence of SITS. On the other hand, the hypertonicity-induced acidification was significantly reduced in the absence of extracellular Na(+) or Ca(2+). However, BAPTA-AM induced an increase in steady-state pH(L) that was independent of V-ATPase inhibition. Moreover, the BAPTA-induced alkalinization was still apparent after depletion of intracellular Ca(2+) using the Ca(2+) ionophore A23187 in Ca(2+)-free medium. We conclude that pH(L) of trout hepatocytes is sensitive to hypertonicity and ionic determinants of hypertonicity. Thus, changes in pH(L) should be considered when studying pH adaptations to hypertonic stress.