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Ionic determinants of pH of acidic compartments under hypertonic conditions in trout hepatocytes
Khaled H. Ahmed, Bernd Pelster


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 (pHL). Pre-exposure of cells to the specific V-ATPase inhibitor bafilomycin A1 (0.3 μmol l–1) increased AO fluorescence – unmasking H+ leaks under steady-state conditions – and partially removed the hypertonicity-induced pHL 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 μmol 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 HCO3-free medium, signifying the possible presence of Cl/HCO3 exchange. Under steady-state conditions, while a slight and brief pHL increase was measured upon exposure of cells to valinomycin, Cl removal, unexpectedly, induced a decrease in pHL, indicating a role for extracellular Cl in limiting luminal acidification. This was confirmed by the substantial pHL 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 Ca2+. However, BAPTA-AM induced an increase in steady-state pHL that was independent of V-ATPase inhibition. Moreover, the BAPTA-induced alkalinization was still apparent after depletion of intracellular Ca2+ using the Ca2+ ionophore A23187 in Ca2+-free medium. We conclude that pHL of trout hepatocytes is sensitive to hypertonicity and ionic determinants of hypertonicity. Thus, changes in pHL should be considered when studying pH adaptations to hypertonic stress.

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