Interdependence of Ca2+ and proton movements in trout hepatocytes

This study was undertaken to investigate possible interrelationships between Ca2+ homeostasis and pH regulation in trout hepatocytes. Exposure of cells to Ca2+ mobilizing agents ionomycin (0.5 micromol l(-1)) and thapsigargin (0.1 micromol l(-1)) induced an increase in intracellular pH (pHi) that was dependent on Ca2+ influx from the extracellular medium as well as Ca2+ release from intracellular pools. Surprisingly, this increase in pHi and intracellular Ca2+ concentration, [Ca2+]i, was not accompanied by any change in proton secretion. By contrast, removal of extracellular Ca2+ (Ca2+e) using EGTA (0.5 mmol l(-1)) briefly increased proton secretion rate with no apparent effect on pHi, while chelation of Ca2+i using BAPTA-AM (25 micromol l(-1)) resulted in a drop in pHi and a sustained increase in proton secretion rate. [Ca2+]i therefore affected intracellular proton distribution and/or proton production and also affected the distribution of protons across the cell membrane. Accordingly, changes in pHi were not always compensated for by proton secretion across the cell membrane. Alteration in pHe below and above normal values induced a slow, continuous increase in [Ca2+]i with a tendency to stabilize upon exposure to high pHe values. Rapid pHi increase induced by NH4Cl was accompanied by an elevation in [Ca2+]i from both extracellular and intracellular compartments. Ca2+e appeared to be involved in pHi regulation following NH4Cl-induced alkalinization whereas neither removal of Ca2+e nor chelation of Ca2+i affected pHi recovery following Na-propionate exposure. Similarly, [Ca2+]i increase induced by hypertonicity appeared to be a consequence of the changes in pHi as Na-free medium as well as cariporide diminished the hypertonicity-induced increase in [Ca2+]i. These results imply that a compensatory relationship between changes in pHi and proton secretion across cell plasma membrane is not always present. Consequently, calculating proton extrusion from buffering capacity and rate of pHi change cannot be taken as an absolute alternative for measuring proton secretion rate, at least in response to Ca2+ mobilizing agents.