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First published online September 14, 2007
Journal of Experimental Biology 210, 3473-3483 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008862

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Interdependence of Ca²⁺ and proton movements in trout hepatocytes

Khaled H. Ahmed and Bernd Pelster^*

Institut für Zoologie, and Center of Molecular Biosciences, Leopold Franzens Universität Innsbruck, Technikerstraße 25, A-6020 Innsbruck, Austria

^* Author for correspondence (e-mail: Bernd.Pelster{at}uibk.ac.at)

Accepted 17 July 2007

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 µmol l^–1) and thapsigargin (0.1 µmol 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 µmol 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 pH_e below and above normal values induced a slow, continuous increase in [Ca²⁺]_i with a tendency to stabilize upon exposure to high pH_e 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.

Key words: trout hepatocyte, intracellular Ca²⁺, intracellular pH, proton secretion, ionomycin, thapsigargin, BAPTA-AM, NH₄Cl, Na-propionate, hypertonicity