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First published online August 3, 2006
Journal of Experimental Biology 209, 3101-3113 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02357

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Signalling pathways involved in hypertonicity- and acidification-induced activation of Na⁺/H⁺ exchange in trout hepatocytes

Khaled H. Ahmed, Bernd Pelster and Gerhard Krumschnabel^*

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

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

Accepted 31 May 2006

In trout hepatocytes, hypertonicity and cytosolic acidification are known to stimulate Na⁺/H⁺ exchanger (NHE) activity, which contributes to recovery of cell volume and intracellular pH (pHi), respectively. The present study investigated the signalling mechanisms underlying NHE activation under these conditions. Exposing trout hepatocytes to cariporide, a specific inhibitor of NHE-1, decreased baseline pHi, completely blocked the hypertonicity-induced increase of pHi and reduced the hypertonicity-induced proton secretion by 80%. Changing extracellular pH (pHe) above and below normal values, and allowing cells to adjust pHi accordingly, significantly delayed alkalinization during hypertonic exposure, whereas following an acid load an enhanced pHi recovery with increasing pHe was seen. Chelating Ca²⁺, and thereby preventing the hypertonicity-induced increase in intracellular Ca²⁺ ([Ca²⁺]i), significantly diminished hypertonic elevation of pHi, indicating that Ca²⁺ signalling might be involved in NHE activation. A reduction in alkalinization and proton secretion was also observed in the presence of the protein kinase A (PKA) inhibitor H-89 or the calmodulin (CaM) inhibitor calmidazolium. A complete inhibition of hypertonic- and acidification-induced changes of pHi concurrent with an increase in hypertonically induced proton efflux was seen with the protein kinase C (PKC) inhibitor chelerythrine. Recovery of pHi following sodium propionate addition was reduced by more than 60% in the presence of cariporide, was sensitive to PKA inhibition, and tended to be reduced by CaM inhibition. In conclusion, we showed that NHE-1 is the main acid secretion mechanism during hypertonicity and recovery following acid loading. In addition, Ca²⁺-, PKA- and CaM-dependent pathways are involved in NHE-1 activation for recovery of cell volume and pHi. On the other hand, PKC appeared to have an impact on NHE-independent pathways affecting intracellular acid-base homeostasis.

Key words: trout hepatocyte, Na⁺/H⁺ exchange, intracellular pH, proton secretion, calcium, PKC, PKA, calmodulin

This article has been cited by other articles:

				K. H. Ahmed and B. Pelster Interdependence of Ca2+ and proton movements in trout hepatocytes J. Exp. Biol., October 1, 2007; 210(19): 3473 - 3483. [Abstract] [Full Text] [PDF]