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1 Institut fuer Zoologie und Limnologie, Leopold Franzens Universitaet Innsbruck, Innsbruck, Austria; Center of Molecular Biosciences, Leopold Franzens Universitaet Innsbruck, Innsbruck, Austria
2 Institut fuer Zoologie und Limnologie, Leopold Franzens Universitaet Innsbruck, Innsbruck, Austria
3 Instituto de Quimica y Fisicoquimica Biologicas (Facultad de Farmacia y Bioquimica), Universidad de Buenos Aires, Buenos Aires, Argentina
* To whom correspondence should be addressed. E-mail: Gerhard.Krumschnabel{at}uibk.ac.at.
The role of cytoskeletal elements in volume regulation was studied in trout hepatocytes by investigating changes in F-actin distribution during anisotonic exposure and by assessing the impact of cytoskeleton disrupting agents on volume regulatory responses. Hypotonic challenge resulted in a significant decrease in the ratio of cortical to cytoplasmic F-actin, whereas this ratio was unaffected in hypertonic saline. Disruption of microfilaments with cytochalasin B (CB) or cytochalasin D (CD) significantly slowed volume recovery following hypo- and hypertonic exposure in both attached and suspended cells. The decrease of net proton release and the intracellular acidification elicited by hypotonic saline were unaltered by CB, whereas the pronounced increase of proton release in hypertonic saline was dramatically reduced by CB. Since amiloride almost completely blocked the hypertonically induced increase of proton release and cytoskeleton disruption diminished the associated increase of intracellular pH (pHi), we suggest that F-actin disruption affected activity of the Na+/H+ exchanger. This is confirmed by the observation that pHi recovery after an ammonium pre-pulse was significantly inhibited in CB-treated cells. The increase of cytosolic Na+ under hypertonic conditions was not diminished, but rather enhanced by F-actin disruption, presumably due to inhibited Na+/K+-ATPase activity and stimulated Na+ channel activity in CB-treated cells. The elevation of cytosolic free Ca2+ in hypertonic medium was significantly reduced by CB. Altogether, our results indicate that the F-actin network is of crucial importance in the cellular responses to anisotonic conditions, possibly via interaction with the activity of ion transporters and with signalling cascades responsible for activation of volume regulatory processes. Disruption of microtubules with colchicin had no effect on any of the parameters investigated.
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