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WATER AND ELECTROLYTE HOMEOSTASIS

Importance of cytoskeletal elements in volume regulatory responses of trout hepatocytes

¹Institut für Zoologie und Limnologie, and ²Center of Molecular Biosciences, Leopold Franzens Universität Innsbruck, Innsbruck, Austria; and ³Instituto de Química y Fisicoquímica Biológicas (Facultad de Farmacia y Bioquímica), Universidad de Buenos Aires, Buenos Aires, Argentina

Submitted 9 March 2005 ; accepted in final form 17 May 2005

The role of cytoskeletal elements in volume regulation was studied in trout hepatocytes by investigating changes in F-actin distribution during anisotonic exposure and assessing the impact of cytoskeleton disruption on volume regulatory responses. Hypotonic challenge caused 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 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 hypotonicity were unaltered by CB, whereas the increase of proton release in hypertonic saline was dramatically reduced. Because amiloride almost completely blocked the hypertonic increase of proton release and cytoskeleton disruption diminished the associated increase of intracellular pH (pH_i), we suggest that F-actin disruption affected Na⁺/H⁺ exchanger activity. In line with this, pH_i recovery after an ammonium prepulse 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. The elevation of cytosolic 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 their activation. Disruption of microtubules with colchicine had no effect on any of the parameters investigated.

Oncorhynchus mykiss; F-actin; microtubule; acid secretion; intracellular pH; intracellular sodium; intracellular free calcium

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