The eel intestine represents a good physiological model for studying cell volume regulation in epithelia. When exposed to hypertonicity it responds with a Regulatory Volume Increase sustained by the stimulation of the Na+-K+-2Cl- cotransporter. This response requires the integrity of the cytoskeleton and is dependent on myosin-driven mechanisms (Lionetto et al., Cell Physiol Biochem, 12: 163–178, 2002).

The aim of the present work was to study the immunolocalization of Na+-K+-2Cl- both in isotonic or hypertonic conditions in order understand the mechanisms of its hypertonicity induced stimulation.

The localization of the contransporter on the brush border was demonstrated by confocal immunofluorescence microscopy. When the tissue was exposed to hypertonic stress, some changes were observed in the fluorescence intensity profile of the brush border. The intensity of the peak of fluorescence was increased and its localization varied along the brush border thickness moving toward the luminal side. This result suggests the possible recruitment of Na+-K+-2Cl- on the luminal membrane from a sub-apical storage pool. This hypertonicity induced response was sensitive to beblistatin, specific myosin II inhibitor, and EHNA, specific dynein inhibitor.

In conclusion, results are consistent with the recruitment of Na+-K+-2Cl- on the luminal membrane following hypertonic stress and suggest a key role of vesicular transport in the activation of cell volume regulation mechanisms.