Signal trasduction pathways in the hypertonic stress response of eel intestinal epithelium

Aim: The intestinal epithelium of the European eel is a good physiological model for cell volume regulation research at the epithelial level. Under hypertonic condition the eel intestine undergoes cellular shrinkage followed by a Regulatory Volume Increase (RVI) sustained by stimulation of Na+-K+-2Cl- cotransport (NKCC). We previously showed this response to be strongly dependent on cytoskeletal integrity. The aim of this study was to elucidate the signalling events involved in activation of RVI in eel intestine, with particular attention to the role of the major F-actin interacting protein, nonmuscle myosin II. Methods: The study was carried out by short circuit current (Isc) measurements and by confocal laser scanning microscopy Results: ML-9 and ML-7, specific inhibitors of myosin light chain kinase, as well as blebbistatin (inhibitor of myosin ATPase) significantly inhibited the NKCC response to hypertonic stress, clearly indicating that the RVI response was dependent on myosin II. Confocal microscopy revealed a specific myosin II localization mainly in the junctional region of the cells. This myosin II immunostaining appeared to markedly increase in the first 5 minutes of hypertonic exposure. Clostridium difficile toxin A, an inhibitor of Rho family small GTPases, and SB203580, an inhibitor of the mitogen-activated protein kinase p38, abolished the hypertonicity induced NKCC activation, suggesting that these proteins, known modulators of the actin-based cytoskeleton, play a key role in the signalling pathways involved in the RVI response. Conclusions. The RVI response to hypertonic stress is accompanied by a transient recruitment of myosin II to the junctional region, probably increasing the resistance of the epithelium to the osmotic stress. Myosin II also appeared to be, directly or indirectly, involved in the hypertonicity-induced NKCC stimulation, possibly downstream of Rho GTPases and p38 MAPK.