Investigation on signal transduction pathways involved in the eel intestine response to hypotonic stress

As previously reported (Lionetto et al., J. Exp. Biol., 208: 749-760, 2005) the eel intestinal epithelium is able to perform an RVD response following hypotonic swelling. The volume response is sustained by the activation of different K+ and anion conductive pathways on the apical and basolateral membranes of the epithelium. This results in the biphasic decrease of basal transepithelial voltage (Vte) and short-circuit current (Isc), which are expression of the transepithelial Cl- absorption. The aim of the present work was to investigate on the intracellular transduction pathways involved in the response to hypotonic stress in eel intestine, by using the electrophysiological technique of short circuit current, confocal microscopy, and light microscopy morphometrical analysis of epithelium height. The electrophysiological response to hypotonic stress was sensitive to the intracellular Ca2+ concentration since chelation of intracellular Ca2+ with 50 M BAPTA-AM produced a significant although not complete inhibition of the response; similar results were obtained with 10 M trifluoroperazine, allowing to hypothesize that the intracellular calcium role is mediated by a Ca2+-calmodulin pathway. The use of specific protein kinases inhibitors revealed that the electrophysiological response to hypotonicity was insensitive to H-89 (specific inhibitor of PKA), partially inhibited by chelerythrine (known to inhibit PKC) and was dramatically decreased by treatment with ML-7, specific inhibitor of myosin light chain kinase. ML-7 was also able to reduce the volume recovery of the epithelium after hypotonic swelling. These results suggest that a MLCK Ca2+-calmodulin mediated phosphorylation of myosin could be a central mechanism in the transduction pathways of hypotonic stress response in eel intestine. By confocal microscopy it was demonstrated the presence of myosin II in eel enterocyte and its localization in the terminal web region.