Hypertonicity Stimulates Cl– Transport in the Intestine of Fresh Water Acclimated EEL, Anguilla Anguilla
Eel intestinal epithelium when bathed symmetrically with normal Ringer solution develops a net Cl– current (short circuit current, Isc) giving rise to a negative transepithelial potential (Vt) at the basolateral side of the epithelium, lower in fresh-water (FW)-acclimated animals with respect to sea-water (SW). The aim of the present work was to study the cell response to hypertonic stress of FW eel intestinal epithelium in relation to Cl– absorption. The hypertonicity of the external bathing solutions produced first a transient increase of Vt and Isc, then followed (after10-15min) by a gradual and sustained increase which reached the maximum value after 40-60 min. The morphometric analysis of the intestine revealed the shrinkage of the cells after 5 min hypertonicity exposure, and then a regulatory volume increase (RVI) response, which parallels the gradual and sustained increase in the electrophysiological parameters. This last phase is inhibited by drugs known to block Cl– absorption in eel intestine, such as luminal bumetanide (10 µM), specific inhibitor of Na+-K+-2Cl– cotransport, or basolateral NPPB (0.5 mM), dichloro-DPC (0.5 mM), inhibitors of basolateral Cl– conductance. Serosal dimethyl-amiloride (100 µM), specific inhibitor of the Na+/H+ antiport, was ineffective on the hyperosmotic response. Bicarbonate revealed a crucial role as a modulator of hypertonicity response, since in bicarbonate-free conditions or in the presence of serosal 0.25 mM SITS, blocker of HCO3– transport systems, the Isc response to hypertonicity was lost. In nominally Ca2+-free conditions the Isc response to hypertonicity was abolished. The same results were obtained by bilateral addition of 100 µM verapamil or 50 µM nifedipine or 1 mM lanthanum, known Ca2+ channel blockers, indicating that extracellular Ca2+ plays a key role for the activation of Cl– current in the response to hypertonic stress. The data show that in the eel intestinal epithelium the hypertonicity of the external medium affects cell volume which in turn might represent the signal to increase the rate of Cl– transport. This response is sustained by the activation of the luminal Na+-K+-2Cl– cotransporter and the functionality of basolateral Cl– channels.