Osmotic regulation of intestinal epithelial Na+-K+-Cl- cotransport:: role of Cl- and F-actin

Previous data indicate that adenosine 3',5'-cyclic monophosphate activates the epithelial basolateral Na+-K+-Cl- cotransporter in microfilament-dependent fashion in part by direct action but also in response to apical Cl- loss (due to cell shrinkage or decreased intracellular Cl-). To further address the actin dependence of Na+-K+-Cl- cotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed. Na+-K+-Cl- cotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellular Cl- concentration and also to activation of K+ and Cl- efflux pathways. Stabilization of actin with phalloidin blunted cotransporter activation by hypotonicity and abolished hypotonic activation of K+ and Cl- efflux. However, phalloidin did not prevent activation of cotransport by hypertonicity or isosmotic reduction of extracellular Cl-. Conversely, hypertonic but not hypotonic activation was attenuated by the microfilament disassembler cytochalasin D. The results emphasize the complex interrelationship among intracellular Cl- activity, cell volume, and the actin cytoskeleton in the regulation of epithelial Cl- transport.