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.