NA-K-2CL COTRANSPORT IN INTESTINAL EPITHELIAL-CELLS - INFLUENCE OF CHLORIDE EFFLUX AND F-ACTIN ON REGULATION OF COTRANSPORTER ACTIVITY AND BUMETANIDE BINDING

Although cAMP-dependent epithelial chloride secretion is largely regul ated via apical membrane chloride channels, cAMP also remodels basolat eral F-actin and activates basolateral Na-K-2Cl cotransport. Whether a ctivation of cotransport is a primary event or secondary to activation of chloride efflux is not established, and the basis for the cytoskel etal dependence is unknown. We studied cotransport in the intestinal l ine HT29 (which lacks cAMP-regulated chloride efflux) and in its subcl one Cl.19A (in which this pathway is present). Cotransporter activity was enhanced by forskolin in both lines but to a considerably greater extent in subclone Cl.19A, in which the number of bumetanide binding s ites was also observed to increase. The F-actin stabilizer phalloidin markedly attenuated cAMP-stimulated cotransport in Cl.19A monolayers, but the increase in bumetanide binding was preserved. These studies id entify two mechanisms for activation of Na-K-2Cl cotransport by cAMP: components independent and dependent of cAMP elicited chloride efflux. Additional Na-K-2Cl cotransporters become accessible to the cell surf ace coincident with the salt efflux-dependent activation of cotranspor t. While F-actin rearrangements influence salt efflux-dependent up-reg ulation of the cotransporter, this influence occurs independently of i ncreases in bumetanide-accessible cotransporters.