title: Na-K-2Cl cotransport in intestinal epithelial cells. Influence of chloride efflux and F-actin on regulation of cotransporter activity and bumetanide binding.
abstract: Although cAMP-dependent epithelial chloride secretion is largely regulated via apical membrane chloride channels, cAMP also remodels basolateral F-actin and activates basolateral Na-K-2Cl cotransport. Whether activation of cotransport is a primary event or secondary to activation of chloride efflux is not established, and the basis for the cytoskeletal dependence is unknown. We studied cotransport in the intestinal line HT29 (which lacks cAMP-regulated chloride efflux) and in its subclone 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 sites 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 identify 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 surface coincident with the salt efflux-dependent activation of cotransport. While F-actin rearrangements influence salt efflux-dependent up-regulation of the cotransporter, this influence occurs independently of increases in bumetanide-accessible cotransporters.
authors.affiliation: Department of Surgery, Beth Israel Hospital, Boston, Massachusetts 02215.
authors.forename: J B
authors.initials: JB
authors.lastname: Matthews
authors.forename: J A
authors.initials: JA
authors.lastname: Smith
authors.forename: K J
authors.initials: KJ
authors.lastname: Tally
authors.forename: C S
authors.initials: CS
authors.lastname: Awtrey
authors.forename: H
authors.initials: H
authors.lastname: Nguyen
authors.forename: J
authors.initials: J
authors.lastname: Rich
authors.forename: J L
authors.initials: JL
authors.lastname: Madara
mesh_terms: D000199:Actins / Q000378:metabolism*; D000230:Adenocarcinoma; D002034:Bumetanide / Q000378:metabolism*; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D002999:Clone Cells; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D012413:Rubidium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D014407:Tumor Cells, Cultured
chemical_list: D000199:Actins; D002352:Carrier Proteins; D002712:Chlorides; D007454:Iodides; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D005576:Colforsin; D012413:Rubidium