title: Microfilament-dependent activation of Na+/K+/2Cl- cotransport by cAMP in intestinal epithelial monolayers.
abstract: cAMP-mediated stimulation of Cl- secretion in the human intestinal cell line T84 is accompanied by significant remodeling of F-actin, and both the secretory and cytoskeletal responses may be largely ablated by previous cell loading with phalloidin derivatives, reagents that prevent dynamic reordering of microfilaments (1991. J. Clin. Invest. 87:1903-1909). In this study, we examined the effect of phalloidin loading on the cAMP-elicited activity of the individual membrane-associated transport proteins involved in electrogenic Cl- secretion. Efflux of 125I and 86Rb was used to assay forskolin-stimulated Cl- and K+ conductances, respectively, and no inhibitory effect of phalloidin could be detected. Na+/K(+)-ATPase pump activity, assessed as bumetanide-insensitive 86Rb uptake and the ability of monolayers to generate a Na+ absorptive current in response to apical addition of a Na+ ionophore, was not different between control and phalloidin-loaded monolayers. Forskolin was found to stimulate Na+/K+/2Cl- cotransport (bumetanide-sensitive 86Rb uptake) in time-dependent fashion. In the absence of any agonist, cotransporter activity was markedly decreased in phalloidin-loaded monolayers. Furthermore, under phalloidin-loaded conditions, the forskolin-elicited increase in bumetanide-sensitive 86Rb uptake was markedly attenuated. These findings suggest that cAMP-induced activity of Cl- channels, K+ channels, and the Na+/K(+)-ATPase are not influenced by F-actin stabilization. However, cAMP-induced activation of the Na+/K+/2Cl- cotransporter appears to be microfilament-dependent, and ablation of this event is likely to account for the inhibition of cAMP-elicited Cl- secretion seen in the phalloidin-loaded state. Such findings suggest that Na+/K+/2Cl- cotransporter is functionally linked to the cytoskeleton and is a regulated site of cAMP-elicited electrogenic Cl- secretion.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
authors.forename: J B
authors.initials: JB
authors.lastname: Matthews
authors.forename: C S
authors.initials: CS
authors.lastname: Awtrey
authors.forename: J L
authors.initials: JL
authors.lastname: Madara
mesh_terms: D008841:Actin Cytoskeleton / Q000502:physiology*; D001692:Biological Transport; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology*; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D010590:Phalloidine / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis
chemical_list: D002712:Chlorides; D010590:Phalloidine; D012964:Sodium; D000242:Cyclic AMP; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium