title: Recruitment of purinergically stimulated Cl- channels from granule membrane to plasma membrane.
abstract: HT29-Cl.16E and HT29-Cl.19A are two different subcloned cell lines derived from the human adenocarcinoma cell line HT-29. They are similar in their ability to grow and differentiate to polarized epithelial cells but differ in that HT29-Cl.16E is goblet cell-like with many mucin granules, whereas HT29-Cl.19A lacks mucin granules. Extracellular ATP stimulates Cl- secretion in both cell lines through luminal purinergic P20 receptors and, in HT29-Cl.16E, also mucin secretion release. To evaluate whether fusion of mucin granules is associated with an increase in Cl- conductance of the plasma membrane, the effects of two fusion inhibitors on luminal Cl- conductance were measured. Blockage of actin depolymerization with phalloidin (1 microM) inhibited purinergically stimulated but not adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated luminal Cl- efflux by 50% in HT29-Cl.16E. The same treatment was without effect in HT29-Cl.19A. The fungal metabolite wortmannin, which is an inhibitor of regulated exocytosis in leukocytes, at 100 nM inhibited Cl- secretion by 70% in HT29-Cl.16E. This inhibition was not a direct effect on purinergically stimulated Cl- channels because wortmannin concentrations of up to 1 microM did not affect the secretory response in HT29-Cl.19A. The wortmannin inhibition of Cl- secretion is associated with an inhibition of granule fusion as judged by electron microscopy. The differential inhibition of Cl- secretion in the related HT-29 clones that differ with respect to the presence of mucin granules indicates that 1) the granule fusion inhibitors, phalloidin and wortmannin, have no direct inhibitory effects on purinergically and cAMP-activated Cl- channels, 2) a major portion of purinergically but not cAMP-activated Cl- channels is associated with granule fusion in HT29-Cl.16E, and 3) the signaling pathways for Cl- secretion and granule fusion are not completely identical.
authors.affiliation: Department of Physiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: D
authors.initials: D
authors.lastname: Merlin
authors.forename: X
authors.initials: X
authors.lastname: Guo
authors.forename: K
authors.initials: K
authors.lastname: Martin
authors.forename: C
authors.initials: C
authors.lastname: Laboisse
authors.forename: D
authors.initials: D
authors.lastname: Landis
authors.forename: G
authors.initials: G
authors.lastname: Dubyak
authors.forename: U
authors.initials: U
authors.lastname: Hopfer
mesh_terms: D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects* / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008854:Microscopy, Electron; D009077:Mucins / Q000378:metabolism; D010590:Phalloidine / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism; D011687:Purines / Q000494:pharmacology*; D011983:Receptors, Purinergic / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000648:ultrastructure; D000077191:Wortmannin
chemical_list: D000199:Actins; D000730:Androstadienes; D018118:Chloride Channels; D002712:Chlorides; D004791:Enzyme Inhibitors; D009077:Mucins; D011108:Polymers; D011687:Purines; D011983:Receptors, Purinergic; D010590:Phalloidine; D000255:Adenosine Triphosphate; D000077191:Wortmannin