title: myo-inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line.
abstract: Does inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)) inhibit apical Ca(2+)-activated Cl(-) conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P(4) levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P(4) levels were therefore specifically elevated, independently of receptor activation, by incubating cells with a cell-permeant bioactivable analogue, 1,2-di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl)ester (Bt(2)Ins (3,4,5,6)P(4)/AM). The latter inhibited Ca(2+)-activated Cl(-) secretion by 60%. We next used nystatin to selectively permeabilize the basolateral membrane to monovalent anions and cations, thereby preventing this membrane from electrochemically dominating ion movements through the apical membrane. Thus, we studied autonomous regulation of apical Cl(-) channels in situ. The properties of Cl(-) flux across the apical membrane were those expected of CaCC: niflumic acid sensitivity, outward rectification, and 2-fold greater permeability of I(-) over Cl(-). Following nystatin-treatment, we elevated intracellular levels of Ins(3,4,5,6)P(4) with either purinergic agonists or with Bt(2)Ins(3,4,5,6)P(4)/AM. Both protocols inhibited Ca(2+)-activated Cl(-) secretion (up to 70%). These studies provide the first demonstration that, in a physiologically relevant context of a polarized monolayer, there is an apical, Ins(3,4,5,6)P(4)-inhibited CaCC.
authors.affiliation: Inositide Signaling Section, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. Carew@niehs.nih.gov
authors.forename: M A
authors.initials: MA
authors.lastname: Carew
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.forename: C
authors.initials: C
authors.lastname: Schultz
authors.forename: S B
authors.initials: SB
authors.lastname: Shears
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D018118:Chloride Channels / Q000187:drug effects*; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism / Q000494:pharmacology*; D008564:Membrane Potentials; D009761:Nystatin / Q000494:pharmacology
chemical_list: D018118:Chloride Channels; D007295:Inositol Phosphates; C081652:inositol-3,4,5,6-tetrakisphosphate; D009761:Nystatin; D002118:Calcium