myo-inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line

Citation
Ma. Carew et al., myo-inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line, J BIOL CHEM, 275(35), 2000, pp. 26906-26913
Citations number
47
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
275
Issue
35
Year of publication
2000
Pages
26906 - 26913
Database
ISI
SICI code
0021-9258(20000901)275:35<26906:M3IAAC>2.0.ZU;2-V
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 C FPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5, 6)P-4 levels were acutely sensitive to purinergic receptor activation, risi ng 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P-4, levels were therefore specifically elevated, independently of receptor acti vation, by incubating cells with a cell-permeant bioactivable analogue, 1,2 -di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl)es ter (Bt(2)Ins (3,4,5,6)P-4/AM). The latter inhibited Ca2+-activated Cl- sec retion by 60%. We next used nystatin to selectively permeabilize the basola teral membrane to monovalent anions and cations, thereby preventing this me mbrane from electrochemically dominating ion movements through the apical m embrane. Thus, we studied autonomous regulation of apical Cl- channels in s itu. The properties of Cl- flux across the apical membrane were those expec ted of CaCC: niflumic acid sensitivity, outward rectification, and 2-fold g reater permeability of I- over Cl-. Following nystatin-treatment, we elevat ed 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 Ca2+-activated C l- secretion (up to 70%). These studies provide the first demonstration tha t, in a physiologically relevant context of a polarized monolayer, there is an apical, Ins(3,4,5,6)P-4-inhibited CaCC.