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