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

Mark A. Carew, Xiaonian Yang, Carsten Schultz, Stephen B. Shears

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Does inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) inhibit apical Ca2+-activated Cl- conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P4 levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P4 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 (Bt2Ins (3,4,5,6)P4/AM). The latter inhibited Ca2+-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)P4 with either purinergic agonists or with Bt2Ins(3,4,5,6)P4/AM. Both protocols inhibited Ca2+-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)P4-inhibited CaCC.

Original languageEnglish (US)
Pages (from-to)26906-26913
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number35
DOIs
StatePublished - Sep 1 2000
Externally publishedYes

Fingerprint

Calcium Chloride
Cystic Fibrosis
Chlorides
Monolayers
Cells
Calcium
Cell Line
Membranes
Nystatin
Purinergic Agonists
Chemical activation
Niflumic Acid
Purinergic Receptors
Monovalent Cations
inositol-3,4,5,6-tetrakisphosphate
Anions
Cations
Permeability
Esters
Demonstrations

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

myo-Inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line. / Carew, Mark A.; Yang, Xiaonian; Schultz, Carsten; Shears, Stephen B.

In: Journal of Biological Chemistry, Vol. 275, No. 35, 01.09.2000, p. 26906-26913.

Research output: Contribution to journalArticle

@article{25ca140efcb144d5b09b3914ce14c6a7,
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)P4) inhibit apical Ca2+-activated Cl- conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P4 levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P4 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 (Bt2Ins (3,4,5,6)P4/AM). The latter inhibited Ca2+-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)P4 with either purinergic agonists or with Bt2Ins(3,4,5,6)P4/AM. Both protocols inhibited Ca2+-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)P4-inhibited CaCC.",
author = "Carew, {Mark A.} and Xiaonian Yang and Carsten Schultz and Shears, {Stephen B.}",
year = "2000",
month = "9",
day = "1",
doi = "10.1074/jbc.M002316200",
language = "English (US)",
volume = "275",
pages = "26906--26913",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "35",

}

TY - JOUR

T1 - myo-Inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line

AU - Carew, Mark A.

AU - Yang, Xiaonian

AU - Schultz, Carsten

AU - Shears, Stephen B.

PY - 2000/9/1

Y1 - 2000/9/1

N2 - Does inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) inhibit apical Ca2+-activated Cl- conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P4 levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P4 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 (Bt2Ins (3,4,5,6)P4/AM). The latter inhibited Ca2+-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)P4 with either purinergic agonists or with Bt2Ins(3,4,5,6)P4/AM. Both protocols inhibited Ca2+-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)P4-inhibited CaCC.

AB - Does inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) inhibit apical Ca2+-activated Cl- conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P4 levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P4 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 (Bt2Ins (3,4,5,6)P4/AM). The latter inhibited Ca2+-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)P4 with either purinergic agonists or with Bt2Ins(3,4,5,6)P4/AM. Both protocols inhibited Ca2+-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)P4-inhibited CaCC.

UR - http://www.scopus.com/inward/record.url?scp=0034282395&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034282395&partnerID=8YFLogxK

U2 - 10.1074/jbc.M002316200

DO - 10.1074/jbc.M002316200

M3 - Article

C2 - 10842174

AN - SCOPUS:0034282395

VL - 275

SP - 26906

EP - 26913

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 35

ER -