Permeabilization via the P2X7 purinoreceptor reveals the presence of a Ca2+-activated Cl- conductance in the apical membrane of murine tracheal epithelial cells.
Calcium-activated Cl(-) secretion is an important modulator of regulated ion transport in murine airway epithelium and is mediated by an unidentified Ca(2+)-stimulated Cl(-) channel. We have transfected immortalized murine tracheal epithelial cells with the cDNA encoding the permeabilizing P2X(7) purinoreceptor (P2X(7)-R) to selectively permeabilize the basolateral membrane and thereby isolate the apical membrane Ca(2+)-activated Cl(-) current. In P2X(7)-R-permeabilized cells, we have demonstrated that UTP stimulates a Cl(-) current across the apical membrane of CF and normal murine tracheal epithelial cells. The magnitude of the UTP-stimulated current was significantly greater in CF than in normal cells. Ion substitution studies demonstrated that the current exhibited a permselectivity sequence of Cl(-) > I(-) > Br(-) > gluconate(-). We have also determined a rank order of potency for putative Cl(-) channel blockers: niflumic acid > or = 5-nitro-2-(3-phenylpropylamino)benzoic acid > 4, 4'-diisothiocyanostilbene-2,2'-disulfonate > glybenclamide >> diphenlyamine-2-carboxylate, tamoxifen, and p-tetra-sulfonato-tetra-methoxy-calix[4]arene. Complete characterization of this current and the corresponding single channel properties could lead to the development of a new therapy to correct the defective airway surface liquid in cystic fibrosis patients.
Gabriel
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SE
Cystic Fibrosis/Pulmonary Research and Clinical Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. sgabriel@med.unc.edu
Makhlina
M
M
Martsen
E
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Thomas
E J
EJ
Lethem
M I
MI
Boucher
R C
RC
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001966:Bromine / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D047250:Calixarenes; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D004848:Epithelium / Q000378:metabolism / Q000502:physiology; D005942:Gluconates / Q000378:metabolism; D005905:Glyburide / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007455:Iodine / Q000378:metabolism; D007477:Ions; D051379:Mice; D009544:Niflumic Acid / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D010636:Phenols / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D013629:Tamoxifen / Q000494:pharmacology; D013997:Time Factors; D014132:Trachea / Q000378:metabolism / Q000502:physiology; D014544:Uridine Triphosphate / Q000378:metabolism; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002614:Chelating Agents; D002712:Chlorides; D018076:DNA, Complementary; D005942:Gluconates; D007004:Hypoglycemic Agents; D007477:Ions; D009579:Nitrobenzoates; C546469:P2rx7 protein, mouse; D010636:Phenols; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C121325:calix(4)arene; D062367:ortho-Aminobenzoates; D013629:Tamoxifen; D047250:Calixarenes; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D009544:Niflumic Acid; C001209:fenamic acid; D007455:Iodine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C030691:gluconic acid; D001966:Bromine; D005905:Glyburide; D002118:Calcium; D014544:Uridine Triphosphate