Increase in intracellular Cl- concentration by cAMP- and Ca2+-dependent stimulation of M1 collecting duct cells.
In the lungs of cystic fibrosis (CF) patients, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) lead to defective Cl- secretion and hyperabsorption of electrolytes. This may be a an important cause for the defective mucociliary clearance in CF lungs. Previous studies have suggested that inhibition of ENaC during activation of CFTR or by purinergic stimulation could be related to an increase in the intracellular [Cl-]i. This was examined in the present study using cultured mouse M1 collecting duct cells transfected with the chloride-sensitive enhanced yellow fluorescent protein YFP(V163S). Calibration experiments showed a linear decrease of YFP fluorescence intensity with increasing [Cl-]i (0-100 mM). Activation of CFTR by isobutyl-1-methylxanthine (IBMX, 100 microM) and forskolin (2 microM) increased [Cl-]i by 9.6+/-1.5 mM (n=35). Similarly, ATP (100 microM) increased [Cl-]i transiently by 9.5+/-2.2 mM (n=17). The increase in [Cl-]i was reduced by the Na+/K+/2 Cl- -cortransporter-1 (NKCC1) blocker azosemide (100 microM), the CFTR blocker SP-303 (50 microM), the blocker of Ca2+-activated Cl- channels DIDS (100 microM) or the ENaC blocker amiloride (10 microM). Changes in YFP(V163S) fluorescence were not due to changes in cell volume or intracellular pH. The present data thus demonstrate an increase in [Cl-]i following stimulation with secretagogues, which could participate in the inhibition of ENaC.
Adam
Gabriele
G
Institut für Physiologie, Universität Regensburg, Universitätsstrasse 31, 93053, Regensburg, Germany.
Ousingsawat
Jiraporn
J
Schreiber
Rainer
R
Kunzelmann
Karl
K
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001704:Biopolymers / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D053503:Epithelial Sodium Channels; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007685:Kidney Tubules, Collecting / Q000166:cytology*; D008164:Luminescent Proteins / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D015222:Sodium Channels / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2; D013424:Sulfanilamides / Q000494:pharmacology; D013777:Tetrazoles; D013876:Thiophenes; D000096926:Benzenesulfonamides; D044945:Proanthocyanidins
D015056:1-Methyl-3-isobutylxanthine; D001426:Bacterial Proteins; D001704:Biopolymers; D002118:Calcium; D002392:Catechin; D002712:Chlorides; D005576:Colforsin; D000242:Cyclic AMP; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004232:Diuretics; D053503:Epithelial Sodium Channels; D008164:Luminescent Proteins; D010726:Phosphodiesterase Inhibitors; D015222:Sodium Channels; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D013424:Sulfanilamides; C578878:Slc12a2 protein, mouse; C054562:yellow fluorescent protein, Bacteria; C018222:azosemide; D013777:Tetrazoles; D013876:Thiophenes; D000096926:Benzenesulfonamides; C546704:crofelemer; D044945:Proanthocyanidins