Regulation of cAMP-dependent chloride channels in DC1 immortalized rabbit distal tubule cells in culture.
Cl- conductances were studied in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule (DCTb). The DC1 clone was obtained after transfection of primary cultures of DCTb with pSV3 neo. RT-PCR experiments showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in the DC1 cell line. Using the whole cell patch-clamp technique, we recorded a linear Cl- conductance activated by forskolin (FK). This conductance was insensitive to DIDS and corresponded to a CFTR-like channel conductance. Fluorescence experiments with 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ) showed that FK induced an increase in Cl- efflux and influx in DC1 cells similar to that observed in cultured DCTb cells. 125I- efflux experiments performed on DC1 cells grown on collagen-coated filters showed that exposure of the monolayer to FK led to an increased 125I- loss through the apical membrane only. The addition of 10 microM adenosine activated a linear conductance identical to that recorded with FK and corresponding to the CFTR-like conductance. This conductance was also activated by 5'-(N-ethylcarboxamido)adenosine and CGS-21680 and inhibited in the presence of 8-cyclopentyl-1, 3-diproxylxanthine (DPCPX). This Cl- conductance could also be activated by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The addition of protein kinase A (PKA) inhibitor to the pipette solution inhibited the development of the current activated by CGS-21680. Finally, 125I- efflux showed that adenosine induced an apical efflux mediated through basolateral A2 receptors. Overall, the data show that the DC1 cell line expressed an apical CFTR Cl- conductance that could be activated by adenosine via A2A receptors located in the basolateral membrane and involving G protein and PKA pathways.
Rubera
I
I
Unite Mixte de Recherche Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, O6108 Nice Cedex 2, France.
Tauc
M
M
Verheecke-Mauze
C
C
Bidet
M
M
Poujeol
C
C
Touret
N
N
Cuiller
B
B
Poujeol
P
P
D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D002461:Cell Line, Transformed; D002463:Cell Membrane Permeability / Q000187:drug effects; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D005456:Fluorescent Dyes; D007454:Iodides / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000378:metabolism*; D008297:Male; D011806:Quinolinium Compounds; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000502:physiology
D018118:Chloride Channels; D002712:Chlorides; D005456:Fluorescent Dyes; D007454:Iodides; D011806:Quinolinium Compounds; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium; D000242:Cyclic AMP; D000241:Adenosine