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 fr om rabbit distal bright convoluted tubule (DCTb). The DC1 clone was obtaine d after transfection of primary cultures of DCTb with pSV3 neo. RT-PCR expe riments showed the presence of cystic fibrosis transmembrane conductance re gulator (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-sulfonato propyl)quinolinium (SPQ) showed that FK induced an increase in Cl- efflux a nd influx in DC1 cells similar to that observed in cultured DCTb cells. I-1 25(-) efflux experiments performed on DC1 cells grown on collagen-coated fi lters showed that exposure of the monolayer to FK led to an increased I-125 (-) loss through the apical membrane only. The addition of 10 mu M adenosin e activated a linear conductance identical to that recorded with FK and cor responding to the CFTR-like conductance. This conductance was also activate d by 5'-(N-ethylcarboxamido)adenosine and CGS-21680 and inhibited in the pr esence of 8-cyclopentyl-1,3-diproxylxanthine (DPCPX). This Cl- conductance could also be activated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S ). The addition of protein kinase A (PKA) inhibitor to the pipette solution inhibited the development of the current activated by CGS-21680. Finally, I-125(-) efflux showed that adenosine induced an apical efflux mediated thr ough basolateral A(2) receptors. Overall, the data show that the DC1 cell l ine expressed an apical CFTR Cl- conductance that could be activated by ade nosine via A(2)A receptors located in the basolateral membrane and involvin g G protein and PKA pathways.