Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) stimulates 36C1- efflux from pancreatic CFPAC-1 cells which bear the ∆F508 genotype common to most cases of cystic fibrosis [Eidelman et al. (1992) Proc. Natl. Acad. Sci. U.SA. 89, 5562-5566]. By contrast, correction of the cystic fibrosis defect by retrovirus-mediated gene transfer renders the resulting CFPAC-PLJ-CFTR cells insensitive to CPX. We now report that CPX also activates chloride efflux from the CF tracheal epithelial cell line IB3-1 bearing a ∆F508 allele, but not if the IB3-1 cells have been repaired by transfection of the wild-type CFTR gene. Similar results were obtained with recombinant NIH 3T3 cells, in which CPX activates 36C1- efflux from cells expressing the CFTR(∆F508) gene product but not from 3T3 cells expressing the wild-type CFTR. In all three cell types expressing CFTR(∆F508), CPX was found to activate 36Cl- efflux in a dose-dependent manner over the concentration range of 1-30 nM and then gradually lose potency at higher CPX concentrations. Six CPX analogues, A1 receptor antagonists of affinity similar to that of CPX, were found to be much less effective than CPX at activating 36C1- efflux from CFPAC-1 cells. These included 2-thio-CPX. CPT (8-cyclopentyl-1,3-dimethylxanthine), 3,4-dehydro- CPX, 3-F-CPX, 3-I-CPX, and KW-3902 (8-noradamantyl- 1,3-dipropylxanthine). We conclude from these studies that CPX can activate chloride efflux from CF epithelial cells in which the CFTR(∆F508) genotype is present and the wild-type CFTR gene is absent. The presence of excess wild-type CFTR in repaired CFPAC-1 and 3T3-CFTR cells was verified by Western blot analysis. In addition, the nature of the gene transfer vehicle does not seem to be important for the loss of sensitivity to CPX. The fact that this relationship can also be demonstrated with mouse 3T3 cells indicates that the CPX effect is not exclusively dependent upon a human epithelial cell substrate. Finally, the comparative data obtained with a variety of selective Ai antagonists lead us to question the hypothesis that the CPX effects on CF cells occur via interactions with a classical A1 receptor. Alternative possibilities include either direct action of CPX upon the ∆F508 mutant of CFTR or action on a yet-to-be-characterized purine binding site common to both human and mouse cells.
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