A(1) RECEPTOR ANTAGONIST 8-CYCLOPENTYL-1,3-DIPROPYLXANTHINE SELECTIVELY ACTIVATES CHLORIDE EFFLUX FROM HUMAN EPITHELIAL AND MOUSE FIBROBLAST CELL-LINES EXPRESSING THE CYSTIC-FIBROSIS TRANSMEMBRANE REGULATOR DELTA-F508 MUTATION

Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mut ations in the cystic fibrosis transmembrane regulator (CFTR). The A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) stimulat es Cl-36(-) efflux from pancreatic CFPAC-1 cells which bear the Delta F508 genotype common to most cases of cystic fibrosis [Eidelman et al, (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5562-5566]. By contrast, cor rection of the cystic fibrosis defect by retrovirus-mediated gene tran sfer renders the resulting CFPAC-PLJ-CFTR cells insensitive to CPX. We now report that CPX also activates chloride efflux from the CF trache al epithelial cell line IB3-1 bearing a Delta F508 allele, but not if the IB3-1 cells have been repaired by transfection of the wild-type CF TR gene. Similar results were obtained with recombinant NIH 3T3 cells, in which CPX activates Cl-36(-) efflux from cells expressing the CFTR (Delta F508) gene product but not from 3T3 cells expressing the wild-t ype CFTR. In all three cell types expressing CFTR(Delta F508), CPX was found to activate Cl-36(-) efflux in a dose-dependent manner over the concentration range of 1-30 nM and then gradually lose potency at hig her CPX concentrations, Six CPX analogues, A(1) receptor antagonists o f affinity similar to that of CPX, were found to be much less effectiv e than CPX at activating Cl-36(-) efflux from CFPAC-1 cells. These inc luded 2-thio-CPX, CPT (8-cyclopentyl-1,3-dimethylxanthine), 3,4-dehydr o-CPX, 3-F-CPX, 3-I-CPX, and KW-3902 (8-noradamantyl-1,3-dipropylxanth ine). We conclude from these studies that CPX can activate chloride ef flux from CF epithelial cells in which the CFTR(Delta F508) genotype i s present and the wild-type CFTR gene is absent. The presence of exces s wild-type CFTR in repaired CFPAC-1 and 3T3-CFTR cells was verified b y Western blot analysis. In addition, the nature of the gene transfer vehicle does not seem to be important for the loss of sensitivity to C PX, The fact that this relationship can also be demonstrated with mous e 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 A(1) antagonists lead us to ques tion the hypothesis that the CPX effects on CF cells occur via interac tions with a classical A(1) receptor. Alternative possibilities includ e either direct action of CPX upon the Delta F508 mutant of CFTR or ac tion on a yet-to-be-characterized purine binding site common to both h uman and mouse cells.