BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 227, 755–761 (1996) ARTICLE NO. 1581 CFTR Expression in C127 Cells Is Associated with Enhanced Cell Shrinkage and ATP Extrusion in Cl 0 -Free Medium Bianca Maria Rotoli,* Ovidio Bussolati,* Valeria Dall’Asta,* Else K. Hoffmann,² Giulio Cabrini,‡ and Gian C. Gazzola* ,1 *Istituto di Patologia Generale, Universita ` degli Studi di Parma, Italy; ² August Krogh Institutet, Københavns Universitet, Copenhagen, Denmark; and ‡Centro Fibrosi Cistica, Ospedale Civile Maggiore, Verona, Italy Received September 16, 1996 In this study we have employed three lines of C127 murine cells, C127 CFTRw/t, C127 CFTRDF508 and C127 mock, transfected with, respectively, wild type, DF508 mutant human CFTR cDNA or the vector only. In the first 10 minutes of a Cl 0 -free incubation the three cell lines exhibit a significant shrinkage due to a loss of K / and Cl 0 . However, C127 CFTRw/t cells shrink more than C127 CFTRDF508 and the mock cells. The supplementation of Cl 0 -free medium with ATP causes a marked decrease in the cell volume of C127 CFTRDF508 and of the mock cells but not of C127 CFTRw/t cells. ATP effect is mimicked by adenosine 5-O-(3-thiotriphosphate), but neither by adenosine nor by UTP. Measurements of extracellular ATP indicate that during the Cl 0 -free incubation C127 CFTRw/t cells extrude more ATP than the other two cell lines. The results are consistent with the hypothesis that CFTR enhances K / and Cl 0 permeabilities by promoting the extrusion of ATP.  1996 Academic Press, Inc. CFTR, the protein mutated in cystic fibrosis, is expressed in a variety of secreting epithelia where it is involved in ion transport (1, 2). It is well established that one major function of the CFTR protein is to serve as a cAMP-activated chloride channel (3-5). However, an enlarging body of experimental data points to additional functions of the protein. In particular, evidence has been obtained in support of the hypothesis that CFTR modulates other chloride channels (6-9). It has been recently proposed that these effects may occur via an autocrine mechanism involving ATP (10) which would be transported by CFTR protein (10, 11). Once at the extracellular side of the membrane, ATP would modulate the channel function either of CFTR itself (11, 12) or of other Cl 0 channels (10). More recently, an activating effect of CFTR on K / permeability has been also described (13). However, data from other Authors do not support a direct role of CFTR in mediating transmembrane ATP fluxes (14). In a variety of cell models, the combined activation of Cl 0 and K / channels is accompanied by a decrease of cell volume (15). Thus, if CFTR activates both Cl 0 and K / channels, it should also influence the rate of cell volume changes. Indeed, Valverde et al. (13) have demonstrated that regulatory cell shrinkage after hypotonic swelling is altered in a mouse model of cystic fibrosis. In order to evaluate the relationships between CFTR expression and the rate of cell volume changes, we have incubated murine C127 cells, transfected with either wild type or DF508 1 To whom correspondence should be addressed. Fax: /39 521 980388. Abbreviations employed are: a,b-MeATP, a,b-methylene ATP; ATPgS, adenosine 5-O-(3-thiotriphosphate); C127 CFTRw/t, C127i cells expressing CFTR wild type; C127 CFTRDF508, C127i cells expressing CFTR bearing DF508 mutation; C127 mock, C127i cells transfected with vector only; CF, cystic fibrosis; CFTR, cystic fibrosis transmem- brane conductance regulator; DMEM, Dulbecco’s modified Eagle medium; EBSS, Earle’s balanced salt solution; FBS, fetal bovine serum. 0006-291X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 755