Purinergic Signalling (2008) 4:125–137 DOI 10.1007/s11302-007-9053-3 ORIGINAL PAPER Endogenous ATP release inhibits electrogenic Na+ absorption and stimulates Cl− secretion in MDCK cells Yi Xie & James A. Schafer Received: 7 December 2006 / Accepted: 2 February 2007 / Published online: 8 March 2007 # Springer Science + Business Media B.V. 2007 Abstract Our previous studies with a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) transfected with FLAG-labeled a, b, and g subunits of epithelial Na+ channel (ENaC) showed that, although most of the short- circuit current (Isc) was amiloride sensitive (AS-Isc), there was also an amiloride-insensitive component (NS-Isc) due to Cl− secretion (Morris and Schafer, J Gen Physiol 120:71– 85, 2002). In the present studies, we observed a progressive increase in NS-Isc and a corresponding decrease in AS-Isc during experiments. There was a significant negative correlation between AS-Isc and NS-Isc both in the presence and absence of treatment with cyclic adenosine mono- phosphate (cAMP). NS-Isc could be attributed to both cystic fibrosis transmembrane conductance regulator (CFTR) and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)- sensitive Ca2+-activated Cl− channel (CaCC). Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions, or addition of hexokinase (6 U/ml), prevented the time-dependent changes and increased AS-Isc by 40–60%, with a proportional decrease in NS-Isc. Addition of 100 μM adenosine Y. Xie : J. A. Schafer Department of Physiology and Biophysics, and Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA J. A. Schafer (*) Departments of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., Rm. 834 MCLM, Birmingham, AL 35294-0005, USA e-mail: jschafer@uab.edu Present address: Y. Xie Department of Physiology, UCLA School of Medicine, 650 Charles Young Dr. S. 3-609 MRL, Los Angeles, CA 90095-1751, USA triphosphate (ATP) in the presence of luminal amiloride produced a transient four-fold increase in NS-Isc that was followed by a sustained increase of 50–60% above the basal level. ATP release from the monolayers, measured by bioluminescence, was found to occur across the apical but not the basolateral membrane, and the apical release was tripled by cAMP treatment. These data show that constitu- tive apical ATP release, which occurs under both basal and cAMP-stimulated conditions, underlies the time-dependent rise in Cl− secretion and the proportional fall in ENaC- mediated Na+ absorption in FL-MDCK cells. Thus, endog- enous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells, and it may underlie at least some of the observed interaction between Cl− secretion and Na+ absorption. Keywords ATP. CFTR . CLC . Calcium-activated Cl− channel . ENaC . Purinergic effects Introduction Madin-Darby canine kidney (MDCK, type I) cells grown as epithelial monolayers have been used extensively as a model of the mammalian collecting duct (CD). These monolayers exhibit many characteristics of the CD, including a high transepithelial resistance and amiloride- sensitive Na+ reabsorption that is mediated by the epithelial Na+ channel (ENaC) and stimulated by agonists that increase intracellular cyclic adenosine monophosphate (cAMP) [1, 2]. Similar cell lines that are derived from the CD, including mouse cortical CD (M-1) and inner medullary CD (mIMCD-K2) cells [3–5] and A6 cells from the Xenopus distal nephron [6, 7] share these character- istics. However, in contrast to the CD, all of these cell lines also have Cl− channels in their apical membranes, and,