126 when studied under short-circuit conditions, exhibit Cl− secretion, which is observed as an amiloride-insensitive component of the short-circuit current (NS-Isc) that is also stimulated by cAMP [2, 3, 5–8]. We previously developed a line of MDCK cells (FL- MDCK) that had been retrovirally transfected with rat ENaC subunits containing the FLAG epitope in their extracellular loops [2]. Monolayers of these cells had higher rates of ENaC-mediated Na+ absorption, measured as the amiloride- sensitive short-circuit current (AS-Isc), than did the original MDCK cell line. Our previous studies with FL-MDCK monolayers in Dulbecco’s modified Eagle’s medium (DMEM) showed that cAMP treatment produced a rapid transient peak in the total short-circuit current (Isc) within 5 min, followed by a broad peak that decayed over 20 min [2]. This biphasic response to cAMP treatment has also been reported in A6 and M-1 cultures and has been attributed to rapid stimulation of Cl− secretion mediated by cystic fibrosis transmembrane conductance regulator (CFTR) followed by a slower activation of ENaC [6–9]. In FL-MDCK cells, the biphasic response to cAMP treatment was followed by a decay of Isc, which reflected a decrease in AS-Isc [2]. Morris and Schafer [2] found that this late fall in AS-Isc in FL-MDCK monolayer was prevented by the omission of Cl− from the bathing solution and attributed the effect to Cl− secretion via CFTR, which has subsequently been demonstrated to be present in this cell line [10]. A large body of evidence indicates that CFTR is a cAMP- regulated Cl− channel as well as a conductance regulator, which is colocalized with ENaC in airway, colonic, and other epithelial tissues [11, 12]. It has been proposed that when cAMP activates CFTR, ENaC is inhibited, and this inhibitory effect of CFTR has been hypothesized to explain the pathophysiology of cystic fibrosis in airway epithelia [13, 14]. In Xenopus oocytes expressing ENaC, coexpression of CFTR reduces the ENaC-mediated Na+ current [15], an effect that might be attributed to a direct effect of CFTR on ENaC activity. However, Kunzelman and his collaborators [16, 17] have shown that the inhibitory effect of CFTR can be mimicked by coexpression of other anion channels or treatment with amphotericin B and is explained by an increase in intracellular Cl− ([Cl−]i). We have shown that an increase in [Cl−]i also inhibits ENaC in FL-MDCK cells; however, this effect cannot explain the effect of Cl− secretion on Na+ absorption with cAMP treatment, because stimulation of Cl− secretion by cAMP results in a fall rather than a rise in [Cl−]i [10]. Moreover, in the Xenopus oocyte expression system, CFTR activation inhibits ENaC at a continuous holding potential at which CFTR mediates inward currents corresponding to Cl− efflux [18]. Under these conditions, activation of CFTR will not result in an increase in [Cl−]i. Therefore, a change in [Cl−]i is unlikely to fully explain the observed reciprocal regulation of ENaC and CFTR. Purinergic Signalling (2008) 4:125–137 In the present studies, we show that adenosine triphos- phate (ATP) accumulation in the apical solution produces a progressive stimulation of NS-Isc and a corresponding decrease in AS-Isc when FL-MDCK cells are studied in Ussing chambers. The possibility of constitutive ATP release raises an important issue when interpreting changes in ion transport in experiments such as these. More important in the present context, ATP release can explain at least part of the inverse relationship between Cl− secretion and Na+ reabsorption, and, as suggested by Wilson et al. [19], it may be involved in the conversion of CD cells from absorptive to secretory in polycystic kidney disease. Materials and methods Cell culture The FL-MDCK cells used were a clone of the type-1 MDCK line that had been retrovirally transfected with flagged rat a-, b-, and gENaC by Morris and Schafer [2]. The cells used were from passages 5–35 and were cultured in T-75 flasks at 37°C in DMEM (Life Technologies) supple- mented with 10% fetal bovine serum (FBS), 50 mM N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4), 1% Pen/strep-fungizone, and the selection anti- biotics G418 (800 μg/ml), hygromycin (300 μg/ml), and puromycin (5 μg/ml). For transepithelial transport studies, cells were seeded onto 24-mm Transwell inserts (Costar; Catalog No. 3412) at a density of ∼105 cells/cm2. For bioluminescence assays, cells were seeded on 12-mm collagen-coated Millicell inserts (Millipore; Catalog No. PHIP 012 50) at the same density. After seeding, cells were fed daily with DMEM containing FBS and HEPES but without selection antibiotics for 5–7 days. Before use in the experiments, the cell monolayers were induced with 1 μM dexamethasone plus 2 mM Na+ butyrate in the culture medium overnight. Ussing chamber experiments FL-MDCK monolayers were carefully cut from the plastic insets and were mounted in Ussing-type chambers. In most experiments, both sides of the monolayers were bathed with Krebs-Ringer bicarbonate (KRB) solution, which contained (in mM): 113 NaCl, 1.2 Na2HPO4, 25 NaHCO3, 1.1 CaCl2, 1.2 MgCl2, 4.5 KCl, and 10 glucose. The KRB solutions were continuously gassed with a mixture of 95% O2-5% CO2 to give a pH of 7.40 at 37°C. As shown by the schematic diagram in Fig. 1a, transepithelial short-circuit currents (Isc, μA/cm2) and voltages (Vte) were measured and conductances were evaluated every 30 s by measuring the current deflections induced by 4-s symmetrical voltage pulses