132 than in the control group (−0.3 ± 0.2 μA/cm2 vs. 3.6 ± 0.7 μA/cm2, P < 0.01). Thus, all of the above data were consistent with the hypothesis that the accumulation of extracellular ATP causes the time-dependent increase of NS- Isc and decrease of AS-Isc that were observed in those experiments in which the bathing solutions were recirculated. Purinergic Signalling (2008) 4:125–137 Measurement of ATP released from MDCK cells ATP released from polarized MDCK cells was determined by a bioluminescence assay, in which a luminometer was used to measure light production from ATP consumption in the luciferin-luciferase reaction [20]. MDCK cells were grown to confluence in 12-mm permeable supports (Milli- cell), and the ATP was measured in the apical or basolateral solution by adding the reagents only to that chamber. As shown in Fig. 10, under basal conditions, apically directed ATP release produced a light emission of 16.4 ± 2.1 ALU (artificial light units), whereas basolaterally directed ATP release was nearly zero (0.7 ± 0.2 ALU). In ten paired experiments, addition of 100 μM cAMP and 100 μM IBMX to the apical solution significantly increased the apically directed ATP release from 16.0 ± 2.1 ALU (basal) to 51.1 ± 9.4 ALU (n = 10, P < 0.005), whereas addition of the same volume of solution without cAMP and IBMX (sham control for the possible effects of manipulating the monolayers) had no significant effect (17.4 ± 3.5 ALU basal vs. 15.1 ± 2.3 ALU sham; n = 4, P= 0.29). Treatment with cAMP had no significant effect on basolaterally directed ATP release (0.8 ± 0.3, n = 8, P > 0.5). Discussion Previous work [10] has shown that the triply transfected FL- MDCK cells used in these experiments (a subclone of αFβ F+F MDCK cells used in the experiments of Morris et al. [2]) expressed all three rat-ENaC subunits and CFTR, and that it was a very useful model epithelium for the study of ENaC regulation. In the current study, we examined in greater detail the relationship between Cl− secretion and Na+ absorption in this epithelium. Time-dependent changes in NS-Isc and AS-Isc FL-MDCK cells uniformly exhibited a high initial Isc, which was almost completely eliminated by 10 μM R Fig. 7 Time course of the short-circuit current (Isc) in experiments with and without recirculation of the bathing solutions. The sequence of treatments was the same in these paired experiments: 100 μM cyclic adenosine monophosphate (cAMP) plus 100 μM isobutylme- thylxanthine (IBMX) were added to both apical and basolateral solutions, followed by 10 μM amiloride to the apical solution. a Apical and basolateral Krebs-Ringer bicarbonate (KRB) solutions were recirculated through the two sides of the Ussing chamber from 10-ml reservoirs. 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS) (300 μM) was added to the apical solution near the end of the experiment. b In this paired experiment, fresh KRB solution was perfused through both sides of the Ussing chamber at the same rate (1 ml/min) as for the recirculated solutions in a. c Summary results for four sets of paired experiments such as those in a and b. *Significantly different from control (with recirculation as in a), P<.001