Purinergic Signalling (2008) 4:125–137 continuously perfused with fresh KRB solutions, as shown in Fig. 1c. The representative control experiment shown in Fig. 7a was conducted in the usual way: the apical and basolateral KRB solutions were recirculated through both sides of the Ussing chamber from 10-ml reservoirs. In the paired experiment in Fig. 7b, we continuously perfused fresh KRB on both sides of the Ussing chamber. In both the recirculation and fresh perfusion experiments, cAMP treatment produced an initial transient spike of Isc lasting less than 6 min, followed by a broader peak; however, Isc decayed more rapidly in the recirculation experiment than it did in the fresh perfusion experiment. In four sets of paired experiments summarized in Fig. 7c, AS- Isc was greater (39.8± 1.1 μA/cm2 vs. 25.2±3.3 μA/cm2, P < 0.01) and NS-Isc was less (2.8± 0.2 μA/cm2 vs. 7.7± 1.6 μA/cm2, P < 0.01) with fresh perfusion than in the recirculation group, suggesting that some substance, which inhibited AS-Isc and stimulated NS-Isc, was accumulating in the recirculated bathing solution. Effect of extracellular ATP on time-dependent changes in Isc We hypothesized that ATP was released by the FL-MDCK cells and accumulated in the recirculated solutions. Extracel- lular nucleotides are known to act as autocrine and paracrine agents that affect Na+ absorption and Cl− secretion, and ATP has been shown to increase intracellular [Ca2+] [24, 25], which might augment the Ca2+-activated component of NS- Isc as well as inhibit ENaC activity. As shown in Fig. 8a, in the presence of amiloride, the addition of 100 μM ATP to the apical solution increased NS-Isc, and, as shown in Fig. 8b, this effect was almost completely abolished when the ATP was added together with hexokinase as an ATP scavenger [26]. (These experiments were conducted using the recircu- lated KRB method.) In five experiments such as that in Fig. 8a, basal NS-Isc (just before ATP addition) averaged 4.1± 0.1 μA/cm2. With the addition of ATP, NS-Isc reached a peak value of 15.4±2.2 μA/cm2 (P< 0.004) within 2–3 min, and then declined gradually to a plateau of 5.9±0.3 μA/cm2, which was still significantly larger than the basal level (P< 0.01). Addition of 300 μM DIDS to the apical solution before the addition of ATP had no effect on the peak amplitude (14.6 ± 0.7 μA/cm2, n = 3), but decreased the plateau value to 4.5±0.2 μA/cm2 (n= 3), which was not significantly different from the basal value. To test whether endogenous ATP production by the epithelium might be involved in the time-dependent increase of NS-Isc and decrease of AS-Isc, we examined the effect of hydrolyzing extracellular ATP with hexokinase on the time course of NS-Isc and AS-Isc. Paired experiments without or with hexokinase were conducted as shown, respectively, in Fig. 9a and b, both using the recirculated KRB method.. With hexokinase in the bathing solutions, the response of Isc 131 Fig. 6 Effect of thapsigargin on the nonamiloride-sensitive compo- nent (NS-Isc) and prevention of that effect by 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS). All experiments were performed in the presence of 10 μM luminal amiloride. a Mean NS-Isc values for seven experiments at four time points: just before adding thapsigargin to the apical and basolateral solutions, 3–5 min after addition of 1 μM thapsigargin to the apical and basolateral solutions (TG-initial), 15– 20 min after thapsigargin addition (TG-late), and 3–5 min after the addition of 300 μM DIDS to the apical solution. * Significantly different compared with NS-Isc before thapsigargin treatment, P<.001; † significantly different compared with both the initial and late NS-Isc after thapsigargin, P<.001. b Mean values of the change in NS-Isc produced by thapsigargin: the initial peak (gray bars) and the late response (open bars) for control experiments (as in a) and in three experiments in which 300 μM DIDS was added before thapsigargin. * Significantly different compared with the corresponding control values, P<.001 to cAMP treatment was exaggerated, and the broader peak was more sustained (Fig. 9b). As shown by the summary of four such sets of paired experiments (Fig. 9c), hexokinase had no effect on the basal Isc measured just before cAMP addition; however, after cAMP, AS-Isc was significantly higher (40.4 ± 2.9 μA/cm2 vs. 29.7 ± 2.8 μA/cm2, P < 0.05) with hexokinase, whereas the DIDS-sensitive NS-Isc was not significantly different from zero and significantly less