134 cultures of rabbit proximal and distal tubule cells [36]. It should also be noted, however, that the molecular composi- tion of CaCC is not as yet know. Thus, we were unable to provide unequivocal evidence for its existence in the FL- MDCK cell line. Purinergic Signalling (2008) 4:125–137 Endogenous ATP release inhibits amiloride-sensitive Na+ absorption When the MDCK monolayers were continuously perfused with fresh apical and basolateral KRB solutions rather than recirculated solutions, the time-dependent increase of NS-Isc and decrease of total Isc was prevented (Fig. 7). AS-Isc was also significantly increased during the continuous perfu- sion, suggesting that inhibition of ENaC was prevented by washing out accumulated metabolites (Fig. 7c). Further- more, our experiments showed that hexokinase, a scavenger of extracellular ATP, produced the same effects on AS-Isc and NS-Isc as the perfusion of fresh KRB solutions (Fig. 9). In the presence of hexokinase, AS-Isc was significantly higher but the DIDS-sensitive NS-Isc was significantly lower than in control experiments, indicating that extracel- lular ATP caused the inhibition of ENaC and activation of NS-Isc, the later possibly via a CaCC. Extracellular ATP is a well-established agonist for purinergic receptors, leading to an increase of intracellular [Ca2+] and activation Ca2+-activated Cl- channels [24, 37, 38]. In parallel, ATP attenuates amiloride-sensitive Na+ absorption in a variety of tissues including airway [39–41] and renal epithelia [42–44]. Similarly, activation of CFTR also increases Cl− secretion and inhibits ENaC in native epithelial tissues [14, 45]. Because CFTR has been suggested to potentiate ATP release [46, 47] and it has been shown to be expressed in this MDCK cell line [10], we examined a possible contribution of ATP release to the time-dependent transport in FL-MDCK cells. As shown in Fig. 8a, addition of 100 μM ATP stimulated anion secretion, possibly via a Ca2+-activated Cl− channel, which was inhibited by DIDS, and this effect was prevented in the presence of hexokinase (Fig. 8b). Endogenous release of ATP by the FL-MDCK mono- layers was demonstrated by the bioluminescence assay experiments shown in Fig. 10. Consistent with measure- ments in cortical CD (see [48]), ATP was shown to be released into the apical solution under basal conditions, and this release was augmented more than three-fold by cAMP Fig. 9 Time course of the short-circuit current (Isc) response to cyclic adenosine monophosphate (cAMP) treatment in the absence and presence of hexokinase. Both types of experiments followed the same general protocol: A mixture of 100 μM (4-chlorophenylthio) (CPT)- cAMP plus 100 μM isobutylmethylxanthine (IBMX) was added as indicated, followed by 10 μM apical amiloride and 300 μM apical 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS). a In this experiment, the Krebs-Ringer bicarbonate (KRB) solution had no hexokinase added. b A paired experiment in which 6 U/ml hexokinase was continuously present in apical and basolateral bathing solution. c Average data for four paired experiments: the mean values of Isc immediately before cAMP was added (Basal-Isc), amiloride-sensitive (AS-Isc) at 30 min after cAMP addition (AS-Isc), and DIDS-sensitive Isc (DS-Isc). * Significantly different compared with the corresponding control values