Purinergic Signalling (2008) 4:125–137 133 Fig. 8 Effect of adenosine triphosphate (ATP) on the nonamiloride- sensitive component (NS-Isc) and prevention of that effect by hexokinase. All experiments were performed in the presence of 10 μM apical amiloride. a Effect of 100 μM ATP added to the apical solution on NS-Isc. Mean values of five such experiments are given in the text. b Effect of 100 μM ATP on NS-Isc. In these experiments, 6 U/ml hexokinase was continuously present in the bathing solution. The same results were obtained in three such experiments amiloride added to the apical solution. The initial Isc fell to a nadir within 15 min and then increased slowly (Figs. 2b and 3b) due to an increase in NS-Isc. This progressive rise of NS-Isc occurred consistently regardless of whether amiloride was present or not and whether the epithelium was continuously short-circuited or was left in the open- circuit condition with only intermittent short-circuiting to measure Isc. Moreover, the time-dependent increase of NS- Isc could not be inhibited by the further addition of amiloride but was partially blocked by apical DIDS, which is consistent with anion secretion (see below). The time course of the Isc response to cAMP using these FL-MDCK cells was like that observed in the previous studies of Morris and Schafer [2] and in other studies of similar epithelia [6, 9, 27]. There was an initial transient increase lasting less than 6 min, followed by a broader peak, which decayed over the next 30 min (Fig. 3). Morris and Schafer [2] have shown that when these experiments are conducted in a Cl−-free solution, the initial transient spike was absent and Isc was stable for at least 30 min. The initial transient and the broader peak of Isc were attributed to, respectively, stimulation of Cl− secretion and Na+ absorption by cAMP [2]. Interestingly, the effect of cAMP on Isc was also time dependent. With late-cAMP treatment (Fig. 3b), the decay of Isc was faster and the magnitude of NS-Isc was larger. As shown in Fig. 4, there was a significant inverse correlation between AS-Isc and NS-Isc both in the absence and in the presence of cAMP. This correlation does not indicate a causal relationship between the rise in NS-Isc and the fall in AS-Isc, but suggests that whatever process is responsible for the time dependence produces reciprocal changes in NS-Isc and AS-Isc. Cl− channels associated with NS-Isc As discussed previously [2], we attributed NS-Isc to Cl− secretion because in the presence of apical amiloride, Cl− was the only ion present in sufficient concentration to account for it. Furthermore, glibenclamide blocked the initial rise in NS-Isc in response to subsequent cAMP treatment, whereas DIDS blocked the broader late transport response but not the early peak (Fig. 5). Glibenclamide is a well-established inhibitor of CFTR at the concentration (200 μM) used in these experiments. Although glib- enclamide also inhibits ATP-activated K+ channels [28, 29], previous studies have shown that the apical membrane of MDCK cells has no measurable K+ conductance [10, 30]. Although DIDS effectively inhibits Cl− channels such as CaCC as well as the outwardly rectifying Cl− channel (ORCC), and the CLC family of Cl− channels, it does not inhibit CFTR [31, 32]. These results indicate that at least two types of Cl− channels contribute to NS-Isc and that they respond differently to cAMP. Based on these data, we have attributed the early, glibenclamide-sensitive, increase in NS-Isc after cAMP treatment to CFTR, which we have demonstrated is expressed in the apical membrane of these FL-MDCK cells [10]. CaCC appears to be responsible for the later increase in NS-Isc after cAMP treatment. As shown in Fig. 6, we found that thapsigargin, which moderately increases [Ca2+]i [22], produced a transient spike and a sustained increase in NS- Isc that could be completely blocked by apical DIDS as expected for a CaCC [33]. Furthermore, cAMP increases [Ca2+]i in MDCK cells [21], and thus a CaCC may contribute to the DIDS-sensitive component of the NS-Isc response to cAMP. A CaCC has also been described in the M-1 and mIMCD-K2 cell lines [34, 35] and in primary