130 responds to cAMP quickly while the other is sensitive to DIDS and responds to cAMP slowly. The Cl− channel that was quickly activated by cAMP and inhibited by glibenclamide has been attributed to CFTR, the expression and function of which has been identified by us in this same cell line [10]. Because cAMP increases intracellular Ca2+ ([Ca2+]i) in MDCK cells [21], we examined the hypothesis that CaCC contributes to the fraction of NS-Isc that is inhibited by DIDS by using thapsigargin to produce a modest but sustained elevation of [Ca2+]I [22, 23]. (Thapsigargin is known to elevate intra- cellular Ca2+ by releasing it from the endoplasmic reticulum [22].) In the experiments shown in Fig. 6, NS-Isc was measured in the continuous presence of luminal amiloride. The addition of 1 μM thapsigargin to both sides of the monolayer produced a transient spike followed by a Purinergic Signalling (2008) 4:125–137 sustained increase in NS-Isc, most of which was sensitive to DIDS (Fig. 6a). When 300 μM DIDS was added to apical solution before thapsigargin, it almost completely blocked the response of NS-Isc to thapsigargin (Fig. 6b). Effect of Ussing chamber perfusion on time-dependent changes in Isc In all of the experiments presented up to this point, the KRB solutions that bathed the MDCK monolayers were recirculated as shown in Fig. 1b. To examine whether the time-dependent changes in AS-Isc and NS-Isc might be caused by the accumulation of a secretagogue or metabolite in these recirculated solutions, we conducted paired experi- ments in which one Ussing chamber was perfused with recirculated KRB solutions and the other chamber was Fig. 5 Effects of glibenclamide and 4, 4'-diisothiocyano-2, 2'- disulfonic acid stilbene (DIDS) on the nonamiloride-sensitive compo- nent (NS-Isc). In these experiments, 10 μM amiloride was continuously present in the apical solution. a Effect of cyclic adenosine mono- phosphate (cAMP) treatment [100 μM cAMP plus 100 μM isobu- tylmethylxanthine (IBMX), lowest bar] on NS-Isc. The middle and upper bars indicate the time of addition of 300 μM DIDS and 200 μM glibenclamide (Gliben.) to the apical solution. b Summary of 14 experiments such as that in a. The mean NS-Isc values immediately before cAMP treatment (point 1 in a), 3–5 min (“initial”, point 2) and 15–20 min (“late”, point 3, just before DIDS or glibenclamide addition) after cAMP treatment, and after DIDS plus glibenclamide (point 4). * Significantly different from value before cAMP treatment, P<.001; † significantly different from the initial and late NS-Isc, P <.001. c Effect of 300 μM apical DIDS added before cAMP treatment. d Effect of 200 μM apical glibenclamide added before cAMP treatment. e Mean values of the change in NS-Isc (the cAMP- stimulated NS-Isc) produced by cAMP treatment in the initial (point 2 in a, c, and d) or late (point 3) response period in three sets of experiments: with no inhibitor (control, from summary of 15 experi- ments in b), DIDS before cAMP (as in c, n=6), and glibenclamide before cAMP (as in d, n=5). * Significant difference compared with the corresponding control value (calculated from the data in b), and between initial and late response to cAMP, P<.001