Purinergic Signalling (2008) 4:125–137 129 Fig. 3 Time course of Isc response to cyclic adenosine monophosphate (cAMP) treatment. A mixture of 100 μM 8-(4-chlorophenylthio) (CPT)- cAMP plus 100 μM isobutylmethylxanthine (IBMX) was added as indicated by the lower bar, either early (a) or late (b) to both sides of the monolayers, followed by 10 μM amiloride addition to the apical solution only (upper bar) between AS-Isc and NS-Isc, both with and without cAMP treatment. In other words, the time-dependent increase in NS- Isc observed in both sets of experiments was correlated with a corresponding decrease in AS-Isc. As expected, the average AS-Isc was significantly greater in those experiments with cAMP treatment (20.2±1.1 μA/cm2, n= 29) than in those without (12.8± 0.6, n = 35), but the correlation with NS-Isc was not significantly different between the two groups. Effect of cAMP and intracellular Ca2+ on NS-Isc In the experiments shown in Fig. 5, NS-Isc was measured throughout the experiments in the continuous presence of 10 μM amiloride in the apical solution. Treatment with cAMP produced an initial rapid peak in NS-Isc, followed by a sustained plateau. We then tested the effects of two inhibitors of Cl− transport pathways: glibenclamide, which is a well-established inhibitor of CFTR; and 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS), which is a less selective inhibitor of Cl− channels including the Ca2+-activated chloride channel (CaCC) and the chloride channel (CLC) family of Cl− channels but not CFTR. (See the Discussion for more detail about these inhibitors and references to their specificity.) Addition of 200 μM glibenclamide followed by 300 μM DIDS to the apical solution ∼20 min after cAMP treatment significantly decreased NS-Isc to or below the magnitude prior to cAMP treatment (Fig. 5a & b). In other experiments, addition of apical DIDS before cAMP treatment reduced the late plateau but not the initial peak of NS-Isc (Fig. 5c and e), whereas apical glibenclamide significantly decreased the initial peak, with no effect on the late plateau (Fig. 5d & e). These data suggest that at least two types of Cl− channels are stimulated by cAMP. One is sensitive to glibenclamide and Fig. 4 Negative correlation between the amiloride-sensitive compo- nent of the short-circuit current ( Isc) (AS-Isc) and the nonamiloride- sensitive component (NS-Isc) in the absence and presence of cyclic adenosine monophosphate (cAMP). The 35 experiments in the first group (open circles, solid line) were conducted using the same protocol as the representative experiments in Fig. 1 with no cAMP addition. The 29 experiments in the second group (solid dots, dashed line) followed the protocol of the representative experiments in Fig. 2, with the addition of 100 μM 8-(4-chlorophenylthio) (CPT)-cAMP plus 100 μM isobutylmethylxanthine (IBMX) ∼30 min before the addition of amiloride. In both groups, there was a significant correlation between AS-Isc and NS-Isc: −cAMP group, r=0.79, P <.001; +cAMP group, r=0.65, P<.001