128 Individual batches of luciferin-luciferase reagent were tested for relative sensitivity by measuring the light output response to serial dilutions of an ATP stock solution with 2 mg/ml luciferin-luciferase reagent in Opti-MEM. Data analysis and statistics StatView for Macintosh (SAS Institute Inc.) was used for statistical analyses. Statistical significance (P < 0.05) was determined by analysis of variance (ANOVA) with Bonferroni/Dunn post hoc testing for multiple comparisons and by paired or nonpaired t-tests, as appropriate, for single comparisons. Time dependence of transport was evaluated by linear regression analysis; the R value and significance of the slope are given. Correlation analysis was used to compare AS-Isc and NS-Isc and calculate a correlation coefficient, r. Materials All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. Results Time-dependent changes in Isc The Isc in FL-MDCK cells was quite high and variable in the first 5 min after recording was begun using continuous short- circuiting. In six experiments, 10 μM amiloride added to the apical solution in the initial 5 min decreased Isc by 91± 2%, indicating that most of the current was due to ENaC- mediated Na+ absorption. The initial high Isc subsequently declined to a nadir at ∼15 min (Fig. 2a and b). Addition of amiloride at this nadir (Fig. 2a) almost completely inhibited Isc; however, when the experiment was continued beyond the nadir, amiloride was less effective in reducing Isc (Fig. 2b). In 35 experiments such as those in Fig. 2a and b, the time interval between the nadir and amiloride addition to the apical solution was varied from 0 to 45 min. In these experiments, the average Isc at the nadir was 15.2 ±0.5 μA/ cm2. Regression analysis showed that NS-Isc increased significantly (R = 0.76, P < .001) and AS-Isc decreased signif- icantly (R = 0.53, P < .002) as a function of the time between the nadir and the addition of amiloride. The same high initial Isc and the time-dependent increase of NS-Isc were also observed in experiments using the intermittent short-circuit technique (see “Materials and methods”), which indicated that neither effect is produced by prolonged short-circuiting of the epithelium (data not shown). As shown in Fig. 3, the effect of cAMP on Isc also depended on whether the treatment occurred early (at the nadir of Isc, Fig. 3a) or late (∼40 min after the nadir, Purinergic Signalling (2008) 4:125–137 Fig. 3b). With both early and late cAMP addition, Isc increased immediately, reaching a transient peak within 5 min; however, the magnitude of this initial peak was exaggerated with late cAMP treatment (Fig. 3b). After the initial response, Isc showed a secondary broader increase that decayed more rapidly after late (Fig. 3b) than after early (Fig. 3a) cAMP treatment. In 29 experiments such as those in Fig. 3, the time interval between the nadir in Isc and cAMP treatment was varied from 2 to 58 min, and 10 μM amiloride was added ∼30 min after cAMP treatment. Regression analysis showed a significant increase in NS-Isc with the time between the nadir and cAMP treatment (R= 0.60, P < .002). Further examination of the data from these experiments (see Fig. 4) revealed a significant negative correlation Fig. 2 Time course of the short-circuit current (Isc) and response to amiloride. Isc was measured across monolayers of FL-Madin-Darby canine kidney (FL-MDCK) cells induced overnight with 2 mM Na butyrate and 1 μM dexamethasone. Amiloride (10 μM) was added to the apical solution either early (a) or late (b) at the point indicated by the arrow. The transepithelial conductance (Gte) is proportional to the height of the deflections in Isc produced by transiently clamping the voltage to ±2 mV, as described in “Materials and methods”