Purinergic Signalling (2008) 4:125–137 of ±2 mV, as described previously [2, 10]. In most experi- ments, KRB was continuously recirculated on both sides of the epithelium from 10-ml reservoirs (Fig. 1b); however, in the experiments involving “fresh perfusion,” as shown in Fig. 1c, both sides of the monolayers were continuously perfused with fresh rather than recirculated KRB. In those experiments involving cAMP treatment, 100 μM 8-(4- chlorophenylthio)-cAMP (CPT-cAMP) plus 100 μM isobu- tylmethylxanthine (IBMX) were added to both the apical 127 and basolateral solutions. AS-Isc was defined as the change in Isc produced by the addition of 10 μM amiloride to the apical solution, and the remaining Isc was defined as NS-Isc. Because we observed time-dependent changes in AS-Isc and NS-Isc in the voltage clamp experiments, we conducted additional experiments in which the monolayers were left under open-circuit conditions during the course of the experiment, and the open-circuit transepithelial voltage (Vte, mV) was measured continuously except for intermittent voltage clamping to 0 mV and ±2 mV for a total of 4 s every 30 s to obtain Isc and conductance measurements. Bioluminescence detection of ATP release ATP released from the MDCK monolayers was measured as described by Taylor et al. [20]. Briefly, culture medium was removed from the inserts, and the cell monolayers were washed three times with phosphate-buffered saline (PBS) solution to remove any FBS present in the culture medium. Opti-minimal essential medium (MEM) with 2 mg/ml luciferin-luciferase reagent (Sigma) was added to the apical or basolateral side of the monolayer. Each insert with cells contained 200 μl of solution, and the average light signal was measured for 10-s, nonintegrated photon collection periods with a TD-20/20 luminometer (Turner Designs; Promega). Treatment with cAMP was applied by adding 1 μl of 20 mM CPT-cAMP and 20 mM IBMX stock solution to the 200 μl apical solution of the inserts (final concentration 100 μM for both), and control experiments were done with addition of the same volume of vehicle alone. The light output was measured for at least 2 min, and the average was taken for the calculation of ATP release. Fig. 1 a Schematic diagram of the electrophysiological circuitry. The Ussing chamber is constructed of two lucite half-chambers, which are clamped together with the Madin-Darby canine kidney (MDCK) cell monolayer (on a Transwell membrane) between them. Voltage-sensing electrodes are positioned close to the center and on either side of the monolayer. Current passing electrodes, made from silver disks with a central hole to accommodate the voltage electrodes, are placed at the back of each half chamber and parallel to the monolayer. The transepithelial voltage (Vte) is measured by the voltage clamp/analog to digital converter (called “the clamp”). When no current is being passed, the voltage recorded is called the “open circuit” Vte. To short- circuit the epithelial monolayer, the clamp passes a current (I) that is sufficient to drive Vte to zero. The current measured in this situation is called the short-circuit current (Isc). b In experiments in which the Krebs-Ringer bicarbonate solutions (KRB) in the Ussing chambers were recirculated, 10-ml syringes were used as reservoirs, and a roller pump was used to continuously move the KRB from each half- chamber to its respective reservoir. c In experiments in which fresh KRB solutions were continuously perfused, large beakers served as the solution reservoirs from which KRB was constantly infused into each half-chamber by a roller pump and constantly removed by suction to a waste reservoir. In both the recirculation and fresh perfusion methods, the solutions in the reservoirs (10-ml syringes or beakers, respectively) were constantly bubbled with a 95% O2–5% CO2 gas mixture