Purinergic Signalling (2008) 4:125–137 135 Fig. 10 Adenosine triphosphate (ATP) release across the apical and basolateral membranes of FL-Madin-Darby canine kidney (MDCK) monolayers. ATP release into the apical and basolateral solutions was measured for FL-MDCK monolayers in 12-mm culture inserts using the luciferin-luciferase assay. To measure apical ATP release, these reagents were present only in the apical solution (n=14), and they were present only in the basolateral solution to measure basolateral release (n=11). The monolayers were first incubated with 200 μl of standard Opti-minimal essential medium (MEM) solution on each side to measure basal ATP production. The monolayers were then removed from the instrument and either 1 μl of a stock solution of 8-(4-chlorophenylthio) (CPT)-cAMP + isobutylmethylxanthine (IBMX) stock solution (cAMP, n=10; final concentrations: 100 μM cAMP and 100 μM IBMX) or 1 μl of plain Opti-MEM medium (Sham, n =4) were added. Average values of the light emission in arbitrary light units (ALU)±standard error of the mean (SEM) are given. * Significantly different from basal and sham, P<0.005 treatment. In contrast, there was no significant release of ATP into the basolateral solution either without or with cAMP treatment. ATP has been shown to inhibit AS-Isc and stimulate NS-Isc in other epithelia of distal nephron origin, including M-1 [43], mIMCK-K2 [42], and A6 cells [49]. Release of endo- genous ATP into the extracellular medium has been proposed as a general response to hypotonicity and mechanical stimulation in many types of cells, including MDCK and A6 cells [49, 50]. Ostrom et al. [51] demonstrated that mechanical stimulation increases ATP release from MDCK cells as well as COS-7 and HEK-293 cells, and they proposed that ATP acts to alter the set point for a variety of signal transduction pathways. Praetorius et al. [50] also observed that mechanical stimulation caused ATP release in MDCK cells and that this release was associated with a transient increase in [Ca2+] . i Because of the opposite actions of ATP in the extracel- lular apical membrane, it can serve to shift the balance between active NaCl absorption and active NaCl secretion in the MDCK cell, as shown by the hypothetical model in Fig. 11. It is also quite important to note that the evidence we have presented for ATP accumulation in the extracellu- lar fluid with longer incubation periods in Ussing chambers raises a caution for all such experiments, and it suggests that recirculation of the bathing solution, which has been in common use, should be avoided. Fig. 11 Model for the role of adenosine triphosphate (ATP) in regulating Na+ absorption and Cl− secretion in Madin-Darby canine kidney (MDCK) epithelia. The epithelial Na+ channel (ENaC) mediates active Na+ absorption and produces the amiloride-sensitive component of the short-circuit current (AS-Isc). Because of the increased Na+ conductance produced by ENaC, it also depolarizes the apical membrane, producing a negative transepithelial voltage (Vte) of −30 mV. The basolateral membrane contains the ubiquitous Na, K-ATPase and, putatively, an Na-K-2Cl cotransporter, which actively accumulates Cl− in the cell. Cl− can then move across the apical membrane down its electrochemical potential gradient through Cl− channels, which include the glibenclamide-sensitive cystic fibrosis transmembrane conductance regulator (CFTR) channel, and a 4, 4'- diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive, calci- um-activated Cl- channel (CaCC). The net secretory movement of Cl− gives rise to the amiloride-insensitive component of the short-circuit current (NS-Isc). Our results show that ATP is secreted across the apical membrane. The pathway for ATP secretion is not known, but it has been proposed that it may also occur via CFTR [46]. When ATP accumulates in the apical extracellular space (e.g., when the solution bathing this space is restricted in volume or is recirculated), ATP inhibits ENaC and stimulates CaCC (and possibly CFTR or other Cl− channels), resulting in a fall in AS-Isc and an increase in NS-Isc