Swelling-Induced, Cftr-Independent Atp Release from a Human Epithelial Cell Line: Lack of Correlation with Volume-Sensitive Cl(−) Channels

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(−) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(−) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56–80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(−) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 μM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(−) channel blocker, 4-acetamido-4′-isothiocyanostilbene at 100 μM. Glibenclamide (500 μM) and arachidonic acid (100 μM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(−) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(−) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach ∼13 μM by a biosensor technique using P2X(2) receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl(−) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 μM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(−) channel; and third, the ATP release is not a prerequisite to activation of the Cl(−) channel.