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Original Article

Lack of Correlation with Volume-Sensitive Cl^– Channels

Akihiro Hazama^a,b,c, Takahiro Shimizu^a,b, Yuhko Ando-Akatsuka^c, Seiji Hayashi^c, Shoko Tanaka^a, Emi Maeno^a,b, and Yasunobu Okada^a,b,c

^a From the Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan
^b Department of Physiological Sciences, School of Life Sciences, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan
^c Core Research for Evolutional Science and Technology of Japan Science and Technology Cooperation (JST), Okazaki 444-8585, Japan
Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki 444-8585, Japan.Fax: 8 1-564-55-7735;

okada{at}nips.ac.jp

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. Gd³⁺, 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 P2X₂ 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.

Key Words: adenosine triphosphate • anion channel • osmotic swelling • cell volume regulation

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