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 microM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 microM. Glibenclamide (500 microM) and arachidonic acid (100 microM), 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 approximately 13 microM 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 microM, 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.
Hazama
A
A
Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan.
Shimizu
T
T
Ando-Akatsuka
Y
Y
Hayashi
S
S
Tanaka
S
S
Maeno
E
E
Okada
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015374:Biosensing Techniques; D002460:Cell Line; D048429:Cell Size / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D004594:Electrophysiology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D049454:Firefly Luciferin / Q000378:metabolism; D005682:Gadolinium / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; C505032:CFTR protein, human; D018118:Chloride Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049454:Firefly Luciferin; D000255:Adenosine Triphosphate; D005682:Gadolinium; D008156:Luciferases