A. Hazama et al., Swelling-induced, CFTR-independent ATP release from a human epithelial cell line - Lack of correlation with volume-sensitive Cl- channels, J GEN PHYSL, 114(4), 1999, pp. 525-533
To examine a possible relation between the swelling-induced ATP release pat
hway and the volume-sensitive Cl- channel, we measured the extracellular co
ncentration of ATP released upon osmotic swelling and whole-cell volume-sen
sitive Cl- currents in a human epithelial cell line, Intestine 407, which l
acks expression of cystic fibrosis transmembrane conductance regulator (CFT
R). 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 mu M. However, swelling-indu
ced STP release was not affected by a stilbene-derivative Cl- channel block
er, 4-acetamido-4'-isothiocyanostilbene at 100 mu M. Glibenclamide (500 mu
M) and arachidonic acid (100 mu M), which are known to block volume-sensiti
ve outwardly rectifying (VSOR) Cl- channels, were also ineffective in inhib
iting the swelling-induced ATP release. Gd3+, a putative blocker of stretch
-activated channels, inhibited swelling-induced ATP release in a concentrat
ion-dependent manner, whereas the trivalent lanthanide failed to inhibit VS
OR Cl- currents. Upon osmotic swelling, the local ATP concentration in the
immediate vicinity of the cell surface was found to reach similar to 13 mu
M by a biosensor technique using P2X(2) receptors expressed in PCl2 cells.
We have raised antibodies that inhibit swelling-induced STP release from In
testine 407 cells. Earlier treatment with the antibodies almost completely
suppressed swelling-induced STP release whereas the activity of VSOR Cl- ch
annel was not affected by pretreatment with the antibodies. Taking the abov
e results together, the following conclusions were reached: first, in a CFT
R-lacking human epithelial cell line, osmotic swelling induces ATP release
and increases the cell surface ATP concentration over 10 mu M, which is hig
h enough to stimulate purinergic receptors; second, the pathway of ATP rele
ase 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.