Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations.
Mechanical stresses release ATP from a variety of cells by a poorly defined mechanism(s). Using custom-designed flow-through chambers, we investigated the kinetics of cell swelling-induced ATP secretion, cell volume and intracellular calcium changes in epithelial A549 and 16HBE14o- cells, and NIH/3T3 fibroblasts. Fifty per cent hypotonic shock triggered transient ATP release from cell confluent monolayers, which consistently peaked at around 1 min 45 s for A549 and NIH/3T3, and at 3 min for 16HBE14o- cells, then declined to baseline within the next 15 min. Whereas the release time course had a similar pattern for the three cell types, the peak rates differed significantly (294 +/- 67, 70 +/- 22 and 17 +/- 2.8 pmol min(-1) (10(6) cells)(-1), for A549, 16HBE14o- and NIH/3T3, respectively). The concomitant volume changes of substrate-attached cells were analysed by a 3-dimensional cell shape reconstruction method based on images acquired from two perpendicular directions. The three cell types swelled at a similar rate, reaching maximal expansion in 1 min 45 s, but differed in the duration of the volume plateau and regulatory volume decrease (RVD). These experiments revealed that ATP release does not correlate with either cell volume expansion and the expected activation of stretch-sensitive channels, or with the activation of volume-sensitive, 5-nitro-2-(3-phenylpropylamino) benzoic acid-inhibitable anion channels during RVD. By contrast, ATP release was tightly synchronized, in all three cell types, with cytosolic calcium elevations. Furthermore, loading A549 cells with the calcium chelator BAPTA significantly diminished ATP release (71% inhibition of the peak rate), while the calcium ionophore ionomycin triggered ATP release in the absence of cell swelling. Lowering the temperature to 10 degrees C almost completely abolished A549 cell swelling-induced ATP release (95% inhibition of the peak rate). These results strongly suggest that calcium-dependent exocytosis plays a major role in mechanosensitive ATP release.
Boudreault
Francis
F
Research Centre, CHUM-Hôtel-Dieu, 3850 Saint-Urbain, Montréal, Québec, Canada.
Grygorczyk
Ryszard
R
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D048429:Cell Size; D006801:Humans; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells
D000255:Adenosine Triphosphate; D002118:Calcium