Cell swelling‐induced ATP release is tightly dependent on intracellular calcium elevations

Abstract

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 swellinginduced 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• 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.

ATP release from non-excitable cells

“…Numerous studies have demonstrated that cellular swelling activates a robust and large ATP release [42,[66][67][68]. This coincides with the fact that all cells in general react to cellular swelling with the activation of volume-sensitive anion channels [69].…”

“…However, mechanically triggered ATP release continues to be present in the absence of pl-VDAC-1 [73]. Another conflicting finding regarding VDAC is its very long lag time before activation of the membrane current (>10 min) [70], a finding which argues against its involvement in swelling-induced ATP release that always occurs promptly [67]. In addition, the other smaller conductance, outwardly rectifying VSOAC, has been suggested to mediate swelling-induced ATP release from endothelial cells.…”

“…Thus, this channel could be a mechano-sensor to trigger an initial Ca 2+ influx which subsequently induces nucleotide release. [67,77,137]. In different cells, it was also found to be inhibited by brefeldin A [77], cytochalasin B [137], the SNARE blocking clostridium toxin F, N-ethylmaleimide [138] and bafilomycin A1 [29].…”

“…Receptor-independent [Ca 2+ ] i elevations with, e.g. ionomycin or A23187 have been shown to trigger ATP release, but these are apparently poor agonists [26,29,67]. Inhibition of signalling events in the IP 3 pathway, such as phospholipase C using U73122, can inhibit agonist-triggered ATP release [26,28].…”

Vesicular and conductive mechanisms of nucleotide release

“…It will now be important to assess the contribution of VNUT to the release of ATP in other tissues in which evidence of exocytotic release of ATP has been reported, e.g., astrocytes [66][67][68][69][70], hepatocytes [64,71,72], alveolar A549 and intestinal 407 epithelial cells [73][74][75][76][77], osteoblasts [78], and esophageal keratinocytes [40]. It will be also important to biochemically and functionally identify the ATP-rich compartment(s) contributing to VNUT-dependent ATP release from non-endocrine/exocrine cell types exhibiting vesicular release of ATP.…”