Ryszard Grygorczyk & Francis Boudreault
Research Centre, Centre hospitalier de l’Université de Montréal – Hôtel-Dieu and Department of Medicine, Université de Montréal, Montréal, Québec, Canada

https://doi.org/10.36866/pn.60.21

The role of ATP as an extracellular signalling molecule is now widely accepted in a broad spectrum of biological systems. The entire field of purinergic signalling is expanding rapidly, as exemplified by the recent launching of a journal entitled Purinergic Signalling (Burnstock, 2004). This progress has been driven by the cloning of several members of the large family of nucleotide-activated cell surface receptors and of the ubiquitously distributed classes of ectoenzymes that catalyze nucleotide breakdown and interconversion (Lazarowski et al. 2003). Recent years have also been marked by growing interest in the mechanisms of nucleotide release, especially from epithelial and other nonexcitable cells, where these mechanisms remain incompletely understood. Because cellular ATP release can be evoked by various stimuli, including mechanical perturbation, cell swelling, hypoxia and a number of agonists via receptor­mediated stimulation, several release mechanisms may be involved. In addition, basal release from unstimulated cells is well-documented and has important physiological implications for the tonic autocrine activation of purinergic signalling pathways.

It is generally accepted that regulated exocytosis is responsible for ATP release from neuronal and secretory cells, in which specialised granules containing ATP, together with other extracellular mediators, have been identified. Such dedicated granules have not been explicitly found in most epithelial cells, and alternative mechanisms of ATP secretion, such as ATP-conducting channels, have been sought. While there is strong evidence against the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, mechano-sensitive or volume-sensitive chloride channels (VSCC), ATP permeability has been convincingly demonstrated for voltage-dependent anion channels (VDAC) and connexin hemichannels. VDAC-like channels have been detected by the patch clamp technique in the plasma membrane of numerous cells, but not always confirmed by immunocytochemical techniques. Because patch clamping inevitably imposes major stresses on cells, the physiological significance of these channels on the cell surface should be viewed with caution. The role of connexins may be limited to specific cells or experimental conditions, e.g. un-physiologically low extracellular Ca2+, and numerous preparations have provided no evidence of their involvement in ATP release (Guyot & Hanrahan, 2002; Lazarowski et al. 2003).

Alternatively, ATP can be released by regulated exocytosis, a Ca2+-dependent process that engages ATP-loaded granules and requires SNARE proteins for membrane fusion. Are these elements present in epithelial cells? In The Journal of Physiology, we (Boudreault & Grygorczyk, 2004) recently provided evidence of a tight correlation between ATP release induced by hypotonic cell swelling and elevation of intracellular Ca2+ ([Ca2+]i). Hypotonicity-evoked ATP release coincided with the peak of [Ca2+]i elevation in the three cell types studied: human lung alveolar A549 cells (Figure 1a), human airway epithelial cells 16HBE14o- and 3T3 fibroblasts. ATP release was almost completely abolished by BAPTA chelation of [Ca2+]i, while Ca2+-ionophore induced ATP release in the absence of hypotonic shock (Figure 1 b, c). Thus, Ca2+ is a necessary and sufficient signal for ATP release, although the magnitude of release can be modulated by other factors. Both hypotonicity-induced ATP release and [Ca2+]i elevations in A549 cells were completely abolished at 10°C, suggesting that exocytotic release is involved and is a dominating mechanism in these cells. Because most ATP was secreted from swollen cells prior to the regulatory volume decrease, a contribution of conductive release via VSCC or exocytotic insertion of other ATP-conducting anion channels could also be excluded with confidence.

Specialised ATP-filled granules have not been definitively identified in non­excitable cells, but other vesicles may contribute to exocytotic ATP release. Due to its fundamental role as an energy source and in protein phosphorylations some ATP is expected in the lumens of most, if not all, cell organelles, including the endoplasmic reticulum (ER), Golgi, endosomes, lysosomes and various membrane­trafficking vesicles. Indeed, ATP was found in the ER lumen and in secretory vesicles from large numbers of non­neuronal cells, and protein transport vesicles were recently shown to contribute to ATP release from oocytes (Maroto & Hamill, 2001). Thus, vesicles of different origins could contribute to ATP secretion, and their relative contributions will require experimental verification.

Further supporting evidence for exocytosis includes the presence of SNARE proteins, key mediators of intracellular membrane fusion events in all eukaryotic cells. Disruption of SNARE complexes by C botulinum toxins abolished ATP release from intestine 407 cells (Van der Wijk et al. 2003). In neuronal and endocrine cells, SNARE complex assembly during exocytosis is tightly coupled to Ca2+, but less is known about these mechanisms in epithelia. Because different SNARE proteins are generally localized to specific organelles and are involved in only specific trafficking pathways, one could speculate that different vesicular pools may contribute to different modes of ATP release, depending on the stimuli. In this context, it is interesting that lysosomes are reported to behave as Ca2+-regulated exocytotic vesicles in fibroblasts and epithelial cells (Rodriguez et al. 1997).

Further studies are required to determine the prevalence and physiological significance of different ATP release mechanisms. While channel-mediated release may be important in some cell types, the recent demonstration that increased [Ca2+]i is a sufficient trigger for ATP secretion from epithelial cells and fibroblasts strongly supports the involvement of exocytosis. Because diverse stimuli could elevate [Ca2+]i, ATP release via regulated exocytosis has the potential to be a widespread mechanism operating in many cell types.

References

Burnstock G (2004). Editorial. Purinergic Signalling 1,1.

Boudreault F & Grygorczyk R (2004). Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations. J Physiol 561, 499-513.

Guyot A & Hanrahan JW (2002). ATP release from human airway epithelial cells studied using a capillary cell culture system. J Physiol 545, 199-206.

Lazarowski ER, Boucher RC & Harden TK (2003). Mechanisms of release of nucleotides and integration of their action as P2X- and P2Y-receptor activating molecules. Mol Pharmacol 64, 785-795.

Maroto R & Hamill, OP (2001). Brefeldin A block of integrin­dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction. J Biol Chem 276, 23867-23872.

Rodriguez A, Webster P, Ortego J & Andrews NW (1997). Lysosomes behave as Ca2+-regulated exocytotic vesicles in fibroblasts and epithelial cells. J Cell Biol 137, 93-104.

Van der Wijk T, Tomassen SFB, Houtsmuller AB, de Jonge HR & Tilly BC (2003). Increased vesicle recycling in response to osmotic cell swelling. J Biol Chem 278, 40020-40025.