Cell culture: complications due to mechanical release of ATP and activation of purinoceptors

There is abundant evidence that ATP (adenosine 5′-triphosphate) is released from a variety of cultured cells in response to mechanical stimulation. The release mechanism involved appears to be a combination of vesicular exocytosis and connexin and pannexin hemichannels. Purinergic receptors on cultured cells mediate both short-term purinergic signalling of secretion and long-term (trophic) signalling such as proliferation, migration, differentiation and apoptosis. We aim in this review to bring to the attention of non-purinergic researchers using tissue culture that the release of ATP in response to mechanical stress evoked by the unavoidable movement of the cells acting on functional purinergic receptors on the culture cells is likely to complicate the interpretation of their data.


Introduction
While it was recognised early that ATP (adenosine 5′triphosphate) is released from damaged or dying cells, it was shown more recently that gentle mechanical perturbation, such as shear stress, membrane stretch and hypo-osmotic cell swelling, leads to release of ATP from most cell types (Bodin and Burnstock 2001;Bodin et al. 1991;Chaudry 1982;Dolovcak et al. 2011;Forrester 1972;Grygorczyk and Guyot 2001;Milner et al. 1990Milner et al. , 1992Leipziger 2009, 2010;Sperlágh et al. 2007;Wang et al. 1996). In the outstanding review by Lazarowski et al. (2011), it was stated that "P2Y receptor expression-dependent formation of second messengers was noted in cultured cells subjected to mechanical stress, for example medium displacement or cell wash (Filtz et al. 1994;Lazarowski et al. 1995;Parr et al. 1994). A vast number of studies have followed, illustrating that nonlytic release of ATP occurred in practically every cell type subjected to physical stresses, such as flow resulting in shear stress, hydrostatic pressure, osmotic swelling or shrinking, compressive stress, mechanical loading, plasma membrane stretch, hypoxia and cell swelling" performed during routine experimental procedures, such as cell rinsing and medium changes. It is unlikely that ATP release caused by gentle mechanical stimulation arises from cell damage, for example mechanical stimulated ATP release occurs without associated membrane conductive changes (Hamill and Martinac 2001). Many novel assays (or sensors) have been developed to detect ATP release from cells, including luciferinluciferase bioluminescence and atomic force microscopy (see Dale and Frenguelli 2012;Furuya et al. 2014;Khlyntseva et al. 2009;Praetorius and Leipziger 2009).
The mechanisms responsible for the transport of ATP from cells have been a matter of intense debate. For most cell types, it appears to be a combination of vesicular exocytosis and connexin or pannexin hemichannels (Dahl 2015;Dubyak 2007;Lazarowski et al. 2011;Li et al. 2011;Lohman and Isakson 2014;Novak 2003;Scemes et al. 2009;Spray et al. 2006), although for some cells ATP-binding cassette transporters or maxi ion channels have been claimed (Sabirov and Okada 2005). It has also been proposed that P2X7 receptors may mediate ATP release (Pellegatti et al. 2005;Suadicani et al. 2006). A vesicular nucleotide transporter has been identified (Sawada et al. 2008).
ATP released from cells is rapidly broken down by ectonucleotidases to adenosine (see Cardoso et al. 2015;Yegutkin 2008;Zimmermann 2006) but both ATP and adenosine will have functional effects on the cells via P1, P2X and P2Y receptors (see Corriden and Insel 2010).

Release of ATP from cultured cells in response to mechanical stimulation
A comprehensive summary is shown in Table 1.

Purinergic receptor expression in cultured cells
A comprehensive summary is shown in Table 2.
When cells are cultured, they de-differentiate, which is associated with changes in receptor expression. If the cell density is high, the cells usually re-differentiate and this again is associated with changes in receptor expression (see, e.g., Chamley et al. 1974). Upregulation of P2Y 2 receptors in rat salivary gland cells during shortterm culture has also been reported (Turner et al. 1997).

Function of purinergic receptors on cultured cells in response to released ATP
A comprehensive review of the functional expression of P2 receptors on a wide range of cell types is available (Burnstock and Knight 2004). Some examples follow. ATP released from retinal epithelial cells acts via P2 receptors to increase the rate of fluid transport or decrease phagocytosis (Mitchell 2001) and regulate neural retinal progenitor cell proliferation (Pearson et al. 2005). ATP released by osteoblasts inhibits bone mineralisation (Orriss et al. 2013). Stretch-released ATP from fibroblasts results in cell proliferation (Wang et al. 2005). ATP released from astrocytes mediates glial calcium waves (Guthrie et al. 1999). ATP released from endothelial cells by shear stress acts on endothelial P2 receptors to release nitric oxide resulting in vasodilatation (Burnstock and Ralevic 2014).
Mechanically-induced Ca 2+ waves have been observed in a variety of cells, including chondrocytes (D'Andrea and Vittur 1996), airways epithelial cells (Boitano et al. 1994;Hansen et al. 1993;Sanderson et al. 1990), glial cells, including Müller cells (Charles et al. 1991(Charles et al. , 1992(Charles et al. , 1993Newman 2001), keratinocytes (Koizumi et al. 2004), endothelial cells (Demer et al. 1993, T cells , mast cells (Osipchuk and Cahalan 1992) and others (see Leybaert and Sanderson 2012). It is likely that they are due to the activation of purinergic receptors by ATP released from the mechanically stimulated cells, mainly via P2Y 1 and P2Y 4 receptors (Frame and de Feijter 1997;Gallagher and Salter 2003;Stamatakis and Mantzaris 2006). Calcium waves are a dynamic intracellular signalling mechanism that allows spatiotemporal information to be rapidly propagated in tissues. ATP released at sites of cell stress signals danger to the immune system.
Conclusion: need for re-interpretation of data derived from cell culture experiments Release of ATP from cultured cells is unavoidable, due to gentle mechanical stimulation. The released ATP acts on purinoceptors expressed by these cells, which mediate both secretion and trophic events, such as cell proliferation, differentiation, death and migration. These events mean that interpreting results from experiments based on tissue culture need to take into account the effects of released ATP and its actions on purinoceptors.     This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
This article does not contain any studies with human participants or animals performed by any of the authors.   (2010) (2015) (2003) Hypertonic stress increases T cell interleukin-2 expression through a mechanism that involves ATP release, P2 receptor, and p38 MAPK activation.