ATP – The Universal Intercellular Signalling Molecule

The molecule of adenosine 5′-triphosphate or ATP was discovered 80 years ago simultaneously in Heidelberg and Boston by Lohman,1 Fiske and SubbaRow.2 Very soon afterwards, the central role of ATP in cell energetics was fully appreciated.3 In fact, the role of ATP in living matter is unique and without ATP we, in all probability, would not witness life in its present forms.

Indeed, the life forms, which we know on earth, are built around the genetic code that is stored in the relatively simple molecules of DNA and RNA, composed from the purine adenine and the pyrimidines, guanine, uracil and thymine. The purines and pyrimidines, as well as ATP and GTP, most likely appeared in the prebiotic period, with adenine derivatives being preferentially synthesised as a result of purely thermal reactions.4, 5 Very early in evolution, ATP was chosen as an energy substrate, thus shaping the metabolism of all forms of life.6 The preponderance of ATP stimulated the evolution of enzymes with preferential binding properties, and adenine nucleotides began to be used in various intracellular signalling cascades, such as, for example, the cAMP cascade.7 At the very same time, ATP probably became the first extracellular signalling molecule, because of its sheer availability. Indeed, as every cell contained high concentrations of ATP, cell damage inevitably results in the appearance of ATP gradients in the surrounding milieu, which thus became a universal ‘danger’ signal. As a result, virtually every known cell or single-cell organism has a form of ATP sensitivity, and purinergic signalling represents the primordial form of chemical intercellular signalling.8

Although the intracellular signalling and metabolic roles for ATP were established quite early, its importance as an extracellular signalling molecule was acknowledged much later. The possible signalling role for AMP was postulated in 19299 and purinergic signalling (i.e., signalling mediated by purines and pyrimidines) was initially suggested in 1970, when ATP was identified as a transmitter in the autonomic nervous system.10 In 1972, the concept of purinergic nerves and purinergic transmission was formulated,11 and after initial resistance is now widely accepted and has a major role in both the nervous system12, 13, 14 and non-neuronal cells.15 Initially, the focus was on short-term purinergic signalling in neurotransmission, neuromodulation and secretion, but more recent studies have also established roles in long-term (trophic) signalling in cell proliferation, differentiation, motility and death in development and regeneration.16, 17

The Omnipresent Purinoceptors

The action of purines and pyrimidines is mediated through an extended family of purinoceptors,18, 19 generally divided into P1 adenosine receptors20 and P2 receptors for ATP and related nucleotides.21, 22 In the early 1990s, receptors for purines and pyrimidines were cloned and characterised12, 23, 24, 25 and it is currently recognised that there are four subtypes of P1 receptors (A1, A2A, A2B and A3), seven subtypes of P2X ligand-gated ion channel receptors (P2X1–7) and eight subtypes of P2Y G-protein-coupled receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y1426). P2 receptors appeared very early in evolution, for example, P2X receptors have been found in early prokaryotes;27, 28, 29 despite little sequence homology with later evolutionary forms of the receptors, the functional properties are very much conserved. Similarly, functional metabotropic (P2Y-like) receptors are present in Protozoa, and in the most primitive plants in which they regulate numerous vital functions.8, 30

The P1 and P2Y receptors are classical 7-transmembrane domain receptors, the action of which is mediated through G-proteins and numerous intracellular second messengers, including the cAMP and InsP3 cascades. In addition, some of these receptors are linked to membrane ion channels, thus mediating plasmalemmal ion fluxes and electrophysiological effects. P2X receptors are archetypal ligand-operated cationic channels,31, 32, 33 many of which have an appreciable Ca2+ permeability.34 The P2X channels are assembled (in a homo- or heteromeric manner) from seven subunits, designated as P2X1–P2X7, which determines the variability of their biophysical and pharmacological properties.

Probably because of their ancient origin, the extensive array of purinoceptors has a unique property of being extraordinarily widely distributed throughout living cells and tissues (Table 1). In contrast to all other chemical transmitters, which are, as a rule, segregated to certain cell types and certain functions, the receptors for purines and pyrimidines are found everywhere and as a matter of fact it is almost impossible to find a cell without sensitivity to ATP and its analogues. Indeed, purinoceptors are extensively present in the central nervous system, where they mediate fast synaptic transmission, provide for presynaptic inhibition and regulation of neuronal excitability and are particularly important for signalling in neuronal–glial circuitry, being one of the most important gliotransmitters.12, 13, 35, 36 In the peripheral nervous system, purinoceptors are involved in sensory37, 38 and autonomic functions.39 Purinoceptors are present in all peripheral tissues, being involved in the regulation of very different functions in the gut, kidneys, in the cardiovascular and respiratory systems, the immunological system, in blood cells, skin, bones and muscles.8, 15 Furthermore, the purinergic signalling system possesses another unique property – the release of the principal mediator, ATP, initiates the appearance of a trail of derivatives, ADP, AMP and adenosine, to which extracellular ATP rapidly degrades due to the activity of ectonucleotidases that represent an important component of purinergic signalling.13 As a result of the single event of ATP release from different cell types (which occurs through different concomitant mechanisms, including Ca2+-regulated exocytosis, membrane transporters and diffusion through large-permeability plasmalemmal channels40, 41), several classes of receptors (sometimes having opposite actions) are activated at effector cells. Finally, purinoceptors are linked to an extensive array of intracellular signalling cascades that underlie their long-term trophic effects (Figure 1).

Table 1 Functional consequences of genetic deletion of purinoceptors
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Figure 1
figure 1

Overview of purinergic signalling mechanisms that regulate long-term, trophic effects. Extracellular nucleotides and nucleosides bind to purinoceptors coupled to signal-transducing effector molecules. Activation of effectors leads to generation of second messengers and/or stimulation of protein kinases that regulate expression of genes needed for long-term, trophic actions. Trophic action of P2X receptors can be mediated by increases in cytosolic Ca2+ concentration; activation of P2X7 receptors can also be coupled to protein kinase cascades and caspases that can mediate proliferation and apoptosis. Cell-specific and/or receptor subtype-specific differences are likely to account for variations in signalling pathways and functional outcomes. It should be noted that the list of elements is not meant to be all-inclusive. Other protein kinases, for example, MEK and PI3K, are upstream of the listed kinases involved in purinergic signalling, whereas others are downstream, for example, p70S6K. In addition, dashed arrows indicate that not all listed elements are activated by the upstream component, for example, not all P1 receptors are coupled to all listed effectors. AC, adenylyl cyclase; AP-1, activator protein-1; CaMK, calcium/calmodulin protein kinase; CREB, cAMP response element binding protein; DG, diacylglycerol; GSK, glycogen synthase kinase; InsP3, inositol trisphosphate; MAPKs, mitogen-activated protein kinases (including extracellular signal-regulated protein kinase (ERK), p38 MAPK, and stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK)); MEK, MAPK/ERK kinase; NO, nitric oxide; PG, prostaglandin; PI3K, phosphoinositide 3-kinase; PLC, phosphatidylinositol-specific phospholipase C; PKA, protein kinase A; PKC, protein kinase C; PLD, phospholipase D; PLA, phospholipase A; STAT3, signal transducer and activator of transcription-3 (based on Figure 11 from Burnstock12 with permission from the American Physiological Society )

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Purinergic Signalling Controls Biological Defence Systems

The ancient ‘damage signaller’ characteristic of ATP is evolutionarily conserved in several systems of biological defence. First, ATP functions as one of the main mediators of pain, both in acute and chronic contexts, as indeed P2X2/3 receptors are involved in fast pain perception,38, 42 whereas P2X4 and P2Y12 receptors assume a leading role in the pathogenesis of neuropathic pain.43, 44, 45

Second, purinergic agonists regulate the immune response in various tissues. In particular, in the brain and spinal cord, activation of several types of purinoceptors (most notably P2X4, P2X7, P2Y6 and P2Y12), which occurs in a highly coordinated temporal sequence, controls motility and activation of microglia, thus being central to the brain immune response.46, 47 In particular, the P2X7 receptor seems to be critical for microglial activation by β-amyloid, being therefore pathologically relevant for Alzheimer's disease.48 Similarly, purinergic signalling is intimately involved in the activation of the peripheral immune response being not only a stimulator but also a precise regulator of the differentiation and function of immunocompetent cells,49, 50 as well as in driving chemotaxis of neutrophiles, eosinophiles, macrophages and mast cells.51, 52, 53

Third, ATP and its analogues are directly involved in tissue remodelling in response to injury and have a key role in the regulation of subsequent repair and regeneration. In the nervous system, stimulation of purinoceptors triggers astrogliosis, the generalised response of astrocytes to brain damage, which is characterised by cell proliferation and remodelling of the neural circuitry.54, 55 Reactive astrogliosis is instrumental for both formation of scar and limitation of the brain damaged area (through anisomorphic astrogliosis), as well as for post-insult remodelling and recovery of neural function (by isomorphic astrogliosis). The initial events in astroglial responses to purinergic signallers are often associated with P2Y1/2 receptor-mediated Ca2+ astroglial signalling in astrocytes,56, 57 which, depending on the context, is instrumental for glial Ca2+ excitability or can initiate long-term effects.58 These trophic/astrogliotic effects of P2 agonists (manifested by proliferative and morphological responses) were found both in vitro, in glial cultures, and in vivo, in nucleus accumbens of rats.17, 59, 60, 61 Similarly, purinergic signalling has a fundamental role in remodelling and healing of lesions in other tissues, including skin and bone. In the next part of this review, we will focus on the long-term trophic roles of purines and pyrimidines in cell proliferation, differentiation and death in the turnover of epithelial cells in skin and in cells lining visceral organs, in restenosis, embryological development, bone formation and resorption and cancer.

Stratified Squamous Epithelia

Stratified squamous epithelia in several sites, including two non-keratinised cell types, namely, rat cornea and oesophagus, and four keratinised cell types, soft palate, foot pad skin, vagina and tongue, showed heavy immunostaining of the P2X5 receptor associated with cell differentiation in spinous and granular cell layers, but not in basal cuboidal or keratinised outer layers. In contrast, there was heavy immunostaining of P2X7 receptors in the outer keratinised layer, perhaps associated with apoptotic cell death.62

Rapid turnover rates are found in the epithelium of the small intestine. The crypts contain undifferentiated progenitor cells from which almost all other epithelial cell types, including goblet cells and enterocytes, arise. The differentiated cells glide towards the villus tips where they are finally ejected into the lumen. In the rat, this whole process takes 3–4 days.63 P2X5 receptors are expressed on the narrow ‘stem’ of villus goblet cells, whereas P2X7 receptor immunoreactivity is seen only on the membranes of enterocytes and goblet cells at the tip of the villus, where cells undergo apoptosis, before shedding into the lumen.64

Skin

The expression of P2X5, P2X7 and P2Y2 receptor subtypes was studied in healthy human epidermal keratinocyes in relation to markers for proliferation (PCNA and Ki-67), differentiation (cytokeratin KIO and involucrin) and apoptosis (TUNEL and anti-caspase-3).65 It was shown that P2Y1 and P2Y2 receptors were immunoreactive in basal and parabasal keratinocytes, P2X5 receptor immunostaining within the stratum spinosum and P2X7 receptor immunostaining in the stratum corneum, associated with cell proliferation, differentiation and apoptotic cell death, respectively (Figure 2). Functional experiments on cultured keratinocytes were also carried out in this study, which showed the following: an increase in cell numbers in response to the P2Y1 receptor agonist 2-methylthio ADP and the P2Y2 receptor agonist UTP; and a significant decrease in cell numbers with the P2X5 receptor agonist ATPγS and the P2X7 receptor agonist BzATP (Figure 3). Later studies from this group examined the purinergic signalling profile in human fetal epidermis.66 They showed P2Y1 receptors in the basal layer of the developing epidermis associated with proliferation; P2X5 receptors predominantly in the basal and intermediate layers associated with differentiation; and P2X7 receptors in the periderm associated with apoptotic cell death. P2Y2 receptors were also found in the periderm, where they may have a role in chloride and fluid secretion into the amniotic fluid.

Figure 2
figure 2

Double labelling of P2Y1 and P2Y2 receptors with markers of proliferation shows colocalisation within a sub-population of basal and parabasal keratinocytes. Double labelling of P2X5 receptors with markers of differentiated keratinocytes shows colocalisation within the stratum spinosum, and double labelling of P2X7 receptors with markers of apoptosis in human leg skin shows colocalisation within the stratum corneum. (a) Ki-67 immunolabelling (a marker for proliferation) stained the nuclei (green) of a sub-population of keratinocytes in the basal and parabasal layers of the epidermis. P2Y1 receptor immunostaining (red) was found in the basal layer on cells also staining for Ki67. Scale bar 30 μm. (b) PCNA immunolabelling (a marker for proliferation) stained the nuclei (green) of a sub-population of keratinocytes. These nuclei were often distributed in clusters and found in the basal and parabasal layers of the epidermis. P2Y2 receptor immunostaining (red) was also expressed in basal and parabasal epidermal cells. Scale bar 30 μm. (c) P2X5 receptor immunostaining (red) showed overlap (yellow) with cytokeratin K10 (green), an early marker of keratinocyte differentiation. P2X5 receptors were present in the basal layer of the epidermis up to the mid-granular layer. Cytokeratin K10 was distributed in most suprabasal keratinocytes. The stratum basale stained only for P2X5 receptors, indicating that no differentiation was taking place in these cells. The colocalisation of P2X5 receptors and cytokeratin K10 appeared mainly in the cytoplasm of differentiating cells within the stratum spinosum and partly in the stratum granulosum. Note that the stratum corneum also stained for cytokeratin K10, which labelled differentiated keratinocytes, even in dying cells. Scale bar 30 μm. (d) P2X5 receptor immunostaining (red) showed overlap (yellow) with involucrin (green). P2X5 receptors were present in the basal layer of the epidermis up to the mid-granular layer. Note that the pattern of staining with involucrin was similar to that seen with cytokeratin K10, except that cells from the stratum basale up to the midstratum spinosum were not labelled with involucrin, which is a late marker of keratinocyte differentiation. Scale bar 30 μm. (e) TUNEL (green) labelled the nuclei of cells at the uppermost level of the stratum granulosum and P2X7 antibody (red) mainly stained cell fragments within the stratum corneum. Scale bar 15 μm. (f) Anti-caspase-3 (green) colocalised with areas of P2X7 receptor immunostaining (red) both at the junction of the stratum granulosum and within the stratum corneum. Areas of colocalisation were yellow. Note that the differentiating keratinocytes in the upper stratum granulosum were also positive for anti-caspase-3. Scale bar 15 μm (reproduced with permission from Greig et al.140)

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Figure 3
figure 3

At 48 h after application of drugs to primary human keratinocyte cultures. (a) ATP (1–10 μM) and UTP (100 μM) cause an increase in cell number, whereas ATPγS (100–500 μM) and ATP (100 μM) cause a significant decrease. Results represent the mean of eight experiments. *P<0.001 compared with that of control. (b) 2MeSADP (500 μM) causes a significant increase in cell number. Results represent the mean of eight experiments. *P<0.05 compared with that of control. (c) BzATP (100–500 μM) causes a significant decrease in cell number. Results represent the mean of nine experiments. *P<0.001 compared with that of control. Error bars represent mean±S.E.M (reproduced with permission from Greig et al.140)

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In a study on purinergic signalling in wound healing67 in regenerating epidermis of denervated wounds, P2Y1 receptor protein expression was significantly increased in keratinocytes, whereas P2Y2 receptor protein expression was significantly decreased. However, NGF treatment of denervated wounds reduced the expression of P2Y1 receptors and enhanced the expression of P2Y2 receptors. In innervated wounds, NGF treatment enhanced both P2X5 and P2Y1 receptor proteins in keratinocytes. P2X7 receptors were absent in all experimental wound healing processes.

P2X5 and P2X7 receptors were shown to be present in human warts and CIN612 organotypic raft cultures of human papillomavirus-infected keratinocytes and may provide a novel approach for the treatment of warts.68 P2Y1, P2Y2 and P2X5 receptors are expressed on human anagen hair follicles, with P2Y1 receptors present in proliferating cells in the outer root sheath and bulb, whereas P2X5 receptors were present on the inner and outer root sheaths and medulla, and were associated with differentiation.65 P2Y2 receptors were found in living cells at the edge of the cortex/medulla. P2X7 receptors were not present.

Cancer

There were early reports of the beneficial effect of ATP in the treatment of cancer,69 and analysis of the purinergic receptor subtypes involved in the development of tumours in the prostate,70 bladder,71 melanoma,72, 73 breast74, 75, 76 and other organs has been described (see review by White and Burnstock77). Again, it was shown that P2Y1 and P2Y2 receptors were expressed and involved in cell proliferation, P2X5 receptors were involved in differentiation (and were therefore antiproliferative) and P2X7 receptors were involved in cell death (Figure 4). Human melanomas express functional P2X7 receptors that mediate the apoptotic functions of ATP,72 whereas P2Y1 and P2Y2 receptor agonists caused a decrease and increase in cell numbers, respectively.73 In human squamous cell carcinoma, P2Y2, P2X5 and P2X7 receptors seem to be associated with proliferation, differentiation and cell death, respectively.78

Figure 4
figure 4

Schematic diagram illustrating the different mechanisms by which P2 receptor subtypes might alter cancer cell function. P2Y1 and P2Y2 receptors could affect the rate of cell proliferation through altering the intracellular levels of cAMP by modulating adenylyl cyclase (AC) or by increasing intracellular calcium levels through the phospholipase C (PLC) pathway. P2X5 and P2Y11 receptor activation might switch the cell cycle from proliferation into a state of differentiation. The P2X7 receptor activates the apoptotic caspase enzyme system (redrawn from White and Burnstock77 with permission from Elsevier)

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In high-grade bladder cancer, using the HT-1376 cell line, P2X5 and P2Y11 receptors were shown to mediate the antineoplastic effects of ATP, whereas P2X7 receptors mediated apoptotic cell death.71 Similar results are described for cell lines of hormone-refractory prostate cancer79 and ATP was shown to reduce the in vivo growth of advanced hormone-refractory prostate cancer implanted into mice.80

Finally, several clinical trials have demonstrated that systemic administration of ATP may have beneficial effects (prolongation of survival and reduced cachexia) in inoperable lung cancer patients (for details see White and Burnstock77).

Long-Term Purinergic Signalling in Embryological Development

The transient appearance of P2 receptors during both embryological and postnatal development suggests that ATP is involved in the sequential proliferation, differentiation, motility and death of cells during the complex events involved in development12, 81, 82. For example, a novel P2Y8 receptor was cloned in Xenopus embryos and was shown to be transiently expressed in the neural plate and tube from stages 13–18 and again at stage 28, when secondary neurulation occurs in the tail bud, suggesting an involvement of this receptor in the development of the nervous system.83 P2Y1 receptors were transiently expressed in the limb buds of chick embryos and were shown to mediate rapid cell proliferation.84 Changes in expression in P2X receptor subtypes during postnatal development of cerebellum have been described.85 Transient changes in P2X receptor subtype expression during the development of skeletal muscle have been described.86 P2X5 receptors were present during the early development of the myotube, followed by P2X6 receptor expression and then, during the development of the neuromuscular junction, P2X2 receptors were expressed. In the chicken retina, ATP-evoked Ca2+ transients were strongest as early as E3 and were drastically reduced at E11–13.5.87 Nucleotide signalling in development probably involves a cross-talk between several other signalling pathways, including growth factors, cytokines and extracellular matrix components.82

Trophic Purinergic Signalling in Bone Formation and Resorption

Activation of P2Y1 receptors by ADP stimulates osteoclast activity and bone resorption (Figure 5), whereas ATP and UTP signalling through P2Y2 receptors in osteoblasts inhibits bone growth and mineralisation.88 More recently, P2X7 receptors have been shown to have trophic regulatory roles in bone formation and resorption.89, 90 P2X7 receptor activation of osteoblasts enhances differentiation and bone formation,91 whereas P2X7 receptor activation of osteoclasts results in apoptosis and bone resorption.92, 93, 94

Figure 5
figure 5

Schematic diagram illustrating the potential functions of extracellular nucleotides and P2 receptors in modulating bone cell function. ATP released from osteoclasts (e.g., through shear stress or constitutively) or from other sources, can be degraded to adenosine 5′-diphosphate (ADP) or converted into uridine 5′-triphosphate (UTP) through ecto-nucleotidases. All three nucleotides can function separately on specific P2 receptor subtypes, as indicated by the colour coding. ATP is a universal agonist, whereas UTP is only active at the P2Y2 receptor and ADP is only active at the P2Y1 receptor. ADP acting on P2Y1 receptors seems to stimulate both the formation (i.e., fusion) of osteoclasts from haematopoietic precursors and the resorptive activity of mature osteoclasts. For the latter, a synergistic action of ATP and protons has been proposed by the P2X2 receptor. ADP could also stimulate resorption indirectly through actions on osteoclasts, which in turn release pro-resorptive factors (e.g., receptor activator of nuclear factor κB ligand, RANKL) ATP at high concentrations might facilitate fusion of osteoclast progenitors through P2X7 receptor pore formation or induce cell death of mature osteoclasts through P2X7 receptors. In osteoblasts, ATP, through P2X5 receptors, might enhance proliferation and/or differentiation. By contrast, UTP, through P2Y2 receptors, is a strong inhibitor of bone formation by osteoblasts. For some receptors (e.g., P2X4 and P2Y2 receptors on osteoclasts or P2X2 receptors on osteoblasts), evidence for expression has been found but their role is still unclear (based on schemes from Hoebertz et al.88)

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Long-Term Trophic Actions of Purines and Pyrimidines in the Pathogenesis of Atherosclerosis and Post-Angioplasty Restenosis

ATP and UTP, acting through P2Y2 receptors, cause proliferation of vascular smooth muscle cells and proliferation of endothelial cells through P2Y1 receptors. Adenosine acting through A2 receptors inhibits smooth muscle proliferation but stimulates endothelial cell proliferation.95 The increase in vascular smooth muscle and endothelial cells in both atherosclerosis and hypertension may be mediated by the trophic actions of purines and pyrimidines released from nerves and endothelial cells96, 97, 98 and in post-angioplasty restenosis.99 P2Y4 receptors seem to be regulators of angiogenesis.100 ATP increases DNA synthesis and migration of vascular endothelial cells in vasa vasorum in diseased pulmonary vessels.101 Diabetic patients express microvascular disease characterised by an increased wall–lumen ratio, mainly because of an increase in vascular smooth muscle cells, and have higher rates of restenosis after angioplasty. High glucose-induced release of ATP exerts an effect on P2Y receptors to stimulate vascular smooth muscle cell growth.102

Conclusions

Purinergic signalling, mediated by ATP, related nucleotides and adenosine, operates in all types of tissues and cells. Purinergic agonists mediate fast cell signalling, and exert numerous long-term trophic effects, involved in regulation of cell replication, proliferation, differentiation and death. It is hoped that there will be further exploration of the roles of this primitive and widespread signalling system in cell biology.

Conflict of interest

The authors declare no conflict of interest.