Gustavo B. Menezes, Laboratório de Imunobiofotônica, Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais. Avenida Antônio Carlos, 6627 – Campus Pampulha – Belo Horizonte, MG, Brazil CEP: 31270-901
The liver plays a vital role in the organism, and thousands of patients suffer and even die from hepatic complications every year. Viral hepatitis is one of the most important causes of liver-related pathological processes. However, sterile liver diseases, such as drug-induced liver injury, cirrhosis and fibrosis, are still a worldwide concern and contribute significantly to liver transplantation statistics. During hepatocyte death, several genuine intracellular contents are released to the interstitium, where they will trigger inflammatory responses that may boost organ injury. Intracellular purines are key molecules to several metabolic pathways and regulate cell bioenergetics. However, seminal studies in early 70s revealed that purines may also participate in cell-to-cell communication, and more recent data have unequivocally demonstrated that the purinergic signalling plays a key role in the recognition of cell functionality by neighbouring cells and also by the immune system. This new body of knowledge has pointed out that several promising therapeutic opportunities may rely on the modulation of purine release and sensing during diseases. Here, we review the most recent data on the physiological roles of purinergic signalling and how its imbalance may contribute to injury progression during sterile liver injury.
Adenosine 5′-triphosphate (ATP) and its metabolites (Fig. 1) constitute important signalling pathways that regulate a broad variety of biological processes [1-3]. In the liver, purinergic signalling can be interpreted as a shifting balance that significantly contributes to tissue homeostasis. Under physiological conditions, intercellular purinergic signalling within hepatic lobules is important for the regulation of key processes, including cell survival, proliferation and cell death, ion homeostasis, bile formation, glucose metabolism and blood flow [4-8]. However, altered signalling mediated by purines shifts the balance towards pathological states , favouring the onset and progression of liver injury even in aseptic situations, a condition known as sterile injury [9, 10].
Sterile injury in the liver may result from different causes, including Wilson's disease, Budd-Chiari syndrome, autoimmune-mediated inflammation and drug-induced liver injury (DILI) [11, 12]. The point of congruence among these conditions is the massive cell stress and death, in which an abrupt increase in extracellular ATP concentration may occur, triggering a robust inflammatory response [10, 13] However, recent data suggest that purinergic signalling may also influence DILI as a result of direct interference with drug metabolism or with cell death pathways . In this review, we discuss the molecular mechanisms of purinergic signalling in the liver, thereby focusing on its participation in the control of cellular events during sterile injury.
Purines as signalling molecules
The hypothesis that purines may act as signalling molecules was originally proposed by Geoffrey Burnstock in 1972 . Cloning of nucleotide receptors allowed the identification of three families of proteins, namely P1, P2X and P2Y receptors. The P1 receptors are specific adenosine, whereas the P2 receptors display high specificity for ATP and adenosine 5′-diphosphate (ADP). The two P2 subfamiles, P2X and P2Y, were defined based on structural similarities (Table 1) .
Table 1. Purinergic receptors: agonists and antagonists. Purinergic receptors are listed with their classes (GPCR or ion channel), agonists and antagonists
The P2X receptors subfamily is composed of seven different proteins (P2X1-P2X7) that act as cation permeable channels . Although these ionotropic receptors do not present specificity for any particular cation, it is noteworthy that the majority of them are highly permeable to calcium after limited ATP stimulation . However, during prolonged, exacerbated or even repeated ATP stimulation, P2X receptor channels become increasingly permeable to large organic molecules . All P2X receptor subtypes have been identified in the liver, with P2X7 and P2X4 being the most widely distributed ones . The distribution of P2X receptors is highly dependent on the cell type. Thus, hepatocytes express P2X1, P2X2 P2X3, P2X4 and P2X7, but not P2X5, P2X6 [8, 16]. Other liver cells, such as cholangiocytes, Kupffer cells, stellate cells and endothelial cells, may also express different P2X receptors [see review in ].
In contrast to the ionotropic P2X receptors, the P2Y receptors subfamily signals via G-protein-coupled metabotropic receptors and their biological action is mediated by second messengers, especially inositol triphosphate (IP3) and protein kinase C (PKC) . As a consequence, calcium is mobilized from intracellular stores (i.e. endoplasmic reticulum) and initiates a series of cellular responses, including gene transcription, cellular growth, secretion and chemotaxis . Little information is available regarding the expression of P2Y receptors within hepatic tissue. It is thought that P2Y2 is the major isoform found in hepatocytes, where its physiological activation favours cell survival . Interestingly, hepatocytes also express P2Y1-like and P2Y2-like receptors in mitochondrial membranes, which act as sensors of cytoplasmic ATP. Stimulation for these receptors requires nanomolars of ATP. Hence, in conditions in which intracellular ATP levels are reduced, these receptors are transiently activated, resulting in increased calcium influx and ATP synthesis stimulation . Furthermore, P2Y receptors can be found in other hepatic cell types, such as stellate cells, in which expression of protein subtypes varies depending on their activating status .
In addition to ATP and ADP signalling via P2 receptors, adenosine may also act as a key mediator with different functions in the organism. These effects are mediated by the activation of distinct adenosine receptor subtypes referred generally as P1R, but subdivided into A1, A2A, A2B and A3 receptors. All P1R belong to the G protein-coupled receptor (GPCR) superfamily, and they promote diverse signalling cascades in the different tissues . In general, A2A receptor activation triggers anti-inflammatory activities, modulating overstimulated immune cells . Contrastingly, A1, A2B and A3 receptors may exert opposite actions, boosting immune response and cytokine release . As immune cells differentially express both A1 and A2 receptors, the opposite actions of the same ligand (adenosine) may offer a more precise mechanism of modulation of the inflammatory response. A seminal review can be found at .
Mechanisms of ATP release during homeostasis and disease
ATP release through exocytosis and transporters
Although ATP can be found in liver fluids and parenchyma, this molecule cannot simply diffuse through plasma membranes because of its molecular weight, size and charge [5, 8].However, the mechanisms involved in ATP release within the liver are still under investigation. With this regard, it has been hypothesized that ATP could be secreted from hepatocytes by exocytosis of vesicles, similar to the secretory pathway observed in sympathetic nerves . In fact, when are cells treated with fluorescent agents with high affinity for ATP, such as quinacrine, foci of punctuated stained granules, compatible with a localization of ATP within intracellular vesicles, become visible . Moreover, stimulation of quinacrine-labelled cells by osmotic stress (i.e. hypotonic medium) results in a rapid and sustained decrease in fluorescence via an exocytotic mechanism that depends on an intact machinery of vesicle formation, acidification and trafficking . However, despite this compelling evidence, a major question that still persists is how ATP, a cytoplasmic molecule, is loaded in such vesicles. Recently, the vesicular nucleotide transporter SLC17A9 has been identified in biliary epithelial cells and was implicated in the transport of ATP into intracellular vesicles . Another putative pathway for ATP release is the involvement of proteins from the ATP-binding cassette (ABC) superfamily. The ABC transporter superfamily comprises proteins that utilize the energy of ATP hydrolysis to promote the movement of different molecules across plasma membranes . Among these proteins, the multidrug resistance 1 (MDR1), also known as P-glycoprotein, and the cystic fibrosis conductance regulator (CFTR) are known to act as specific channels for the exit of ATP in the liver . However, this concept is controversial, as a number of studies failed to demonstrate the ability of ABC transporters to conduct ATP across plasma membranes  or even showed that the release of this purine could occur independently of MDR1 or CFTR . Regardless of this controversy, it is not possible to completely rule out the involvement of ABC transporters in ATP release, as these proteins may associate with other channels or even regulate other cellular processes (i.e. exocytosis) and therefore indirectly impact ATP transport towards plasma membranes [32, 33].
ATP release through connexin and pannexin hemichannels
Connexin hemichannels. In the last decade, connexin hemichannels have emerged as mediators of extracellular ATP release. They are built up by six connexin (Cx) proteins, of which more than 20 different tissue-specific isotypes have been characterized in humans and rodents [34-38]. In the liver, hepatocytes mainly express Cx32, whereas most non-parenchymal liver cells harbour Cx43 [34-37]. In the classical view, connexin hemichannels at the plasma membrane surface of adjacent cells dock to form a gap junction, which controls direct intercellular communication. It has now become clear that undocked connexin hemichannels also provide a pathway for cellular signalling, albeit between the intracellular compartment and the extracellular environment [35, 38-40]. Connexin hemichannels remain mostly closed in physiological conditions, although they can be opened by a number of stimuli, which are typically of pathological origin, such as pronounced decreases in extracellular calcium concentration, decreases in pH, membrane depolarization, mechanical stress, ischemia/reperfusion insults, oxidative stress and metabolic inhibition [38, 40, 41]. Post-translational modifications, such as phosphorylation and S-nitrosylation, are known to lie at the basis of connexin hemichannel opening and closure [40, 42].
Although surrounded by controversy, functional connexin hemichannels have been identified in a plethora of cell types [39, 43], including hepatocytes [36, 44]. Open connexin hemichannnels allow the entry of below 1–1.5 kDa substances, such calcium and sodium ions, or the escape of essential metabolites, like nicotinamide adenine dinucleotide, prostaglandins, glutathione, glutamate and ATP [38, 40, 45]. Regarding the latter, considerable evidence now points to a role for connexin hemichannels as a paracrine pathway in ATP release for the propagation of intercellular calcium waves [39, 40, 42]. The trigger for connexin hemichannel opening in this process is IP3 and associated changes in intracellular calcium concentration . ATP liberation through connexin hemichannels is believed to act in concert with other ATP release systems and plays a role in proliferation [35, 39], such as shown in radial glial cells in the developing brain and in neural progenitor cells in the retina. Vice versa, extracellularly conveyed ATP through connexin hemichannels may affect also cell death [38-40]. This been demonstrated in the context of ischemic preconditioning in the heart, whereby it was simultaneously shown that connexin hemichannels can reside at subcellular localizations other than the plasma membrane, such as in mitochondria [38, 47]. In other tissues, ATP release via connexin hemichannels is of relevance for more specific functions. In the cochlea, for instance, this process affects the electromotility of hair cells . Connexin hemichannel-based ATP signalling, particularly in polymorphonuclear leukocytes, has been involved in immune responses, as occurring during hypoxia or acute inflammation [45, 49]. Furthermore, connexin hemichannels fulfil a role in bacterial infection. Indeed, Shigella flexneri invasion in colonic mucosa induces the opening of Cx26 hemichannels, allowing extracellular release of ATP, which in turn favours bacterial dissemination . Similarly, the bacterial wall component peptidoglycan induces an open status of Cx43 hemichannels in endothelial cells and subsequent ATP release . Other bacterial pathogens as well as their toxins, including lipopolysaccharide (LPS), have also been found to induce connexin hemichannel opening . Recently, it was reported that the calcium-sequestrating agent 2-aminoethoxydipenylborate (2APB) is able to inhibit Cx32-based channels, which results in the protection of the liver against chemical-induced injury and cell death upon administration to mice .
Pannexin hemichannels. A novel class of connexin-like proteins has been identified in the year 2000, namely the pannexins . Pannexins share no sequence homology with connexins, but have a similar topology [55-58]. The pannexin family consists of three members, of which Panx1 and Panx2 are expressed in liver tissue . Pannexins gather in a connexin hemichannel-like configuration and are unlikely to form gap junctions [41, 55-57]. Pannexin hemichannels are permeable to small molecules, including ATP, and are controlled by post-translational modifications [40, 42, 55, 56]. In contrast to their connexin counterparts, extracellular ATP release through pannexin hemichannels serves some clear physiological purposes [41, 55, 56, 58]. Thus, Panx1-mediated ATP liberation controls mucociliary clearance in airway epithelial cells , gustatory afferent nerves during tastant-evoked signalling  and serves as a costimulator for T-cell proliferation . In addition, pannexin hemichannel-based ATP signalling is thought to mediate chemical coupling in neurons  and has been linked to excitation-transcription regulation during tetanic contractions in muscle cells .
From the pathological perspective, pannexin hemichannels are essentially involved in inflammatory responses, in casu experimentally evoked by LPS [55, 58]. The latter binds to a Toll-like receptor to initiate the expression of inactive precursor IL-1β. Activation of precursor IL-1β occurs through cleavage by caspase 1, which itself becomes activated by processing within a cryopyrin-containing inflammasome. The subsequent extracellular release of active proinflammatory IL-1β requires the presence of ATP, which acts via the P2X7 receptor. In fact, Panx1 hemichannels are crucial for the processing of caspase 1 and release of IL-1β. Panx1 hemichannel opening is hereby induced by ATP-stimulation of P2X7 receptors, as shown in mouse and human macrophages exposed to LPS [65-67], which may burgeon into the onset of cell death . Indeed, Panx1 hemichannels have been found to mediate the release of ‘find me’ signals, including ATP, for phagocyte recruitment and membrane permeability during apoptosis . In addition, it has been suggested that Panx1 hemichannels fulfil a critical role in the recognition and the intracellular delivery of bacterial molecules, including subsequent activation of the cryopyrin-mediated caspase 1 cleavage, independently of Toll-like receptor signalling [70, 71]. Although the involvement of Panx1 hemichannels in inflammation has now been well accepted and has been demonstrated in several cell types, information regarding the liver is scarce. Only one report showed that administration of LPS to mice induces the inflammasome in the liver, which is associated with upregulated Panx1 levels .
ATP release and signalling during liver injury
Drug-induced liver injury (DILI). DILI is a consequence of acute hepatocyte death and may range from an asymptomatic lesion to life-threatening cases that require liver transplantation . However, almost half of eligible patients die before liver transplantation because of multiorgan failure and hepatic encephalopathy , indicating that novel therapies aimed to control the progression of liver damage are urgently needed. There is a growing body of evidence demonstrating that, following massive hepatocyte necrosis, a robust inflammatory response amplifies the original organ injury [13, 74-76]. In fact, liver-resident and infiltrating leukocytes, especially neutrophils, orchestrate a reactive inflammatory response to cell death products that may be experimentally manipulated to reduce drug-induced hepatotoxicity [74, 76]. Furthermore, necrosis-derived mediators may be collected to systemic circulation and trigger a systemic inflammatory response, which may cause remote organ injury during acute liver failure [74, 77].
Moreover, following acute hepatocyte death as a result of acetaminophen (APAP) administration, ATP is released from the liver and may bind to P2Y2 receptors to modulate immune responses and promote hepatocyte death . Consequently, ATP conversion into adenosine is an anti-inflammatory event and drives cytoprotection mainly via the adenosine receptor A2A (A2A) activation [6-8]. In addition, ATP metabolism by ectonucleotidases, mainly NTPDases (nucleoside triphosphate diphosphohydrolases) and adenosine kinase could regulate liver inflammatory responses through either removing the excessive extracellular ATP or transferring a phosphate group from ATP to adenosine . Recent data have shown that ATP may play a role in liver sterile inflammation by independently affecting inflammasome activation. It was shown that the protective effect of the P2X7 selective antagonist A438079 against APAP hepatotoxicity in vivo can was owing to its direct effect on metabolic activation and cell death pathways in hepatocytes .
Neutrophils are leukocytes involved in the defence against microorganisms. Since their discovery in 1893, these cells have been extensively associated not only with tissue damage observed during acute infections, but also with sterile inflammatory responses . Circulating neutrophils are rapidly recruited from the blood into tissues during sterile injury. Our group has previously demonstrated that, following liver sterile necrosis, the local release of extracellular ATP enhances neutrophil adhesivity to endothelial cells, mainly via β2-integrins, which will allow these cells to crawl precisely to necrotic sites following an intravascular gradient of CXC-chemokine receptor-2 (CXCR2) ligands and mitochondria-derived formyl peptides . Such purinergic actions were almost totally derived from signalling via P2X7 receptors and inflammassome activation. However, extracellular ATP was not necessary for neutrophil chemotaxis towards dead cells. These data suggest that extracellular ATP may activate resident cells to guide infiltrating immune cells to initially localize necrotic sites, but it is not a major chemotactic signal. Strikingly, blockage of neutrophil infiltration to the liver following APAP overdose dramatically reduced hepatotoxicity [74, 76], which is also reproduced by dampening purinergic signalling via P2X7 receptors . Despite the concerns related to the actual contribution of neutrophils to sterile liver inflammation [81-83], pharmacological strategies aimed at reducing both neutrophil infiltration and activation within injured liver are considered promising for the clinical management of DILI (Fig. 2).
Liver fibrosis, cirrhosis and transplantation
Liver fibrosis, and its progress to cirrhosis, results from chronic liver damage with concomitant accumulation of extracellular matrix (ECM) proteins . Liver fibrosis may be a consequence of excessive alcohol intake, chronic hepatitis C virus infection and nonalcoholic steatohepatitis. The unique hepatic architecture is disfigured by accumulation of ECM proteins and fibrous tissue leading to formation of nodules that contain regenerating hepatocytes . A massive loss of cell viability and a reduction in intrahepatic blood flow culminate in hepatic insufficiency and portal hypertension during cirrhosis.
Amongst liver-resident cells, special interest has been devoted to the role of stellate cells in hepatic collagen deposition. The hepatic stellate cell has emerged in the past decades as multipurpose mesenchymal cell playing a critical role in both liver homeostasis and disease . In fact, the paradigm of stellate cell activation into contractile myofibroblasts as the major pathway in hepatic fibrogenesis has opened new perspectives to treat liver fibrosis. Consistent with this, the approach of purinergic signalling during liver fibrosis has already being reported. Administration of adenosine partially reverts carbon tetrachloride (CCL4)-induced liver fibrosis, and the mechanism underlying this effect may be explained by adenosine signalling through A2A receptors to modulate procollagen-1 transcription .
Severe cases of liver fibrosis and cirrhosis necessitate liver transplantation as the only life-saving procedure. The post-operatory survival rate is intimately associated with graft viability. The University of Wisconsin preservation solution is one of the most established protocols to preserve graft, and contains a high concentration of adenosine . Despite the expression of purinergic receptors on a variety of immune cells, data concerning hepatic graft survival are still scarce. In contrast, ATP-based strategies to enhance liver regeneration have gained special attention. Thus, ATP activates proliferation of hepatocytes in vitro and in vivo and enhances hepatocyte responsiveness to growth factors via P2Y2 receptor activation and calcium influx [87-90].
Chronic liver disease, including cirrhosis, frequently burgeons in to liver cancer. Several disturbances in liver functions are observed during tumorigenesis, including enhanced cell multiplication, modulation of immune response and altered metabolic behaviour. Interestingly, all these actions may be also controlled by P2 receptor signalling, which substantiate the growing body of studies showing that the purinergic system regulates cell proliferation rate in both physiological and pathological conditions. It is well accepted that ATP has a potent antiproliferative effect, and tumours implanted in CD39-deficient mice, which display lower extracellular ATP metabolism ability, exhibit increased necrosis in association with P2X7 expression . Therefore, higher levels of extracellular ATP may reduce tumour cell proliferation, suggesting that dampening CD39-mediated ATP cleavage may be useful in clinical liver tumour management. Previous in vivo data have shown that daily intraperitoneal injection of ATP inhibited tumour growth in mice [92, 93], which also enhanced the antitumoral effects of radiotherapy or chemotherapy [94, 95]. The mechanisms underlying these effects are still under debate. However, while ATP might act directly on P2R to promote anticancer effect, ATP metabolism to adenosine by ectonucleotidases might also enhance P1R activation and reduce tumour proliferation. In fact, ATP and ADP degradation to adenosine by hepatoma cells in vitro (Li-7A cells) led to inhibition of cell growth and caspase 3 activation, indicating that ATP metabolism may controversially cause apoptosis in tumour cells . A seminal review on P2 receptors signalling and cancer development can be found at .
Conclusions and perspectives
Facing ATP beyond its most famous role as the prototypical energy molecule has transformed our knowledge about how different cells can recognize intracellular molecules and how these cells react to them. ATP is constantly released within the liver during homeostasis via different mechanisms and its signalling through P2 receptors influence liver regeneration, bile secretion and metabolism. However, disturbances in purinergic discharge and signalling may be crucial for hepatopathies even in the absence of infection, as during DILI, liver cancer and fibrosis. It is nowadays believed that three conditions, namely the location (i.e. extracellular or intracellular), the concentration and the target cell will profoundly influence the biological effects that purines will promote in the systems. Along the same line, a successful therapeutic approach by manipulating the purinergic system may be challenging mainly owing to the different and paradoxical effects of their components. Thus, further studies will not only be key to expand our knowledge on the biology of cell-to-cell communication, but will equally accelerate the translation of the purinergic signalling management to the clinics.
This study was supported by CNPq, CAPES, PRONEX, FAPEMIG, FAPESP, The Fund for Scientific Research (FWO) Flanders-Belgium and The Research Council of the Vrije Universiteit Brussel (VUB)-Belgium.