Role of purinergic P2X receptors in the control of liver homeostasis

Abstract

It is now accepted that extracellular ATP and other nucleotides are potent signaling molecules, akin to neurotransmitters, hormones and lipid mediators. In the liver, several clues support a significant role for extracellular ATP-induced signaling pathways in the control of tissue homeostasis. First, ATP and other nucleotides are physiologically detected in extracellular fluids within the liver, including sinusoidal blood and intraductular bile, in various mammalian species including human and rodents. Moreover, finely tuned mechanisms of ATP release by different liver cell types have been described, under physiological cellular changes. In addition, most hepatic cells constitutively express, at the membrane level, several ATP-metabolizing ectoenzymes and ATP-sensitive receptors that modulate and transduce these mediator signals respectively. Finally, hepatic cells also express numerous membrane transporters that actively contribute to purinergic salvage pathways. Once released in the extracellular medium, unmetabolised ATP molecules can bind to purinergic P2X and P2Y receptors, and subsequently trigger various intracellular signal transduction pathways collectively referred to as purinergic signaling. In the liver, purinergic signaling has been shown to regulate key basic cellular functions, such as glucose/lipid metabolism, protein synthesis and ionic secretion, and homeostatic processes, such as cell cycle, inflammatory response and immunity. Whilst the functional relevance of P2Y receptors in liver physiology has been well documented, limited information is available regarding the potential role of hepatic P2X receptors in the modulation of liver homeostasis.

P2X receptor signaling

Identification and cloning of P2X receptors

Burnstock advanced in 1972 the concept of purinergic signaling, hypothesizing that extracellular ATP acts as a signaling molecule, such as neurotransmitters adrenaline and acetylcholine, based on early studies on excitable tissues by Szent-Györgyi and Drury, Holton et al. as well as his own work (1). Before long, surface-located membrane receptors called purinoreceptors including adenosine-activated P1 and ATP-activated P2 receptor classes were described for the first time as receptors that mediate purinergic signaling in tissues (2). In 1985, a new classification system (using functional criteria, such as structure, pharmacology, and mechanism of action) for purinergic receptors was proposed, wherein P2 receptors were divided into ionotropic P2X and metabotropic P2Y receptors (3). In the mid 90s, P2X1 and P2X2 were the first P2X family members identified by cDNA cloning in rat species (4–5). Hitherto, the P2X receptor gene family comprises seven distinct genes P2RX1-7, each encoding for P2X1-7 protein subunits and found in various mammalian species including human and rodents (6).

Molecular structure of P2X receptors (P2XRs)

P2X receptors are non-selective ligand-gated ion channels that are activated by extracellular ATP. Each P2X channel is formed by the hetero-/oligomeric assembly of three P2X protein subunits (7). This long-debated minimal trimeric stoichiometry was recently confirmed by a study describing the solved crystal structure of zebrafish P2×R4 ion channel in closed state (8). The basic P2X subunit is a protein 379 (P2×6) to 595 (P2×7) amino acids long, with the following structural topology: intracellular N- and C-termini, two transmembrane spanning regions that delimits a large ectodomain (9). This extracellular loop contains the so-called ATP-binding domain of ten conserved cysteine residues forming disulfide bonds, and several phosphorylation, glycosylation, and palmitoylation sites likely involved channel activity modulation (6). Biochemical and biophysical studies in heterologous expression systems, such as Xenopus oocytes or mammalian HEK293 and 1321N1 cell lines, have shown that all P2X receptors can form both hetero- and homomeric channels. Accordingly, all seven homomeric channels: P2X1-7 and, at least, a dozen heteromeric channels have been identified and pharmacologically characterised (9–13). However, additional functional evidences from studies in native tissues as well as genetically modified mouse models are needed to determine or confirm the existence and physiological relevance of the combinations identified in biochemical studies. This is well illustrated by the debate surrounding the existence of native homomeric P2X6 receptors (14–15), although functional highly glycosylated P2X6 receptors have been described upon transient transfection of HEK293 cells with recombinant rat P2X6 receptor cDNA (11). Also, unknown are the factors influencing the stoichiometry of each partner P2X subunit for a given heteromeric channel (13–14).

Activation mechanism of P2X receptors (P2XRs)

The basic activation mechanism of P2X receptor involves conformational changes of the ion channel from a “closed” to an “open” state, upon binding of (at least) three ATP molecules (7). These structural changes lead to the opening of a pore with an increased permeability to Ca²⁺, Na⁺, K⁺ and Cl⁻ ions. The changes in P2X ion channel permeability trigger a fast membrane depolarization, which is followed by the generation of cellular Ca²⁺ (and to a less extent Na⁺) influxes that activate still poorly understood downstream intracellular coupling mechanisms. P2X receptors are characterised by different pharmacological properties. First, the potency of ATP ligand for P2X receptors is ranging from nanomolars (ex: P2×1) to millimolars (ex: P2×7) (6, 16). Second, prolonged exposure to ATP leads to a desensitization phase of P2X receptors that varies from fast (milliseconds for P2×1) to fairly sustained (more than twenty seconds for P2X2-4 and P2×7) (6). Third, pharmacokinetics of P2X receptors can be distinguished using various synthetic ATP analogues that act as agonists (ex: α,β-MeATP; ATPγS) or antagonists (ex: TNP-ATP; PPDADS) (15, 17). For instance, both P2X1 and P2X3 receptors are selectively activated by α,β-MeATP and inhibited by TNP-ATP. Finally, P2X receptor channel activity can be affected by physiological parameters, such as extracellular pH, divalent ions (ex: Zn²⁺, Cu²⁺, Hg²⁺, Ni²⁺ or Cd²⁺) and interactions with other membrane receptors. For example, the activation of the P2X2 receptor is potentiated by Zn²⁺ and blocked by Ca²⁺ (15).

Now that the reader is hopefully more familiar with the P2X receptor pharmacology, the following section will recapitulate the existing data about the liver distribution of these receptors.

Distribution of hepatic P2X receptors

All P2X receptors are expressed in the liver, with P2X4 and P2X7 being the most widely distributed proteins (18–19). The distribution of hepatic P2X receptors is summarized in Table1. Parenchymal cells or hepatocytes express all P2X receptor subtypes, except P2X5 and P2X6. Non-parenchymal cells including cholangiocytes, liver fibroblasts, endothelial cells, immune cells and smooth muscle cells express also multiple P2X receptors. To the best of our knowledge, there are no data available in the current literature regarding the expression of P2X receptors in liver stem or precursor cells (including hepatoblasts and oval cells). It is interesting to note the expression of multiple P2X receptors in various functionally specialised hepatic cells, which suggests that ATP-mediated signaling pathways through P2X receptors likely modulate different aspects of liver physiology.

Table 1.

Pharmacology and Liver Distribution of P2X receptors

Receptor	Agonists¶	Antagonists¶	Modulators	Transduction mechanism¶	Hepatic distribution§
P2X1	ATP, α,β-MeATP, 2- MeSATP	IP5I, MRS2159, NF023, NF279, NF449, PPNDS	Cd2+, Gd2+, Zn2+	Ion channel (Ca2+ and Na+)	Hepatocytes, Kupffer cells, lymphocytes, portal vein myocytes smooth muscle cells
P2X2	ATP, ATPγS, AP4A	isoPPADS, NF279, NF770, RB2, Suramin	Ca2+, Gd2+, Hg2+, Zn2+	Ion channel (Ca2+)	Cholangiocytes, hepatocytes, lymphocytes, mesenteric artery cells
P2X3	ATP, α,β-MeATP, 2- MeSATP, AP5A	IP4I, NF110, RO-3, RO-85, Spinorphin, TNP-ATP	Cibracon blue, ethanol, Zn2+	Intrinsic ion channel	Cholangiocytes, hepatocytes, portal vein myocytes, smooth muscle cells
P2X4	ATP, α,β-MeATP, BzATP, CTP	5-BDBD, Coomassie blue, oATP, paroxetine, NF023	Cd2+, Hg2+, hydrogen peroxide, toluene	Ion channel (Ca2+ and Na+)	cholangiocytes, cyst epithelial cells, dendritic cells), hepatic stellate cells, hepatocytes (apical and basolateral membranes, Kupffer cells, lymphocytes, portal fibroblasts, portal vein myocytes, smooth muscle cells, vascular endothelium
P2X5	ATP, α,β-MeATP, ATPγS	BBG, Coomassie blue, PPADS, Suramin	Zn2+	Intrinsic ion channel (Cl−)	Portal vein myocytes, smooth muscle cells
P2X6	ATP, BzATP	isoPPADS, TNP- ATP	N.D.	Intrinsic ion channel	Cholangiocytes, Kupffer cells
P2X7	ATP, BzATP, β,γ-MeATP, ATPγS	A740003, AZ 11645373, KN62, MRS2427, MRS2159, oATP	Calmodulin, Mg2+, Zn2+	Intrinsic ion channel (Ca2+) and pore	cholangiocytes, dendritic cells, hepatic stellate cells, hepatocytes, lymphocytes, mesenteric artery cells, portal fibroblasts, smooth muscle cells, vascular endothelium

N.D., not determined.

^¶Data obtained from mRNA and/or protein expression studies using native tissues, primary and/or immortalised cell lines (11, 19, 23, 32, 38, 40, 48, 62, 68–71).

^§Data obtained from pharmacological and functional studies in native tissues, primary and/or immortalised cell lines (6–7, 12, 15, 52, 71–81).

Finally, the next section will address the contribution of P2X receptor signaling pathways to liver homeostasis.

Modulation of liver functions by P2X receptors

Bile formation

Several lines of evidence have supported a role for extracellular ATP in the regulation of bile formation by hepatocytes and cholangiocytes (20–22). First, studies have shown that extracellular ATP is detectable in bile samples collected downstream at the common bile duct level in human and rodents as well as in the supernatants of primary and/or immortalised cholangiocytes, as mentioned above. For instance, a recent study using primary mouse intrahepatic bile duct units showed evidence of ATP release by these cells, when subjected to mechanosensitive (ex: fluid flow) stimulation (23). Interestingly, measurements of nucleotide release by small cholangiocytes (located upstream along the biliary tree) were greater than by large ones (located downstream) following stimulation. These findings suggest that purinergic signaling triggered in cholangiocytes can be actively involved in bile formation and that biliary ATP release mechanism is differently regulated by morphologically and functionally distinct cholangiocytes. Second, ATP stimulation induces in both primary and immortalised murine cholangiocytes Cl⁻ currents that are essential to both alkalinization and bile formation processes. The P2X4 receptor has been recently identified as key regulator of this mechanism in cholangiocytes (23). Finally, numerous ectoenzymes and transporters that can modify greatly extracellular ATP bioavailability are expressed in the canalicular membrane domain of hepatocytes (24–25). However, there has been, thus far, no direct evidence of canalicular ATP release by parenchymal cells, as bile sample collection in the lumen of bile canaliculi is technically challenging. Yet, because of the anatomic continuity between hepatocytes and intrahepatic bile duct cells, it is reasonable to hypothesize that unmetabolized ATP upon release in the canalicular lumen by hepatocytes could possibly stimulate downstream purinergic P2X and P2Y receptors expressed apically in cholangiocytes and regulate their physiological functions, for instance bile formation. While the contribution of potent mediator secretin and metabotropic P2Y2 (26) and P2Y12 receptors (27) to bile formation process has been established, the role of P2X receptors in this important hepatic function needs to be further investigated.

Glucose metabolism

Early studies using the isolated perfused liver model showed that infusion of exogenous ATP and UTP nucleotides stimulates hepatic glycogenolysis and glucose release by rat hepatocytes (28–29), suggesting a role for P2 receptor signaling in hepatic glucose metabolism. A more recent study has shown that exposure to P2X-selective BzATP agonist causes decreases in glycogen contents in isolated rat and human hepatocytes (30). In the same study, an interesting observation showed that P2X4 mRNA expression is increased in glycogen-rich hepatocytes from carbohydrate-activated transcription factor (ChREBP)-deficient mice, suggesting a functional link between P2X4 and glucose metabolism in hepatocytes. Another recent study has shown that, upon a glucose tolerance test, plasma glucose levels are higher in ectonucleotidase CD39/NTPDase1-deficient (Cd39^−/−) mice (wherein extracellular ATP hydrolysis is considerably diminished) than in wild-type mice (31). Interestingly, increases in plasma glucose levels could also be induced in wild-type mice by pre-administration of ecto-ATPase inhibitor ARL-67156 or administration of exogenous nucleotide ATP (but not of ADP, UTP or UDP) species, suggesting functional involvement of P2X receptor-mediated signaling in glucose homeostasis.

Inflammation and Immunity

Immune cell functions within the liver are also modulated by P2X receptors. In developing post-natal rat livers, intraperitoneal injection of lipopolysaccharide causes increases of P2X6 gene transcriptional activity in resident Kupffer cells, suggesting that extracellular ATP-induced signaling mediates immune functions in this cell type (32). In a mouse concavalin A-induced hepatitis model, P2X7 receptor signaling exacerbates pro-inflammatory activities in natural killer T (NKT) cells (33). In the same model of hepatitis, genetic deletion of ectoenzyme CD39 is associated with reduced liver injury, because of increased NKT cell apoptosis resulting from unimpeded activation of P2X7 expressed on these cells (34). A role for purinergic signaling in the modulation of liver anti-viral response has been recently uncovered by a number of studies showing that pharmacological antagonism of P2X7 receptors with synthetic compounds, such as suramin and brilliant blue G causes reduced hepatitis B and/or D virus infection, in human primary and immortalised hepatocytes (35–36). Also, reduced parasite burden and necrotic areas are observed in livers of mice with functional P2X7 receptors, following Toxoplasma gondii infection (37).

Ionic secretion

A number of pharmacological studies have shown that P2X receptor stimulation can regulate ionic secretion in hepatocytes and cholangiocytes. In murine and human hepatocytes, activation of P2X4 and P2X7 receptor leads to increased Ca²⁺ and Na⁺ influxes (30, 38). In rat hepatoma HTC cells, P2X4 activation modulates cellular regulatory volume decrease response by controlling the opening of volume-sensitive rectifying outwardly Cl⁻ channels (39). P2X receptors are also involved in the control of ionic secretion by biliary epithelium. In Mz-Cha-1 cholangiocytes, BzATP-induced Cl⁻ currents can be inhibited by addition of Cu²⁺ ions in the extracellular medium, suggesting ionic secretion is mediated, at least in part, by P2X4 receptors (40).

Liver cell proliferation and death

In liver, purinergic signaling plays an important role in the maintenance of organ cell mass, by modulating both cellular growth and apoptosis mechanisms. Functional studies in vitro and in vivo have demonstrated that proliferation of liver cell types, such as hepatocytes and liver sinusoidal cells is mainly mediated by ATP/UTP-sensitive P2Y receptors (41–44). On the other hand, programmed cell death in hepatic cells, such as hepatocytes and NKT cells, is mediated by ionotropic P2X7 receptor. In primary rat hepatocytes, the purinergic P2Z receptor, now known as P2X7 receptor, mediates Ca²⁺-dependent cell death induced by extracellular adenosine triphosphate (45). Another functional study has reported that isolated rat hepatocytes exposed to high concentrations of P2X4/P2X7-selective agonist BzATP exhibit several classical hallmarks of apoptosis: membrane blebbing and pore formation (38). In addition, these mechanisms could be inhibited by addition of P2X receptor antagonist oxidised ATP in the medium, suggesting these mechanisms are regulated by P2X7 receptors expressed on hepatocytes. Moreover, in livers from Cd39^−/− mice, resulting prolonged ATP exposure leads to an increased activation of P2X7 receptors expressed on tumor cells and causes reduced tumor progression, when compared to wild-type animals (46).

Vasculature

The role of P2X receptors in the modulation of liver vasculature function(s) has not been addressed extensively. Using pharmacological and genetic approaches, a number of studies have demonstrated that extracellular ATP signaling, mainly through P2X1 receptor activation, modulates adrenergic-mediated control of vascular tone in both rat hepatic artery and portal vein systems (47–49). Also, it has been recently shown that ATP stimulation increases portal vein pressure in Cd39^−/− mice upon administration of nitric oxide (NO) inhibitor L-NG-Nitroarginine, suggesting a P2X-dependent vasoconstrictive effect, in absence of P2Y-mediated NO release (50).

Conclusion

The contribution of purinergic signaling to liver homeostasis is now well established. This notion is supported by the identification in most hepatic cells of functional components of purinergic signaling: first, mechanisms involving constitutive and/or regulated release of nucleotides and nucleosides; second, cell surface ectoenzymes and membrane transporters that are involved in the metabolism of nucleotides and salvage pathways of nucleosides; third and finally, membrane receptors that transduce signals induced by extracellular nucleotides and nucleosides. Among the latter, ATP-gated P2X receptor ion channels are emerging as new potential players in modulating liver functions. Indeed, the expression of all seven P2X receptors has been described in the liver and their involvement in the modulation of several key hepatic functions, such as glucose metabolism, bile formation, cell proliferation, apoptosis, inflammation and immunity has been demonstrated (18–19). Yet, future investigations will be required to fully understand the involvement of P2X receptor signaling in liver homeostasis and, as a result, develop innovative and effective treatments of liver diseases. In that respect, the development of new potent pharmacological compounds (51–53) and molecular tools (54) targeting P2X receptors will be essential in achieving these goals.

Sidebar. Sources of extracellular nucleotides in the liver.

In the liver, there are various identified sources of extracellular ATP, the natural ligand for P2X receptors. Because of their central role in metabolism, nucleotides are found virtually in every cell (55). For instance, in hepatocytes, the intracellular ATP concentration is estimated between 3 and 5 millimolars (21). Thus, any active (ex: cell apoptosis) (56) or passive (ex: tissue injury) (44) process leading to loss of cell membrane integrity will cause the release of large amounts of ATP in the extracellular medium. A number of studies have also shown that various hepatic physiological fluids contain nucleotides and derivatives. For instance, biochemical analyses of normal bile samples collected from human, pig and rat donors detected the presence of physiologically relevant ATP, ADP and AMP nucleotides levels (57). In the same study, the authors estimated that the ATP concentration in human bile was 5.2 +/− 0.9 μM. In another study using the isolated perfused rat liver model, ATP levels were measured in the nanomolar range in sus-hepatic venous blood (58). Moreover, several studies have reported that nanomolar concentrations of ATP can be measured in the supernatants of various hepatic primary cells (isolated hepatocytes; isolated bile duct units) or immortalised cell lines (HTC, hepatoma; Mz-ChA-1, cholangiocarcinoma), consistent with a constitutive release of ATP (44, 57–62). On the other hand, hepatic cells can liberate ATP molecules in the extracellular medium upon stimulation and several examples of this mechanism have been reported in studies using pharmacological (ex: purinergic antagonist PPADS) and/or biochemical (ex; apyrase) approaches. Mechanical pressure (isolated hepatocytes; WB-F344 liver epithelial cell line; HTC) (39, 63–64), hypo-osmotic shock (isolated hepatocytes; Mz-ChA-1; NRC, isolated normal rat cholangiocytes) (59, 65), fluid flow (Mz-Cha-1; NRC; isolated mouse cholangiocytes) (23, 66) and exposure to biologically potent endogenous molecules such as bile acids (isolated hepatocytes) (67) can trigger the release of ATP molecules in the extracellular milieu. Thus, all these studies indicate that ATP release by hepatic cells is a fine-tuned mechanism and clearly support a second messenger role for extracellular ATP in different liver compartments.