P2X7 receptors in the central nervous system.

For the past three decades, our laboratory has conducted pioneering research to elucidate the complexity of purinergic signaling in the CNS, alone and in collaboration with other groups, inspired by the ground-breaking efforts of Geoffrey Burnstock. This review summarizes our contribution to understand the nucleotide receptor signaling in the CNS with a special focus on the P2X7 receptor.

The P2Y/P2X divide: How it began.

Extracellular purine and pyrimidine nucleotides produce their pharmacological effects through P2 receptors. These were first named by Geoff Burnstock in an extensive review in 1978. They were then subdivided into P2X and P2Y purinoceptors by Burnstock and Kennedy in 1985, based on applying pharmacological criteria to data generated by functional studies in smooth muscle tissues. Several other P2 subtypes, P2T, P2Z, P2U and P2D were subsequently identified in the following years, again using pharmacological criteria. The number and identity of subtypes were clarified and simplified by the cloning of seven ATP-sensitive ligand-gated ion channel subunits and eight adenine and/or uracil nucleotide-sensitive G protein-coupled receptors from 1993 onwards. The former were all classified as members of the P2X receptor family and the latter as members of the P2Y receptor family. More recently, high resolution imaging of the tertiary and quaternary structures of several P2X and P2Y receptor subtypes has provided a much greater understanding of how and where agonists and antagonists bind to the receptors and how this leads to changes in receptor conformation and activity. In addition, medicinal chemistry has produced a variety of subtype-selective agonists and antagonists, some of which are now in clinical use. This progress and success is a testimony to the foresight, intelligence, enthusiasm and drive of Geoff Burnstock, who led the field forward throughout his scientific life.

P2X7 receptor and the NLRP3 inflammasome: Partners in crime.

Adenosine triphosphate (ATP) is a molecule that on one hand plays a central role in cellular energetics and which on the other is a ubiquitous signaling molecule when released into the extracellular media. Extracellular ATP accumulates in inflammatory environments where it acts as a damage-associated molecular pattern and activates the purinergic P2X receptor 7 (P2X7) in immune cells. P2X7 receptor activation induces the formation of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) inflammasome and the activation of the inflammatory caspase-1. Caspase-1 causes an inflammatory type of cell death called pyroptosis through the release of pro-inflammatory cytokines and intracellular content. Consequently, intense research efforts have been devoted to the design of novel anti-inflammatory therapies, focusing in particular on the P2X7 receptor and the NLRP3 pathway and the introduction of new blocking molecules in early phase clinical trials.

Nociceptive signaling mediated by P2X3, P2X4 and P2X7 receptors.

Chronic pain is a debilitating condition that often occurs following peripheral tissue inflammation and nerve injury. This pain, especially neuropathic pain, is a significant clinical problem because of the ineffectiveness of clinically available drugs. Since Burnstock proposed new roles of nucleotides as neurotransmitters, the roles of extracellular ATP and P2 receptors (P2Rs) in pain signaling have been extensively studied, and ATP-P2R signaling has subsequently received much attention as it can provide clues toward elucidating the mechanisms underlying chronic pain and serve as a potential therapeutic target. This review summarizes the literature regarding the role of ATP signaling via P2X3Rs (as well as P2X2/3Rs) in primary afferent neurons and via P2X4Rs and P2X7Rs in spinal cord microglia in chronic pain, and discusses their respective therapeutic potentials.

P2X receptors in cancer growth and progression.

It is increasingly appreciated that ion channels have a crucial role in tumors, either as promoters of cancer cell growth, or modulators of immune cell functions, or both. Among ion channels, P2X receptors have a special status because they are gated by ATP, a common and abundant component of the tumor microenvironment. Furthermore, one P2X receptor, i.e. P2X7, may also function as a conduit for ATP release, thus fuelling the increased extracellular ATP level in the tumor interstitium. These findings show that P2X receptors and extracellular ATP are indissoluble partners and key regulators of tumor growth, and suggest the exploitation of the extracellular ATP-P2X partnership to develop innovative therapeutic approaches to cancer.

The P2X7 receptor: A main player in inflammation.

Damage associated molecular patterns (DAMPs) are intracellular molecules released from infected or injured cells to activate inflammatory and reparatory responses. One of the most ancient and conserved DAMPs is extracellular ATP that exerts its phlogistic activity mainly through activation of the P2X7 receptor (P2X7R). The P2X7R is an ATP gated ion channel, expressed by most immune cells, including the monocyte-derived cell lineages, T and B lymphocytes and their precursors. Here we give an overview of recent and established literature on the role of P2X7R in septic and sterile inflammation. P2X7R ability in restraining intracellular bacteria and parasite infection by modulation of the immune response are described, with particular focus on Mycobacteria and Plasmodium. Emerging literature on the role of P2X7 in viral infections such as HIV-1 is also briefly covered. Finally, we describe the numerous intracellular pathways related to inflammation and activated by the P2X7R, including the NLRP3 inflammasome, NF-kB, NFAT, GSK3ß and VEGF, and discuss the involvement of P2X7R in chronic diseases. The possible therapeutic applications of P2X7R antagonists are also described.

The P2X7 receptor links mechanical strain to cytokine IL-6 up-regulation and release in neurons and astrocytes.

Mechanical strain in neural tissues can lead to the up-regulation and release of multiple cytokines including interleukin 6 (IL-6). In the retina, the mechanosensitive release of ATP can autostimulate P2X7 receptors on both retinal ganglion cell neurons and optic nerve head astrocytes. Here, we asked whether the purinergic signaling contributed to the IL-6 response to increased intraocular pressure (IOP) in vivo, and stretch or swelling in vitro. Rat and mouse eyes were exposed to non-ischemic elevations in IOP to 50-60 mmHg for 4 h. A PCR array was used to screen cytokine changes, with quantitative (q)PCR used to confirm mRNA elevations and immunoblots used for protein levels. P2X7 antagonist Brilliant Blue G (BBG) and agonist (4-benzoyl-benzoyl)-ATP (BzATP) were injected intravitreally. ELISA was used to quantify IL-6 release from optic nerve head astrocytes or retinal ganglion cells. Receptor identity was confirmed pharmacologically and in P2X7-/- mice, acute elevation of IOP altered retinal expression of multiple cytokine genes. Elevation of IL-6 was greatest, with expression of IL1rn, IL24, Tnf, Csf1, and Lif also increased more than twofold, while expression of Tnfsf11, Gdf9, and Tnfsf4 were reduced. qPCR confirmed the rise in IL-6 and extracellular ATP marker ENTPD1, but not pro-apoptotic genes. Intravitreal injection of P2X7 receptor antagonist BBG prevented the pressure-dependent rise in IL-6 mRNA and protein in the rat retina, while injection of P2X7 receptor agonist BzATP was sufficient to elevate IL-6 expression. IOP elevation increased IL-6 in wild-type but not P2X7R knockout mice. Application of mechanical strain to isolated optic nerve head astrocytes increased IL-6 levels. This response was mimicked by agonist BzATP, but blocked by antagonists BBG and A839977. Stretch or BzATP led to IL-6 release from both astrocytes and isolated retinal ganglion cells. The mechanosensitive up-regulation and release of cytokine IL-6 from the retina involves the P2X7 receptor, with both astrocytes and neurons contributing to the response.

A317491 relieved HIV gp120-associated neuropathic pain involved in P2X3 receptor in dorsal root ganglia.

Glycoprotein 120 (gp120) is an HIV envelope glycoprotein. Gp120 can directly stimulate the primary sensory afferent neurons and cause hyperalgesia. The P2X3 receptor in dorsal root ganglia (DRG) is involved in the transmission of pain. In this study, we aimed to explore the role of the P2X3 receptor in gp120-induced neuropathic pain. Our data showed that mechanical and thermal hyperalgesia in rats treated with gp120 were increased compared to those in the control group. The expression levels of the P2X3 mRNA and protein in rats treated with gp120 were higher than those in the control group. The P2X3 antagonist A317491 decreased mechanical hyperalgesia and thermal hyperalgesia and the up-regulated expression levels of P2X3 mRNA and protein in rats treated with gp120. A317491 decreased ERK1/2 phosphorylation levels in the gp120-treated rat DRG. In addition, P2X3 agonist α,ß-methylene ATP (α,ß-meATP)-activated currents in DRG neurons cultured with gp120 were higher than those in control neurons. The inhibitory effect of A317491 on α,ßme-ATP-induced currents in DRG neurons from the gp120-treated neurons was larger than that for control neurons. Molecular docking data showed that A317491 may be acted in the gp120 protein to inhibit the gp120 initiated the P2X3 activation, decrease the sensitizing DRG primary afferents and reduce the signal transmission of neuropathic pain in gp120-treated rats. Therefore, the inhibition of the P2X3 receptor in rat DRG neurons relieved gp120-induced mechanical hyperalgesia.

Contribution of nerve growth factor to upregulation of P2X3 expression in DRG neurons of rats with femoral artery occlusion.

Femoral artery occlusion augments the sympathetic nerve and pressor responses to muscle contraction and muscle metabolites injected into the arterial blood supply of the hindlimb muscles in rats. The underlying mechanism by which these reflex responses are enhanced after muscle vascular insufficiency is unclear. Purinergic P2X(3) receptor has been reported to contribute to the metabolic component of the exercise pressor reflex. Thus the purpose of this study was to examine if chronic femoral occlusion would alter the expression of P2X(3) in dorsal root ganglion (DRG) neurons of rats. Also, P2X(3)-mediated sympathetic responsiveness was examined after femoral occlusion. In addition, the role played by nerve growth factor (NGF) in regulating the expression and response of P2X(3) was examined. Western blot analysis showed that 24 h of femoral ligation increased the levels of P2X(3) (optical density: 0.93 ± 0.07 in control and 1.37 ± 0.10 after occlusion; P < 0.05 vs. control). The fluorescence immunohistochemistry further demonstrated that the occlusion elevated P2X(3) expression in DRG neurons (percentage of P2X(3)-positive cells: 33 ± 3% in control and 51 ± 3% in occlusion; P < 0.05 vs. control). Furthermore, the results showed that responses of renal sympathetic nerve activity and blood pressure to stimulation of P2X were greater in occluded rats than responses in control rats by injection of α,ß-methylene ATP into the arterial blood supply of the hindlimb muscle. Finally, infusion of NGF in the hindlimb muscles of healthy rats increased P2X(3) (optical density: 0.98 ± 0.12 in control and 1.37 ± 0.16 with NGF; P < 0.05 vs. control). The pressor response to injection of α,ß-methylene ATP was increased in the rats with NGF infusion. Likewise, blocking NGF attenuated exaggeration of the reflex response induced by α,ß-methylene ATP in occluded rats. The findings of this study suggest that the levels of P2X(3) in primary afferent neurons are upregulated as the blood supply to the hindlimb is deficient under ischemic conditions, leading to augmentation of the muscle reflex. NGF is closely related to increases in P2X(3) receptor expression and response.

P2X7 receptor agonists pre- and postcondition the heart against ischemia-reperfusion injury by opening pannexin-1/P2X7 channels.

Protection of the heart from ischemia-reperfusion injury can be achieved by ischemic preconditioning and ischemic postconditioning. Previous studies revealed that a complex of pannexin-1 with the P2X(7) receptor forms a channel during ischemic preconditioning and ischemic postconditioning that results in the release of endogenous cardioprotectants. ATP binds to P2X(7) receptors, inducing the formation of a channel in association with pannexin-1. We hypothesized that this channel would provide a pathway for the release of these same cardioprotectants. Preconditioning-isolated perfused rat hearts with 0.4 µM ATP preceding 40 min of ischemia minimized infarct size upon subsequent reperfusion (5% of risk area) and resulted in >80% recovery of left ventricular developed pressure. Postconditioning with ATP after ischemia during reperfusion was also protective (6% infarct and 72% recovery of left ventricular developed pressure). Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X(7) receptors (brilliant blue G and A438079) blocked ATP pre- and postconditioning, indicating that ATP protection was elicited via the opening of a pannexin-1/P2X(7) channel. An antagonist of binding of the endogenous cardioprotectant sphingosine 1-phosphate to its G protein-coupled receptor diminished protection by ATP, which is also consistent with an ATP-dependent release of cardioprotectants. Suramin, an antagonist of binding of ATP (and ADP) to P2Y receptors, was without effect on ATP protection. Benzoyl benzoyl-ATP, a more specific P2X(7) agonist, was also a potent pre- and postconditioning agent and sensitive to blockade by pannexin-1/P2X(7) channel antagonists. The data point out for the first time the potential of P2X(7) agonists as cardioprotectants.

Purinergic receptor agonists modulate phagocytosis and clearance of apoptotic cells in macrophages.

Phagocytosis plays an important role in controlling inflammation and antigen cross-presentation through the uptake of apoptotic bodies from dying cells. As dying cells are known to release nucleotides and other "danger signals", we investigated whether extracellular nucleotides may affect phagocytosis through binding to P2 purinergic receptors on phagocytic cells. We here show that the purinergic receptor agonists, ATP, ADP, α,ß-methylene ATP (α,ß-meATP), 3'-O-(4-benzoyl)benzoyl ATP, UTP and UDP, increased phagocytosis of latex beads, and some of them increased endocytosis and/or macropinocytosis of dextran by macrophages. The enhanced phagocytosis could be inhibited by pre-treatment with the P2X and P2Y antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid and suramin, and the P2Y1-selective antagonist, MRS2179. The nucleotides induced upregulation in macrophages of the ß2 integrin CD11b/CD18 (Mac-1) and the vitronectin receptor (α(v)ß3, CD51/CD61), both of which are involved in recognition and internalization of apoptotic cells. In addition, ATP and α,ß-meATP increased adhesion of apoptotic cells to macrophages, both in vitro and in vivo, and α,ß-meATP had a small effect on adhesion of necrotic cells. The nucleotides had no effect on adhesion of viable cells. We propose that engagement of the P2 receptors (P2X1, or P2X3) by extracellular nucleotides released from dying cells increases the ability of macrophages to bind apoptotic bodies, thus enhancing their ability to internalize and present antigens from the dying cells.