P2X7 receptor is a member of ATP-gated non-selective cation channels. P2X7 receptor is widely distributed in vivo, and its expression is always observed to be up-regulated in the pathological inflammatory circumstances. P2X7 receptor has an unusual property of forming membrane pore during prolonged agonist exposure or high concentrations of agonist activation, different from other members of P2X receptors (P2X1-6). Because of this property, P2X7 receptor has been implicated in inflammatory cytokine release, and is closely related to inflammatory diseases. With the wide application of the P2X7-knockout animal model and specific P2X7 receptor antagonists in inflammatory disease research, P2X7 receptor is emerging as a new target for the treatment of inflammatory diseases. This article will review the recent progress regarding the effect of P2X7 receptor on inflammatory diseases and its mechanism.
Animals ; Cytokines ; metabolism ; Disease Models, Animal ; Inflammation ; metabolism ; Purinergic P2X Receptor Antagonists ; pharmacology ; Receptors, Purinergic P2X7 ; metabolism

OBJECTIVE: Accumulating evidence suggests that inflammation plays a role in the pathophysiology of major depression. The adenosine triphosphate (ATP)-sensitive P2X7 receptor (P2X7R) plays a crucial role in microglial activation caused by inflammation. The dye brilliant blue G (BBG) is a P2X7R antagonist. This study examined whether BBG shows antidepressant effects in an inflammation-induced model of depression. METHODS: We examined the effects of BBG (12.5, 25, or 50 mg/kg) on serum tumor necrosis factor-alpha (TNF-alpha) levels after administering the bacterial endotoxin lipopolysaccharide (LPS; 0.5 mg/kg) and the effects of BBG (50 mg/kg) on depression-like behavior in the tail-suspension test (TST) and forced swimming test (FST). RESULTS: Pretreatment with BBG (12.5, 25, or 50 mg/kg) significantly blocked the increase in serum TNF-alpha levels after a single dose of LPS (0.5 mg/kg). Furthermore, BBG (50 mg/kg) significantly attenuated the increase in immobility time in the TST and FST after LPS (0.5 mg/kg) administration. CONCLUSION: The results suggest that BBG has anti-inflammatory and antidepressant effects in mice after LPS administration. Therefore, P2X7R antagonists are potential therapeutic drugs for inflammation-related major depression.
Adenosine Triphosphate ; Animals ; Depression ; Inflammation ; Mice* ; Physical Exertion ; Receptors, Purinergic P2X7 ; Tumor Necrosis Factor-alpha*

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1~2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 micrometer Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 micrometer of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca2+]i response; a transient [Ca2+]i peak and sustained [Ca2+]i increase secondary to ATP-stimulated Ca2+ influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.
Adenosine Triphosphate ; Blister ; Caspase 3 ; Cell Death ; Epithelial Cells ; Membranes ; Phosphorylation ; Receptors, Purinergic P2X7 ; Salivary Glands

We investigated the role of central P2X receptors in inflammatory pain transmission in the orofacial area in rats. Experiments were carried out using male Sprague-Dawley rats weighing 230-280g. Complete Freund's adjuvant (CFA, 40 microL) was applied subcutaneously to the vibrissa pad to produce inflammatory pain. The intracisternal administration of iso-PPADS tetrasodium salt, a non-selective P2X receptor antagonist, A317491 sodium salt hydrate, a P2X2/3 receptor antagonist, 5-BDBD, a P2X4 receptor antagonist, or A438079 hydrochloride, a P2X7 receptor antagonist, was performed 5 days after CFA injection. Subcutaneous injections of CFA produced increases in thermal hypersensitivity. Intracisternal injections of iso-PPADS (25 microg) or A438079 (25 or 50 microg) produced significant anti-hyperalgesic effects against thermal stimuli compared to the vehicle group. A317491 or 5-BDBD did not affect the head withdrawal latency times in rats showing an inflammatory response. Subcutaneous injections of CFA resulted in the up-regulation of OX-42, a microglia marker, and GFAP, an astrocyte marker, in the medullary dorsal horn. The intracisternal administration of A438079 reduced the numbers of activated microglia and astrocytes in the medullary dorsal horn. These results suggest that a blockade of the central P2X7 receptor produces antinociceptive effects, mediated by inhibition of glial cell function in the medullary dorsal horn. These data also indicate that central P2X7 receptors are potential targets for future therapeutic approaches to inflammatory pain in the orofacial area.
Animals ; Astrocytes ; Freund's Adjuvant ; Head ; Horns ; Humans ; Hyperalgesia ; Hypersensitivity ; Inflammation ; Injections, Subcutaneous ; Male ; Microglia ; Neuroglia ; Rats* ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X4 ; Receptors, Purinergic P2X7* ; Sodium ; Up-Regulation

OBJECTIVETo clone the entire coding sequence and analyze the function of P2X7 receptor of J6-1 human leukemia cells.

METHODSThe entire coding sequence of P2X7 receptor was amplified by RT-PCR and then inserted into pTARGET plasmid to construct an eukaryotic expressing plasmid followed by DNA sequencing. HEK293 cells stably expressing P2X7 receptor were obtained after transfection and screening, and confirmed by RT-PCR and Western blotting. The bleb formation upon agonist stimulation was observed under phase contrast microscope.

RESULTSThe entire coding sequence of P2X7 receptor of J6-1 cells was successfully cloned. DNA sequencing analysis revealed a substitution of G559, for A559, causing a substitution of Glu187 for Gln187. The P2X7 receptor derived from J6-1 cells could be functionally expressed in HEK293 cells, in which bleb formation could be detected upon stimulation.

CONCLUSIONSThe entire coding sequence of P2X7 receptors was successfully cloned from J6-1 leukemia cells. Other unknown mechanism may contribute to the dysfunction of P2X7 receptor in these cells.

Cell Line, Tumor ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Expression ; Humans ; Leukemia ; genetics ; metabolism ; Receptors, Purinergic P2 ; genetics ; physiology ; Receptors, Purinergic P2X7 ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVE: Purinergic 2X7 receptor (P2X7R) activation is known to be involved in pathogenesis of depression. Our aims were to investigate P2X7R-activated inflammasome pathways in parallel with induction of depression and to test the antidepressant-like effects of the selective P2X7R antagonist Brilliant Blue G (BBG) in a rat model of chronic unpredictable mild stress (CUMS). METHODS: Male Wistar albino rats were divided into control, CUMS, CUMS+BBG25 (25 mg/kg/day) and CUMS+BBG50 (50 mg/kg/day) groups (n=10 for each group). Various stressors were applied to rats for 6 weeks to establish the CUMS model and daily BBG treatment was started at the end of 3rd week. Sucrose preference test and forced swim test (FST) were performed to assess antidepressant-like effects. Brain samples were obtained for real-time polymerase chain reaction and immunohistochemistry analysis. RESULTS: In FST, duration of immobility was reduced in the CUMS+BBG50 group. Also, BBG treatment significantly enhanced sucrose preference. While NLRP3 gene expression levels were unchanged in rats exposed to the CUMS protocol, expression levels of other inflammasome pathway factors NLRP1, caspase-1, ASC, NF-κB, IL-1β, IL-6 and P2X7R were increased. BBG treatment reduced expression levels of these factors. Likewise, Iba-1 and GFAP immunoreactivities were enhanced by the CUMS protocol and this action was reversed by BBG treatment. CONCLUSION: Chronic administration of BBG in CUMS model results in antidepressant-like activity in a dose dependent manner. Molecular and histological results show that these effects might be at least partially related to the suppression of inflammasome-related neuroinflammatory responses and suggest involvement of NLRP1 in depression.
Animals ; Brain ; Depression ; Gene Expression ; Humans ; Immunohistochemistry ; Inflammasomes ; Interleukin-6 ; Male ; Models, Animal ; Rats ; Real-Time Polymerase Chain Reaction ; Receptors, Purinergic P2 ; Receptors, Purinergic P2X7 ; Sucrose

Microglia are involved in the inflammatory response and retinal ganglion cell damage in glaucoma. Here, we investigated how microglia proliferate and migrate in a mouse model of chronic ocular hypertension (COH). In COH retinas, the microglial proliferation that occurred was inhibited by the P2X7 receptor (P2X7R) blocker BBG or P2X7R knockout, but not by the P2X4R blocker 5-BDBD. Treatment of primary cultured microglia with BzATP, a P2X7R agonist, mimicked the effects of cell proliferation and migration in COH retinas through the intracellular MEK/ERK signaling pathway. Transwell migration assays showed that the P2X4R agonist CTP induced microglial migration, which was completely blocked by 5-BDBD. In vivo and in vitro experiments demonstrated that ATP, released from activated Müller cells through connexin43 hemichannels, acted on P2X7R to induce microglial proliferation, and acted on P2X4R/P2X7R (mainly P2X4R) to induce microglial migration. Our results suggest that inhibiting the interaction of Müller cells and microglia may attenuate microglial proliferation and migration in glaucoma.
Adenosine Triphosphate/pharmacology* ; Animals ; Cell Proliferation ; Glaucoma/metabolism* ; Mice ; Microglia/metabolism* ; Receptors, Purinergic P2X4/metabolism* ; Receptors, Purinergic P2X7/metabolism* ; Retinal Ganglion Cells/metabolism*

BACKGROUND: This study was aimed at investigating the relation of P2X7 receptor (P2X7R) expression with the clinicopathological features of papillary thyroid carcinoma (PTC) coexisting with Hashimoto's thyroiditis (HT). METHODS: We examined 170 patients (84, PTC with HT; 86, PTC without HT). P2X7R expression was examined by immunohistochemical methods. The staining intensity and patterns were evaluated and scored using a semi-quantitative method. RESULTS: The PTC with HT group was more likely to contain women and had less extrathyroid extension, lymph node (LN) metastasis, lymphovascular invasion, and recurrence than the PTC without HT group. Patients positive for P2X7R had significantly higher frequencies of lymphovascular invasion, extrathyroid extension, LN metastasis, and absence of HT. As shown by multivariate analysis, the expression of P2X7R was significantly higher if HT was absent and extrathyroid extension was present. In the PTC with HT group, the expression of P2X7R was significantly higher in patients with tumor multifocality, lymphovascular invasion, and extrathyroid extension. In the PTC without HT group, the expression of P2X7R was significantly higher in women and those having tumor multifocality. CONCLUSIONS: Coexistence of PTC with HT is associated with good prognostic factors, and P2X7R expression in PTC was correlated with poor prognostic factors and the absence of HT.
Female ; Hashimoto Disease ; Humans ; Lymph Nodes ; Methods ; Multivariate Analysis ; Neoplasm Metastasis ; Receptors, Purinergic P2X7* ; Recurrence ; Thyroid Gland* ; Thyroid Neoplasms* ; Thyroiditis*

Interactions between microbes and epithelial cells in the gastrointestinal tract are closely associated with regulation of intestinal mucosal immune responses. Recent studies have highlighted the modulation of mucosal immunity by microbe-derived molecules such as ATP and short-chain fatty acids. In this study, we undertook to characterize the expression of the ATP-gated P2X7 receptor (P2X7R) on M cells and its role in gastrointestinal mucosal immune regulation because it was poorly characterized in Peyer's patches, although purinergic signaling via P2X7R and luminal ATP have been considered to play an important role in the gastrointestinal tract. Here, we present the first report on the expression of P2X7R on M cells and characterize the role of P2X7R in immune enhancement by ATP or LL-37.
Adenosine Triphosphate ; Epithelial Cells ; Fatty Acids, Volatile ; Gastrointestinal Tract ; Immunity, Mucosal ; Peyer's Patches ; Phenobarbital ; Receptors, Purinergic P2X7*

This study aimed to construct the dual expression vectors of wide type or N187D mutant P2X7 receptor and intracellular domain of Notch1 (ICN1) linked by 2A peptide to coexpress them in leukemia cells so as to lay a foundation for further investigating the role of P2X7 in development of leukemia. Overlap PCR was used to construct the dual expression vectors encoding wide type or N187D mutant type P2X7 receptor and ICN1 linked by the self-cleaving 2A sequence. The results showed that stable expressing cell lines were obtained by retroviral infection followed by cell sorting after DNA sequence analysis. RT-PCR, Western blot, intracellular free calcium concentration analysis were used to verify the functionally successful construction of K562 cell line expressing P2X7 receptor alone or with ICN1. DNA sequence analysis revealed that all construction were right. The infection efficiency of packaged constructed virus ranged from 40% to 70% for K562 cells. Stable infected cell line was obtained by cell sorting. RT-PCR analysis revealed that P2X7 receptor and/or ICN1 could be detected at high level in their stable infected cell lines, respectively. Western blot analysis also showed that P2X7 receptor was highly expressed in cell line infected by virus with P2X7 receptor. Sustained increase in intracellular free calcium concentration ([Ca(2+)]i) could be observed in K562 cells overexpressing either type of P2X7 receptor upon stimulation with BzATP. It is concluded that the wide type or N187D mutant P2X7 receptor and ICN1 are simultaneously and functionally over-express in leukemia cells, which lay a foundation for further studying the role of P2X7 receptor in the development of leukemia.
Gene Expression ; Genetic Vectors ; Humans ; K562 Cells ; RNA, Messenger ; genetics ; Receptor, Notch1 ; genetics ; Receptors, Purinergic P2X7 ; genetics ; Retroviridae ; genetics