Apically Sorted P2X7 Receptors Mediate Purinergic-Induced Pore Formation Preferentially in Apical Domains of the Plasma Membrane

Treatment of human epithelial cervical cells CaSki attached on filters with the P2X7-receptor (P2X7-R) agonist BzATP induced acute transient influx of calcium, most likely the result of P2X7-R channel activation, followed by slower sustained calcium influx. In cultures incubated in the presence of ethidium bromide (EB), BzATP induced slow and sustained influx of the dye with a time-course similar to the late slow calcium influx, suggesting P2X7-R pore formation. The acute and late calcium effects of BzATP were greater if the agonist was added to the luminal solution, facing the apical membrane of the cells. The EB effect of BzATP initially occurred in the apical membrane, while effects in the basolateral membrane were delayed and smaller. These results suggest that in polarized epithelial cells under steady-state conditions the P2X7-R is located in the apical membrane, and activation of the receptor induces formation of P2X7-R pores preferentially in the apical membrane.     

Plasticizer DBP Activates NLRP3 Inflammasome through the P2X7 Receptor in HepG2 and L02 Cells

Ditubyl phthalate (DBP), one of the most widely used plasticizers, can migrate out to contaminate our bodies and environment. A number of studies have showed that DBP is closely related to liver pathological changes and diseases. Inflammasomes are multiprotein complexes composed of procaspase and pattern recognition receptors such as Nucleotide oligomerization domain (NOD) like receptor family, pyrin domain containing 3 (NLRP3). Activation of NLRP3 inflammasome is implicated in the pathogeneses of liver damage. The aim of this study was to determine the effects of DBP on NLRP3 inflammasome. We found that DBP triggered the activation of NLRP3 inflammasome in hepatocyte cell lines. By using Ca‐074‐Me, N‐acetylcysteine and KN‐62, we observed that the P2X₇ receptor participated in the DBP‐induced activation of NLRP3 inflammasome. DBP could also trigger the ATP release. In conclusion, we demonstrated that DBP is one of the activator of NLRP3 inflammasome and may play an important role in liver damage.     

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2’,3’-O-(2,4,6-trinitrophenyl)-adenosine 5’-triphosphate), a nonspecific P2X1–7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4–L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.     

Gated Access to the Pore of a P2X Receptor: STRUCTURAL IMPLICATIONS FOR CLOSED-OPEN TRANSITIONS*

P2X receptors are ligand-gated cation channels that transition from closed to open states upon binding ATP. The crystal structure of the closed zebrafish P2X4.1 receptor directly reveals that the ion-conducting pathway is formed by three transmembrane domain 2 (TM2) α-helices, each being provided by the three subunits of the trimer. However, the transitions in TM2 that accompany channel opening are incompletely understood and remain unresolved. In this study, we quantified gated access to Cd²⁺ at substituted cysteines in TM2 of P2X2 receptors in the open and closed states. Our data for the closed state are consistent with the zebrafish P2X4.1 structure, with isoleucines and threonines (Ile-332 and Thr-336) positioned one helical turn apart lining the channel wall on approach to the gate. Our data for the open state reveal gated access to deeper parts of the pore (Thr-339, Val-343, Asp-349, and Leu-353), suggesting the closed channel gate is between Thr-336 and Thr-339. We also found unexpected interactions between native Cys-348 and D349C that result in tight Cd²⁺ binding deep within the intracellular vestibule in the open state. Interpreted with a P2X2 receptor structural model of the closed state, our data suggest that the channel gate opens near Thr-336/Thr-339 and is accompanied by movement of the pore-lining regions, which narrow toward the cytosolic end of TM2 in the open state. Such transitions would relieve the barrier to ion flow and render the intracellular vestibule less splayed during channel opening in the presence of ATP.     

Neuroprotection of exercise: P2X4R and P2X7R regulate BDNF actions

The neurotrophin brain-derived neurotrophic factor (BDNF), which acts as a transducer, is responsible for improving cerebral stroke, neuropathic pain, and depression. Exercise can alter extracellular nucleotide levels and purinergic receptors in central nervous system (CNS) structures. This inevitably activates or inhibits the expression of BDNF via purinergic receptors, particularly the P2X receptor (P2XR), to alleviate pathological progression. In addition, the significant involvement of sensitive P2X4R in mediating increased BDNF and p38-MAPK for intracerebral hemorrhage and pain hypersensitivity has been reported. Moreover, archetypal P2X7R blockade induces mouse antidepressant-like behavior and analgesia by BDNF release. This review summarizes BDNF-mediated neural effects via purinergic receptors, speculates that P2X4R and P2X7R could be priming molecules in exercise-mediated changes in BDNF, and provides strategies for the protective mechanism of exercise in neurogenic disease.     

P2X7受体在病理性疼痛中的研究进展

病理性疼痛主要包括组织损伤或炎症引起的炎症痛、神经系统损伤或疾病引起的神经病理性疼痛和恶性肿瘤及治疗引起的癌症痛三大类。病理性疼痛对常规的镇痛药物反应不理想,迫切需要寻找新的对病理性疼痛更有效和更特异的治疗手段。P2X7受体作为离子通道型嘌呤能受体,在炎症痛、神经病理性疼痛和癌症痛中都具有重要作用。靶向P2X7受体的新药物将为病理性疼痛的治疗带来新的希望。该文综述了P2X7受体在三类病理性疼痛中的研究进展。     

柚皮苷通过P2X7受体减轻HIV-1包膜糖蛋白gp120导致大鼠神经功能损害的的作用及机制

人类免疫缺陷病毒-1(Human immunodeficiency virus-1,HIV-1)包膜糖蛋白gp120能够引起大鼠出现学习记忆障碍的行为学改变并增加海马中P2X7受体的表达;柚皮苷对gp120引起的行为学改变及P2X7表达增多具有改善作用。为了研究柚皮苷通过减少P2X7受体表达减轻gp120导致大鼠神经功能损害的作用及机制,本实验选择SD大鼠并分为假手术操作的对照组、脑室注射gp120的gp120组、脑室注射gp120及不同剂量柚皮苷灌胃的柚皮苷组、脑室注射BzATP的BzATP组、脑室注射gp120、BzATP及90mg/kg柚皮苷灌胃的90mg/kg/d柚皮苷+BzATP组。Morris水迷宫检测逃避潜伏期及错误次数,TUNEL法检测海马中细胞凋亡率,Elisa法检测海马中肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白介素-1β(Interleukin-1β,IL-1β)、白介素-6(Interleukin-6,IL-6)的含量,Western blot检测海马中P2X7受体的表达。结果显示:与对照组比较,gp120组大鼠的逃避潜伏期延长,错误次数及海马中细胞凋亡率、TNF-α、IL-1β、IL-6的含量、P2X7受体的表达水平增加(P<0.05);与gp120组比较,不同剂量柚皮苷组大鼠的逃避潜伏期缩短,错误次数及海马中细胞凋亡率、TNF-α、IL-1β、IL-6的含量、P2X7受体的表达水平减少(P<0.05);与90mg/kg/d柚皮苷组比较,90mg/kg/d柚皮苷+BzATP组大鼠的逃避潜伏期延长,错误次数及海马中细胞凋亡率、TNF-α、IL-1β、IL-6的含量、P2X7受体的表达水平增加(P<0.05)。本研究的结果表明柚皮苷减轻HIV-1包膜糖蛋白gp120诱导的神经功能损害,这一作用与抑制海马组织中P2X7受体表达并减轻炎症反应及细胞凋亡有关。创新在于首次阐明了柚皮苷减轻HIV-1包膜糖蛋白gp120诱导神经功能损害的分子机制。在gp120引起大鼠行为学改变及海马组织中细胞凋亡、炎症反应激活的过程中,柚皮苷通过抑制P2X7受体的表达来改善行为学改变、抑制细胞凋亡及炎症反应的激活。     

Tension Force‐Induced ATP Promotes Osteogenesis Through P2X7 Receptor in Osteoblasts

Orthodontic tooth movement induces alveolar bone resorption and formation by mechanical stimuli. Force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators that respond to bone cells by mechanical stimuli. However, the effect of tension force (TF)‐induced ATP on osteogenesis is inadequately understood. Accordingly, we investigated the effect of TF on ATP production and osteogenesis in MC3T3‐E1 cells. Cells were incubated in the presence or absence of P2X7 receptor antagonist A438079, and then stimulated with or without cyclic TF (6% or 18%) for a maximum of 24 h using Flexercell Strain Unit 3000. TF significantly increased extracellular ATP release compared to control. Six percent TF had maximum effect on ATP release compared to 18% TF and control. Six percent TF induced the expression of Runx2 and Osterix. Six percent TF also increased the expression of extracellular matrix proteins (ECMPs), ALP activity, and the calcium content in ECM. A438079 blocked the stimulatory effect of 6% TF on the expression of Runx2, Osterix and ECMPs, ALP activity, and calcium content in ECM. This study indicated that TF‐induced extracellular ATP is released in osteoblasts, suggesting that TF‐induced ATP promotes osteogenesis by autocrine action through P2X7 receptor in osteoblasts. J. Cell. Biochem. 116: 12–21, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Synthesis and SAR development of quinoline analogs as novel P2X7 receptor antagonists

The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, suggesting that the human P2X7R (hP2X7R) is an attractive therapeutic target. In the present study, the synthesis and structure-activity relationship (SAR) of a novel series of quinoline derivatives as P2X7R antagonists are described herein. These compounds exhibited mechanistic activity (YO PRO) in an engineered HEK293 expressing hP2X7R as well as a functional response (IL-1β) in human THP-1 (hTHP-1) cellular assays. Compound 19 was identified as the most promising compound in this series with excellent cellular potency, low liver microsomal clearance, good permeability and low efflux ratio. In addition, this compound also displayed good pharmacokinetic properties and acceptable brain permeability (K_p,uu of 0.37).     

Synthesis and activity of N-cyanoguanidine-piperazine P2X7 antagonists

A novel series of cyanoguanidine-piperazine P2X₇ antagonists were identified and structure–activity relationship (SAR) studies described. Compounds were assayed for activity at human and rat P2X₇ receptors in addition to their ability to inhibit IL-1β release from stimulated human whole blood cultures. Compound 27 possesses potent activity (0.12 μM) in this latter assay and demonstrates moderate clearance in-vivo.     

Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

P2X7 receptors are nonselective cation channels gated by high extracellular ATP, but with sustained activation, receptor sensitization occurs, whereby the intrinsic pore dilates, making the cell permeable to large organic cations, which eventually leads to cell death. P2X7 receptors associate with cholesterol-rich lipid rafts, but it is unclear how this affects the properties of the receptor channel. Here we show that pore-forming properties of human and rodent P2X7 receptors are sensitive to perturbations of cholesterol levels. Acute depletion of cholesterol with 5 mm methyl-β-cyclodextrin (MCD) caused a substantial increase in the rate of agonist-evoked pore formation, as measured by the uptake of ethidium dye, whereas cholesterol loading inhibited this process. Patch clamp analysis of P2X7 receptor currents carried by Na⁺ and N-methyl-d-glucamine (NMDG⁺) showed enhanced activation and current facilitation following cholesterol depletion. This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes. Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating. These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.     

Diadenosine Homodinucleotide Products of ADP-ribosyl Cyclases Behave as Modulators of the Purinergic Receptor P2X7

ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5′,5′"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509–14514). P24, but not P18, proved to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca²⁺ through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca²⁺ influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC₅₀ of ∼1 μm. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca²⁺]_i has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146–23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca²⁺]_i to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A.     

C-terminal Calmodulin-binding Motif Differentially Controls Human and Rat P2X7 Receptor Current Facilitation

P2X₇ receptors (P2X₇R) are ATP-gated calcium-permeable cationic channels structurally unique among the P2X family by their much longer intracellular C-terminal tail. P2X₇Rs show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X₇R resulted from a Ca²⁺-calmodulin-dependent process and a distinct calcium-independent process. However, P2X₇Rs show striking species differences; thus, this study compared the properties of ATP-evoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X₇R as well as rat/human, human/rat chimeric, and mutated P2X₇Rs. Facilitation at the human P2X₇R was 5-fold slower than at the rat P2X₇R. Facilitation did not resulting from an increase of receptor addressing the plasma membrane. We found the human P2X₇R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C terminus of rat P2X₇R. Replacement of three critical residues of this binding domain from the rat into the human P2X₇R (T541I, C552S, and G559V) reconstituted the Ca²⁺-calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X₇Rs. The absence of Ca²⁺-dependent facilitation at the human P2X₇R may represent a protective adaptation of the innate immune response in which P2X₇R plays significant roles.     

Somatic and axonal effects of ATP via P2X2 but not P2X7 receptors in rat thoracolumbar sympathetic neurones

Excitatory ATP responses in rat cultured thoracolumbar sympathetic neurones are mediated by somatic P2X(2) receptors. The present study investigated a possible role of axonal P2X(2) as well as P2X(7) receptors on the same preparation. Confocal laser scanning microscopy demonstrated P2X(2) and P2X(7) immunoreactivity along the axons as well as P2X(7) immunoreactivity surrounding the cell nuclei. P2X(7) mRNA expression was detected in individual neurones using a single-cell RT-PCR approach. Adenosine triphosphate (ATP) caused a significant increase in axonal Ca(2+) concentration which was dependent on external Ca(2+) but insensitive to depletion of the cellular Ca(2+) pools by cyclopiazonic acid. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 micro m) virtually abolished the ATP response, whereas brilliant blue G (0.1 micro m), a selective P2X(7) receptor antagonist, had no effect. Dibenzoyl-ATP (BzATP; 100 micro m) induced a much smaller increase in axonal [Ca(2+)] concentration than ATP at equimolar concentrations. The response to BzATP was distinctly reduced by PPADS but not by brilliant blue G. The overall pharmacological profile of the axonal P2X receptors resembled closely that of the somatic P2X(2) receptors. In conclusion, the present data suggest the occurrence of axonal excitatory P2X(2) receptors in thoracolumbar sympathetic neurones. However, the functional significance of axonal and (peri)-nuclear P2X(7) receptors has still to be proven.     

Physiological and pathological functions of P2X7 receptor in the spinal cord

ATP-mediated signaling has widespread actions in the nervous system from neurotransmission to regulation of proliferation. In addition, ATP is released during injury and associated to immune and inflammatory responses. Still, the potential of therapeutic intervention of purinergic signaling during pathological states is only now beginning to be explored because of the large number of purinergic receptors subtypes involved, the complex and often overlapping pharmacology and because ATP has effects on every major cell type present in the CNS. In this review, we will focus on a subclass of purinergic-ligand-gated ion channels, the P2X7 receptor, its pattern of expression and its function in the spinal cord where it is abundantly expressed. We will discuss the mechanisms for P2X7R actions and the potential that manipulating the P2X7R signaling pathway may have for therapeutic intervention in pathological events, specifically in the spinal cord.     

Genetics of the P2X7 receptor and human disease

The P2RX7 gene is highly polymorphic, and many single nucleotide polymorphisms (SNPs) underlie the wide variation observed in P2X7 receptor responses. We review the discovery of those non-synonymous SNPs that affect receptor function and compare their frequencies in different ethnic populations. Analysis of pairwise linkage disequilibrium (LD) predicts a limited range of haplotypes. The strong LD between certain functional SNPs provides insight into published studies of the association between SNPs and human disease.