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.     

P2X4 purinoceptor signaling in chronic pain

ATP, acting via P2 purinergic receptors, is a known mediator of inflammatory and neuropathic pain. There is increasing evidence that the ATP-gated P2X4 receptor (P2X4R) subtype is a locus through which activity of spinal microglia and peripheral macrophages instigate pain hypersensitivity caused by inflammation or by injury to a peripheral nerve. The present article highlights the recent advances in our understanding of microglia-neuron interactions in neuropathic pain by focusing on the signaling and regulation of the P2X4R. We will also develop a framework for understanding converging lines of evidence for involvement of P2X4Rs expressed on macrophages in peripheral inflammatory pain.     

Combinations of TLR and NOD2 ligands stimulate rat microglial P2X4R expression

As ATP-gated ion channels, P2X4 receptors (P2X4R) of microglial cells play a crucial role in central nervous system (CNS) inflammation. In this study, we used rat microglial cell cultures to examine P2X4R expression in response to stimulation by combination of toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) receptors. Various TLR1-9 ligands and NOD2 agonist muramyldipeptide (MDP) were investigated. Our results showed that certain combination of ligands had additive effects on upregulating microglial P2X4R at both mRNA and protein levels, and induced nitric oxide increase and tumor necrosis factor-α production. Thus TLRs and NOD2 combinations are contributors to the signaling cascades resulting in purinergic microglial activation.     

NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响

目的：观察硫化氢（H₂S）供体硫氢化钠（NaHS）对ATP致伤的大鼠小胶质细胞细胞活力、细胞膜通透性及P2X7受体表达的影响。方法：实验取对数期形态结构及生长分化良好的大鼠小胶质细胞,随机分4组,每组设3个复孔。①正常对照组：常规培养,不进行ATP处理。②ATP组：接种细胞24 h后ATP处理。③NaHS+ATP组：NaHS预先孵育30 min后再用ATP处理,并且NaHS始终存在于反应体系中。④KN-62（P2X₇受体阻断剂）+ATP组：KN-62预先孵育30 min,其余同NaHS+ATP组。MTT检测各组细胞活力,荧光染料YO-PRO-1检测各组相对荧光单位（RFU）反映膜的通透性,Western blot检测各组P2X₇受体表达水平。结果：①与对照组相比,不同浓度的ATP （1、3、5、10 mmol/L）作用3 h均可明显降低大鼠小胶质细胞活力,NaHS （200 μmol/L）干预后大鼠小胶质细胞活力较ATP组明显增加（P<0.01）,但NaHS达400 μmol/L浓度时,其保护作用未进一步增加。②随着ATP浓度的增加,大鼠小胶质细胞内YO-PRO-1的荧光强度显著增加,NaHS预处理可明显减少细胞对YO-PRO-1的摄取（P<0.01）。③ATP （3 mmol/L）能上调P2X₇受体蛋白表达水平,而NaHS （200 μmol/L）预孵育则可明显抑制ATP引起的P2X₇受体蛋白表达的上调（P<0.01）。结论：NaHS可减少ATP致伤的大鼠小胶质细胞的P2X₇受体表达、降低通透性、增加细胞活力,提示调控P2X₇受体的表达和功能可能是H₂S神经保护作用的重要环节。     

Zinc and copper modulate differentially the P2X4 receptor

The rat ATP P2X4 receptor was expressed in Xenopus laevis oocytes to assess the effect of zinc and copper as possible regulators of purinergic mechanisms. ATP applied for 20 s evoked an inward cationic current with a median effective concentration (EC50) of 21.4+/-2.8 microM and a Hill coefficient (nH) of 1.5+/-0.1. Coapplication of ATP plus 10 microM zinc displaced leftward, in a parallel fashion, the ATP concentration-response curve, reducing the EC50 to 8.4+/-1.8 microM (p < 0.01) without altering the receptor nH. The zinc potentiation was fast in onset, easily reversible, and voltage-independent and did not require metal preexposure. The zinc EC50 was 2-5 microM, with a bell-shaped curve. At concentrations of 100-300 microM, zinc produced less potentiation, and at 1 mM, it inhibited 50% the ATP current. The effect of zinc was mimicked by cadmium. In contrast, copper inhibited the ATP-evoked currents in a time- and concentration-dependent fashion, reducing the maximal current (Imax) without altering the EC50. The copper-induced inhibition was slow in onset, slowly reversible, and voltage-independent. Whereas coapplication of 300 microM copper plus ATP reduced Imax to 36.2+/-5%, the coapplication of, or 60-s preexposure by, 10 microM copper reduced Imax to 79+/-9.2% (p < 0.05) and 39.6+/-8.7% (p < 0.01), respectively. The inhibition was noncompetitive in nature and mimicked by mercury. Cobalt, barium, and manganese did not modify significantly the ATP-evoked current, demonstrating metal specificity. The simultaneous 1-min preapplication of both metals revealed that the 10 microM zinc-induced potentiation was obliterated by 10 microM copper, whereas 30 microM copper not only reduced the potentiation, but inhibited the ATP response. Following coapplication of both metals for 20 s with ATP, at least 100 microM copper was required to counteract the 10 microM zinc-induced potentiation. The simultaneous preincubation with both metals provided evidence for a noncompetitive interaction. We hypothesize the existence of metal binding site(s), which are most likely localized in the extracellular domain of the P2X4 receptor structure. These sites are selective and accessible to extracellular metal applications and bind micromolar concentrations of metals. The present results are compatible with the working hypothesis that trace metals, such as copper and zinc, are physiological modulators of the P2X4 receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted.     

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.     

Lack of functional P2X7 receptor aggravates brain edema development after middle cerebral artery occlusion

Effective therapeutic measures against the development of brain edema, a life-threatening complication of cerebral ischemia, are necessary to improve the functional outcome for the patient. Here, we identified a beneficial role of purinergic receptor P2X7 activation in acute ischemic stroke. Involvement of P2X7 in the development of neurological deficits, infarct size, brain edema, and glial responses after ischemic cerebral infarction has been analyzed. Neurologic evaluation, magnetic resonance imaging, and immunofluorescence assays were used to characterize the receptor’s effect on the disease progress during 72 h after transient middle cerebral artery occlusion (tMCAO). Sham-operated animals were included in all experiments for control purposes. We found P2X7-deficient mice to develop a more prominent brain edema with a trend towards more severe neurological deficits 24 h after tMCAO. Infarct sizes, T2 times, and apparent diffusion coefficients did not differ significantly between wild-type and P2X7^?/? animals. Our results show a characteristic spatial distribution of reactive glia cells with strongly attenuated microglia activation in P2X7^?/? mice 72 h after tMCAO. Our data indicate that P2X7 exerts a role in limiting the early edema formation, possibly by modulating glial responses, and supports later microglia activation.     

Nociceptive signaling of P2X receptors in chronic pain states

Purinergic Signalling - P2X3 monomeric receptors (P2X3Rs) and P2X2/3 heteromeric receptors (P2X2/3Rs) in primary sensory neurons and microglial P2X4 monomeric receptors (P2X4Rs) in the spinal...     

Fluvastatin suppresses native and recombinant human P2X4 receptor function

Statins have both cholesterol lowering and anti-inflammatory activities, whether mechanisms underlying their activities are independent remains unclear. The ATP-gated P2X(4) receptor is a pro-inflammatory mediator. Here, we investigate the action of fluvastatin and other cholesterol depleting agents on native and recombinant human P2X(4) receptor. Fluvastatin and mβCD suppressed P2X(4)-dependent calcium influx in THP-1 monocytes, without affecting P2Y receptor responses. mβCD or filipin III suppressed the current density of recombinant human P2X(4) receptors. Human P2X(2) was insensitive to cholesterol depletion. Cholesterol depletion had no effect on intrinsic P2X(4) receptor properties as judged by ATP concentration-response relationship, receptor rundown or current decay during agonist occupancy. These data suggest fluvastatin suppresses P2X(4) activity in monocytes through cholesterol depletion and not by modulating intrinsic channel properties.     

P2X7 receptor activation leads to increased cell death in a radiosensitive human glioma cell line

Gliomas are the most lethal tumors of central nervous system. ATP is an important signaling molecule in CNS and it is a selective P2X7 purinergic receptor ligand at high concentrations. Herein, we investigated whether the activation of P2X7R might be implicated in death of a radiosensitive human glioma lineage. The effects of P2X7R agonists (ATP and BzATP) and irradiation (2 Gy) on glioma cells were analyzed by MTT assay and annexin-V/PI determination, whereas mRNA and protein P2X7R expression was assessed by qRT-PCR and flow cytometry, respectively. P2X7R pore formation was functionality examined by analyzing ethidium bromide uptake. The human glioma cells U-138 MG and U-251 MG were resistant to death when treated with either ATP (5 mM) or BzATP (100 μM), but the radiosensitive M059J glioma cells displayed a significant decrease of cell viability (32.4 ± 4.1 % and 25.6 ± 3.3 %, respectively). The M059J lineage expresses significantly higher mRNA P2X7R levels when compared to the U-138 MG and U-251 cell lines (0.40 ± 0.00; 0.28 ± 0.01, and 0.31 ± 0.01, respectively), and irradiation upregulated P2X7R expression (0.55 ± 0.08) in this lineage. Noteworthy, P2X7R protein doubled after irradiation on M059J lineage, and increased in 50 % and 42.6 % when comparing M059J-irradiated to irradiated U-138 MG and U-251 MG cells, respectively. Ethidium bromide uptake was significantly increased in 104 % and 77.8 % when comparing M059J to U-138 MG and U-251MG, respectively. Finally, the selective P2X7R antagonist A740003 significantly decreased the cell death caused by irradiation. We provide novel evidence indicating that M059J human glioma cell line is ATP-P2X7R sensitive, pointing out the relevance of the purinergic P2X7R on glioma radiosensitivity.     

Contribution of P2X4 receptor in pain associated with rheumatoid arthritis: a review

Purinergic Signalling - Pain is the most common symptom reported by patients with rheumatoid arthritis (RA) even after the resolution of chronic joint inflammation. It is believed that...     

P2X receptors regulate adenosine diphosphate release from hepatic cells

Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2–4 h. Nucleotide release from hepatic cells is stimulated by the Ca²⁺ ionophore, ionomycin, and by the P2 receptor agonist, 2′3′-O-(4-benzoyl-benzoyl)-adenosine 5′-triphosphate (BzATP). Ionomycin (10 μM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10–100 μM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca²⁺ levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9419-2) contains supplementary material, which is available to authorized users.     

Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin

The P2X4 receptor is an ATP-gated ion channel expressed in neurons, endothelia and immune cells. Plasma membrane expression of P2X4 is regulated by dynamin-dependent endocytosis, and this study identifies a Rab5-dependent pathway of receptor internalisation. Expression of Rab5 constructs altered the distribution of P2X4 in HEK-293 cells, and both constitutive internalisation and agonist-induced desensitisation of P2X4 were increased by co-expression of wild-type Rab5 or constitutively active Rab5 (Q79L). Expression of inactive dynamin K44A and Rab5 S34N constructs abolished agonist-induced desensitisation, suggesting internalisation as the underlying mechanism. Blocking P2X4 internalisation in this way also abolished potentiation of ATP-induced currents by the allosteric modulator ivermectin. This suggests that the dynamin-Rab5 internalisation pathway is essential for the ivermectin potentiation effect. In agreement with this hypothesis, the co-expression of wild-type dynamin, wild-type Rab5 or active Rab5 (Q79L) could increase the potentiation of the ATP-induced P2X4 response by ivermectin. These findings highlight Rab5 GTPase as a key regulator of P2X4 receptor cell surface expression and internalisation.     

Activation and desensitization of the recombinant P2X1 receptor at nanomolar ATP concentrations

Activation and desensitization kinetics of the rat P2X1 receptor at nanomolar ATP concentrations were studied in Xenopus oocytes using two-electrode voltage-clamp recording. The solution exchange system used allowed complete and reproducible solution exchange in <0.5 s. Sustained exposure to 1-100 nM ATP led to a profound desensitization of P2X1 receptors. At steady-state, desensitization could be described by the Hill equation with a K1/2 value of 3.2 +/- 0.1 nM. Also, the ATP dependence of peak currents could be described by a Hill equation with an EC50 value of 0.7 microM. Accordingly, ATP dose-effect relationships of activation and desensitization practically do not overlap. Recovery from desensitization could be described by a monoexponential function with the time-constant tau = 11.6 +/-1.0 min. Current transients at 10-100 nM ATP, which elicited 0.1-8.5% of the maximum response, were compatible with a linear three-state model, C-O-D (closed-open-desensitized), with an ATP concentration-dependent activation rate and an ATP concentration-independent (constant) desensitization rate. In the range of 18-300 nM ATP, the total areas under the elicited current transients were equal, suggesting that P2X1 receptor desensitization occurs exclusively via the open conformation. Hence, our results are compatible with a model, according to which P2X1 receptor activation and desensitization follow the same reaction pathway, i.e., without significant C to D transition. We assume that the K1/2 of 3.2 nM for receptor desensitization reflects the nanomolar ATP affinity of the receptor found by others in agonist binding experiments. The high EC50 value of 0.7 microM for receptor activation is a consequence of fast desensitization combined with nonsteady-state conditions during recording of peak currents, which are the basis of the dose-response curve. Our results imply that nanomolar extracellular ATP concentrations can obscure P2X1 receptor responses by driving a significant fraction of the receptor pool into a long-lasting refractory closed state.     

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca²⁺]_i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca²⁺]_i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9415-6) contains supplementary material, which is available to authorized users.     

Activation kinetics of single P2X receptors

After the primary structure of P2X receptors had been identified, their function had to be characterized on the molecular level. Since these ligand-gated ion channels become activated very quickly after binding of ATP, methods with adequate time resolution have to be applied to investigate the early events induced by the agonist. Single-channel recordings were performed to describe conformational changes on P2X₂, P2X₄, and P2X₇ receptors induced by ATP and also by allosteric receptor modifiers. The main results of these studies and the models of P2X receptor kinetics derived from these observations are reviewed here. The investigation of purinoceptors by means of the patch clamp technique following site-directed mutagenesis will probably reveal more details of P2X receptor function at the molecular level.     

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.     

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.     

Quercetin relieved diabetic neuropathic pain by inhibiting upregulated P2X4 receptor in dorsal root ganglia

The upregulation of nociceptive ion channels expressed in dorsal root ganglia (DRG) contributes to the development and retaining of diabetic pain symptoms. The flavonoid quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a component extracted from various fruits and vegetables and exerts anti-inflammatory, analgesic, anticarcinogenic, antiulcer, and antihypertensive effects. However, the exact mechanism underlying quercetin's analgesic action remains poorly understood. The aim of this study was to investigate the effects of quercetin on diabetic neuropathic pain related to the P2X₄ receptor in the DRG of type 2 diabetic rat model. Our data showed that both mechanical withdrawal threshold and thermal withdrawal latency in diabetic rats treated with quercetin were higher compared with those in untreated diabetic rats. The expression levels of P2X₄ messenger RNA and protein in the DRG of diabetic rats were increased compared with the control rats, while quercetin treatment significantly inhibited such enhanced P2X₄ expression in diabetic rats. The satellite glial cells (SGCs) enwrap the neuronal soma in the DRG. Quercetin treatment also lowered the elevated coexpression of P2X₄ and glial fibrillary acidic protein (a marker of SGCs) and decreased the upregulation of phosphorylated p38 mitogen-activated protein kinase (p38MAPK) in the DRG of diabetic rats. Quercetin significantly reduced the P2X₄ agonist adenosine triphosphate-activated currents in HEK293 cells transfected with P2X₄ receptors. Thus, our data demonstrate that quercetin may decrease the upregulation of the P2X₄ receptor in DRG SGCs, and consequently inhibit P2X₄ receptor-mediated p38MAPK activation to relieve the mechanical and thermal hyperalgesia in diabetic rats.