P2X7 Receptor Expression Research Articles

Chronic Hemodynamic Overload of the Atria is an Important Factor for Shear Signaling Remodeling in Rat Hearts

Hemodynamic disturbances induced by hypertension, heart failure, and valve diseases lead atrial fibrillation. We have previously shown that shear stress induces proarrhythmic Ca2+ waves via connexin43 (Cx43) hemichannel opening, and that ATP release through the Cx43, in turn, activates P2Y1 receptors (P2Y1Rs) and P2X4 receptors (P2X4Rs) in rat atrial myocytes. To understand cellular mechanism for mechanically-induced atrial arrhythmia and role of shear signaling in this pathogenesis, we compared atrial shear signaling between sham-operated rats and the rats subjected to prolonged transverse aortic constriction (TAC). Atrial cell hypertrophy with higher fractional shortening and dilation of atrial chamber were observed at 5 weeks (wk) after TAC surgery. On 4-month (mon) after TAC surgery, more severe dilation in atrium with lower fractional shortening was detected. Whole-cell patch clamp studies revealed that shear-induced nonselective cation currents that are mediated by P2X4Rs (about 50%) and Cx43 (about 20%), were enhanced with hypertrophy, but they were no longer enhanced during atrial failure. Total Cx43 proteins and phosphorylated Cx43 at its ser-368 residue were gradually increased by TAC, which was consistent with larger shear-induced NMDG+ currents in TAC rat atrial myocytes. P2X4R expression increased in 5-wk-TAC atrial cells, but decreased in 4-mon-TAC atrial cells. On the other hand, P2Y1R-dependent longitudinal Ca2+ waves and P2Y1R expression were significantly increased by 4-mon-TAC. Our data suggest that Cx43-P2 receptor-mediated shear signaling is significantly altered by the progress of atrial dilation and volume overload, and that biphasic changes in P2X4R function and expression versus TAC duration may play a role in the progression from hypertrophy to failure in atrium subjected to aortic stenosis and increased afterload.

Protein-Protein Interactions of Human P2X7 in Microglia and Human ASIC1a in Kidney Cells

Acid-sensing ion channel 1a (ASIC1a) and P2X7 receptors (P2X7Rs) are trimeric cation channels activated by extracellular protons and ATP, respectively. ASIC1a is expressed in kidney and neuronal tissues and has recently been suggested as a target to treat acute kidney injury, pain and stroke. Expression and activation of P2X7Rs in microglia triggers neuroinflammation, and P2X7Rs thus represent potential drug targets for mental and neurodegenerative disorders. To target these receptors therapeutically, it is necessary to understand the downstream effects of activation and modulation by cell type-specific protein-protein interactions (PPIs).

Osthole relieves diabetics cardiac autonomic neuropathy associated with P2X3 receptor in ratstellate ganglia

Diabetic cardiac autonomic neuropathy (DCAN) is a serious complication of diabetes mellitus, which often leads to cardiac dysfunction and even threatens patients’ life. Osthole, a natural coumarin derivative, has anti-inflammatory, anti-oxidant and antihypertensive effects. The P2X3 receptor is related to DCAN. The objective of this study will investigate whether osthole relieves DCAN associated with the P2X3 receptor in the stellate ganglia of diabetic rats. A type 2 diabetes mellitus rat model was induced by a combination of diet and streptozotocin. Our results showed that osthole improved the abnormal changes of blood pressure, heart rate, and heart rate variability in diabetic rats and significantly reduced the up-regulated expression levels of the P2X3 receptor, tumor necrosis factor-α and interleukin-1β in stellate ganglia of diabetic rats. Meanwhile, osthole significantly decreased the elevated serum adrenaline concentration and phosphorylation level of extracellular regulated protein kinase 1/2. In addition, the molecular docking result indicated that osthole was a perfect fit for interacting with the P2X3 receptor. Overall, osthole alleviates the sympathetic relative excitation via inhibiting the expression of P2X3 receptors in the stellate ganglia, to achieve a balance between sympathetic and parasympathetic nerves, relieves the DCAN.

Effects of single-dose antipurinergic therapy on behavioral and molecular alterations in the valproic acid-induced animal model of autism

Autism spectrum disorder (ASD) is characterized by deficits in communication and social interaction, restricted interests, and stereotyped behavior. Environmental factors, such as prenatal exposure to valproic acid (VPA), may contribute to the increased risk of ASD. Since disturbed functioning of the purinergic signaling system has been associated with the onset of ASD and used as a potential therapeutic target for ASD in both clinical and preclinical studies, we analyzed the effects of suramin, a non-selective purinergic antagonist, on behavioral, molecular and immunological in an animal model of autism induced by prenatal exposure to VPA. Treatment with suramin (20 mg/kg, intraperitoneal) restored sociability in the three-chamber apparatus and decreased anxiety measured by elevated plus maze apparatus, but had no impact on decreased reciprocal social interactions or higher nociceptive threshold in VPA rats. Suramin treatment did not affect VPA-induced upregulation of P2X4 and P2Y2 receptor expression in the hippocampus, and P2X4 receptor expression in the medial prefrontal cortex, but normalized an increased level of interleukin 6 (IL-6). Our results suggest an important role of purinergic signaling modulation in behavioral, molecular, and immunological aberrations described in VPA model, and indicate that the purinergic signaling system might be a potential target for pharmacotherapy in preclinical studies of ASD.

Therapeutic effects and modulatory mechanism of Alpiniae oxyphyllae Fructus in chronic intermittent hypoxia induced enuresis in rats.

The objective of this study was to explore the effect of Alpiniae oxyphyllae Fructus (AOF) on a rat model of chronic intermittent hypoxia (CIH)-induced enuresis. Findings of this study may help identify therapeutic targets in children with nocturnal enuresis (NE). Female rats were randomly divided into a control group (saline gavage, 4 weeks of normal air), CIH group (saline gavage, 4 weeks of CIH), and AOF group (AOF gavage, 4 weeks of CIH). The variables measured in this study included water intake, urine output, bladder leak point pressure (BLPP), malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activity. The expression levels of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3-adrenergic receptor (ß3-AR) in the bladder were also measured. The bladder was subjected to haematoxylin and eosin (HE) and Weigert staining, and histological changes were observed under a light microscope to evaluate the morphological changes in the bladder in each group. Compared with the control group, urine output was increased, and the BLPP was decreased in the CIH group, but AOF administration decreased urine output and increased BLPP. In addition, the serum MDA level increased and the SOD activity decreased in the CIH group compared with the control group. Administration of AOF decreased the MDA level and increased the SOD activity. Additionally, compared with the control group, HE and Weigert staining in the CIH group showed that the bladder detrusor muscle bundles were disordered and loose, some muscle bundles were broken, the content of collagen fibres in the gap was reduced, and the gap was significantly widened. However, following the administration of AOF, the bladder detrusor muscle bundles were neatly arranged, and the content of collagen fibres in the gap was increased. Furthermore, compared with the control group, the purinergic P2X3 receptor and muscarinic M3 receptor were expressed at higher levels, and ß3-AR was expressed at lower levels in the CIH group, but AOF administration decreased the expression of the purinergic P2X3 receptor and muscarinic M3 receptor and increased the expression of the ß3-AR. AOF improves enuresis by inhibiting oxidative stress and regulating the expression of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3 adrenergic receptor.

Minocycline Attenuates Experimental Subarachnoid Hemorrhage in Rats.

BackgroudThe aim of this study was to evaluate the therapeutic effect of minocycline on treating experimental subarachnoid hemorrhage (SAH) in rats and to explore its possible molecular mechanism.MethodsSAH was induced in male Sprague-Dawley rats by endovascular perforation. The rats were treated with minocycline (25 mg/kg or 50 mg/kg) or saline at 2 hand 12 h post SAH. Neurological function, cerebral hemorrhage, and edema were scored at 48 h post SAH. Cell death and P2X4 receptor (P2X4R) expression were observed in the prefrontal cortex (PFC).ResultsTreatment with a highdose of minocycline significantly improved the neurological function score, and attenuated cerebral hemorrhage and edema. Low-dose minocycline could reduce hemorrhage, but the effect on neurological deficits and brain edema was not obvious. Minocycline treatment could alleviate neuronal apoptosis in the PFC, which was related to reduced expression of inflammatory cytokines. Immunofluorescence showed that P2X4R on microglia was activated after SAH. Minocycline treatment inhibited P2X4R activation and further suppressed the phosphorylation of downstream p38 MAPK.ConclusionsMinocycline plays a neuroprotective role by attenuating early brain injury after experimental SAH. The therapeutic mechanism of minocycline may be mediated by the inhibition of P2X4R on microglia.

2 Hz EA Reduces Heroin Withdrawal-Induced Hyperalgesia and Heroin Relapse by Downregulating P2X3 Receptors in DRG Neurons.

Electroacupuncture (EA) has effective analgesic effects. Our previous study demonstrated that the upregulation of P2X3 receptors in the dorsal root ganglia (DRG) might participate in heroin withdrawal-induced hyperalgesia. The aim of this study is to further explore whether 2 Hz EA reduces heroin relapse associated with its analgesic effect and whether P2X3 receptors in the DRG are involved in this process. 2 Hz EA was adopted to treat the heroin SA rats in the present study. Heroin-seeking and pain sensitivity were evaluated. The expression of P2X3 receptors in the DRG was detected. Our results showed that compared with the control group, the reinstatement, thermal hyperalgesia, and mechanical allodynia of the heroin-addicted group were increased significantly. The expression of P2X3 receptors in the DRG was increased markedly. After being treated using 2 Hz EA, reinstatement was reduced, hyperalgesia was decreased, and the upregulated expression of P2X3 receptors in the DRG had decreased significantly compared to that in the heroin-addicted group. Consequently, our results indicated that 2 Hz EA was an effective method for treating heroin-induced hyperalgesia and helping prevent relapse, and the potential mechanism might be related to the downregulation of P2X3 receptor expression in the DRG.

Expression of P2X receptors in the rat anterior pituitary.

In this study, the distribution patterns of P2X1 to P2X7 receptors in the anterior pituitary cells of rat were studied with single-, double-, and triple-labeling immunofluorescence, combined method of immunofluorescence and in situ hybridization, and Western blot. The results showed that the expression level of the P2X4 receptor protein was highest, followed by P2X5, P2X3, P2X2, P2X6, and P2X7 receptor proteins, but no P2X1 receptor protein was detected. Strong P2X4 receptor-immunoreactivity was detected in almost all the anterior pituitary cells. Different combinations of P2X receptors were detected in each individual cell type of the rat anterior pituitary. Gonadotrophs express P2X4, P2X5, and P2X6 receptors. Corticotrophs express P2X3 and P2X4 receptors. Folliculo-stellate cells express P2X2 and P2X4 receptors, and somatotrophs, lactotrophs, and thyrotrophs express only P2X4 receptors. The macrophages with Iba-1-ir expressed P2X7 receptors. The possible functions of these P2X receptors in each individual cell type of the rat anterior pituitary are discussed.

The P2X7 Receptor in the Maintenance of Cancer Stem Cells, Chemoresistance and Metastasis.

Metastasis is the worst prognosis predictor in the clinical course of cancer development. Features of metastatic cancer cells include migratory ability, low degree of differentiation, self-renewal and proliferation potentials, as well as resistance to therapies. Metastatic cells do not present all of the necessary characteristics at once. Indeed, they have a unique phenotypic plasticity, allowing the acquisition of features that make them successful in all steps of metastasis. Cancer stem cells (CSC), the most undifferentiated cells in the tumor mass, display highest metastatic potential and resistance to radio- and chemotherapy. Growing tumors exhibit marked upregulation of P2X7 receptor expression and secrete ATP. Since the P2X7 receptor plays an important role in the maintenance of undifferentiated state of pluripotent cells, its importance on cell fate regulation in the tumor mass is suggested. Considering the extensive crosstalk between CSCs, epithelial-mesenchymal transition, drug resistance and metastasis, current knowledge implicating P2X7 receptor function in these phenomena and new avenues for therapeutic strategies to control metastasis are reviewed.

Novel Evidence That Extracellular Adenosine Triphosphate (ATP), As a Purinergic Signaling Mediator, Activates Mobilization By Engaging a P2X4 Ligand-Gated Cation Channel Receptor Expressed on the Surface of Hematopoietic and Innate Immunity Cells

Background . Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but it is also released from activated cells into the extracellular space as a crucial component of the purinergic signaling network. Purinergic signaling is an ancient form of extracellular signaling that is mediated by ATP and other extracellular nucleotides (EXNs). Purinergic receptors for EXNs are expressed on the surface of all cells in the body; are represented by several families of P1, P2X, and P2Y receptors; and are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for ATP signaling and consists of seven members (P2X1-7). We recently reported that by activating the P2X7 receptor ATP activates the Nlrp3 inflammasome in hematopoietic cells and significantly enhances G-CSF-induced mobilization of hematopoietic stem progenitor cells (HSPCs, Leukemia 2018, 32:1920-1931). Hypothesis. Since P2X7-KO mice still mobilize some HSPCs, we hypothesized that, in addition to the P2X7 receptor, other receptors from this family also play a role in the mobilization process.Materials and Methods. RT-PCR and FACS were employed to phenotype human and murine bone marrow (BM) mononuclear cells (MNCs) as well as sorted HSCs for expression of P2X receptors. Mobilization studies were performed in wild type mice in which the P2X4 receptor was blocked by the specific small-molecule inhibitor PSB12054 and in P2X7-KO animals. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. We also evaluated activation of the Nlrp3 inflammasome as well as other inflammasomes expressed in hematopoietic cells, including Aim2, Nlrp1a, Nlrp1b, and Nlrc4. Results. We found that, of all the receptors of the P2X family, two members, P2X7 and P2X4, are highly expressed on the surface of murine and human MNCs and HSPCs (Figure 1a). Activation of both receptors on the surface of innate immunity CD11b+Gr-1+ cells resulted in activation of the Nlrp3, Nlrp1a, Nlrp1b, and Aim2 inflammasomes. Inhibition of the P2X4 receptor by a specific small-molecule inhibitor decreased G-CSF- and AMD3100-induced mobilization in wild type mice (Figure 1b). Interestingly, we observed differences during AMD3100 mobilization, namely that while P2X7-KO deficiency resulted in decreased G-CSF-induced mobilization and did not affect AMD3100 mobilization efficiency, P2X4 inhibition, by contrast, led to a profound decrease in both G-CSF- and AMD3100-induced mobilization efficiency. Conclusions. Our results further support an important role for purinergic signaling in the mobilization of HSPCs. We also demonstrated that P2X7 and P2X4 receptors, which are highly expressed on human and murine hematopoietic cells, are involved in G-CSF-induced mobilization by activating the Nlrp3, Nlrp1a, Nlrp1b and Aim2 inflammasomes. By contrast, AMD3100-induced mobilization requires expression of P2X4 but not P2X7 receptors, which implies the involvement of different mechanisms and indicates that P2X7 deficiency can be compensated by the P2X4 receptor. Currently, we are performing RNASeq studies to identify differences in the G-CSF- and AMD3100-induced pathways in the mobilization process. These investigations will shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in cases of poor mobilizers. Figure 1 Disclosures No relevant conflicts of interest to declare.

Heterologous Expression and Patch-Clamp Recording of P2X Receptors in HEK293 Cells.

P2X receptors (P2XRs) are ligand-gated ion channels gated by extracellular adenosine 5'-triphosphate (ATP) and play a critical role in mediating ATP-induced purinergic signaling in physiological and pathological processes. Heterologous expression of P2XR in human embryonic kidney 293 (HEK293) cells and measurement of P2XR-mediated currents using patch-clamp recording technique have been widely used to study the biophysical and pharmacological properties of these receptors. Combination of electrophysiology with site-directed mutagenesis and structural information has shed light on the molecular basis for receptor activation and mechanisms of actions by receptor antagonists and modulators. It is anticipated that such methodologies will continue helping us to provide more mechanistic understanding of P2XRs and to test novel receptor antagonists and allosteric modulators for therapeutical purposes. In this chapter, we describe protocols of transiently or stably expressing the P2XR in HEK293 cells and measuring P2XR-mediated currents by using whole-cell recording.

Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X7 Receptor.

Diabetic neuropathic pain (DNP) and major depressive disorder (MDD) are common complications of diabetes mellitus and mutually affect each other. As a member of the ATP-gated ion channel family, P2X7 receptor is associated with the transduction of pain signal and the onset of depression. The aim of this study was to investigate the effects of dihydromyricetin (DHM) on rats with comorbid DNP and MDD. After the comorbid model was established, rat behavior changes were monitored by measuring the mechanical withdrawal threshold, thermal withdrawal latency, sugar water preference, immobility time in the forced-swim test, and open-field test parameters. The expressions of P2X7 receptor in the dorsal root ganglia (DRGs), spinal cord, and hippocampus were assessed by quantitative real-time PCR, Western blotting, and double immunofluorescence. We found that hyperalgesia, allodynia, and depressive behaviors of rats with comorbid DNP and MDD were relieved by treatment with DHM or application of a short-hairpin RNA for P2X7 receptor. The expression levels of P2X7, phosphorylated extracellular signal–regulated kinase 1/2, tumor necrosis factor α, and interleukin 1ß were increased in the DRGs, spinal cord, and hippocampus of rats in the model group but restored after DHM or P2X7 short-hairpin RNA treatment. In conclusion, P2X7 receptor in the DRGs, spinal cord, and hippocampus participates in the transduction of DNP and MDD signals. DHM seems to relieve comorbid DNP and MDD by reducing the expression of P2X7 receptor in the DRGs, spinal cord, and hippocampus and may be an effective new drug for the treatment of patients with both DNP and MDD.

Microencapsulated olfactory ensheathing cell transplantation reduces P2X4 receptor overexpression and inhibits neuropathic pain in rats

ObjectivesThe aim of this study was to determine the role of microencapsulated olfactory ensheathing cells (MC-OECs) transplantation in rats with sciatic nerve injury-induced neuropathic pain, and its relationship with P2X4 receptor expression in the L4-5 spinal cord segment. MethodsOlfactory bulb tissues of healthy Sprague Dawley (SD) rats were collected to culture olfactory ensheathing cells (OECs) using differential attachment methods. Seventy-two healthy SD rats were randomly assigned to four groups: the sham, chronic constriction injury (CCI), OECs, and MC-OECs groups. Mechanical paw withdrawal thresholds were measured on days 7, 14 and 21 after surgery. The expression of P2X4 receptor genes in the L4-5 spinal cord segment was detected by in situ hybridization, western blotting and RT-PCR. ResultsOn post-surgical days 7, 14 and 21. The mechanical paw withdrawal thresholds of rats from low to high were the CCI, OECs, MC-OECs and sham groups. The expression level of P2X4 receptor mRNA and protein from low to high were the sham, MC-OECs, OECs and CCI groups. Compared with the OECs group, the mechanical paw withdrawal thresholds of rats were increased, and the expression level of P2X4 receptor mRNA and protein were decreased in the MC-OECs group. In addition, the mechanical paw withdrawal thresholds of rats were higher and the expression level of P2X4 receptor were lower in the MC-OECs group on post-surgical days 21 than that on days 7 and 14. All differences between groups were statistically significant (P value < 0.05). ConclusionsOECs and MC-OECs transplantation can reduce the expression level of P2X4 receptor and relieve pain. The therapeutic efficacy was better in the MC-OECs group than in the OECs group. MC-OECs transplantation have the better effects of treatment with the increase of MC-OECs transplantation time.

Contribution of adenosine and ATP to the carotid body chemosensory activity in ageing.

Adenosine and ATP are excitatory neurotransmitters involved in the carotid body (CB) response to hypoxia. During ageing the CB exhibits a decline in its functionality, demonstrated by decreased hypoxic responses. In aged rats (20-24months old) there is a decrease in: basal and hypoxic release of adenosine and ATP from the CB; expression of adenosine and ATP receptors in the petrosal ganglion; carotid sinus nerve (CSN) activity in response to hypoxia; and ventilatory responses to ischaemic hypoxia. There is also an increase in SNAP25, ENT1 and CD73 expression. It is concluded that, although CSN activity and ventilatory responses to hypoxia decrease with age, adjustments in purinergic metabolism in the CB in aged animals are present aiming to maintain the contribution of adenosine and ATP. The possible significance of the findings in the context of ageing and in CB-associated pathologies is considered. During ageing the carotid body (CB) exhibits a decline in its functionality. Here we investigated the effect of ageing on functional CB characteristics as well as the contribution of adenosine and ATP to CB chemosensory activity. Experiments were performed in 3-month-old and 20- to 24-month-old male Wistar rats. Ageing decreased: the number of tyrosine hydroxylase immune-positive cells, but not typeII cells or nestin-positive cells in the CB; the expression of P2X2 and A2A receptors in the petrosal ganglion; and the basal and hypoxic release of adenosine and ATP from the CB. Ageing increased ecto-nucleotidase (CD73) immune-positive cells and the expression of synaptosome associated protein 25 (SNAP25) and equilibrative nucleoside transporter 1 (ENT1) in the CB. Additionally, ageing did not modify basal carotid sinus nerve (CSN) activity or the activity in response to hypercapnia, but decreased CSN activity in hypoxia. The contribution of adenosine and ATP to stimuli-evoked CSN chemosensory activity in aged animals followed the same pattern of 3-month-old animals. Bilateral common carotid occlusions during 5, 10 and 15s increased ventilation proportionally to the duration of ischaemia, an effect decreased by ageing. ATP contributed around 50% to ischaemic-ventilatory responses in young and aged rats; the contribution of adenosine was dependent on the intensity of ischaemia, being maximal in ischaemias of 5s (50%) and much smaller in 15s ischaemias. Our results demonstrate that both ATP and adenosine contribute to CB chemosensory activity in ageing. Though CB responses to hypoxia, but not to hypercapnia, decrease with age, the relative contribution of both ATP and adenosine for CB activity is maintained.

Magnesium sulfate inhibits inflammation through P2X7 receptors in human umbilical vein endothelial cells.

Introduction:Magnesium sulfate (MgSO4) is utilized for fetal neuroprotection in preterm birth but its mechanism of action is still poorly understood. P2X7 receptor (P2X7R) is required for secretion of IL-1β, and can be blocked by divalent cations such as magnesium (Mg) and its own antagonist, Brilliant Blue G (BBG). We sought to determine, whether during inflammation MgSO4 can block endothelial IL-1β secretion, using an in-vitro model.Methods:Human umbilical vein endothelial cell (HUVEC) cultures were treated with varying doses of LPS, 2’(3)-O-(4-Benzoylbenzoyl) adenosine-5’-triphosphate (BzATP), BBG and MgSO4 for 3- or 24-hours. We determined cell cytotoxicity, apoptosis, IL-1β mRNA expression, IL-1β production and secretion and P2X7R expression on HUVECs.Results:We demonstrated that MgSO4 is efficacious in blocking IL-1β-mediated-inflammation in HUVECs, at both the initiation and propagation phases of inflammation. MgSO4 exerts these anti-inflammatory effects via downregulation of P2X7Rs on HUVECs.Conclusion:LPS-exposure increases IL-1β production and secretion in HUVECs, which is further intensified by P2X7R agonist, BzATP while MgSO4 inhibits IL-1β in both presence and absence of BzATP. This effect is similar to the results of P2X7R antagonist, BBG, suggesting that the anti-inflammatory effects of MgSO4is through P2X7R.

Human Peripheral Blood Eosinophils Express High Levels of the Purinergic Receptor P2X4.

Extracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation, and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterized the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils.

The P2X7 Receptor Is Overexpressed in the Lesional Skin of Subjects Affected by Hidradenitis Suppurativa: A Preliminary Study

Background: P2X receptors (P2XRs) are plasma membrane channels involved in the modulation of immune responses. The role of the P2X7 receptor (P2X7R) has never been investigated in hidradenitis suppurativa (HS), which is a recurrent skin disease characterized by inflammatory nodules, scarring, and suppuration. Objective: Our aim was to investigate by immunohistochemistry (IHC) P2X7R, NLRP3 (NOD-like receptor family, pyrin domain-containing 3), and interleukin-1β (IL-1β) expression in HS lesions compared to healthy control (HC) skin. Method: The intensity of IHC immunostaining was semi-quantitatively graded for keratinocytes, neutrophils, lymphocytes, and monocytes. Statistical significance was assessed by the Mann-Whitney U test, Cohen’s κ coefficient, and χ² test. Results: A total of 59 samples, 31 from HS and 28 from HC, were collected and analysed. In skin keratinocytes, lymphocytes, and monocytes, but not in neutrophils, P2X7R and NLRP3 protein expression was significantly increased in HS versus the HC group. IL-1β protein expression was also higher in HS versus the HC group both in skin keratinocytes and in the inflammatory infiltrate. Cohen’s κ correlation coefficients for the expression of P2X7R versus NLRP3 or IL-1β in skin keratinocytes were significant (κ = 0.43 and 0.34, respectively). The same association between P2X7R and NLRP3 or IL-1β was confirmed by χ² tests. Conclusion: P2X7R, NLRP3, and IL-1β are overexpressed, and therefore the entire P2X7R/NLRP3/IL-1β pro-inflammatory axis is likely overactive in the skin of HS patients. This observation might provide clues to the pathogenesis of this disease and suggest novel therapies and markers of disease activity.

Extracellular Nucleotides and P2 Receptors in Renal Function.

The understanding of the nucleotide/P2 receptor system in the regulation of renal hemodynamics and transport function has grown exponentially over the last 20 yr. This review attempts to integrate the available data while also identifying areas of missing information. First, the determinants of nucleotide concentrations in the interstitial and tubular fluids of the kidney are described, including mechanisms of cellular release of nucleotides and their extracellular breakdown. Then the renal cell membrane expression of P2X and P2Y receptors is discussed in the context of their effects on renal vascular and tubular functions. Attention is paid to effects on the cortical vasculature and intraglomerular structures, autoregulation of renal blood flow, tubuloglomerular feedback, and the control of medullary blood flow. The role of the nucleotide/P2 receptor system in the autocrine/paracrine regulation of sodium and fluid transport in the tubular and collecting duct system is outlined together with its role in integrative sodium and fluid homeostasis and blood pressure control. The final section summarizes the rapidly growing evidence indicating a prominent role of the extracellular nucleotide/P2 receptor system in the pathophysiology of the kidney and aims to identify potential therapeutic opportunities, including hypertension, lithium-induced nephropathy, polycystic kidney disease, and kidney inflammation. We are only beginning to unravel the distinct physiological and pathophysiological influences of the extracellular nucleotide/P2 receptor system and the associated therapeutic perspectives.

Role of purinergic receptors in hepatobiliary carcinoma in Pakistani population: an approach towards proinflammatory role of P2X4 and P2X7 receptors.

The primary malignancy of liver, known as hepatocellular carcinoma (HCC), comprises 9% of all hepatobiliary carcinomas. A steady rise has also been observed in adenocarcinoma (ADC) of the liver and ampullary carcinoma (AMC), ascending to 0.5% of gastrointestinal malignancies. Hepatobiliary carcinomas consist of 13% of all cancer occurrences worldwide. Purinergic receptor-based signaling holds the therapeutic potential based on its role in cell proliferation of several carcinomas. An altered ATP concentration in nanomoles may lead towards crucial changes in cancer growth patterns in liver tissue. A total of 40 tissue samples were collected (20 samples of HCC, 10 samples of ADC, and 10 samples of AMC) from patients that underwent surgery. P2X4 and P2X7 receptors exhibited significantly increased expression in HCC, ADC, and AMC samples as compared with the control tissue samples. While ADC and AMC samples showed higher expression of P2X4 and P2X7 than the control, statistically, HCC samples exhibited the most significant expression of both P2X4 and P2X7 receptors than control tissues. It may be inferred that higher expression of P2X4 and P2X7 receptors is significantly associated with the upregulated cellular stress leading to inflammation and it is plausible that both these receptors may be used in diagnostic, prognostic, and therapeutic tools for carcinoma studiesin the future.

Role of the P2X7 receptor in in vitro and in vivo glioma tumor growth.

Human glioblastoma cells are strikingly refractory to ATP-stimulated, P2X7 receptor (P2X7R)-mediated cytotoxicity. To elucidate the mechanistic basis of this feature, we investigated P2X7R-dependent responses in wild type and P2X7R-transfected U138 cells. Mouse GL261 glioma cells were used as an additional control. Here, we report that wild type U138 glioma cells expressed the P2X7R to very low level. Contrary to human U138 cells, mouse GL261 cells showed strong P2X7R expression and P2X7R-dependent responses. Transfection of wild type P2RX7 into U138 cells fully restored P2X7R-dependent responses. P2RX7 transfection conferred a negligible in vitro growth advantage to U138 cells, while strongly accelerated in vivo growth. In silico analysis showed that the P2RX7 gene is seldom mutated in specimens from glioblastoma multiforme (GBM) patients. These observations suggest that the P2X7R might be an important receptor promoting GBM growth.