P2X7 Receptor Expression Research Articles

Repeated administration of a subanesthetic dose of ketamine results in impaired motor and cognitive behavior and differential expression of hippocampal P2X1 and P2X7 receptors in adult mice.

Ketamine hydrochloride serves multiple purposes, including its use as a general anesthetic, treatment for depression, and recreational drug. In studies involving rodents, ketamine is utilized as a model for schizophrenia. However, it is unclear whether age affects the behavioral response induced by repeated ketamine administration and if it modifies the expression levels of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and purinergic receptors (P2X1, P2X4, P2X7). In the present study, we evaluated the effect of intraperitoneal administration of subanesthetic ketamine dose (30mg/Kg) for fourteen days on young (35 days of age) and adult (76 days of age) mice on different behavioral tests. Nest-building behavior was evaluated during the fourteen-day treatment; short-term memory and social interaction tests were assessed twenty-four hours after the last administration of ketamine. Interestingly, only adult mice treated with ketamine showed impaired nest-building and novel object recognition tests. In the hippocampus, an area related to memory and cognition, ketamine administration showed no changes in the relative expression of GluN1, P2X4, and P2X7 while increasing GluA2 and P2X1 only in young mice. In contrast, when assessing the protein levels of P2X1 and P2X7 in the hippocampus following ketamine treatment, young mice exhibited a decrease in P2X1 levels while P2X7 levels increased. In contrast, adult mice showed the opposite pattern; P2X1 levels were higher, and P2X7 levels decreased. These results suggest that adult mice are more vulnerable to repeated ketamine administration than young mice and that a differential response of P2X1 and P2X7 might contribute to ketamine-induced behavioral changes.

Naphtho[1,2-b][1,4]diazepinedione-Based P2X4 Receptor Antagonists from Structure-Activity Relationship Studies toward PET Tracer Development.

The P2X4 receptor is implicated in various pathological conditions, including neuropathic pain and cancer. This study reports the development of 1,4-naphthodiazepinedione-based P2X4 receptor antagonists aimed at both therapeutic applications and potential use as PET tracers for imaging P2X4 receptor expression in cancer. Structure-activity relationship studies aided by docking studies and molecular dynamics simulations led to a series of compounds with potent P2X4 receptor antagonism, promising in vitro inhibition of interleukin-1β release in THP-1 cells and suitability for radiolabeling with fluorine-18. The most potent compounds were further evaluated for their physicochemical properties, metabolic stability, and in vivo biodistribution using PET imaging in mice. While these antagonists exhibited strong receptor binding and serum stability, rapid in vivo metabolism limited their potential as PET tracers, highlighting the need for further structural optimization. This study advances the understanding of P2X4 receptor antagonism and underscores the challenges in developing effective PET tracers for this target.

Inhibition of P2X7 receptor mitigates atrial fibrillation susceptibility in isoproterenol-induced rats.

Atrial fibrillation (AF) is a common cardiac arrhythmia that is characterized by atrial electrical remodeling. The P2X7 receptor (P2X7R), an ATP-gated ion channel, has been implicated in cardiovascular pathologies; however, its role in atrial electrical remodeling remains unclear. This study investigated whether inhibition of P2X7R could mitigate isoproterenol (ISO)-induced atrial electrical remodeling in rats and explored the underlying mechanisms. Two gene expression profiles related to AF (GSE79768 and GSE10598) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened using GEO2R. Mendelian randomization (MR) investigated the causal relationship between P2X7R expression and AF. Enrichment analysis was also conducted. An animal model was established via intraperitoneal injection of ISO for 2 weeks. The rats were divided into three groups: control (CTL), ISO, and ISO+Brilliant Blue G (BBG). Cardiac electrophysiological parameters were assessed using programmed electrical stimulation. Myocardial fibrosis and hypertrophy were evaluated using Sirius Red and Wheat Germ Agglutinin staining, respectively. P2X7R abundance was assessed using immunofluorescence, and relevant proteins were detected by Western blotting. GEO2R and MR analyses indicated a correlation between P2X7R expression and AF. Rats in the ISO group exhibited increased P2X7R levels, abnormal cardiac electrophysiology, altered ion channel protein expression, myocardial hypertrophy, and fibrosis. Enrichment analysis indicated that oxidative stress responses might be involved, and Western blotting showed significantly elevated levels of NOX, CaMKII, and associated proteins. BBG (P2X7R inhibitor) treatment mitigated these effects. P2X7R was associated with AF, and inhibition of P2X7R curbed electrical and structural remodeling in ISO-induced AF, potentially via the NOX/CaMKII pathway.

P2X7R antagonism suppresses long-lasting brain hyperexcitability following traumatic brain injury in mice.

Purpose: Post-traumatic epilepsy (PTE) is one of the most common life-quality reducing consequences of traumatic brain injury (TBI). However, to date there are no pharmacological approaches to predict or to prevent the development of PTE. The P2X7 receptor (P2X7R) is a cationic ATP-dependent membrane channel that is expressed throughout the brain. While increasing evidence suggests a role for the P2X7R during seizures and epilepsy, it is unclear if changes in P2X7R expression can predict TBI-induced epilepsy development, and whether P2X7R antagonism can protect against long-lasting brain hyperexcitability caused by TBI. Methods: TBI was induced in adult male mice using the controlled cortical impact model (CCI). To test the anti-epileptogenic effects of P2X7R antagonism, mice were treated with brain-penetrant P2X7R antagonists JNJ-54175446 (30 mg/kg) or AFC-5128 (30 mg/kg) for 7 days post-CCI. The cell-type specific effects of P2X7Rs on TBI-induced hyperexcitability were analyzed in mice lacking exon 2 of the P2rx7 gene selectively in microglia (P2rx7:Cx3cr1-Cre). Static positron emission tomography (PET) via an intravenous injection of the P2X7R radioligand 18F-JNJ-64413739 and magnetic resonance imaging (MRI) were conducted twice during the first- and third-week post-injury. Results: Following TBI, while there were no obvious changes in P2X7R protein levels in the ipsilateral hippocampus post-injury, there was a delayed increase in P2X7R protein levels in the ipsilateral cortex at 3 months post-injury. Treatment with P2X7R antagonists shortly after TBI reduced long-lasting brain hyperexcitability, reduced cortical contusion volume, and normalized injury-induced hyperactivity to control sham-levels at 3 weeks post-TBI. Notably, mice lacking P2rx7 in microglia had an increased seizure threshold after TBI, suggesting that P2X7R contributed to brain hyperexcitability via its effects on microglia. Finally, P2X7R radioligand uptake after TBI correlated with seizure threshold at 3 weeks post-injury. Conclusions: Our results demonstrate the antiepileptogenic potential of P2X7R antagonism to prevent TBI-induced epilepsy and indicate that P2X7R-based PET imaging may be a useful diagnostic tool to identify people at risk of developing PTE.

The role and mechanism of P2X7R in cirrhotic cardiomyopathy

In the context of liver cirrhosis, the incidence of myocardial inflammation and apoptosis escalates, contributing to the development and progression of cirrhotic cardiomyopathy. The P2X7 receptor, a purinergic receptor linked to inflammatory processes, has been identified in the etiology of a range of autoinflammatory, autoimmune, chronic inflammatory, and metabolic disorders. Despite this, the specific role of the P2X7 receptor in the etiology of cirrhotic cardiomyopathy remains to be elucidated. In our research, a cirrhotic cardiomyopathy animal model was established using mice subjected to bile duct ligation. The expression of the P2X7 receptor was suppressed via intraperitoneal administration of Brilliant Blue G. Cardiac function was evaluated using echocardiographic techniques, while histopathological examination and enzyme-linked immunosorbent assays were employed to assess the presence of inflammation and apoptosis in liver and cardiac tissues. The expression of key proteins, including P2X7, NLRP3, and IL-1β, in the myocardial tissue was quantified by Western blot analysis. Our research has unveiled significant findings in a murine model of liver fibrosis induced by two weeks of bile duct ligation. Notably, we detected escalated levels of liver fibrosis coupled with disruptions in liver blood flow dynamics. Concurrently, there was a marked increase in myocardial inflammation and apoptosis, which adversely affected heart function. Intriguingly, the expression of P2X7 receptors (P2X7R) in cardiac and hepatic tissues was found to be significantly elevated. Targeting and inhibiting the expression of P2X7R not only alleviated myocardial inflammation and apoptosis but also enhanced cardiac performance. Furthermore, this intervention resulted in a noticeable reduction in liver fibrosis. The interplay between the P2X7 and NLRP3 pathways emerges as a pivotal mechanism in the etiology and progression of cirrhotic cardiomyopathy. Our findings suggest that modulating the P2X7-NLRP3 axis could offer promising therapeutic avenues for managing cirrhotic cardiomyopathy.

Targeted therapy: P2X3 receptor silencing in bone cancer pain relief.

Bone cancer pain remains a significant clinical challenge, often refractory to conventional treatments. The upregulation of the P2X3 receptor in the dorsal root ganglia has been implicated in the pathogenesis of bone cancer pain. This study aimed to elucidate the role of the P2X3 receptor in this context and assess the therapeutic potential of receptor silencing. Utilizing a rat model with Walker 256 cells to simulate bone cancer pain, researchers conducted molecular analyses, including semi-quantitative RT-PCR and Western Blot, to investigate P2X3 receptor expression in the dorsal root ganglia. Results demonstrated a marked increase in P2X3 receptor levels in the dorsal root ganglia of the bone cancer pain model. Targeted silencing of the P2X3 receptor using specific shRNA delivered via lentiviral vectors significantly reduced pain sensitivity, underscoring the receptor's potential as a valuable therapeutic target. In addition, a comprehensive gene expression analysis leveraging the GEO data set GSE249443 was performed to explore the underlying biological pathways linked to bone cancer pain. This analysis provided insights into the intricate interplay between bone cancer pain and associated biological processes, offering a deeper understanding of the mechanisms involved in pain modulation and progression. In conclusion, this research identifies the P2X3 receptor as a critical molecular target for mitigating bone cancer pain. The selective silencing of the P2X3 receptor emerges as a promising and innovative therapeutic strategy, presenting novel avenues for managing bone cancer pain and potentially revolutionizing treatment approaches in this challenging domain.

The analgesic effect and mechanism of the active components screening from Corydalis yanhusuo by P2X3 receptors

Ethnopharmacological relevanceCavidine (CAV) is the main bioactive ingredient of Corydalis ternata f. yanhusuo (Y.H.Chou & Chun C.Hsu) Y.C.Zhu, which is a traditional Chinese herbal containing a variety of uses such as analgesic, anticancer, and anti-inflammatory properties. Aim of the studyThe goal is to screen Corydalis yanhusuo for anti-central sensitization active components and investigate and clarify the pharmacological mechanism and therapeutic efficacy of the active ingredient CAV in the treatment of chronic pain. Material and methodsFirst, cell membrane immobilized chromatography was used to screen the bioactive ingredients in Corydalis yanhusuo. Spare nerve injury (SNI) model and complete Freund's adjuvant (CFA) mice model were constructed to identify the analgesic effect of CAV. RNA-seq and bioinformatics analyses were used to explore the potential targets of CAV in CFA mice and SNI mice. HE staining was used to observe the infiltration of inflammatory cells in the dorsal root ganglion (DRG) and spinal cord(SC) of CFA mice and SNI mice. WB and qPCR were used to detect the level of inflammatory factors TNF-α, IL-1β, and IL-6 in DRG and SC of mice. SNI and CFA mice were used to study the effect and mechanism of CAV on microglial activation. Results9 potential active ingredients were screened out from Corydalis yanhusuo that can regulate P2X3 receptors. CAV showed good analgesic effects, increased the mechanical pain and thermal pain thresholds of CFA mice and SNI mice, inhibited the expression of DRG and SC inflammatory factors, downregulated IBA-1, and inhibited microglial activation. Further in vivo and in vitro experiments showed that CAV significantly inhibited the expression of P2X3 receptors and the activation of its downstream MAPK pathway in DRG neurons and SC. ConclusionThis study is the first to indicate that CAV exerts an analgesic effect by inhibiting microglia activation via the P2X3 signaling pathway axis, providing the clinical utility of CAV in chronic pain therapy.

The P2X7R is a crucial target for Angiotensin II-induced myocardial ferroptosis and remodeling

AbstractOngoing cardiac remodeling can lead to negative outcomes, such as cardiac failure and diminished myocardial function, although the remodeling process initially protects the heart as a compensatory mechanism[1] . Importantly, ferroptosis appears to be a critical process in the development of cardiac disease. In a recent publication in Redox Biology, (Zhong et al. [2] showed that reactive oxygen species (ROS) generation and cardiac ferroptosis may be the mechanisms underlying angiotensin II (Ang II)-induced cardiac remodeling, as well as that ferroptosis is required for heart impairment and cardiac dysfunction induced by Ang II. Moreover, this study provides evidence that Ang II increases the expression of P2X7 receptors (P2X7R) in cardiac tissues and that both silencing and pharmacological inhibition of P2X7R significantly inhibited Ang II-induced ferroptosis and hypertrophy. Also, this work confirmed that P2X7R deficiency mitigated the Ang II-induced deterioration of cardiac injury in mice fed an iron-rich diet. Most interestingly, this study revealed that Ang II directly interacts with the P2X7R to activate and induce nucleocytoplasmic shuttling of human antigen R (HuR), which in turn controls the stability of the mRNA of heme oxygenase 1 (HO-1) and GPX4 and subsequent ROS production, which translated to induction of myocardial ferroptosis and remodeling.

Activation of P2X7 Receptor Mediates the Abnormal Ovulation Induced by Chronic Restraint Stress and Chronic Cold Stress.

Chronic stress has become a major problem that endangers people's physical and mental health. Studies have shown that chronic stress impairs female reproduction. However, the related mechanism is not fully understood. P2X7 receptor (P2X7R) is involved in a variety of pathological changes induced by chronic stress. Whether P2X7R is involved in the effect of chronic stress on female reproduction has not been studied. In this study, we established a chronic restraint stress mouse model and chronic cold stress mouse model. We found that the number of corpora lutea was significantly reduced in the two chronic stress models. The number of corpora lutea indirectly reflects the ovulation, suggesting that chronic stress influences ovulation. P2X7R expression was significantly increased in ovaries of the two chronic stress models. A superovulation experiment showed that P2X7R inhibitor A-438079 HCL partially rescued the ovulation rate of the two chronic stress models. Further studies showed that activation of P2X7R signaling inhibited the cumulus expansion and promoted the expression of NPPC in granulosa cells, one key negative factor of cumulus expansion. Moreover, sirius red staining showed that the ovarian fibrosis was increased in the two chronic stress models. For the fibrosis-related factors, TGF-β1 was increased and MMP2 was decreased. In vitro studies also showed that activation of P2X7R signaling upregulated the expression of TGF-β1 and downregulated the expression of MMP2 in granulosa cells. In conclusion, P2X7R expression was increased in the ovaries of the chronic restraint-stress and chronic cold-stress mouse models. Activation of P2X7R signaling promoted NPPC expression and cumulus expansion disorder, which contributed to the abnormal ovulation of the chronic stress model. Activation of P2X7R signaling is also associated with the ovarian fibrosis changes in the chronic stress model.

The role of P2X4R in regulating CA1 hippocampal synaptic impairment in LPS-induced depression

AimTo study the role of the P2X4 receptor (P2X4R) in regulating hippocampal synaptic impairment in lipopolysaccharide (LPS)-induced depression. MethodsA rat model of depression was established by LPS injection. P2X4R expression was inhibited by 5-(3-bromophenyl)-1, 3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD). Depressive symptoms were identified through behavioral tests. P2X4R and cytokine mRNA levels were measured by qRT–PCR, while synaptic protein levels were measured by Western blotting. Synaptic ultrastructure was assessed by transmission electron microscopy, and the colocalization of brain-derived neurotrophic factor (BDNF) with microglia, astrocytes, and neurons was determined by double immunofluorescence staining. ResultsInjection of 5-BDBD alleviated LPS-induced depressive symptoms. LPS injection significantly increased the mRNA levels of P2X4R and proinflammatory cytokines in the hippocampus, especially in the CA1 region. The levels of synaptic proteins (BDNF, PSD95, and synapsin I) in the CA1 region were significantly lower than those in the other two regions of the hippocampus, and the synaptic ultrastructure in the hippocampal CA1 region was significantly altered. As expected, the Pearson's correlation R and the overlap coefficient R for the hippocampal colocalization of IBA-1 with BDNF were decreased, and 5-BDBD injection reversed these trends. Injection of 5-BDBD increased hippocampal BDNF mRNA expression. ConclusionsP2X4R may induce synaptic impairment in the hippocampal CA1 region by influencing microglial BDNF expression in the context of LPS-induced depression in rats.

The P2X7 Hypothesis of Central Post-Stroke Pain.

The present study examined how P2X7 receptor knockout (KO) modulates central post-stroke pain (CPSP) induced by lesions of the ventrobasal complex (VBC) of the thalamus in behaviors, molecular levels, and electrical recording tests. Following the experimental procedure, the wild-type and P2X7 receptor KO mice were injected with 10 mU/0.2 μL type IV collagenase in the VBC of the thalamus to induce an animal model of stroke-like thalamic hemorrhage. Behavioral data showed that the CPSP group induced thermal and mechanical pain. The P2X7 receptor KO group showed reduced thermal and mechanical pain responses compared to the CPSP group. Molecular assessments revealed that the CPSP group had lower expression of NeuN and KCC2 and higher expression of GFAP, IBA1, and BDNF. The P2X7 KO group showed lower expression of GFAP, IBA1, and BDNF but nonsignificant differences in KCC2 expression than the CPSP group. The expression of NKCC1, GABAa receptor, and TrkB did not differ significantly between the control, CPSP, and P2X7 receptor KO groups. Muscimol, a GABAa agonist, application increased multiunit numbers for monitoring many neurons and [Cl-] outflux in the cytosol in the CPSP group, while P2X7 receptor KO reduced multiunit activity and increased [Cl-] influx compared to the CPSP group. P2X4 receptor expression was significantly decreased in the 100 kDa but not the 50 kDa site in the P2X7 receptor KO group. Altogether, the P2X7 hypothesis of CPSP was proposed, wherein P2X7 receptor KO altered the CPSP pain responses, numbers of astrocytes and microglia, CSD amplitude of the anterior cingulate cortex and the medial dorsal thalamus, BDNF expression, [Cl-] influx, and P2X4 expression in 100 kDa with P2X7 receptors. The present findings have implications for the clinical treatment of CPSP symptoms.

Exploring the Therapeutic Potential of Baicalin: Mitigating Anxiety and Depression in Epileptic Rats.

Epilepsy is a serious neurological disorder that affects millions of people each year, often leading to cognitive issues and reduced quality of life. Medication is the main treatment, but many patients experience negative side effects. Male Sprague-Dawley (SD) rats were chosen as experimental animals for this experiment due to their physiological and genetic similarities to humans, cost-effectiveness, and ease of handling in a laboratory setting. The objective of this study was to assess the neuroprotective properties of baicalin (BA) in relation to its impact on anxiety and depressive-like behaviors in the epilepsy model. Thirty male Sprague-Dawley (SD) rats were selected for this experiment. Pentylenetetrazol (PTZ) kindling (40 mg/kg; i.p.) was utilized to establish an epilepsy model. The effect of BA (50 mg/kg; gavage) on seizure severity (assessed using the Racine scale), anxiety, and depressive- like behaviors (evaluated through open field experiments and forced swimming tests) was examined. Histological examinations, including hematoxylin and eosin (HE) staining and Nissl staining, were conducted to assess neuronal damage. Furthermore, the neuroprotective properties of BA were examined through the analysis of Doublecortin (DCX), MKI67 (KI67), and Brain-Derived Neurotrophic Factor (BDNF) levels in the hippocampus of rats. The inhibitory impact of BA on neuroinflammation was assessed via dual labeling for NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and the microglial marker ionized calcium- binding adapter molecule 1 (Iba-1). The influence of BA on the expression of P2X7 receptor (P2X7R), NLRP3, and Interleukin-1β (IL-1β) was also assessed by reverse transcription quantitative PCR (RT-qPCR) in the brain. Finally, we employed a molecular docking model to assess the extent of receptor-ligand binding. Epilepsy models exhibited significant anxiety and depressive-like behaviors, and BA significantly reduced the severity of seizures in these rats while also alleviating their anxiety and depressive-like behaviors. Moreover, neuronal loss and damage were observed in the hippocampus of epileptic rats, but BA was able to effectively counteract this issue by enhancing BDNF expression and promoting neurogenesis within the hippocampus, especially in the DG region. The co-localization of Iba-1 with NLRP3 indicated the activation of NLRP3 inflammasome in microglia. Subsequent RT-PCR revealed that BA may alleviate anxiety and depressive-like behaviors in epileptic rats by activating the P2RX7/NLRP3/ IL-1β signaling pathway. The final molecular docking results indicated that BA had a good binding affinity with proteins, such as P2RX7, NLRP3, and IL-1β. This study confirmed the effectiveness of BA in improving anxiety and depressivelike behaviors associated with epilepsy. Moreover, it provides theoretical support for the neuroprotective role demonstrated by BA.

Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice

BackgroundThe purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. MethodsEffects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. ResultsRNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. ConclusionsOur results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

The P2X7 Receptor is a Master Regulator of Microparticle and Mitochondria Exchange in Mouse Microglia.

Microparticles (MPs) are secreted by all cells, where they play a key role in intercellular communication, differentiation, inflammation, and cell energy transfer. P2X7 receptor (P2X7R) activation by extracellular ATP (eATP) causes a large MP release and affects their contents in a cell-specific fashion. We investigated MP release and functional impact in microglial cells from P2X7R-WT or P2X7R-KO mice, as well as mouse microglial cell lines characterized for high (N13-P2X7RHigh) or low (N13-P2X7RLow) P2X7R expression. P2X7R stimulation promoted release of a mixed MP population enriched with naked mitochondria. Released mitochondria were taken up and incorporated into the mitochondrial network of the recipient cells in a P2X7R-dependent fashion. NLRP3 and the P2X7R itself were also delivered to the recipient cells. Microparticle transfer increased the energy level of the recipient cells and conferred a pro-inflammatory phenotype. These data show that the P2X7R is a master regulator of intercellular organelle and MP trafficking in immune cells.

Endothelin-1-mediated Brainstem Glial Activation Produces Asthmatic Airway Vagal Hypertonia Via Enhanced ATP-P2X4 Receptor Signaling in Sprague-Dawley Rats.

The occurrence of major asthma symptoms is largely attributed to airway vagal hypertonia, of which the central mechanisms remain unclear. This study tests the hypotheses that endothelin-1-mediated brainstem glial activation produces asthmatic airway vagal hypertonia via enhanced action of adenosine 5'-triphosphate on neuronal purinergic P2X4 receptors. A rat model of asthma was prepared using ovalbumin. Airway vagal tone was evaluated by the recurrent laryngeal discharge and plethysmographic measurement of pulmonary function. The changes in the brainstem were examined using ELISA, Western blot, luciferin-luciferase, quantitative reverse transcription-polymerase chain reaction, enzyme activity assay and immunofluorescent staining, respectively. The results showed that in the medulla of rats, endothelin receptor type B and P2X4 receptors were primarily expressed in astrocytes and neurons, respectively, and both of which, along with endothelin-1 content, were significantly increased after ovalbumin sensitization. Ovalbumin sensitization significantly increased recurrent laryngeal discharge, which was blocked by acute intracisternal injection of P2X4 receptor antagonist 5-BDBD, knockdown of brainstem P2X4 receptors, and chronic intraperitoneal injection of endothelin receptor type B antagonist BQ788, respectively. Ovalbumin sensitization activated microglia and astrocytes and significantly decreased ecto-5'-nucleotidase activity in the medulla, and all of which, together with the increase of medullary P2X4 receptor expression and decrease of pulmonary function, were reversed by chronic BQ788 treatment. These results demonstrated that in rats, allergic airway challenge activates both microglia and astrocytes in the medulla via enhanced endothelin-1/endothelin receptor type B signaling, which subsequently causes airway vagal hypertonia via augmented adenosine 5'-triphosphate/P2X4 receptor signaling in central neurons of airway vagal reflex.

Interstitial Cells of Cajal and P2X3 Receptors at Ureteropelvic Junction Obstruction and Their Relationship with Pain Response.

Introduction: Etiopathogenesis and the symptomatology of ureteropelvic junction obstruction (UPJO) in the pediatric population has not yet been definitely clarified, suggesting a multifactorial nature of the condition. The aim was to analyze the association between the number of Interstitial Cells of Cajal (ICCs), as well as P2X3 receptors in ureteropelvic junction (UPJ) and the pain response in pediatric patients with hydronephrosis. Methods: 50 patients with congenital hydronephrosis underwent open or laparoscopic pyeloplasty at one of two departments of pediatric surgery and urology in Poland. Patients were divided into two groups according to the pain symptoms before surgery. A total of 50 samples of UPJ were obtained intraoperatively and underwent histopathological and immunohistochemical (IHC) analysis. Quantitative assessment of ICCs was based on the number of CD117(+) cells of adequate morphology in the subepithelial layer and the muscularis propria. Expression of P2X3 receptors was evaluated as the intensity of IHC staining. Results: Patients with hydronephrosis and accompanying pain were on average 60 months older (77 vs. 17 months) than children with asymptomatic hydronephrosis (p = 0.017). Symptomatic children revealed higher numbers of ICCs in both the subepithelial layer and in the lamina muscularis propria. In particular, symptomatic patients aged 2 years or more exhibited significantly higher numbers of ICCs in the subepithelial layer. Significant differences in the distribution of ICCs between the subepithelial layer and the lamina muscularis propria were observed in both groups. Expression of P2X3 receptors was limited to the urothelium and the muscle layer and correlated between these structures. There was no relationship between pain response and the expression of P2X3 receptors. Conclusions: ICCs and P2X3 receptors may participate in the pathogenesis of UPJO and in the modulation of pain response to a dilatation of the pyelocaliceal system. Explanation of the role of ICCs and P2X3 receptors in propagation of ureteral peristaltic wave and the modulation of pain stimuli requires further studies.

The NFATc1/P2X7 receptor relationship in human intervertebral disc cells.

A comprehensive understanding of the molecules that play key roles in the physiological and pathological homeostasis of the human intervertebral disc (IVD) remains challenging, as does the development of new therapeutic treatments. We recently found a positive correlation between IVD degeneration (IDD) and P2X7 receptor (P2X7R) expression increases both in the cytoplasm and in the nucleus. Using immunocytochemistry, reverse transcription PCR (RT-PCR), overexpression, and chromatin immunoprecipitation, we found that NFATc1 and hypoxia-inducible factor-1α (HIF-1α) are critical regulators of P2X7R. Both transcription factors are recruited at the promoter of the P2RX7 gene and involved in its positive and negative regulation, respectively. Furthermore, using the proximity ligation assay, we revealed that P2X7R and NFATc1 form a molecular complex and that P2X7R is closely associated with lamin A/C, a major component of the nuclear lamina. Collectively, our study identifies, for the first time, P2X7R and NFATc1 as markers of IVD degeneration and demonstrates that both NFATc1 and lamin A/C are interaction partners of P2X7R.

ATP-P2X7 signaling mediates brain pathology while contributing to viral control in perinatal Zika virus infection

Zika virus (ZIKV), the causative agent of Zika fever, is a flavivirus transmitted by mosquitoes of the Aedes genus. Zika virus infection has become an international concern due to its association with severe neurological complications such as fetal microcephaly. Viral infection can induce the release of ATP in the extracellular environment, activating receptors sensitized by extracellular nucleotides, such as the P2X7 receptor. This receptor is the primary purinergic receptor involved in neuroinflammation, neurodegeneration, and immunity. In this work, we investigated the role of ATP-P2X7 receptor signaling in Zika-related brain abnormalities. Wild-type mice (WT) and P2X7 receptor-deficient (P2X7-/-) C57BL/6 newborn mice were subcutaneously inoculated with 5 × 106plaque-forming units of ZIKV or mock solution. P2X7 receptor expression increased in the brain of Zika virus-infected mice compared to the mock group. Comparative analyses of the hippocampi from WT and P2X7-/-mice revealed that the P2X7 receptor increased hippocampal damage in CA1/CA2 and CA3 regions. Doublecortin expression decreased significantly in the brains of ZIKV-infected mice. WT ZIKV-infected mice showed impaired motor performance compared to P2X7-/- infected mice. WT ZIKV-infected animals showed increased expression of glial markers GFAP (astrocytes) and IBA-1 (microglia) compared to P2X7-/- infected mice. Although the P2X7 receptor contributes to neuronal loss and neuroinflammation, WT mice were more efficient in controlling the viral load in the brain than P2X7 receptor-deficient mice. This result was associated with higher induction of TNF-α, IFN-β, and increased interferon-stimulated gene expression in WT mice than P2X7-/-ZIKV-infected. Finally, we found that the P2X7 receptor contributes to inhibiting the neuroprotective signaling pathway AKT/mTOR while stimulating the caspase-3 activation, possibly two distinct pathways contributing to neurodegeneration. These findings suggest that ATP-P2X7 receptor signaling contributes to the antiviral response in the brain of ZIKV-infected mice while increasing neuronal loss, neuroinflammation, and related brain abnormalities.

Glial reactivity in a mouse model of beta-amyloid deposition assessed by PET imaging of P2X7 receptor and TSPO using [11C]SMW139 and [18F]F-DPA

BackgroundP2X7 receptor has emerged as a potentially superior PET imaging marker to TSPO, the gold standard for imaging glial reactivity. [11C]SMW139 is the most recently developed radiotracer to image P2X7 receptor. The aim of this study was to image reactive glia in the APP/PS1-21 transgenic (TG) mouse model of Aβ deposition longitudinally using [11C]SMW139 targeting P2X7 receptor and to compare tracer uptake to that of [18F]F-DPA targeting TSPO at the final imaging time point. TG and wild type (WT) mice underwent longitudinal in vivo PET imaging using [11C]SMW139 at 5, 8, 11, and 14 months, followed by [18F]F-DPA PET scan only at 14 months. In vivo imaging results were verified by ex vivo brain autoradiography, immunohistochemical staining, and analysis of [11C]SMW139 unmetabolized fraction in TG and WT mice.ResultsLongitudinal change in [11C]SMW139 standardized uptake values (SUVs) showed no statistically significant increase in the neocortex and hippocampus of TG or WT mice, which was consistent with findings from ex vivo brain autoradiography. Significantly higher [18F]F-DPA SUVs were observed in brain regions of TG compared to WT mice. Quantified P2X7-positive staining in the cortex and thalamus of TG mice showed a minor increase in receptor expression with ageing, while TSPO-positive staining in the same regions showed a more robust increase in expression in TG mice as they aged. [11C]SMW139 was rapidly metabolized in mice, with 33% of unmetabolized fraction in plasma and 29% in brain homogenates 30 min after injection.Conclusions[11C]SMW139, which has a lower affinity for the rodent P2X7 receptor than the human version of the receptor, was unable to image the low expression of P2X7 receptor in the APP/PS1-21 mouse model. Additionally, the rapid metabolism of [11C]SMW139 in mice and the presence of several brain-penetrating radiometabolites significantly impacted the analysis of in vivo PET signal of the tracer. Finally, [18F]F-DPA targeting TSPO was more suitable for imaging reactive glia and neuroinflammatory processes in the APP/PS1-21 mouse model, based on the findings presented in this study and previous studies with this mouse model.

Age-Dependent Activation of Purinergic Transmission Contributes to the Development of Epileptogenesis in ADSHE Model Rats.

To explore the developmental processes of epileptogenesis/ictogenesis, this study determined age-dependent functional abnormalities associated with purinergic transmission in a genetic rat model (S286L-TG) of autosomal-dominant sleep-related hypermotor epilepsy (ADSHE). The age-dependent fluctuations in the release of ATP and L-glutamate in the orbitofrontal cortex (OFC) were determined using microdialysis and ultra-high-performance liquid chromatography with mass spectrometry (UHPLC-MS). ATP release from cultured astrocytes was also determined using UHPLC-MS. The expressions of P2X7 receptor (P2X7R), connexin 43, phosphorylated-Akt and phosphorylated-Erk were determined using capillary immunoblotting. No functional abnormalities associated with purinergic transmission could be detected in the OFC of 4-week-old S286L-TG and cultured S286L-TG astrocytes. However, P2X7R expression, as well as basal and P2X7R agonist-induced ATP releases, was enhanced in S286L-TG OFC in the critical ADSHE seizure onset period (7-week-old). Long-term exposure to a modest level of P2X7R agonist, which could not increase astroglial ATP release, for 14 d increased the expressions of P2X7R and connexin 43 and the signaling of Akt and Erk in astrocytes, and it enhanced the sensitivity of P2X7R to its agonists. Akt but not Erk increased P2X7R expression, whereas both Akt and Erk increased connexin 43 expression. Functional abnormalities, enhanced ATP release and P2X7R expression were already seen before the onset of ADSHE seizure in S286L-TG. Additionally, long-term exposure to the P2X7R agonist mimicked the functional abnormalities associated with purinergic transmission in astrocytes, similar to those in S286L-TG OFC. Therefore, these results suggest that long-term modestly enhanced purinergic transmission and/or activated P2X7R are, at least partially, involved in the development of the epileptogenesis of ADSHE, rather than that of ictogenesis.