Purine Release, Metabolism, and Signaling in the Inflammatory Response.

ATP, NAD+, and nucleic acids are abundant purines that, in addition to having critical intracellular functions, have evolved extracellular roles as danger signals released in response to cell lysis, apoptosis, degranulation, or membrane pore formation. In general ATP and NAD+ have excitatory and adenosine has anti-inflammatory effects on immune cells. This review focuses on recent advances in our understanding of purine release mechanisms, ectoenzymes that metabolize purines (CD38, CD39, CD73, ENPP1, and ENPP2/autotaxin), and signaling by key P2 purinergic receptors (P2X7, P2Y2, and P2Y12). In addition to metabolizing ATP or NAD+, some purinergic ectoenzymes metabolize other inflammatory modulators, notably lysophosphatidic acid and cyclic GMP-AMP (cGAMP). Also discussed are extracellular signaling effects of NAD+ mediated by ADP-ribosylation, and epigenetic effects of intracellular adenosine mediated by modification of S-adenosylmethionine-dependent DNA methylation.

Inflammation and thrombosis in COVID-19 pathophysiology: proteinase-activated and purinergic receptors as drivers and candidate therapeutic targets.

Evolving information has identified disease mechanisms and dysregulation of host biology that might be targeted therapeutically in coronavirus disease 2019 (COVID-19). Thrombosis and coagulopathy, associated with pulmonary injury and inflammation, are emerging clinical features of COVID-19. We present a framework for mechanisms of thrombosis in COVID-19 that initially derive from interaction of SARS-CoV-2 with ACE2, resulting in dysregulation of angiotensin signaling and subsequent inflammation and tissue injury. These responses result in increased signaling by thrombin (proteinase-activated) and purinergic receptors, which promote platelet activation and exert pathological effects on other cell types (e.g., endothelial cells, epithelial cells, and fibroblasts), further enhancing inflammation and injury. Inhibitors of thrombin and purinergic receptors may, thus, have therapeutic effects by blunting platelet-mediated thromboinflammation and dysfunction in other cell types. Such inhibitors include agents (e.g., anti-platelet drugs) approved for other indications, and that could be repurposed to treat, and potentially improve the outcome of, COVID-19 patients. COVID-19, caused by the SARS-CoV-2 virus, drives dysregulation of angiotensin signaling, which, in turn, increases thrombin-mediated and purinergic-mediated activation of platelets and increase in inflammation. This thromboinflammation impacts the lungs and can also have systemic effects. Inhibitors of receptors that drive platelet activation or inhibitors of the coagulation cascade provide opportunities to treat COVID-19 thromboinflammation.

Dangerously Fit: Extracellular ATP Aids Memory T Cell Metabolism.

In a recent issue of Nature, Borges da Silva et al. (2018) reveal that P2RX7, a receptor for extracellular ATP, promotes CD8 T cell memory by enhancing metabolic fitness. This work links an ancient "danger" signal with long-term immunity.

The Raf-like MAPKKK INTEGRIN-LINKED KINASE 5 regulates purinergic receptor-mediated innate immunity in Arabidopsis.

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to an elevated plant defense response. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we show that in Arabidopsis thaliana, P2K1 phosphorylates the Raf-like MAP kinase kinase kinase (MAPKKK) INTEGRIN-LINKED KINASE 5 (ILK5) on serine 192 in the presence of eATP. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses. ILK5 interacts with and phosphorylates the MAP kinase MKK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type (WT). The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to WT plants. Plants expressing only the mutant ILK5S192A protein, with decreased kinase activity, did not activate the MAPK cascade upon ATP addition. These results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MPK3/6 associated with an elevated innate immune response.

Crosstalk between purinergic receptor P2Y11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages.

P2Y11 is a G protein-coupled ATP receptor that activates IL-1 receptor (IL-1R) in a cyclic AMP dependent manner. In human macrophages, P2Y11/IL-1R crosstalk with CCL20 as a prime target is controlled by phosphodiesterase 4 (PDE4), which mediates breakdown of cyclic AMP. Here, we used gene expression analysis to identify activation of CXCR4 and CXCR7 as a hallmark of P2Y11 signaling. We found that PDE4 inhibition with rolipram boosts P2Y11/IL-1R-induced upregulation of CXCR7 expression and CCL20 production in an epidermal growth factor receptor dependent manner. Using an astrocytoma cell line, naturally expressing CXCR7 but lacking CXCR4, P2Y11/IL-1R activation effectively induced and CXCR7 agonist TC14012 enhanced CCL20 production even in the absence of PDE4 inhibition. Moreover, CXCR7 depletion by RNA interference suppressed CCL20 production. In macrophages, the simultaneous activation of P2Y11 and CXCR7 by their respective agonists was sufficient to induce CCL20 production with no need of PDE4 inhibition, as CXCR7 activation increased its own and eliminated CXCR4 expression. Finally, analysis of multiple CCL chemokines in the macrophage secretome revealed that CXCR4 inactivation and CXCR7 activation selectively enhanced P2Y11/IL-1R-mediated secretion of CCL20. Altogether, our data establish CXCR7 as an integral component of the P2Y11/IL-1R-initiated signaling cascade and CXCR4-associated PDE4 as a regulatory checkpoint.

Distribution of P2Y and P2X purinergic receptor expression within the intestine.

Nucleotides are potent extracellular signaling molecules during homeostasis, infection, and injury due to their ability to activate purinergic receptors. The nucleotide ATP activates P2X receptors (P2RXs), whereas the nucleotides ADP, ATP, UTP, and UDP-glucose selectively activate different P2Y receptors (P2RYs). Several studies have established crucial roles for P2 receptors during intestinal inflammatory and infectious diseases, yet the most extensive characterization of purinergic signaling has focused on immune cells and the central and enteric nervous systems. As epithelial cells serve as the first barrier against irritants and infection, we hypothesized that the gut epithelium may express multiple purinergic receptors that respond to extracellular nucleotide signals. Using the Human Protein Atlas and Gut Cell Survey, we queried single-cell RNA sequencing (RNAseq) data for the P2 purinergic receptors in the small and large intestines. In silico analysis reveals robust mRNA expression of P2RY1, P2RY2, P2RY11, and P2RX4 throughout the gastrointestinal tract. Human intestinal organoids exhibited a similar expression pattern with a prominent expression of P2RY1, P2RY2, and P2RX4, but this purinergic receptor repertoire was not conserved in T84, Caco2, and HT29 intestinal epithelial cell lines. Finally, P2YR1 and P2YR2 agonists elicited robust calcium responses in human intestinal organoids, but calcium responses were weaker or absent in the cell lines. These findings suggest that the gastrointestinal epithelia respond to extracellular purinergic signaling via P2RY1, P2RY2, P2RY11, and P2RX4 receptors and highlight the benefit of using intestinal organoids as a model of intestinal purinergic signaling.NEW & NOTEWORTHY Several studies have revealed crucial roles for P2 receptors during inflammatory and infectious diseases, however, these have largely been demonstrated in immune cells and the enteric nervous system. Although epithelial cells serve as the first barrier against infection and inflammation, the role of purinergic signaling within the gastrointestinal tract remains largely unknown. This work expands our knowledge of purinergic receptor distribution and relative expression along the intestine.

Mathematical Modeling of PI3K/Akt Pathway in Microglia.

The motility of microglia involves intracellular signaling pathways that are predominantly controlled by changes in cytosolic Ca2+ and activation of PI3K/Akt (phosphoinositide-3-kinase/protein kinase B). In this letter, we develop a novel biophysical model for cytosolic Ca2+ activation of the PI3K/Akt pathway in microglia where Ca2+ influx is mediated by both P2Y purinergic receptors (P2YR) and P2X purinergic receptors (P2XR). The model parameters are estimated by employing optimization techniques to fit the model to phosphorylated Akt (pAkt) experimental modeling/in vitro data. The integrated model supports the hypothesis that Ca2+ influx via P2YR and P2XR can explain the experimentally reported biphasic transient responses in measuring pAkt levels. Our predictions reveal new quantitative insights into P2Rs on how they regulate Ca2+ and Akt in terms of physiological interactions and transient responses. It is shown that the upregulation of P2X receptors through a repetitive application of agonist results in a continual increase in the baseline [Ca2+], which causes the biphasic response to become a monophasic response which prolongs elevated levels of pAkt.

María Teresa Miras Portugal: a pioneer in the study of purinoceptors in chromaffin cells.

María Teresa Miras Portugal devoted most of her scientific life to the study of purinergic signalling. In an important part of her work, she used a model system: the chromaffin cells of the adrenal medulla. It was in these cells that she identified diadenosine polyphosphates, from which she proceeded to the study of adrenomedullary purinome: nucleotide synthesis and degradation, adenosine transport, nucleotide uptake into chromaffin granules, exocytotic release of nucleotides and autocrine regulation of chromaffin cell function via purinoceptors. This short review will focus on the current state of knowledge of the purinoceptors of adrenal chromaffin cells, a subject to which María Teresa made seminal contributions and which she continued to study until the end of her scientific life.

Crosstalk between P2Y receptors and cyclooxygenase activity in inflammation and tissue repair.

The role of extracellular nucleotides as modulators of inflammation and cell stress is well established. One of the main actions of these molecules is mediated by the activation of purinergic receptors (P2) of the plasma membrane. P2 receptors can be classified according to two different structural families: P2X ionotropic ion channel receptors, and P2Y metabotropic G protein-coupled receptors. During inflammation, damaged cells release nucleotides and purinergic signaling occurs along the temporal pattern of the synthesis of pro-inflammatory and pro-resolving mediators by myeloid and lymphoid cells. In macrophages under pro-inflammatory conditions, the expression and activity of cyclooxygenase 2 significantly increases and enhances the circulating levels of prostaglandin E2 (PGE2), which exerts its effects both through specific plasma membrane receptors (EP1-EP4) and by activation of intracellular targets. Here we review the mechanisms involved in the crosstalk between PGE2 and P2Y receptors on macrophages, which is dependent on several isoforms of protein kinase C and protein kinase D1. Due to this crosstalk, a P2Y-dependent increase in calcium is blunted by PGE2 whereas, under these conditions, macrophages exhibit reduced migratory capacity along with enhanced phagocytosis, which contributes to the modulation of the inflammatory response and tissue repair.

Lipopolysaccharide-mediated ATP signaling regulates interleukin-6 mRNA expression via the P2-purinoceptor in human dental pulp cells.

Dental pulp cells play a crucial role in maintaining the balance of the pulp tissue. They actively respond to bacterial inflammation by producing proinflammatory cytokines, particularly interleukin-6 (IL-6). While many cell types release adenosine triphosphate (ATP) in response to various stimuli, the mechanisms and significance of ATP release in dental pulp cells under inflammatory conditions are not well understood. This study aimed to investigate ATP release and its relationship with IL-6 during the inflammatory response in immortalized human dental pulp stem cells (hDPSC-K4DT) following lipopolysaccharide (LPS) stimulation. We found that hDPSC-K4DT cells released ATP extracellularly when exposed to LPS concentrations above 10 µg/mL. ATP release was exclusively attenuated by N-ethylmaleimide, whereas other inhibitors, including clodronic acid (a vesicular nucleotide transporter inhibitor), probenecid (a selective pannexin-1 channel inhibitor), meclofenamic acid (a selective connexin 43 inhibitor), suramin (a nonspecific P2 receptor inhibitor), and KN-62 (a specific P2X7 antagonist), did not exhibit any effect. Additionally, LPS increased IL-6 mRNA expression, which was mitigated by the ATPase apyrase enzyme, N-ethylmaleimide, and suramin, but not by KN-62. Moreover, exogenous ATP induced IL-6 mRNA expression, whereas ATPase apyrase, N-ethylmaleimide, and suramin, but not KN-62, diminished ATP-induced IL-6 mRNA expression. Overall, our findings suggest that LPS-induced ATP release stimulates the IL-6 pathway through P2-purinoceptor, indicating that ATP may function as an anti-inflammatory signal, contributing to the maintenance of dental pulp homeostasis.

ATP Increases Ciliary Beat Frequency and Ciliary Bend Angle through Distinct Purinergic Receptors in Bronchial Ciliary Cells Isolated from Mice.

Airway ciliary cells are components of the mucociliary transport system and play an important role in sweeping out small particles, such as bacteria and viruses, towards the oropharynx by the action of beating cilia. Several lines of evidence have shown that the ciliary beat is under the regulation of the purinergic system; however, the subtype of receptor and the intracellular signaling pathways involved in the activation of ciliary movement remain to be elucidated. In addition, although the activity of ciliary movement comprises two parameters, the ciliary beat frequency (CBF) and ciliary bend angle (CBA), few reports have analyzed CBA. In this study, we examined the effects of ATP and other purinergic ligands on both CBF and CBA and demonstrated that the purinergic signaling requirements for CBF and CBA are different, with CBF mediated by P2Y1 receptor activation and CBA mediated by the P2X4 receptor.

ATP-Induced Contractile Response of Esophageal Smooth Muscle in Mice.

The tunica muscularis of mammalian esophagi is composed of striated muscle and smooth muscle. Contraction of the esophageal striated muscle portion is mainly controlled by cholinergic neurons. On the other hand, smooth muscle contraction and relaxation are controlled not only by cholinergic components but also by non-cholinergic components in the esophagus. Adenosine triphosphate (ATP) is known to regulate smooth muscle contraction and relaxation in the gastrointestinal tract via purinergic receptors. However, the precise mechanism of purinergic regulation in the esophagus is still unclear. Therefore, the aim of the present study was to clarify the effects of ATP on the mechanical responses of the esophageal muscle in mice. An isolated segment of the mouse esophagus was placed in a Magnus's tube and longitudinal mechanical responses were recorded. Exogenous application of ATP induced contractile responses in the esophageal preparations. Tetrodotoxin, a blocker of voltage-dependent sodium channels in neurons and striated muscle, did not affect the ATP-induced contraction. The ATP-evoked contraction was blocked by pretreatment with suramin, a purinergic receptor antagonist. RT-PCR revealed the expression of mRNA of purinergic receptor genes in the mouse esophageal tissue. The findings suggest that purinergic signaling might regulate the motor activity of mouse esophageal smooth muscle.

Microglial remodeling of actin network by Tau oligomers, via G protein-coupled purinergic receptor, P2Y12R-driven chemotaxis.

Alzheimer's disease (AD) is associated with age-related neurodegeneration, synaptic deformation and chronic inflammation mediated by microglia and infiltrated macrophages in the brain. Tau oligomers can be released from damaged neurons via various mechanisms such as exosomes, neurotransmitter, membrane leakage etc. Microglia sense the extracellular Tau through several cell-surface receptors and mediate chemotaxis and phagocytosis. The purinergic receptor P2Y12R recently gained interest in neurodegeneration for neuro-glial communication and microglial chemotaxis towards the site of plaque deposition. To understand the effect of extracellular Tau oligomers in microglial migration, the P2Y12R-mediated actin remodeling, reorientation of tubulin network and rate of migration were studied in the presence of ATP. The extracellular Tau species directly interacted with P2Y12R and also induced this purinoceptor expression in microglia. Microglial P2Y12R colocalized with remodeled membrane-associated actin network as a component of migration in response to Tau oligomers. As an inducer of P2Y12R, ATP facilitated the localization of P2Y12R in lamellipodia and filopodia during accelerated microglial migration. The direct interaction of extracellular Tau oligomers with microglial P2Y12R would facilitate the signal transduction in both way, directional chemotaxis and receptor-mediated phagocytosis. These unprecedented findings emphasize that microglia can modulate the membrane-associated actin structure and incorporate P2Y12R to perceive the axis and rate of chemotaxis in Tauopathy.

Age-related changes in P2Y receptor signalling in mouse cochlear supporting cells.

Our sense of hearing depends on the function of a specialised class of sensory cells, the hair cells, which are found in the organ of Corti of the mammalian cochlea. The unique physiological environment in which these cells operate is maintained by a syncitium of non-sensory supporting cells, which are crucial for regulating cochlear physiology and metabolic homeostasis. Despite their importance for cochlear function, the role of these supporting cells in age-related hearing loss, the most common sensory deficit in the elderly, is poorly understood. Here, we investigated the age-related changes in the expression and function of metabotropic purinergic receptors (P2Y1 , P2Y2 and P2Y4 ) in the supporting cells of the cochlear apical coil. Purinergic signalling in supporting cells is crucial during the development of the organ of Corti and purinergic receptors are known to undergo changes in expression during ageing in several tissues. Immunolabelling and Ca2+ imaging experiments revealed a downregulation of P2Y receptor expression and a decrease of purinergic-mediated calcium responses after early postnatal stages in the supporting cells. An upregulation of P2Y receptor expression was observed in the aged cochlea when compared to 1 month-old adults. The aged mice also had significantly larger calcium responses and displayed calcium oscillations during prolonged agonist applications. We conclude that supporting cells in the aged cochlea upregulate P2Y2 and P2Y4 receptors and display purinergic-induced Ca2+ responses that mimic those observed during pre-hearing stages of development, possibly aimed at limiting or preventing further damage to the sensory epithelium. KEY POINTS: Age-related hearing loss is associated with lower hearing sensitivity and decreased ability to understand speech. We investigated age-related changes in the expression and function of metabotropic purinergic (P2Y) receptors in cochlear non-sensory supporting cells of mice displaying early-onset (C57BL/6N) and late-onset (C3H/HeJ) hearing loss. The expression of P2Y1 , P2Y2 and P2Y4 receptors in the supporting cells decreased during cochlear maturation, but that of P2Y2 and P2Y4 was upregulated in the aged cochlea. P2Y2 and P2Y4 receptors were primarily responsible for the ATP-induced Ca2+ responses in the supporting cells. The degree of purinergic expression upregulation in aged supporting cells mirrored hearing loss progression in the different mouse strains. We propose that the upregulation of purinergic-mediated signalling in the aged cochlea is subsequent to age-related changes in the hair cells and may act as a protective mechanism to limit or to avoid further damage to the sensory epithelium.

Oncomodulin regulates spontaneous calcium signalling and maturation of afferent innervation in cochlear outer hair cells.

Cochlear outer hair cells (OHCs) are responsible for the exquisite frequency selectivity and sensitivity of mammalian hearing. During development, the maturation of OHC afferent connectivity is refined by coordinated spontaneous Ca2+ activity in both sensory and non-sensory cells. Calcium signalling in neonatal OHCs can be modulated by oncomodulin (OCM, ß-parvalbumin), an EF-hand calcium-binding protein. Here, we investigated whether OCM regulates OHC spontaneous Ca2+ activity and afferent connectivity during development. Using a genetically encoded Ca2+ sensor (GCaMP6s) expressed in OHCs in wild-type (Ocm+/+ ) and Ocm knockout (Ocm-/- ) littermates, we found increased spontaneous Ca2+ activity and upregulation of purinergic receptors in OHCs from Ocm-/- cochlea immediately following birth. The afferent synaptic maturation of OHCs was delayed in the absence of OCM, leading to an increased number of ribbon synapses and afferent fibres on Ocm-/- OHCs before hearing onset. We propose that OCM regulates the spontaneous Ca2+ signalling in the developing cochlea and the maturation of OHC afferent innervation. KEY POINTS: Cochlear outer hair cells (OHCs) exhibit spontaneous Ca2+ activity during a narrow period of neonatal development. OHC afferent maturation and connectivity requires spontaneous Ca2+ activity. Oncomodulin (OCM, ß-parvalbumin), an EF-hand calcium-binding protein, modulates Ca2+ signals in immature OHCs. Using transgenic mice that endogenously expressed a Ca2+ sensor, GCaMP6s, we found increased spontaneous Ca2+ activity and upregulated purinergic receptors in Ocm-/- OHCs. The maturation of afferent synapses in Ocm-/- OHCs was also delayed, leading to an upregulation of ribbon synapses and afferent fibres in Ocm-/- OHCs before hearing onset. We propose that OCM plays an important role in modulating Ca2+ activity, expression of Ca2+ channels and afferent innervation in developing OHCs.

Purinergic receptors in cognitive disturbances.

Purinergic receptors (Rs) of the ATP/ADP, UTP/UDP (P2X, P2Y) and adenosine (A1, A2A)-sensitive classes broadly interfere with cognitive processes both under quasi normal and disease conditions. During neurodegenerative illnesses, high concentrations of ATP are released from the damaged neuronal and non-neuronal cells of the brain; then, this ATP is enzymatically degraded to adenosine. Thus, the primary injury in neurodegenerative diseases appears to be caused by various protein aggregates on which a superimposed damage mediated by especially P2X7 and A2AR activation develops; this can be efficiently prevented by small molecular antagonists in animal models of the above diseases, or are mitigated in the respective knockout mice. Dementia is a leading symptom in Alzheimer's disease (AD), and accompanies Parkinson's disease (PD) and Huntington's disease (HD), especially in the advanced states of these illnesses. Animal experimentation suggests that P2X7 and A2ARs are also involved in a number of psychiatric diseases, such as major depressive disorder (MDD), obsessive compulsive behavior, and attention deficit hyperactivity disorder. In conclusion, small molecular antagonists of purinergic receptors are expected to supply us in the future with pharmaceuticals which are able to combat in a range of neurological/psychiatric diseases the accompanying cognitive deterioration.

P2Y2 purinergic receptor gene deletion protects mice from bacterial endotoxin and sepsis-associated liver injury and mortality.

The liver plays a significant role in regulating a wide range of metabolic, homeostatic, and host-defense functions. However, the impact of liver injury on the host's ability to control bacteremia and morbidity in sepsis is not well understood. Leukocyte recruitment and activation lead to cytokine and chemokine release, which, in turn, trigger hepatocellular injury and elevate nucleotide levels in the extracellular milieu. P2Y2 purinergic receptors, G protein-coupled and activated by extracellular ATP/UTP, are expressed at the cell surface of hepatocytes and nonparenchymal cells. We sought to determine whether P2Y2 purinergic receptor function is necessary for the maladaptive host response to bacterial infection and endotoxin-mediated inflammatory liver injury and mortality in mice. We report that P2Y2 purinergic receptor knockout mice (P2Y2-/-) had attenuated inflammation and liver injury, with improved survival in response to LPS/galactosamine (LPS/GalN; inflammatory liver injury) and cecal ligation and puncture (CLP; polymicrobial sepsis). P2Y2-/- livers had attenuated c-Jun NH2-terminal kinase activation, matrix metallopeptidase-9 expression, and hepatocyte apoptosis in response to LPS/GalN and attenuated inducible nitric oxide synthase and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 protein expression in response to CLP. Implicating liver injury in the disruption of amino acid homeostasis, CLP led to lower serum arginine and higher bacterial load and morbidity in the WT mice, whereas serum arginine levels were comparable to sham-operated controls in P2Y2-/- mice, which had attenuated bacteremia and improved survival. Collectively, our studies highlight the pathophysiological relevance of P2Y2 purinergic receptor function in inflammatory liver injury and dysregulation of systemic amino acid homeostasis with implications for sepsis-associated immune dysfunction and morbidity in mice.NEW & NOTEWORTHY Our studies provide experimental evidence for P2Y2 purinergic receptor-mediated potentiation of inflammatory liver injury, morbidity, and mortality, in two well-established animal models of inflammatory liver injury. Our findings highlight the potential to target P2Y2 purinergic signaling to attenuate the induction of "cytokine storm" and prevent its deleterious consequences on liver function, systemic amino acid homeostasis, host response to bacterial infection, and sepsis-associated morbidity and mortality.

Purinergic receptor P2X7 activates NOX2/JNK signaling to participate in granulosa cell inflammation and apoptosis in polycystic ovary syndrome.

Increasing evidence shows that polycystic ovary syndrome (PCOS) is often accompanied by an inflammatory response, hence, appropriately managing granulosa cell inflammation is critical to regaining ovarian function in PCOS. In this study, the differential levels of purinergic receptor P2X7 between the control and PCOS samples in the dataset GSE34526 were assessed, then PCOS mouse models were established. Following evaluating the fluctuations in hormone levels, inflammatory cytokines, and P2X7, mice received treatment with the P2X7 antagonist A740003. Its effects on hormones, inflammation, apoptosis, and NOX2 signaling in mice were examined. Afterward, primary mouse granulosa cells were isolated, and the mediating role of NOX2 signaling in the P2X7 regulatory pathway was confirmed by transfection of NOX2 overexpression plasmids. The results demonstrated that P2X7 was significantly elevated in the PCOS samples in the dataset. Compared with the control group, PCOS mice had significant differences in the follicle-stimulating hormone, luteinizing hormone, testosterone, anti-Müllerian hormone, inflammatory factors, and P2X7. Treatment with A740003 partially restored these parameter levels, including NOX2 signaling. Based on in vitro experiments on primary mouse granulosa cells, the above findings were re-verified, and the overexpression of NOX2 could reverse the regulatory function of P2X7. The present study highlights that P2X7 level increases in PCOS, and inhibition of P2X7 can reduce disease symptoms. It is involved in inflammation and apoptosis in granulosa cells through NOX2/JNK signaling.

A systematic review and in silico study of potential genetic markers implicated in cases of overactive bladder.

OBJECTIVE: The contribution of genetic factors to the presence of an overactive bladder is recognized. This study aimed to (1) assemble and synthesize available data from studies assessing differential gene expression in patients with overactive bladder vs controls without overactive bladder and (2) determine possible correlations and functional pathways between genes. DATA SOURCES: We searched PubMed, Ovid or Medline, and Wiley Cochrane Central Register of Controlled Trials databases between January 1, 2000, and December 15, 2021. STUDY ELIGIBILITY CRITERIA: Studies were included if gene expression was detected and quantified using molecular approaches performed on human bladder tissue specimens directly and excluded if the gene expression analysis was carried out from blood and urine specimens alone. METHODS: A systematic review was completed to identify publications that reported differently expressed gene candidates among patients with overactive bladder vs healthy individuals. Gene networking connections and pathway analysis were performed employing Metascape software, where inputs were identified from our systematic review of differentially expressed genes in overactive bladder. RESULTS: A total of 9 studies were included in the final analysis and 11 genes were identified as being up-regulated (purinergic receptor P2X 2 [P2RX2], smoothelin [SMTN], growth-associated protein 43 [GAP43], transient receptor potential cation channel subfamily M member 8 [TRPM8], cadherin 11 [CDH1], gap junction protein gamma 1 [GJC1], cholinergic receptor muscarinic 2 [CHRM2], cholinergic receptor muscarinic 3 [CHRM3], and transient receptor potential cation channel subfamily V member 4 [TRPV4]) or down-regulated (purinergic receptor P2X 2 [P2RX3] and purinergic receptor P2X 5 [P2RX5]) in patients with overactive bladder. Gene network analysis showed that genes are involved in chemical synaptic transmission, smooth muscle contraction, blood circulation, and response to temperature stimulus. Network analysis demonstrated a significant genetic interaction between TRPV4, TRPM8, P2RX3, and PR2X2 genes. CONCLUSION: Outcomes of this systematic review highlighted potential biomarkers for treatment efficacy and have laid the groundwork for developing future gene therapies for overactive bladder in clinical settings.

Purinergic receptors mediate endothelial dysfunction and participate in atherosclerosis.

Atherosclerosis is the main pathological basis of cardiovascular disease and involves damage to vascular endothelial cells (ECs) that results in endothelial dysfunction (ED). The vascular endothelium is the key to maintaining blood vessel health and homeostasis. ED is a complex pathological process involving inflammation, shear stress, vascular tone, adhesion of leukocytes to ECs, and platelet aggregation. The activation of P2X4, P2X7, and P2Y2 receptors regulates vascular tone in response to shear stress, while activation of the A2A, P2X4, P2X7, P2Y1, P2Y2, P2Y6, and P2Y12 receptors promotes the secretion of inflammatory cytokines. Finally, P2X1, P2Y1, and P2Y12 receptor activation regulates platelet activity. These purinergic receptors mediate ED and participate in atherosclerosis. In short, P2X4, P2X7, P2Y1, and P2Y12 receptors are potential therapeutic targets for atherosclerosis.