Ginsenosides enhance P2X7-dependent cytokine secretion from LPS-primed rodent macrophages.

The activation of P2X7 is a well-known stimulus for the NLRP3-caspase 1 inflammasome and subsequent rapid IL-1ß secretion from monocytes and macrophages. Here we show that positive allosteric modulators of P2X7, ginsenosides, can enhance the release of three important cytokines, IL-1ß, IL-6 and TNF-α from LPS-primed rodent macrophages using the J774 mouse macrophage cell line and primary rat peritoneal macrophages. We compared the immediate P2X7 responses in un-primed and LPS-primed macrophages and found no difference in calcium response amplitude or kinetics. These results suggest that under inflammatory conditions positive allosteric modulators are capable of increasing cytokine secretion at lower concentrations of ATP, thus boosting the initial pro-inflammatory signal. This may be important in the control of intracellular infections.

Screening herbal and natural product libraries to aid discovery of novel allosteric modulators of human P2X7.

 P2X7 is an emerging therapeutic target for several disorders and diseases due to its role in inflammatory signalling. This study aimed to exploit the unique chemical libraries of plants used in traditional medicinal practices to discover novel allosteric modulators from natural sources. We identified several compounds from the NCI Natural Product library as P2X7 antagonists including confertifolin and digallic acid (IC50 values 3.86 µM and 4.05 µM). We also identified scopafungin as a novel positive allosteric modulator of hP2X7. Screening a traditional medicinal plant extract library revealed 39 plant species with inhibitory action at hP2X7 and 17 plant species with positive allosteric modulator activity. Using computational docking to filter identified components from these plant species and determine potential antagonists, we investigated nine purified chemicals including flavonoids quercetin, kaempferol, ECG, and EGCG. These were shown to inhibit ATP-induced YO-PRO-1 uptake into HEK-hP2X7 cells; however, we also showed that all four flavonoids demonstrated significant assay interference using a cell-free DNA YO-PRO-1 fluorescence test. One plant extract, Dioscorea nipponica, demonstrating positive modulator activity was investigated, and dioscin was identified as a glycoside with PAM activity in ATP-induced YO-PRO-1 uptake assay and whole-cell patch-clamp recordings. However, membrane permeabilisation was observed following application > 10 min limiting the use of dioscin as a pharmacological tool. This work describes a useful workflow with multiple assays for the identification of novel allosteric modulators for human P2X7.

Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors.

P2X7 is an important ligand-gated ion channel expressed in multiple immune cell populations. This study aimed to investigate the chemical requirements of triterpenoid glycosides within a new binding pocket to characterize the structure-activity relationship. A set of glycosides were screened for positive modulator activity at human P2X7 using a YO-PRO-1 dye uptake assay in HEK-293 cells stably expressing the wild-type human P2X7 variant (HEK-hP2X7 cells). The highest positive modulator activity was with ginsenoside-compound K (CK), containing a monosaccharide (glucose) attached at carbon-20. Ginsenoside-20(S)-Rg3, containing a disaccharide group (glucose-glucose) at carbon-3, displayed positive modulator activity with a reduced EC50 for ATP and increased maximal response at human P2X7. The epimer 20(R)-Rg3 was inactive. A similar stereo-specific pattern was observed for 20(S)-Rh2. Ginsenoside-F1, highly similar to ginsenoside-CK but containing a single additional hydroxyl group, was also inactive at P2X7. Computational docking suggests hydrophobic residues in the pocket are involved in steric discrimination between triterpenoids, whereas the position and identity of the carbohydrate group are important for positive modulator activity at human P2X7. Ginsenosides containing monosaccharide attachments perform better than di- or trisaccharide glycosides. Additional modifications to the triterpenoid scaffold at carbon-6 are not tolerated. Gypenosides from plant sources other than Panax ginseng (gypenoside XVII, gypenoside XLIX, stevenleaf) can also act as positive allosteric modulators of P2X7. We also investigated the effect of positive allosteric modulators on endogenous P2X7 in THP-1 monocytes and confirmed our findings in a calcium response assay. A cell viability assay showed potentiation of ATP-induced cell death with ginsenoside-CK in THP-1 and HEK-hP2X7 cells. SIGNIFICANCE STATEMENT: Ginsenosides are active as positive allosteric modulators at P2X7, and this study determines the chemical features important for mediating this effect. The position and identity of the sugar group is important for activity, as is the position of a number of hydroxyl groups on the triterpenoid scaffold. Diastereomers of ginsenoside-Rg3 and ginsenoside-Rh2 demonstrate the importance of the location of hydroxyl groups relative to the hydrophobic face of the predicted binding pocket.

A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function.

P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele-specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C-terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP-binding site. P2X7L-transfected HEK 293 cells have phagocytic but not channel, pore, or membrane-blebbing function, and double-transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP-binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.

Ginsenosides Act As Positive Modulators of P2X4 Receptors.

We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP- or ATP+ ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain.

Development of High-Throughput Fluorescent-Based Screens to Accelerate Discovery of P2X Inhibitors from Animal Venoms.

Animal venoms can play an important role in drug discovery, as they are a rich source of evolutionarily tuned compounds that target a variety of ion channels and receptors. To date, there are six FDA-approved drugs derived from animal venoms, with recent work using high-throughput platforms providing a variety of new therapeutic candidates. However, high-throughput methods for screening animal venoms against purinoceptors, one of the oldest signaling receptor families, have not been reported. Here, we describe a variety of quantitative fluorescent-based high-throughput screening (HTS) cell-based assays for screening animal venoms against ligand-gated P2X receptors. A diverse selection of 180 venoms from arachnids, centipedes, hymenopterans, and cone snails were screened, analyzed, and validated, both analytically and pharmacologically. Using this approach, we performed screens against human P2X3, P2X4, and P2X7 using three different fluorescent-based dyes on stable cell lines and isolated the active venom components. Our HTS assays are performed in 96-well format and allow simultaneous screening of multiple venoms on multiple targets, improving testing characteristics while minimizing costs, specimen material, and testing time. Moreover, utilizing our assays and applying them to the other natural product libraries, rather than venoms, might yield other novel natural products that modulate P2X activity.

Correlation between thyroidal and peripheral blood total T cells, CD8+ T cells, and CD8+ T- regulatory cells and T-cell reactivity to calsequestrin and collagen XIII in patients with Graves' ophthalmopathy.

Purpose/aim of the study: Graves' ophthalmopathy (GO) is closely related to the thyroid autoimmune disorder Graves' disease. Previous studies have suggested roles for thyroidal CD8+ T cells and autoimmunity against calsequestrin-1 (CASQ)-1 in the link between thyroidal and orbital autoimmune reactions in GO. A role for autoimmunity against CollXIII has also been suggested. In this study, we aimed to investigate correlations between some thyroidal and peripheral blood T-cell subsets and thyroidal T-cell reactivity against CASQ1 and CollXIII in patients with GO. MATERIALS AND METHODS: Fresh thyroid tissues were processed by enzyme digestion and density gradient to isolate mononuclear cells (MNCs). Peripheral blood MNCs were also isolated using density gradient. Flow-cytometric analysis was used to identify the various T-cell subsets. T -cell reactivity to CASQ1 and CollXIII was measured by a 5-day culture of the MNCs and BrdU uptake method. RESULTS: We found a positive correlation between thyroidal CD8+  T cells and CD8+ T-regulatory (T-reg) cells in patients with GO. Thyroidal T cells from two out of the three patients with GO tested (66.7%) showed a positive response to CASQ1, while thyroidal T cells from none of the six Graves' Disease patients without ophthalmopathy (GD) tested showed a positive response to this antigen. Thyroidal T cells from these patient groups however, showed no significant differences in their response to CollXIII. CONCLUSIONS: Our observations provide further evidence for a possible role of thyroidal CD8+ T cells, CD8+ T-reg cells and the autoantigen CASQ1 in the link between thyroidal and orbital autoimmune reactions of GO.

The P2X7 receptor channel: recent developments and the use of P2X7 antagonists in models of disease.

The P2X7 receptor is a trimeric ATP-gated cation channel found predominantly, but not exclusively, on immune cells. P2X7 activation results in a number of downstream events, including the release of proinflammatory mediators and cell death and proliferation. As such, P2X7 plays important roles in various inflammatory, immune, neurologic and musculoskeletal disorders. This review focuses on the use of P2X7 antagonists in rodent models of neurologic disease and injury, inflammation, and musculoskeletal and other disorders. The cloning and characterization of human, rat, mouse, guinea pig, dog, and Rhesus macaque P2X7, as well as recent observations regarding the gating and permeability of P2X7, are discussed. Furthermore, this review discusses polymorphic and splice variants of P2X7, as well as the generation and use of P2X7 knockout mice. Recent evidence for emerging signaling pathways downstream of P2X7 activation and the growing list of negative and positive modulators of P2X7 activation and expression are also described. In addition, the use of P2X7 antagonists in numerous rodent models of disease is extensively summarized. Finally, the use of P2X7 antagonists in clinical trials in humans and future directions exploring P2X7 as a therapeutic target are described.

Paroxetine suppresses recombinant human P2X7 responses.

P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1ß (IL-1ß) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC(50) of 24 µM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC(50) of 6.4 µM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1ß secretion from lipopolysaccharide (LPS)-primed human CD14(+) monocytes was suppressed with trifluoperazine and paroxetine.

R270C polymorphism leads to loss of function of the canine P2X7 receptor.

The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.

Neonatal overfeeding alters hypothalamic microglial profiles and central responses to immune challenge long-term.

The early life period is one of significant vulnerability to programming effects from the environment. Given the sensitivity of microglial cells to early life programming and to adult diet, we hypothesized overfeeding during the neonatal period would acutely alter microglial profiles within the developing brain, predisposing the individual to a lasting central pro-inflammatory profile that contributes to overactive immune responses long-term. We tested this idea by manipulating litter sizes in which Wistar rat pups were raised, so the pups were suckled in litters of 4 (neonatally overfed) or 12 (control). This manipulation induces obesity and susceptibility to lipopolysaccharide (LPS) long-term. We then examined microglial and central pro-inflammatory profiles during development and in adulthood as well as susceptibility to neuroimmune challenge with LPS. Neonatally overfed rats have evidence of microgliosis in the paraventricular nucleus of the hypothalamus (PVN) as early as postnatal day 14. They also show changes in hypothalamic gene expression at this time, with suppressed hypothalamic interleukin 1ß mRNA. These effects persist into adulthood, with basal PVN microgliosis and increased hypothalamic toll-like receptor 4, nuclear factor κB, and interleukin 6 gene expression. These neonatally overfed rats also have dramatically exacerbated microglial activation in the PVN 24h after an adult LPS challenge, coupled with changes in inflammatory gene expression. Thus, it appears neonatal overfeeding sensitizes PVN microglia, contributing to a basal pro-inflammatory profile and an altered response to a neuroimmune challenge throughout life. It remains to be seen if these effects can be reversed with early interventions.

Methods for studying P2X4 receptor ion channels in immune cells.

The P2X4 receptor is a trimeric ligand-gated ion channel activated by adenosine 5'-triphosphate (ATP). P2X4 is present in immune cells with emerging roles in inflammation and immunity, and related disorders. This review aims to provide an overview of the methods commonly used to study P2X4 in immune cells, focusing on those methods used to assess P2RX4 gene expression, the presence of the P2X4 protein, and P2X4 ion channel activity in these cells from humans, dogs, mice and rats. P2RX4 gene expression in immune cells is commonly assessed using semi-quantitative and quantitative reverse-transcriptase-PCR. The presence of P2X4 protein in immune cells is mainly assessed using anti-P2X4 polyclonal antibodies with immunoblotting or immunochemistry, but the use of these antibodies, as well as monoclonal antibodies and nanobodies to detect P2X4 with flow cytometry is increasing. Notably, use of an anti-P2X4 monoclonal antibody and flow cytometry has revealed that P2X4 is present on immune cells with a rank order of expression in eosinophils, then neutrophils and monocytes, then basophils and B cells, and finally T cells. P2X4 ion channel activity has been assessed mainly by Ca2+ flux assays using the cell permeable Ca2+-sensitive dyes Fura-2 and Fluo-4 with fluorescence microscopy, spectrophotometry, or flow cytometry. However, other methods including electrophysiology, and fluorescence assays measuring Na+ flux (using sodium green tetra-acetate) and dye uptake (using YO-PRO-12+) have been applied. Collectively, these methods have demonstrated the presence of functional P2X4 in monocytes and macrophages, microglia, eosinophils, mast cells and CD4+ T cells, with other evidence suggestive of functional P2X4 in dendritic cells, neutrophils, B cells and CD8+ T cells.

Anti-Inflammatory Activities of Yataprasen Thai Traditional Formulary and Its Active Compounds, Beta-Amyrin and Stigmasterol, in RAW264.7 and THP-1 Cells.

Yataprasen (YTPS) remedy formulary, a national Thai traditional medicine formulary, comprises 13 herbal plants. It has been extensively prescribed to relieve osteoarthritis and musculoskeletal pain in the Thai traditional medicine healthcare system. The aim of this study was to investigate the antioxidant and anti-inflammatory properties of the bioactive compounds (ß-amyrin and stigmasterol) of YTPS remedy formulary ethanolic extract, along with its composition. The YTPS formulary extract contains 70.30 nM of ß-amyrin and 605.76 nM of stigmasterol. The YTPS formulary extract exhibited ABTS and DPPH free radical scavenging activity, with IC50 values of 144.50 ± 2.82 and 31.85 ± 0.18 µg/mL, respectively. The ethanolic extract of YTPS at a concentration of 1000 µg/mL showed a significant (p < 0.01) anti-inflammatory effect, mainly by reducing IL-6 and TNF-α release in response to LPS. NO production was prominently lowered by 50% at 24.76 ± 1.48 µg/mL, 55.52 ± 24.40 µM, and more than 570 µM of YTPS formulary extract, ß-amyrin, and stigmasterol, respectively. Major components of YTPS, ß-amyrin, and stigmasterol exerted significant anti-inflammatory effects by inhibiting LPS-induced IL-1ß, IL-6, TNF-α secretion in THP-1 cells. Our findings suggest that the ethanolic extract from YTPS holds promise as an alternative topical treatment for osteoarthritis and inflammatory disorders, potentially with fewer side effects than non-steroidal anti-inflammatory medications (NSAIDs).

Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin.

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

P2X receptors: Insights from the study of the domestic dog.

Fifty years ago, the late Geoffrey Burnstock described the concept of purinergic nerves and transmission bringing into existence the broader concepts of purinergic signaling including P2X receptors. These receptors are trimeric ligand-gated cation channels activated by extracellular adenosine 5'-triphosphate (ATP). P2X receptors have important roles in health and disease and continue to gain interest as potential therapeutic targets in inflammatory, neurological, cardiovascular and many other disorders including cancer. Current understanding of P2X receptors has largely arisen from the study of these receptors in humans and rodents, but additional insights have been obtained from the study of P2X receptors in the domestic dog, Canis familiaris. This review article will briefly introduce purinergic signaling and P2X receptors, before detailing the pharmacological profiles of the two recombinant canine P2X receptors studied to date, P2X7 and P2X4. The article will then describe the current state of knowledge concerning the distribution and function of the P2X receptor family in dogs. The article will also discuss the characterization of single nucleotide polymorphisms in the canine P2RX7 gene, and contrast this variation to the canine P2RX4 gene, which is largely conserved between dogs. Finally, this article will outline published examples of the use of dogs to study the pharmacokinetics of P2X7 and P2X3 antagonists, and how they have contributed to the preclinical testing of antagonists to human P2X7, CE-224,535, and human P2X3, Gefapixant (AF-219, MK-7264) and Eliapixant (BAY, 1817080), with Gefapixant gaining recent approval for use in the treatment of refractory chronic cough in humans. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'.

P2X receptor antagonists and their potential as therapeutics: a patent review (2010-2021).

INTRODUCTION: Purinergic receptors play a critical role in neurotransmission, and modulation of complex physiological functions and thus have implications in numerous disease states. The past decade has seen substantial progress in the design of novel chemical compounds that act on the P2X class of receptors and warrants an updated review of this field. AREAS COVERED: This review provides a summary of the patent literature describing the discovery and clinical uses of P2X receptor antagonists published between 2010 and September 2021. The reader will gain information on structural claims, representative structures, and biological data of recently reported P2X antagonists. EXPERT OPINION: Despite continual advancement in both crystallography and chemical biology strengthening our understanding of purinergic signalling, there remains an absence of clinically approved chemotypes. A testament to both the therapeutic potential and academic perseverance in purinergic research is the multitude of research initiatives that maintain active P2X receptor programs that have spanned decades. Very recently, the FDA declined Merck Pharmaceuticals application for Gefapixant, a P2X3 selective inhibitor as a treatment for chronic cough, requesting additional data. This unfortunate setback will ultimately be insignificant considering the long history of P2X investigation and the preclinical and clinical development that will undoubtedly occur over the next decade.

Two haplotypes of the P2X(7) receptor containing the Ala-348 to Thr polymorphism exhibit a gain-of-function effect and enhanced interleukin-1beta secretion.

The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.

Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation.

ATP-gated P2X(4) receptors (P2X(4)R) in macrophages and microglia have been implicated in neuropathic and inflammatory pain by currently unidentified mechanisms. P2X(4)R are found predominantly in intracellular lysosomal compartments but can be rapidly trafficked to the surface membrane by procedures that induce endolysosomal secretion. We studied total and surface membrane P2X(4)R protein expression by Western blot and biotinylation assays and functional expression by whole-cell patch clamp assays in human and rat alveolar macrophages in response to phagocytosis of zymosan and opsonized zymosan bioparticles and to classical and alternative macrophage activation. Unstimulated macrophages showed high total protein expression but very low functional expression. Phagocytosis rapidly (within 4 h) increased functional P2X(4)R expression by 2- to 7-fold as did chloroquine, an agent known to induce lysosomal secretion. In contrast, classical activation of macrophage for 48 h with IFN-gamma and TNF-alpha or IFN-gamma and LPS reduced surface and functional P2X(4)R expression by 3-fold without altering total P2X(4)R protein levels. Alternative activation with IL-4 or IL-13 did not alter total, surface or functional expression of P2X(4)R. This is the first study of the regulation of P2X(4)R in macrophages by physiological stimuli and presents a picture whereby P2X(4)R become functional in response to initial phagocytic stimuli but return to a non-functional state during sustained activation by classical macrophage activation.

Recording P2X Receptors Using Whole-Cell Patch Clamp from Native Monocytes and Macrophages.

Investigating ion channels in their native cell type is important when striving to understand their regulation and function, but this comes with added complexities due to the plethora of channels and receptors present. Details of recording ATP-gated ion channels in macrophages are presented together with information on how to prepare the primary cells for electrophysiological analysis.