Biochemistry, Genetics and Molecular Biology › Physiology

Adenosine and Purinergic Signaling

Works Count: 43298

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Description

This cluster of papers focuses on the molecular physiology and pharmacology of purinergic signalling, including the role of adenosine receptors, P2X receptors, and nucleotide signalling in immune suppression, inflammation, neuroprotection, and the tumor microenvironment. It explores the potential therapeutic targets related to purinergic signalling.

Keywords

Purinergic Signalling; Adenosine Receptors; P2X Receptors; Nucleotide Signalling; Immune Suppression; Inflammation; Neuroprotection; Extracellular ATP; Tumor Microenvironment; Therapeutic Targets

Cardiac nucleotides in hypoxia: possible role in regulation of coronary blood flow

1963-02-01

Robert M. Berne

Experiments were performed on isolated cat hearts perfused with Tyrode's solution and intact hearts of open-chest dogs. Cardiac hypoxia resulted in a decrease in coronary vascular resistance and a release … Experiments were performed on isolated cat hearts perfused with Tyrode's solution and intact hearts of open-chest dogs. Cardiac hypoxia resulted in a decrease in coronary vascular resistance and a release of significant amounts of inosine and hypoxanthine from the myocardium. From 3 to 27 times more inosine and hypoxanthine were released from the heart during myocardial hypoxia than were required to double the coronary blood flow when infused as adenosine into the left coronary artery. Based on the assumption that with hypoxia the nucleotide derivatives leave the myocardial cell as adenosine, an hypothesis is proposed for the metabolic regulation of coronary blood flow.

Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor

1997-08-14

Catherine Ledent , Jean‐Marie Vaugeois , Serge N. Schiffmann +7 more

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Intracellular ATP levels determine cell death fate by apoptosis or necrosis.

1997-05-15

Yutaka Eguchi , S Shimizu , Yoshihide Tsujimoto

Although apoptosis and necrosis are morphologically distinct manifestations of cell death, apoptosis and some necroses share common features in the death signaling pathway involving functional steps of death-driving interleukin 1beta-converting … Although apoptosis and necrosis are morphologically distinct manifestations of cell death, apoptosis and some necroses share common features in the death signaling pathway involving functional steps of death-driving interleukin 1beta-converting enzyme family proteases and anti-cell death protein Bcl-2. One evident physiological difference in cells undergoing apoptosis versus necrosis is in intracellular levels of ATP. In this study, we specifically addressed the question of whether apoptosis depends on intracellular ATP levels, since longer incubation under ATP-depleting conditions results in necrotic cell death. Incubation of cells in glucose-free medium with an inhibitor of mitochondrial F0F1-ATPases reduces intracellular ATP levels and completely blocks Fas/Apo-1-stimulated apoptosis. ATP supplied through glycolysis or oxidative phosphorylation restores the apoptotic cell death pathway. ATP depletion also leads to a block in Fas-induced activation of CPP32/Yama(-like) proteases, and when ATP is depleted after the activation of the proteases, subsequent apoptosis is significantly blocked. Thus, ATP-dependent steps exist both upstream and downstream of CPP32/Yama(-like) protease activation in apoptotic signal transduction. Treatment with the calcium ionophore induces apoptosis under ATP-supplying conditions but induces necrotic cell death under ATP-depleting conditions, indicating that ATP levels are a determinant of manifestation of cell death.

Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels

1996-04-15

Ginetta Collo , North Ra , Eric Kawashima +4 more

Two new P2X receptor cDNAs (P2X5 and P2X6) were isolated and expressed. All six proteins are 36-48 percent identical and seem to have two transmembrane segments with a large extracellular … Two new P2X receptor cDNAs (P2X5 and P2X6) were isolated and expressed. All six proteins are 36-48 percent identical and seem to have two transmembrane segments with a large extracellular loop. Functionally, P2X5 and P2X6 receptors most resemble P2X2 and P2X4; they desensitize only slowly and do not respond to alpha beta methylene-ATP. P2X6 receptors, like P2X4, receptors, are not blocked by the antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. P2X6 and P2X5 receptors express at lower levels than P2X1-P2X4 receptors do, perhaps indicating that they do not normally form homomultimeric channels. P2X6 and P2X4 are the receptors expressed most heavily in brain, where their RNAs have a widespread and extensively overlapping distribution. The spinal cord expresses all receptors except P2X3. P2X2, P2X4, and P2X6, are the most abundant in the dorsal horn. Sensory neurons of the trigeminal, dorsal root, and nodose ganglia express all six RNAs; P2X3 is found only there. The functional properties and tissue distribution of these six P2X receptors indicate new roles for ATP-gated ion channels.

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Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides

1993-09-01

George Dubyak , Chakib El-Moatassim

Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic … Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.

The P2X7 Receptor: A Key Player in IL-1 Processing and Release

2006-04-01

Davide Ferrari , Cinzia Pizzirani , Elena Adinolfi +5 more

Abstract Human IL-1 family proteins are key mediators of the host response to infections, injury, and immunologic challenges. The mechanism by which IL-1 activates proinflammatory responses in target cells, and … Abstract Human IL-1 family proteins are key mediators of the host response to infections, injury, and immunologic challenges. The mechanism by which IL-1 activates proinflammatory responses in target cells, and the plasma membrane receptors involved, is fairly well known. This has led to the development of innovative drugs that block IL-1 downstream to its synthesis and secretion. On the contrary, the mechanism of IL-1 and other IL-1 family members (e.g., IL-18) maturation and release is incompletely understood. Accruing evidence points to a plasma membrane receptor for extracellular ATP, the P2X7 receptor, as a key player in both processes. A deeper understanding of the mechanism by which the P2X7 receptor triggers IL-1 maturation and exteriorization may suggest novel avenues for the treatment of inflammatory diseases and provide a deeper insight in the fundamental mechanism of protease activation and cellular export of proteins lacking a leader sequence.

Nomenclature and classification of purinoceptors.

1994-06-01

Bertil B. Fredholm , Maria P. Abbracchio , Geoffrey Burnstock +5 more

“Receptors recognize a distinct chemical entity and translate information from that entity into a form that the cell can read to alter its state” (Kenakin et al., 1992). Even though … “Receptors recognize a distinct chemical entity and translate information from that entity into a form that the cell can read to alter its state” (Kenakin et al., 1992). Even though the receptors are often pharmacologically defined on the basis of synthetic compounds, they are assumed to have developed to respond to endogenous molecules. Therefore, receptors are generally named on the basis of their natural ligands. Hence, it is appropriate to very briefly summarize the evidence that purine nucleotides and nucleosides are natural ligands for a wide class of receptors. In a seminal paper, Drury and Szent-Gyorgyi (1929) showed that adenosine exerted a large number of biological effects, including bradycardia and vasodilation. A wider interest in the role of adenosine followed from the demonstration in 1963 that adenosine can be produced by the hypoxic heart. Two groups independently formulated the hypothesis that adenosine may be involved in the metabolic regulation of coronary blood flow (Berne, 1963; Gerlach et al., 1963). The observation by de Gubareff and Sleator (1965) that the actions of adenosine in heart tissue could be blocked by caffeine suggested the existence of an adenosine receptor. The potent cardiovascular effects of adenosine led to an interest in the synthesis of new adenosine analogs, and careful dose-response studies with a number of these drugs (Cobbin et al., 1974) strongly suggested the presence of a receptor for adenosine-like compounds. Sattin and Rall (1970) reported that adenosine increased cyclic AMP accumulation in slices of rodent brain and that this adenosine-induced second-messenger response was blocked by methylxanthines. Their findings suggested that adenosine receptors exist in the central nervous system. The essentially simultaneous findings by Mcilwain (1972), that such brain slices actually elaborate adenosine in concentrations that would be sufficient to elevate cyclic AMP, provided support that these putative receptors were physiologically occupied by adenosine. Thus, in the 1970s there was good evidence that there were receptors for adenosine at which methylxanthines acted as antagonists. Biochemical evidence for the existence of multiple adenosine receptors was subsequently provided by the demonstration that adenosine analogs increased cyclic AMP production in some preparations and decreased it in others. Because the relative agonist potency for a variety of adenosine analogs was different for these two types of effects, the presence of two classes of receptors, called A1 and A2 (van Calker et al., 1979) or Ri and Ra (Londos et al., 1980), was proposed. The A1/A2 nomenclature is now generally used. The presence of receptors for ADP, particularly on blood platelets, was also recognized several decades ago. Studies of the factors in blood that induce platelet aggregation led to the identification of ADP as an active component present in red blood cell extracts (Gaarder et al., 1961). The evidence that ADP and adenosine (presumably A2) receptors exist on platelets was summarized by Haslam and Cusack (1981). Four decades ago, ATP was shown to produce important cardiovascular effects (Green and Stoner, 1950) and to be released from sensory nerves (Holton and Holton, 1954; Holton, 1959), hinting at a role in neural transmission. In his landmark review of purinergic nerves, Burnstock (1972) postulated the existence of specific ATP receptors. Although evidence in support of this idea was not overwhelming at the time, many subsequent studies have supported the existence of receptors for extracellular ATP (Burnstock and Brown, 1981; Gordon, 1986; O’Connor et al., 1991). Similarly, the evidence is now compelling that ATP plays important physiological and/ or pathophysiological roles in a variety of biological systems, including that of a neurotransmitter in peripheral and central neurons. Finally, diadenosinetetraphosphate is a dinucleotide stored in synaptic vesicles and chromaffin granules (Flodgaard and Klenow, 1982; Rodriguez del Castillo et al., 1988) and released therefrom (Pintor et al., 1991a, 1992). The purine dinucleotide also binds with subnanomolar affinity to receptors (Pintor et al., 1991b, 1993) and exerts biological effects (Pintor et al., 1993), indicating that it is an endogenous purinoceptor ligand. Thus, strong evidence for the presence of receptors for the endogenous ligands adenosine, ADP, ATP, and dia-denosinetetraphosphate had accumulated. This group of receptors is called the purinoceptors. If at some future time there is compelling evidence that UTP, or another pyrimidine nucleotide, is an endogenous ligand at receptors that respond poorly or not at all to ATP, then this terminology may need revision.

Extracellular ATP: effects, sources and fate

1986-01-15

John L. Gordon

Research Article| January 15 1986 Extracellular ATP: effects, sources and fate J L Gordon J L Gordon Search for other works by this author on: This Site PubMed Google Scholar … Research Article| January 15 1986 Extracellular ATP: effects, sources and fate J L Gordon J L Gordon Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1986) 233 (2): 309–319. https://doi.org/10.1042/bj2330309 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation J L Gordon; Extracellular ATP: effects, sources and fate. Biochem J 15 January 1986; 233 (2): 309–319. doi: https://doi.org/10.1042/bj2330309 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1986 London: The Biochemical Society1986 Article PDF first page preview Close Modal You do not currently have access to this content.

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Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis

2010-10-01

Faraaz B. Chekeni , Michael R. Elliott , Joanna K. Sandilos +7 more

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A proton-gated cation channel involved in acid-sensing

1997-03-01

Rainer Waldmann , Guy Champigny , Frédéric Bassilana +2 more

Adenosine receptors as therapeutic targets

2006-03-01

Kenneth A. Jacobson , Zhan‐Guo Gao

Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage

2001-12-01

Akio Ohta , Michail V. Sitkovsky

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Cellular function and molecular structure of ecto-nucleotidases

2012-05-03

Herbert Zimmermann , M. Zebisch , Norbert Sträter

Purine and pyrimidine receptors

2007-03-19

Geoffrey Burnstock

Cannabis use in adolescence and risk for adult psychosis: longitudinal prospective study

2002-11-21

Louise Arseneault , Mary Cannon , R. Poulton +3 more

A mycoplasma-encoded purine nucleoside phosphorylase (designated PNPHyor) has been cloned and characterized for the first time. Efficient phosphorolysis of natural 6-oxopurine and 6-aminopurine nucleosides was observed, with adenosine the preferred … A mycoplasma-encoded purine nucleoside phosphorylase (designated PNPHyor) has been cloned and characterized for the first time. Efficient phosphorolysis of natural 6-oxopurine and 6-aminopurine nucleosides was observed, with adenosine the preferred natural substrate (Km = 61 µM). Several cytostatic purine nucleoside analogs proved to be susceptible to PNPHyor-mediated phosphorolysis, and a markedly decreased or increased cytostatic activity was observed in Mycoplasma hyorhinis–infected human breast carcinoma MCF-7 cell cultures (MCF-7.Hyor), depending on the properties of the released purine base. We demonstrated an ∼10-fold loss of cytostatic activity of cladribine in MCF-7.Hyor cells and observed a rapid and complete phosphorolysis of this drug when it was exposed to the supernatant of mycoplasma-infected cells. This conversion (inactivation) could be prevented by a specific PNP inhibitor. These findings correlated well with the high efficiency of PNPHyor-catalyzed phosphorolysis of cladribine to its less toxic base 2-chloroadenine (Km = 80 µM). In contrast, the cytostatic activity of nucleoside analogs carrying a highly toxic purine base and being a substrate for PNPHyor, but not human PNP, was substantially increased in MCF-7.Hyor cells (∼130-fold for fludarabine and ∼45-fold for 6-methylpurine-2′-deoxyriboside). Elimination of the mycoplasma from the tumor cell cultures or selective inhibition of PNPHyor by a PNP inhibitor restored the cytostatic activity of the purine-based nucleoside drugs. Since several studies suggest a high and preferential colonization or association of tumor tissue in cancer patients with different prokaryotes (including mycoplasmas), the data presented here may be of relevance for the optimization of purine nucleoside–based anticancer drug treatment.

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ADENOSINE REGULATES VIA TWO DIFFERENT TYPES OF RECEPTORS, THE ACCUMULATION OF CYCLIC AMP IN CULTURED BRAIN CELLS

1979-11-01

Dietrich van Calker , Margarete Müller , Bernd Hamprecht

Abstract— In cell cultures of glial character derived from perinatal mouse brain adenosine elicits two effects. (a) At submicromolar concentrations It inhibits the increase in the intracellular level of cyclic … Abstract— In cell cultures of glial character derived from perinatal mouse brain adenosine elicits two effects. (a) At submicromolar concentrations It inhibits the increase in the intracellular level of cyclic AMP caused by β‐adrenoceptor agonists. (b) At concentrations above micromolar it increases the level of cyclic AMP in the cultures. These two effects are mediated by two different adenosine receptors present on the outer surface of the cells. This is concluded from the following evidence. (a) Both effects are antagonized by methylxanthines but not by blockage of adenosine uptake or inhibition of phosphodiesterase activity. (b) In both cases activity depends on the integrity of the ribose moiety of the nucleotide. Substituents of the purine system are tolerated comparatively well. (c) The order of potency of adenosine analogues is different for the two effects. We suggest the name A1 receptors for those that mediate the inhibition and A2 for those that mediate the stimulation of cyclic AMP accumulation.

Adenosine receptors: therapeutic aspects for inflammatory and immune diseases

2008-09-01

György Haskó , Joel Linden , Bruce N. Cronstein +1 more

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Subclasses of external adenosine receptors.

1980-05-01

Constantine Londos , Dermot M.F. Cooper , J. Wolff

Cell surface adenosine receptors mediate either stimulation or inhibition of adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], and the receptors that mediate these different responses can be discriminated with … Cell surface adenosine receptors mediate either stimulation or inhibition of adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], and the receptors that mediate these different responses can be discriminated with selected adenosine analogs. 5'-N-Ethylcarboxamide-adenosine is a more potent agonist at stimulatory receptors (Ra) than is N6-phenylisopropyladenosine, whereas the reverse potency order is seen with inhibitory receptors (Ri). The potency of adenosine is intermediate between the potencies of these two analogs. The relative potencies of adenosine receptor agonists are maintained in physiological responses in intact cells, such as steroidogenesis and inhibition of lipolysis. As with adrenergic receptors, subclasses of adenosine receptors differ functionally and pharmacologically.

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ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

2012-07-01

Tatsuo Hashimoto , Thomas Perlot , Ateequr Rehman +7 more

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New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor

1994-10-01

Anthony J. Brake , Michael Wagenbach , David Julius

Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade

2008-02-13

Gennady G. Yegutkin

A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP

1994-10-01

Soledad Valera , Nicolas Hussy , Richard J. Evans +4 more

Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation

2006-06-20

Martijn J.L. Bours , Els L.R. Swennen , Francesco Di Virgilio +2 more

Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons

1995-10-01

Carolyn J. Lewis , S. Neidhart , C. Holy +3 more

ATP drives lamina propria TH17 cell differentiation

2008-08-20

Koji Atarashi , Junichi Nishimura , Tatsuichiro Shima +7 more

Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression

2007-04-21

Giovanna Borsellino , Markus Kleinewietfeld , Diletta Di Mitri +7 more

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Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities

2003-02-01

Go Saito , Joel A. Swanson , Kyung‐Dall Lee

Physiology and Pathophysiology of Purinergic Neurotransmission

2007-04-01

Geoffrey Burnstock

This review is focused on purinergic neurotransmission, i.e., ATP released from nerves as a transmitter or cotransmitter to act as an extracellular signaling molecule on both pre- and postjunctional membranes … This review is focused on purinergic neurotransmission, i.e., ATP released from nerves as a transmitter or cotransmitter to act as an extracellular signaling molecule on both pre- and postjunctional membranes at neuroeffector junctions and synapses, as well as acting as a trophic factor during development and regeneration. Emphasis is placed on the physiology and pathophysiology of ATP, but extracellular roles of its breakdown product, adenosine, are also considered because of their intimate interactions. The early history of the involvement of ATP in autonomic and skeletal neuromuscular transmission and in activities in the central nervous system and ganglia is reviewed. Brief background information is given about the identification of receptor subtypes for purines and pyrimidines and about ATP storage, release, and ectoenzymatic breakdown. Evidence that ATP is a cotransmitter in most, if not all, peripheral and central neurons is presented, as well as full accounts of neurotransmission and neuromodulation in autonomic and sensory ganglia and in the brain and spinal cord. There is coverage of neuron-glia interactions and of purinergic neuroeffector transmission to nonmuscular cells. To establish the primitive and widespread nature of purinergic neurotransmission, both the ontogeny and phylogeny of purinergic signaling are considered. Finally, the pathophysiology of purinergic neurotransmission in both peripheral and central nervous systems is reviewed, and speculations are made about future developments.

Purinoceptors: Are there families of P2X and P2Y purinoceptors?

1994-01-01

Maria P. Abbracchio , Geoffrey Burnstock

The Cytolytic P 2Z Receptor for Extracellular ATP Identified as a P 2X Receptor (P2X 7 )

1996-05-03

Annmarie Surprenant , François Rassendren , Eric Kawashima +2 more

The P 2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P 2X receptors, … The P 2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P 2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X 7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P 2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X 7 (or P 2Z ) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells.

A2A adenosine receptor protects tumors from antitumor T cells

2006-08-18

Akio Ohta , Elieser Gorelik , Simon J. Prasad +7 more

The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR … The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Here we confirm this hypothesis by showing that genetic deletion of A2AR in the host resulted in rejection of established immunogenic tumors in approximately 60% of A2AR-deficient mice with no rejection observed in control WT mice. The use of antagonists, including caffeine, or targeting the A2 receptors by siRNA pretreatment of T cells improved the inhibition of tumor growth, destruction of metastases, and prevention of neovascularization by antitumor T cells. The data suggest that effects of A2AR are T cell autonomous. The inhibition of antitumor T cells via their A2AR in the adenosine-rich tumor microenvironment may explain the paradoxical coexistence of tumors and antitumor immune cells in some cancer patients (the "Hellstrom paradox"). We propose to target the hypoxia-->adenosine-->A2AR pathway as a cancer immunotherapy strategy to prevent the inhibition of antitumor T cells in the tumor microenvironment. The same strategy may prevent the premature termination of immune response and improve the vaccine-induced development of antitumor and antiviral T cells. The observations of autoimmunity during melanoma rejection in A2AR-deficient mice suggest that A2AR in T cells is also important in preventing autoimmunity. Thus, although using the hypoxia-->adenosine-->A2AR pathway inhibitors may improve antitumor immunity, the recruitment of this pathway by selective drugs is expected to attenuate the autoimmune tissue damage.

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Is there a basis for distinguishing two types of P2-purinoceptor?

1985-01-01

Geoffrey Burnstock , Charles Kennedy

Altered Cytokine Production in Mice Lacking P2X7Receptors

2001-01-01

Mike Solle , Jeff M. Labasi , David G. Perregaux +5 more

The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by monocytes and macrophages. To directly address the role of this receptor in interleukin (IL)-1β post-translational processing, we have generated … The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by monocytes and macrophages. To directly address the role of this receptor in interleukin (IL)-1β post-translational processing, we have generated a P2X7R-deficient mouse line.P2X7R −/− macrophages respond to lipopolysaccharide and produce levels of cyclooxygenase-2 and pro-IL-1β comparable with those generated by wild-type cells. In response to ATP, however, pro-IL-1β produced by the P2X7R −/− cells is not externalized or activated by caspase-1. Nigericin, an alternate secretion stimulus, promotes release of 17-kDa IL-1β from P2X7R −/− macrophages. In response to in vivo lipopolysaccharide injection, both wild-type and P2X7R −/− animals display increases in peritoneal lavage IL-6 levels but no detectable IL-1. Subsequent ATP injection to wild-type animals promotes an increase in IL-1, which in turn leads to additional IL-6 production; similar increases did not occur in ATP-treated, LPS-primed P2X7R −/− animals. Absence of the P2X7R thus leads to an inability of peritoneal macrophages to release IL-1 in response to ATP. As a result of the IL-1 deficiency,in vivo cytokine signaling cascades are impaired in P2X7R-deficient animals. Together these results demonstrate that P2X7R activation can provide a signal that leads to maturation and release of IL-1β and initiation of a cytokine cascade. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by monocytes and macrophages. To directly address the role of this receptor in interleukin (IL)-1β post-translational processing, we have generated a P2X7R-deficient mouse line.P2X7R −/− macrophages respond to lipopolysaccharide and produce levels of cyclooxygenase-2 and pro-IL-1β comparable with those generated by wild-type cells. In response to ATP, however, pro-IL-1β produced by the P2X7R −/− cells is not externalized or activated by caspase-1. Nigericin, an alternate secretion stimulus, promotes release of 17-kDa IL-1β from P2X7R −/− macrophages. In response to in vivo lipopolysaccharide injection, both wild-type and P2X7R −/− animals display increases in peritoneal lavage IL-6 levels but no detectable IL-1. Subsequent ATP injection to wild-type animals promotes an increase in IL-1, which in turn leads to additional IL-6 production; similar increases did not occur in ATP-treated, LPS-primed P2X7R −/− animals. Absence of the P2X7R thus leads to an inability of peritoneal macrophages to release IL-1 in response to ATP. As a result of the IL-1 deficiency,in vivo cytokine signaling cascades are impaired in P2X7R-deficient animals. Together these results demonstrate that P2X7R activation can provide a signal that leads to maturation and release of IL-1β and initiation of a cytokine cascade. interleukin lipopolysaccharide P2X7 receptor embryonic stem intraperitoneal cyclooxygenase-2, LDH, lactate dehydrogenase phosphospecific luminescence fetal bovine serum phosphate-buffered saline human embryonic kidney cells enzyme-linked immunoadsorbent assay Interleukin (IL)1-1 is a multipotential inflammatory mediator produced in abundance by activated monocytes and macrophages (1Dinarello C.A. Int. Rev. Immunol. 1998; 16: 457-499Crossref PubMed Scopus (682) Google Scholar). When released from producing cells, IL-1 binds to receptors on target cells and elicits signaling cascades leading to the up-regulation of gene products that contribute to an inflammatory state including matrix metalloproteinases, cyclooxygenase-2 (Cox-2), IL-6, and cellular adhesion molecules (2Flannery C.R. Little C.B. Caterson B. Hughes C.E. Matrix Biol. 1999; 18: 225-237Crossref PubMed Scopus (103) Google Scholar, 3Guzn Z. Buckman S.Y. Miller B.W. Springer L.D. Morrison A.R. J. Biol. Chem. 1998; 273: 28670-28676Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 4Allen M. Svensson L. Roach M. Hambor J. McNeish J. Gabel C.A. J. Exp. Med. 2000; 191: 859-869Crossref PubMed Scopus (256) Google Scholar, 5Bevilacqua M.P. Stengelin S. Gimbrone Jr., M.A. Seed B. Science. 1989; 243: 1160-1164Crossref PubMed Scopus (1910) Google Scholar). Two distinct gene products, IL-1α and IL-1β, contribute to IL-1 biological activity (6Auron P.E. Webb A.C. Rosenwasser L.J. Mucci S.F. Rich A. Wolff S.M. Dinarello C.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7907-7911Crossref PubMed Scopus (880) Google Scholar, 7March C.J. Mosley B. Larsen A. Cerretti D.P. Braedt G Price V. Gillis S. Henney C.S. Kroneeim S.R. Grabstein K. Conlon P.J. Hopp T.P. Cosman D. Nature. 1985; 315: 641-647Crossref PubMed Scopus (1333) Google Scholar). The amino acid sequences of IL-1α and IL-1β are <30% identical yet these two polypeptides bind to the same receptors on target cells (8Slack J. McMahan C.J. Waugh S. Schooley K. Spriggs M.K. Sims J.E. Dower S.K. J. Biol. Chem. 1993; 268: 2513-2524Abstract Full Text PDF PubMed Google Scholar). Human IL-1α and IL-1β both are initially produced as 31-kDa procytokines containing amino-terminal extensions; these extensions subsequently are removed by proteolysis. In the case of pro-IL-1α, the propolypeptide and its 17-kDa cleavage product display equivalent signaling activity, indicating that proteolytic cleavage is not necessary to generate a receptor-competent ligand (9Mosley B. Urdal D.L. Prickett K.S. Larsen A. Cosman D. Conlon P.J. Gillis S. Dower S.K. J. Biol. Chem. 1987; 262: 2941-2944Abstract Full Text PDF PubMed Google Scholar). In contrast, pro-IL-1β does not bind to the signaling IL-1 receptor (9Mosley B. Urdal D.L. Prickett K.S. Larsen A. Cosman D. Conlon P.J. Gillis S. Dower S.K. J. Biol. Chem. 1987; 262: 2941-2944Abstract Full Text PDF PubMed Google Scholar), and cleavage by caspase-1 is necessary to generate the mature 17-kDa signaling-competent form of this cytokine (10Cerretti D.P. Kozlosky C.J. Mosley B. Nelson N. Van Ness K. Greenstreet T.A. March C.J. Kronheim S.R. Druck T. Cannizzaro L.A. Huebner K. Black R.A. Science. 1992; 256: 97-100Crossref PubMed Scopus (1040) Google Scholar,11Thornberry N.A. Bull H.G Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.-F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2298) Google Scholar). The two forms of IL-1 share another very unusual attribute; both pro-IL-1α and pro-IL-1β are synthesized without a signal sequence (7March C.J. Mosley B. Larsen A. Cerretti D.P. Braedt G Price V. Gillis S. Henney C.S. Kroneeim S.R. Grabstein K. Conlon P.J. Hopp T.P. Cosman D. Nature. 1985; 315: 641-647Crossref PubMed Scopus (1333) Google Scholar), the peptide epitope required to direct nascent polypeptides to the endoplasmic reticulum (12Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (729) Google Scholar). As a result, newly synthesized pro-IL-1α and pro-IL-1β accumulate within the cytoplasmic compartment of producing cells (13Singer I.I. Scott S. Hall G.L. Limjuco G. Chin J. Schmidt J.A. J. Exp. Med. 1988; 167: 389-407Crossref PubMed Scopus (135) Google Scholar) rather than being sequestered to the secretory apparatus. Caspase-1 also is produced as a cytosol-localized proenzyme; the 45-kDa propolypeptide must be proteolytically processed to generate the 20- and 10-kDa subunits that constitute the mature active protease (11Thornberry N.A. Bull H.G Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.-F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.-T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2298) Google Scholar, 14Miller D.K. Ayala J.M. Egger L.A. Raju S.M. Yamin T.-T. Ding G.J.-F. Gaffney E.P. Howard A.D. Palyha O.C. Rolando A.M. Salley J.P. Thornberry N.A. Weidner J.R. Williams J.H. Chapman K.T. Jackson J. Kostura M.J. Limjuco G. Molineaux S.M. Mumford R.A. Calaycay J.R. J. Biol. Chem. 1993; 268: 18062-18069Abstract Full Text PDF PubMed Google Scholar, 15Ayala J.M. Yamin T.-T. Egger L.A. Chin J. Kostura M.J. Miller D.K. J. Immunol. 1994; 153: 2592-2599PubMed Google Scholar). In activated monocytes and macrophages, therefore, pro-IL-1β and procaspase-1 co-exist within the cytoplasm. Mechanisms that control activation of procaspase-1, and in turn cleavage of pro-IL-1β, are not well understood. Recent studies, however, have provided evidence that proteolytic processing of IL-1β and release of the mature cytokine product extracellularly do not proceed constitutively. Rather, the post-translational processing of pro-IL-1 requires that lipopolysaccharide (LPS)-activated monocytes and/or macrophages encounter an external stimulus that promotes activation of procaspase-1, cleavage of pro-IL-1β, and release of the 17-kDa cytokine (16Miller B.E. Krasney P.A. Gauvin D.M. Holbrook K.B. Koonz D.J. Abruzzese R.V. Miller R.E. Pagani K.A. Dolle R.E. Ator M.A. Gilman S.C. J. Immunol. 1995; 154: 1331-1338PubMed Google Scholar, 17Laliberte R.E. Eggler J. Gabel C.A. J. Biol. Chem. 1999; 274: 36944-36951Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 18Sanz J.M. Di Virgilio F. J. Immunol. 2000; 164: 4893-4898Crossref PubMed Scopus (237) Google Scholar). Stimuli that function in vitro to promote IL-1 post-translational processing by LPS-activated monocytes and/or macrophages include ATP, nigericin, cytolytic T-cells, bacterial toxins, and hypotonic stress (19Bhakdi S. Muhly M. Korom S. Schmidt G. J. Clin. Invest. 1990; 85: 1746-1753Crossref PubMed Scopus (115) Google Scholar, 20Hogquist K.A. Nett M.A. Unanue E.R. Chaplin D.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8485-8489Crossref PubMed Scopus (448) Google Scholar, 21Perregaux D. Barberia J. Lanzetti A.J. Geoghegan K.F. Carty T.J. Gabel C.A. J. Immunol. 1992; 149: 1294-1303PubMed Google Scholar, 22Perregaux D. Gabel C.A. J. Biol. Chem. 1994; 269: 15195-15203Abstract Full Text PDF PubMed Google Scholar, 23Perregaux D. Svensson L. Gabel C.A. J. Immunol. 1996; 157: 57-64PubMed Google Scholar, 24Walev I. Reske K. Palmer M. Valeva A. Bhakdi S. EMBO J. 1995; 14: 1607-1614Crossref PubMed Scopus (248) Google Scholar). This requirement for a secretion stimulus is not restricted to cells in culture; mouse peritoneal macrophages produce pro-IL-1β in response to intraperitoneal (ip) injection of LPS, but release little cytokine extracellularly (25Griffiths R.J. Stam E.J. Downs J.T. Otterness I.G. J. Immunol. 1995; 154: 2821-2828PubMed Google Scholar). Subsequent ip injection of ATP, however, stimulates generation of large quantities of extracellular mature IL-1β (25Griffiths R.J. Stam E.J. Downs J.T. Otterness I.G. J. Immunol. 1995; 154: 2821-2828PubMed Google Scholar). The mechanism by which ATP activates IL-1β post-translational processing is believed to involve the P2X7 purinergic receptor (17Laliberte R.E. Eggler J. Gabel C.A. J. Biol. Chem. 1999; 274: 36944-36951Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 18Sanz J.M. Di Virgilio F. J. Immunol. 2000; 164: 4893-4898Crossref PubMed Scopus (237) Google Scholar, 26Laliberte R. Perregaux D. Svensson L. Pazoles C.J. Gabel C.A. J. Immunol. 1994; 153: 2168-2179PubMed Google Scholar, 27Buell G. Chessell I.P. Michel A.D. Collo G. Salazzo M. Herren S. Gretener D. Grahames C. Kaur R. Kosco-Vilbois M.H. Humphrey P.P.A. Blood. 1998; 92: 3521-3528Crossref PubMed Google Scholar). Like other members of the P2X receptor family, the P2X7 receptor (P2X7R) is an ATP-gated ion channel (28Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1547) Google Scholar, 29Rassendren F. Buell G.N. Virginio C. Collo G. North R.A. Surprenant A. J. Biol. Chem. 1997; 272: 5482-5486Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 30Michel A.D. Chessell I.P. Hibell A.D. Simon J. Humphrey P.P.A. Br. J. Pharmacol. 1998; 125: 1194-1201Crossref PubMed Scopus (38) Google Scholar). The P2X7R, however, demonstrates attributes that clearly distinguish it from other members of the family. For example, the P2X7R requires levels of ATP in excess of 1 mm to achieve activation, whereas other P2X receptors activate at ATP concentrations of ≤100 μm(31Greenberg S. Di Virgilio F. Steinberg T.H. Silverstein S.C. J. Biol. Chem. 1988; 263: 10337-10343Abstract Full Text PDF PubMed Google Scholar, 32Steinberg T.H. Newman A.S. Swanson J.A. Silverstein S.C. J. Biol. Chem. 1987; 262: 8884-8888Abstract Full Text PDF PubMed Google Scholar); the higher concentration requirement reflects, in part, the preference of the P2X7R for ATP4− as its ligand and the relatively low abundance of this species in media containing physiological concentrations of divalent cations (e.g. Ca2+ and Mg2+). An additional unique feature of the P2X7R is found in its conductance properties. All P2X receptors demonstrate non-selective channel-like properties following ligation, but the channels formed by the P2X7R rapidly transform to pores that allow passage of solutes as large as 900 Da (32Steinberg T.H. Newman A.S. Swanson J.A. Silverstein S.C. J. Biol. Chem. 1987; 262: 8884-8888Abstract Full Text PDF PubMed Google Scholar, 33Virginio C. Mackenzie A. North R.A. Surprenant A. J. Physiol. (Lond.). 1999; 519: 335-346Crossref Scopus (330) Google Scholar). Molecular details of this transformation remain to be described, but domain swapping and deletion experiments have suggested that the carboxyl-terminal domain of the P2X7R participates in pore complex formation (28Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1547) Google Scholar, 29Rassendren F. Buell G.N. Virginio C. Collo G. North R.A. Surprenant A. J. Biol. Chem. 1997; 272: 5482-5486Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar); the carboxyl-terminal domain of the P2X7R is significantly longer than the comparable domains in the other P2X receptors (34North R.A. Curr. Opin. Cell Biol. 1996; 8: 474-483Crossref PubMed Scopus (138) Google Scholar). Possibly as a consequence of this pore-like activity, continuous ligation of the P2X7 receptor for times of >15 min can lead to cell death (35Di Virgilio F. Immunol. Today. 1995; 16: 524-528Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 36Murgia M. Pizzo P. Steinberg T.H. Di Virgilio F. Biochem. J. 1992; 288: 897-901Crossref PubMed Scopus (89) Google Scholar, 37Ferrari D. Los M. Bauer M.K.A. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. FEBS Lett. 1999; 447: 71-75Crossref PubMed Scopus (244) Google Scholar). In this study we employ a genetic approach to inactivate the P2X7R. Mice lacking the P2X7R are healthy and fertile and demonstrate no overt phenotype. However, in contrast to their wild-type counterparts, LPS-activated peritoneal macrophages from P2X7R −/− animals fail to generate mature IL-1β when challenged with ATP. This defect is not because of an inability of the macrophages to produce pro-IL-1β but rather to an inability of the cytokine producing cells to respond to the purinoceptor agonist. As a consequence of their inability to produce mature IL-1β post-ATP challenge,P2X7R −/− animals generate reduced quantities of IL-6 relative to their wild-type controls. Therefore, the knockout animals establish that the P2X7R is a necessary component of ATP-induced IL-1 post-translational processing, and demonstrate that this receptor can serve as an important element of an inflammatory cascade mechanism. A cDNA probe specific to the mouse P2X7R gene was synthesized by reverse transcription-polymerase chain reaction using primers P2X7-F1 (5′-CGGCGTGCGTTTTGACATCCT-3′) and P2X7-R2 (5′-AGGGCCCTGCGGTTCTC-3′), which were designed based on the published rat cDNA sequence of the P2X7R gene (28Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1547) Google Scholar). Total RNA isolated from the J774 A.1 mouse monocyte/macrophage cell line was used as the template RNA. This polymerase chain reaction product was 401 base pairs long and was cloned and sequenced to verify that it corresponded to the mouse P2X7R gene. The probe was used to screen a 129/Sv mouse genomic library and to isolate a single positive genomic clone. Sequence analysis of BamHI subcloned fragments confirmed that this clone corresponded to the mouse P2X7R gene. A targeting vector was constructed that inserted the neomycin resistance gene from the pJNS2 plasmid directly after the Arg505 codon, deleting from Cys506 to Pro532, which is in the carboxyl-terminal domain of the P2X7R gene product (38Chessell I.P. Simon J. Hibell A.D. Barnard E.A. Humphrey P.P.A. FEBS Lett. 1998; 439: 260-300Crossref Scopus (134) Google Scholar). 129/Ola-derived E14Tg2a ES cells (39Hooper M. Hardy K. Handyside A. Hunter S. Monk M. Nature. 1987; 326: 292-295Crossref PubMed Scopus (968) Google Scholar) were grown, transformed, and screened using standard methods (40Mohn A. Koller B.H. DNA Cloning 4. Oxford University Press, New York1995Google Scholar). Targeted ES cells and mice carrying the mutant allele were identified using a probe specific to a genomic region upstream of the targeted locus. Chimeric mice derived from targeted ES cells were mated with B6D2 (C57 BL/6 × DBA/2 F1) or C57BL/6 mice. Mouse peritoneal macrophages were harvested by injecting 5 ml of RPMI medium containing 5% FBS into each peritoneal cavity; immediately prior to injection, the animals were euthanized. The injected medium was dispersed throughout the peritoneal cavity, after which a hole in the skin covering the peritoneum was introduced and the injected fluid was recovered with the aid of a transfer pipette. Lavage fluids from multiple animals were pooled and the cells were collected by centrifugation (300 × g). These cell pellets were washed twice by centrifugation in RPMI containing 5% fetal calf serum. A cell count was performed before the final wash. Peritoneal macrophage cell pellets were washed once in cavitation buffer (25 mm Hepes, pH 7, 30 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol, 1 μg/ml leupeptin, and 1 μg/ml pepstatin) by centrifugation. Cell pellets then were suspended in 2.5 ml of cavitation buffer, and the cells were disrupted by nitrogen cavitation (15 min on ice at 750 psi). The resulting cell lysates were adjusted to 0.1% saponin, incubated on ice for 30 min, and cell membranes subsequently were recovered by centrifugation (50,000 rpm for 30 min at 4 °C in a Beckman Ti70 rotor). The membrane pellet was suspended in 2 ml of cavitation buffer with the aid of a glass tube-teflon pestle homogenizer, and an aliquot of the suspension was set aside for analysis of total protein (Pierce, Rockford, IL). The membranes again were collected by centrifugation after which the pellets were suspended in 100 μl of 2× Laemmli sample buffer (41Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (218082) Google Scholar). 40 μg of protein were loaded into wells of a 4–20% Tris-glycine gel (Novex, San Diego, CA), and after separation the proteins were transferred to nitrocellulose. These blots were blocked overnight at 4 °C in 1× Western blocking reagent (Roche Molecular Biochemicals, Indianapolis, IN) in TBS-T (10 mm Tris, pH 8, 150 mm NaCl, 0.1% Tween 20). Blots then were incubated for 2 h at room temperature in a TBS-T solution containing a 1:200 dilution of anti-P2X7R serum (Alomone, Jerusalem, Israel) and 1× Western blocking reagent. Blots were washed in TBS-T (three rinses, 5 min each) and then incubated for 1 h with TBS-T containing a 1:2000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (New England BioLabs, Beverly, MA) and 1× Western blocking reagent. Blots were washed in TBS-T (three rinses, 5 min each), and then developed with Super Signal (Pierce) and imaged with a Lumi-imager (Roche Molecular Biochemicals). For Cox-2 Western analysis, peritoneal macrophages (in RPMI, 5% FBS) were seeded into 6-well plates (1 × 106 cells/well) and incubated overnight at 37 °C in a 5% CO2environment. Cells were washed twice with RPMI, 5% FBS and then 1 ml of medium containing 100 ng/ml LPS (type 055:B5, Sigma) was added to each well and the cells were incubated at 37 °C for 4 h. Medium supernatants then were discarded, the adherent cells were washed twice with PBS, and then they were solubilized by addition of 200 μl of 2× Laemmli sample buffer; the resulting samples were boiled for 3 min. 20 μl of each sample were fractionated on a 4–20% Tris-glycine gel, after which the proteins were transferred to nitrocellulose. These blots were processed as described above except that the primary antibody employed was anti-prostaglandin synthase-2 (Oxford, Oxford, MI; 1:2000 dilution) and the secondary antibody was horseradish peroxidase-conjugated rabbit anti-IgG (New England BioLabs, 1:2000 dilution). Peritoneal macrophages were washed with isotonic medium (15 mm Hepes, pH 7.2, 135 mmNaCl, 5 mm KCl, 1.8 mm CaCl2, 0.8 mm MgCl2) by centrifugation, and the resulting cell pellet was suspended in isotonic medium to achieve a final cell concentration of 1 × 106 cells/ml. 50 μl of this cell suspension then was placed into wells of a Microfluor “B” U-bottom plate (Dynatech, Chantilly, VA), and 50 μl of 2 μm YoPro Yellow (Molecular Probes, Eugene, OR) was introduced; the fluorescent dye was dissolved in isotonic medium. Each well then was adjusted to 5 mm ATP or 0.0075% saponin by addition of concentrated stock solutions of these agents. Fluorescence was monitored as a function of time at 37 °C; excitation, 450 nm; emission, 530 nm. Macrophages from wild-type and P2X7R−/− animals (2 × 106cells seeded per well of 6-well cluster plates) were stimulated with 1 μg/ml LPS for 75 min and then rinsed with 2 ml of methionine-free RPMI medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, 1% dialyzed FBS, 1 μg/ml LPS, and 25 mmHepes, pH 7.3 (pulse medium). One ml of pulse medium containing 83 μCi/ml of [35S]methionine (Amersham Pharmacia Biotech) then was added to each well, and the cells were labeled at 37 °C for 1 h. These labeled cells subsequently were rinsed twice with RPMI 1640 medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, 1% FBS, 2 mm glutamine, 1 μg/ml LPS, and 25 mm Hepes, pH 7.3 (chase medium). One ml of chase medium containing no effector, 5 mm ATP, or 20 μmnigericin then was added to each well, and the cells were chased at 37 °C for 30 min. Media were harvested and clarified by centrifugation (6000 × g for 5 min) to remove cells and/or cell debris. Cell monolayers were suspended in 1 ml of a lysis buffer composed of 1% Triton X-100, 150 mm NaCl, 25 mm Hepes, pH 7, 0.1 mm phenylmethylsulfonyl fluoride, 1 mg/ml ovalbumin, 1 mm iodoacetic acid, 1 μg/ml pepstatin, and 1 μg/ml leupeptin. Clarified medium samples were adjusted to the same final Triton X-100 and protease inhibitor concentrations by addition of concentrated stocks of these reagents. After a 30-min incubation on ice, all samples were clarified by centrifugation at 45,000 rpm for 30 min in a TLA-45 rotor (Beckman). The resulting supernatants were recovered and IL-1β was immunoprecipitated from these samples using a goat anti-murine IL-1β serum obtained from Dr. Ivan Otterness (Pfizer Central Reseach, Groton, CT). Immunoprecipitates were fractionated by SDS-gel electrophoresis; the quantity of radioactivity associated with individual IL-1β polypeptide species was determined by scanning dried gels with a phosphorimager. Groups of mice were injected ip with 1 μg of LPS. Two hours after this LPS injection, mice were injected ip with either 0.5 ml of 30 mm ATP (adjusted to pH 7) or PBS. Mice were euthanized 30 min or 120 min after the ATP or PBS injection, and each peritoneal cavity was lavaged with 3 ml of media. Individual lavages were centrifuged, supernatants were collected and tested by ELISA for the presence of IL-1β (Amersham Pharmacia Biotech) and IL-6 (Endogen, Inc. Woburn, MA). All procedures involving mice were approved by the Institutional Animal Care and Use Committee at Pfizer Inc. Mouse ES cells in which the P2X7R gene was disrupted by homologous recombination were generated using the scheme shown in Fig.1. Integration of the targeting vector into the mouse genome by homologous recombination results in replacement of the region of the gene encoding Cys506 to Pro532 with the neomycin resistance gene. ES cells containing the mutant P2X7R allele were identified by Southern blot analysis and used to generate the P2X7R Δ506–532 mouse line. Homozygous null animals were recovered in the F2 generation with the expected Mendelian frequencies.P2X7R Δ506–532(P2X7R −/−) animals were viable and fertile and could not be identified among littermates by observation alone. Northern analysis of RNA isolated from cultured bone marrow mast cells signified that the transcript for the P2X7R was present in wild-type (+/+) but not knockout (−/−) animals (Fig. 1 C). To further demonstrate that the mutation introduced into the P2X7R locus resulted in loss of expression of this gene, peritoneal macrophages obtained from wild-type or P2X7R-deficient mice were compared by Western analysis for the presence of the receptor polypeptide. An equivalent number of macrophages were recovered from wild-type and P2X7R-deficient animals, suggesting that absence of the receptor did not alter macrophage development. Membranes isolated from wild-type peritoneal macrophages contained a 76-kDa polypeptide that cross-reacted with the P2X7R antiserum. The size of the murine polypeptide is comparable with that displayed by the human P2X7R when overexpressed in HEK293 cells (Fig.2). In contrast, macrophage membranes prepared from cells isolated from the P2X7R-deficient animals did not contain a similarly sized polypeptide (Fig. 2). In addition, no smaller cross-reacting polypeptides were observed in the Western blot of the P2X7R −/−cells, suggesting that the mutation introduced into the P2X7R gene did not lead to expression of a truncated version of the receptor (Fig. 2). However, because the antibody employed was prepared against a peptide epitope that resides at the extreme carboxyl terminus of the receptor polypeptide, we cannot exclude the possibility that a truncated form of the receptor lacking the entire carboxyl terminus is present and not detected by the analysis. A hallmark of the P2X7R is its ability to facilitate translocation of large organic molecules such as the fluorescent dye YoPro Yellow in response to ATP activation (32Steinberg T.H. Newman A.S. Swanson J.A. Silverstein S.C. J. Biol. Chem. 1987; 262: 8884-8888Abstract Full Text PDF PubMed Google Scholar, 33Virginio C. Mackenzie A. North R.A. Surprenant A. J. Physiol. (Lond.). 1999; 519: 335-346Crossref Scopus (330) Google Scholar, 42Buisman H.P. Steinberg T.H. Fischbarg J. Silverstein S.C. Vogelzang S.A. Ince C. Ypey D.L. Leijh P.C.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7988-7992Crossref PubMed Scopus (119) Google Scholar, 43Hickman S.E. El Khoury J. Greenberg S. Schieren I. Silverstein S.C. Blood. 1994; 84: 2452-2456Crossref PubMed Google Scholar). When mouse peritoneal macrophages isolated from wild-type animals were activated with 5 mm ATP in the presence of extracellular YoPro Yellow, a time-dependent increase in fluorescence intensity was observed (Fig. 3). This increase in fluorescence results from internalization of the dye molecules followed by their binding to DNA; when bound to DNA, their fluorescence intensity increases (43Hickman S.E. El Khoury J. Greenberg S. Schieren I. Silverstein S.C. Blood. 1994; 84: 2452-2456Crossref PubMed Google Scholar). In the absence of ATP, no significant increase in fluorescence intensity was observed, indicating that YoPro Yellow is impermeable to the plasma membrane in the absence of the nucleotide triphosphate. In contrast, addition of ATP to macrophages isolated from P2X7R −/− animals did not result in a time-dependent increase in fluorescence intensity (Fig. 3). Macrophages isolated from the P2X7R −/− animals demonstrated the same low fluorescence in the absence and presence of extracellular ATP. In the presence of saponin, a detergent that permeabilizes the plasma membrane, YoPro Yellow accumulates to the same extent in both wild-type and P2X7R −/− macrophages (Fig. 3). This demonstrates that the P2X7R mediates ATP-dependent YoPro Yellow accumulation. Peritoneal macrophages isolated from wild-type and P2X7R −/− animals were stimulated with LPS and labeled with [35S]methionine. These radiolabeled cells then were chased in the absence or presence of a secretory stimulus, after which cells and media were harvested separately, cells were solubilized by detergent extraction, and IL-1β was recovered from the medium and cell extracts by immunoprecipitation. Radiolabeled 35-kDa pro-IL-1β was recovered from cell extracts derived from both wild-type and P2X7R −/− macrophages (Fig.4). The amount of [35S]methionine recovered as the cell-associated 35-kDa polypeptide (assessed by phosphorimager analysis) after the chase in the absence of a secretory stimulus was 43,900 PSL/LDH equivalent and 44,100 PSL/LDH equivalent, respectively, from the wild-type and knockout macrophages. This similarity suggests that the two cell-types generated comparable amounts of pro-IL-1β in response to LPS activation. Neither the wild-type nor the P2X7R −/− macrophages released radiolabeled IL-1β to the medium in the absence of a secretory stimulus (Fig. 4). Treatment of LPS-activated [35S]methionine-labeled wild-type macrophages with extracellular ATP promoted formation and release of a 17-kDa IL-1β (Fig. 4). Cytokine that remained cell-assoc

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There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes … There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors deorphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review.

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The study of T regulatory cells (T reg cells) has been limited by the lack of specific surface markers and an inability to define mechanisms of suppression. We show that … The study of T regulatory cells (T reg cells) has been limited by the lack of specific surface markers and an inability to define mechanisms of suppression. We show that the expression of CD39/ENTPD1 in concert with CD73/ecto-5′-nucleotidase distinguishes CD4+/CD25+/Foxp3+ T reg cells from other T cells. These ectoenzymes generate pericellular adenosine from extracellular nucleotides. The coordinated expression of CD39/CD73 on T reg cells and the adenosine A2A receptor on activated T effector cells generates immunosuppressive loops, indicating roles in the inhibitory function of T reg cells. Consequently, T reg cells from Cd39-null mice show impaired suppressive properties in vitro and fail to block allograft rejection in vivo. We conclude that CD39 and CD73 are surface markers of T reg cells that impart a specific biochemical signature characterized by adenosine generation that has functional relevance for cellular immunoregulation.

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2001-12-01

Bertil B. Fredholm , Adriaan P. IJzerman , Kenneth A. Jacobson +2 more

Four adenosine receptors have been cloned and characterized from several mammalian species. The receptors are named adenosine A(1), A(2A), A(2B), and A(3). The A(2A) and A(2B) receptors preferably interact with … Four adenosine receptors have been cloned and characterized from several mammalian species. The receptors are named adenosine A(1), A(2A), A(2B), and A(3). The A(2A) and A(2B) receptors preferably interact with members of the G(s) family of G proteins and the A(1) and A(3) receptors with G(i/o) proteins. However, other G protein interactions have also been described. Adenosine is the preferred endogenous agonist at all these receptors, but inosine can also activate the A(3) receptor. The levels of adenosine seen under basal conditions are sufficient to cause some activation of all the receptors, at least where they are abundantly expressed. Adenosine levels during, e.g., ischemia can activate all receptors even when expressed in low abundance. Accordingly, experiments with receptor antagonists and mice with targeted disruption of adenosine A(1), A(2A), and A(3) expression reveal roles for these receptors under physiological and particularly pathophysiological conditions. There are pharmacological tools that can be used to classify A(1), A(2A), and A(3) receptors but few drugs that interact selectively with A(2B) receptors. Testable models of the interaction of these drugs with their receptors have been generated by site-directed mutagenesis and homology-based modelling. Both agonists and antagonists are being developed as potential drugs.

The P2Y12 receptor regulates microglial activation by extracellular nucleotides

2006-11-19

Sharon E Haynes , Gunther Hollopeter , Guang Yang +4 more

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Extracellular metabolism of ATP and other nucleotides

2000-08-19

Herbert Zimmermann

A SIMPLE, SENSITIVE METHOD FOR THE ASSAY OF ADENYL CYCLASE

1968-10-01

Gopal Krishna , Benjamin Weiss , Bernard B. Brodie

A simple method for the amay of adenyl cyclase in tissue homogenates is described that uses either α-P82-, C14-or H3-labeled adenosine triphosphate (ATP) as substrate. Cyclic 3’, 5’- adenosine monophosphate … A simple method for the amay of adenyl cyclase in tissue homogenates is described that uses either α-P82-, C14-or H3-labeled adenosine triphosphate (ATP) as substrate. Cyclic 3’, 5’- adenosine monophosphate (cyclic 3’, S’-AMP) formed during the incubation was isolated by chromatography on Dowex 50-H+ columns followed by precipitation of all nucleotides and inorganic phosphates by ZnSO4-Ba(OH)2, treatment, which left cyclic 3’, S’-AMP in the supernatant fluid. The purity of the cyclic 3’, 5’-AMP fraction was determined with the use of ion-exchange chromatography and paper chromatographic and electrophoretic techniques, as well as crystallization to constant specific activity. Furthermore, the purity of the nucleotide has been assessed enzymatically, by utilizing a purified cyclic nucleotide phosphodiesterase. The method described here makes possible the measurement of enzyme activities in tissue weighing less than 1 mg. Moreover, the entire procedure can be completed less than 3 hr.

The Role and Regulation of Adenosine in the Central Nervous System

2001-03-01

Thomas V. Dunwiddie , Susan A. Masino

Adenosine is a modulator that has a pervasive and generally inhibitory effect on neuronal activity. Tonic activation of adenosine receptors by adenosine that is normally present in the extracellular space … Adenosine is a modulator that has a pervasive and generally inhibitory effect on neuronal activity. Tonic activation of adenosine receptors by adenosine that is normally present in the extracellular space in brain tissue leads to inhibitory effects that appear to be mediated by both adenosine A1 and A2A receptors. Relief from this tonic inhibition by receptor antagonists such as caffeine accounts for the excitatory actions of these agents. Characterization of the effects of adenosine receptor agonists and antagonists has led to numerous hypotheses concerning the role of this nucleoside. Previous work has established a role for adenosine in a diverse array of neural phenomena, which include regulation of sleep and the level of arousal, neuroprotection, regulation of seizure susceptibility, locomotor effects, analgesia, mediation of the effects of ethanol, and chronic drug use.

Cloning of the β Cell High-Affinity Sulfonylurea Receptor: a Regulator of Insulin Secretion

1995-04-21

Lydia Aguilar‐Bryan , Colin G. Nichols , Sérgio Wechsler +7 more

Sulfonylureas are a class of drugs widely used to promote insulin secretion in the treatment of non-insulin-dependent diabetes mellitus. These drugs interact with the sulfonylurea receptor of pancreatic β cells … Sulfonylureas are a class of drugs widely used to promote insulin secretion in the treatment of non-insulin-dependent diabetes mellitus. These drugs interact with the sulfonylurea receptor of pancreatic β cells and inhibit the conductance of adenosine triphosphate (ATP)-dependent potassium (K ATP ) channels. Cloning of complementary DNAs for the high-affinity sulfonylurea receptor indicates that it is a member of the ATP-binding cassette or traffic ATPase superfamily with multiple membrane-spanning domains and two nucleotide binding folds. The results suggest that the sulfonylurea receptor may sense changes in ATP and ADP concentration, affect K ATP channel activity, and thereby modulate insulin release.

Molecular Physiology of P2X Receptors

2002-01-10

R. Alan North

P2X receptors are membrane ion channels that open in response to the binding of extracellular ATP. Seven genes in vertebrates encode P2X receptor subunits, which are 40–50% identical in amino … P2X receptors are membrane ion channels that open in response to the binding of extracellular ATP. Seven genes in vertebrates encode P2X receptor subunits, which are 40–50% identical in amino acid sequence. Each subunit has two transmembrane domains, separated by an extracellular domain (∼280 amino acids). Channels form as multimers of several subunits. Homomeric P2X 1 , P2X 2 , P2X 3 , P2X 4 , P2X 5 , and P2X 7 channels and heteromeric P2X 2/3 and P2X 1/5 channels have been most fully characterized following heterologous expression. Some agonists (e.g., αβ-methylene ATP) and antagonists [e.g., 2′,3′- O-(2,4,6-trinitrophenyl)-ATP] are strongly selective for receptors containing P2X 1 and P2X 3 subunits. All P2X receptors are permeable to small monovalent cations; some have significant calcium or anion permeability. In many cells, activation of homomeric P2X 7 receptors induces a permeability increase to larger organic cations including some fluorescent dyes and also signals to the cytoskeleton; these changes probably involve additional interacting proteins. P2X receptors are abundantly distributed, and functional responses are seen in neurons, glia, epithelia, endothelia, bone, muscle, and hemopoietic tissues. The molecular composition of native receptors is becoming understood, and some cells express more than one type of P2X receptor. On smooth muscles, P2X receptors respond to ATP released from sympathetic motor nerves (e.g., in ejaculation). On sensory nerves, they are involved in the initiation of afferent signals in several viscera (e.g., bladder, intestine) and play a key role in sensing tissue-damaging and inflammatory stimuli. Paracrine roles for ATP signaling through P2X receptors are likely in neurohypophysis, ducted glands, airway epithelia, kidney, bone, and hemopoietic tissues. In the last case, P2X 7 receptor activation stimulates cytokine release by engaging intracellular signaling pathways.

International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and Classification of Adenosine Receptors—An Update

2011-02-08

Bertil B. Fredholm , Adriaan P. IJzerman , Kenneth A. Jacobson +2 more

In the 10 years since our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes … In the 10 years since our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes in the recommendations. However, there have been so many other developments that an update is needed. The fact that the structure of one of the adenosine receptors has recently been solved has already led to new ways of in silico screening of ligands. The evidence that adenosine receptors can form homo- and heteromultimers has accumulated, but the functional significance of such complexes remains unclear. The availability of mice with genetic modification of all the adenosine receptors has led to a clarification of the functional roles of adenosine, and to excellent means to study the specificity of drugs. There are also interesting associations between disease and structural variants in one or more of the adenosine receptors. Several new selective agonists and antagonists have become available. They provide improved possibilities for receptor classification. There are also developments hinting at the usefulness of allosteric modulators. Many drugs targeting adenosine receptors are in clinical trials, but the established therapeutic use is still very limited.

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The P2X7 Receptor in Infection and Inflammation

2017-07-01

Francesco Di Virgilio , Diego Dal Ben , Alba Clara Sarti +2 more

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A simple and sensitive saturation assay method for the measurement of adenosine 3′:5′-cyclic monophosphate

1971-02-01

B. L. Brown , J. D. M. Albano , Roger Ekins +2 more

Research Article| February 01 1971 A simple and sensitive saturation assay method for the measurement of adenosine 3′:5′-cyclic monophosphate B L Brown; B L Brown Search for other works by … Research Article| February 01 1971 A simple and sensitive saturation assay method for the measurement of adenosine 3′:5′-cyclic monophosphate B L Brown; B L Brown Search for other works by this author on: This Site PubMed Google Scholar J D M Albano; J D M Albano Search for other works by this author on: This Site PubMed Google Scholar R P Ekins; R P Ekins Search for other works by this author on: This Site PubMed Google Scholar A M Sgherzi; A M Sgherzi Search for other works by this author on: This Site PubMed Google Scholar W Tampion W Tampion Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1971) 121 (3): 561–562. https://doi.org/10.1042/bj1210561 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation B L Brown, J D M Albano, R P Ekins, A M Sgherzi, W Tampion; A simple and sensitive saturation assay method for the measurement of adenosine 3′:5′-cyclic monophosphate. Biochem J 1 February 1971; 121 (3): 561–562. doi: https://doi.org/10.1042/bj1210561 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search © 1971 The Biochemical Society1971 Article PDF first page preview Close Modal You do not currently have access to this content.

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Structure–activity relationship of the allosteric effects of ivermectin at human P2X4 and GABAA receptors

2025-06-25

Jessica Lauren Meades , Marcella Bassetto , Marius C. Staiculescu +2 more | British Journal of Pharmacology

Abstract Background and purpose Positive allosteric modulation of the P2X4 receptor is a potential route to providing cardiovascular benefit through enhancing flow‐dependent arterial vasodilation and providing cardioprotection. However, ligands that … Abstract Background and purpose Positive allosteric modulation of the P2X4 receptor is a potential route to providing cardiovascular benefit through enhancing flow‐dependent arterial vasodilation and providing cardioprotection. However, ligands that selectively enhance P2X4 activity are absent. The broad‐spectrum antiparasitic ivermectin (MK‐933) is a known positive allosteric modulator of P2X4, but not selective for P2X4, acting to enhance the activity of other ion channels including the GABA A receptor through which its neurotoxic and anti‐convulsant properties are mediated. Here, we combine complementary methodology to investigate the structure–activity relationship of ivermectin at human P2X4 and GABA A receptors. Experimental approach and key results Intracellular Ca 2+ and membrane potential assays in cell lines expressing human P2X4 or human GABA A α1β3γ2 receptor are used, respectively. A chemical library of ivermectin analogues are pharmacologically characterised at both receptors, and in silico techniques are used to identify ligand binding modes in human P2X4 to interpret pharmacological properties. We identify ivermectin‐B1a as a positive allosteric modulation of P2X4, but full agonist at the GABA A α1β3γ2 receptor. We discover that the large disaccharide moiety of ivermectin‐B1a is not required for activity. We identify an intersubunit transmembrane domain binding mode for ivermectin‐B1a in P2X4 supported by the structure–activity relationship of ivermectin analogues. A series of novel compounds with selectivity for P2X4 over GABA A receptor are identified. Conclusions and implications Ivermectin‐B1a enhances P2X4 and GABA A α1β3γ2 receptor activity but through differing pharmacological mechanisms. We identify pharmacophore information for the development of positive allosteric modulators selective for human P2X4 over GABA A receptors.

Characterization of CDDP-Resistant OSCC with Enhanced Drug Efflux and Autophagy

2025-06-24

Makoto Adachi , Naoki Umemura , Yusuke Shibuya | International Journal of Oral and Maxillofacial Surgery

HIF-1α Stabilization Hampers the Chondrogenic Differentiation of Aged Bone Marrow Mesenchymal Stem Cells by Adenosine A2A/A2B Receptors Imbalance

2025-06-24

Rui Pinto-Cardoso , Catarina Bessa-Andrês , Flávio Pereira-Costa +7 more | ACS Pharmacology & Translational Science

Synthesis, Labeling, and Biological Evaluation of P2Y12 Receptor Radioligands for Positron Emission Tomography Imaging of Neuroinflammation

2025-06-24

Eugénie Pincemail , Marie‐Anne Peyronneau , Caroline Denis +7 more | ACS Chemical Neuroscience

The P2Y12 receptor (P2Y12R) is a G-protein-coupled receptor whose expression level is directly correlated to microglial activation. Herein, we report on the design of a series of new P2Y12R ligands … The P2Y12 receptor (P2Y12R) is a G-protein-coupled receptor whose expression level is directly correlated to microglial activation. Herein, we report on the design of a series of new P2Y12R ligands and the radiolabeling and characterization of two positron emission tomography (PET) tracers, [11C]37 and [18F]41. These compounds were evaluated by autoradiography studies on rat brain slices exhibiting overexpression of human P2Y12Rs (AAV-hP2Y12R). Metabolism and biodistribution of [18F]41 were evaluated ex vivo in healthy rats and indicated good metabolic stability with 41% of unchanged radioligand 1 h post injection and a limited crossing of the blood-brain barrier with a brain uptake of 0.02%ID/g 1 h post injection. In vivo PET imaging performed in the AAV-hP2Y12R rat model confirmed this low brain uptake, and no significant difference was found in the transfected (SUVmean 0.14 ± 0.01) versus contralateral (SUVmean 0.13 ± 0.01) striatum of the AAV-hP2Y12R model. Similar results were observed in healthy rats and in nonhuman primates. Additional studies in the presence of tariquidar led to a 3-4-fold increase in the [18F]41 brain concentration, suggesting that [18F]41 is a P-glycoprotein substrate. Future work will focus on improving radioligand design to enhance blood-brain barrier permeation and to reduce efflux transport.

Setdb1 ablation in macrophages attenuates fibrosis in heart allografts

2025-06-24

Zhibo Ma , Xi Zhou , Wenjun Jia +7 more | Proceedings of the National Academy of Sciences

Tissue fibrosis is commonly associated with organ malfunction and is strongly associated with the development of chronic rejection, cardiovascular diseases, and other chronic diseases. Fibrosis also contributes to immune exclusion … Tissue fibrosis is commonly associated with organ malfunction and is strongly associated with the development of chronic rejection, cardiovascular diseases, and other chronic diseases. Fibrosis also contributes to immune exclusion in tumor tissues. Targeting fibrosis might be a strategy for prolonging allograft survival while suppressing cancer development. Here, single-cell transcriptomes of human and mouse heart allografts showed that macrophages accumulated in grafts with fibrosis were reprogrammed via histone methylation regulated by Setdb1, an H3K9 methyltransferase. Myeloid-specific deletion of Setdb1 prolonged heart allograft survival but reversed immune exclusion in tumor tissues. Interestingly, myeloid-specific Setdb1-knockout led to lower fibrosis in heart allografts and tumor tissues in mice. Our single-cell sequencing data showed that Setdb1 ablation impaired Fn1+ and SPP1+ profibrogenic macrophage reprogramming. Mechanistically, Fn1, which was induced by the CCR2-Creb/Setdb1 axis, upregulated the expression of genes related to fibrosis in fibroblasts and macrophages via ITGA5 and PIRA receptors. Blocking the interaction between FN1 and these receptors inhibited fibrosis in allograft and tumor tissues. Our results reveal a target, histone methylation in macrophages, for the treatment of fibrosis-related disease.

P2RX7 regulates tauopathy progression via tau and mitochondria loading in extracellular vesicles

2025-06-23

Tsuneya Ikezu , Victor Bodart‐Santos , Arun Reddy Ravula +7 more

<title>Abstract</title> P2x purinoreceptor 7 (P2RX7), an ATP-gated ion channel, is known to play pivotal roles in the progression of Alzheimer’s disease (AD), although its cell type-specific pathological mechanisms have yet … <title>Abstract</title> P2x purinoreceptor 7 (P2RX7), an ATP-gated ion channel, is known to play pivotal roles in the progression of Alzheimer’s disease (AD), although its cell type-specific pathological mechanisms have yet to be elucidated. Here, we show that genetic deletion of P2rx7 mitigates brain atrophy, tau accumulation and cognitive impairment in PS19 tauopathy mice. Specific deletion of P2rx7 in microglia, but not astrocytes, significantly suppresses tau propagation from the entorhinal cortex to CA1 in the hippocampus, an early event in AD pathology. Single-cell (sc)-RNA sequencing of mouse brains revealed specific P2rx7 expression in microglia, inducing inflammatory changes accompanied by elevated extracellular vesicles (EVs) secretion in PS19 mice. Brain-derived EVs (BDEVs) proteome demonstrated that P2RX7 increases EV cargo loading of tau and mitochondrial molecules in BDEVs from PS19 mice, which was further validated by single-molecule super-resolution. Notably, following the injection of BDEVs isolated from PS19 mice with or without P2rx7 deficiency, the microglial transcriptome of recipient mice revealed enriched DNA-sensing and type II interferon signaling in response to BDEVs from PS19 mice, which was diminished in the group injected with P2rx7-deficient BDEVs. Thus, our results indicate that P2RX7 regulates EV-mediated tau and mitochondrial transfer and inflammatory activation in microglia with increased EV secretion, thereby contributing to tauopathy and neurodegeneration, highlighting the therapeutic potential of targeting the P2RX7-EV axis in AD.

Humanized scFv Molecule Specific to an Extracellular Epitope of P2X4R as Therapy for Chronic Pain Management

2025-06-22

Adinarayana Kunamneni , Karin N. Westlund | Cells

Chronic pain affects a significant portion of the population, with fewer than 30% achieving adequate relief from existing treatments. This study describes the humanization methodology and characterization of an effective … Chronic pain affects a significant portion of the population, with fewer than 30% achieving adequate relief from existing treatments. This study describes the humanization methodology and characterization of an effective non-opioid single-chain fragment variable (scFv) biologic that reverses pain-related behaviors, in this case by targeting P2X4. After nerve injury, ATP release activates/upregulates P2X4 receptors (P2X4R) sequestered in late endosomes, triggering a cascade of chronic pain-related events. Nine humanized scFv (hscFv) variants targeting a specific extracellular 13-amino-acid peptide fragment of human P2X4R were generated via CDR grafting. ELISA analysis revealed nanomolar binding affinities, with most humanized molecules exhibiting comparable or superior affinity compared to the original murine antibody. Octet measurements confirmed that the lead, HC3-LC3, exhibited nanomolar binding kinetics (KD = 2.5 × 10−9 M). In vivo functional validation with P2X4R hscFv reversed nerve injury-induced chronic pain-related behaviors with a single dose (0.4 mg/kg, intraperitoneal) within two weeks. The return to naïve baseline remained durably reduced > 100 days. In independent confirmation, the spared nerve injury (SNI) model was similarly reduced. This constitutes an original method whereby durable reversals of chronic nerve injury pain, anxiety and depression measures are accomplished.

The enzymatic degradation of ATP to adenosine by microglial CD39 regulates neurovascular coupling and metabolic supply to the brain

2025-06-20

Jing Guo , Yong Tang , Peter Illéš | Purinergic Signalling

Adenosine A2B receptor mediates cyclosporine counteraction of inflammatory and renal consequences of sepsis in rats

2025-06-19

Sara Salama , Marwa Y. Sallam , Sahar M. El‐Gowilly | Immunopharmacology and Immunotoxicology

The immunosuppressant drug cyclosporine A (CsA) demonstrates anti-inflammatory properties in numerous pathological conditions. It acts through modulating T-cell receptor signaling, reducing the expression of inflammatory cytokines, and inhibiting mitochondrial permeability, … The immunosuppressant drug cyclosporine A (CsA) demonstrates anti-inflammatory properties in numerous pathological conditions. It acts through modulating T-cell receptor signaling, reducing the expression of inflammatory cytokines, and inhibiting mitochondrial permeability, besides modulating vascular response. These features make it a potential drug to prevent or treat septic acute kidney injury (AKI). In this study, we investigated whether CsA exerts a protective effect against hemodynamic, inflammatory, and renovascular consequences of sepsis and whether these effects are modulated by adenosine receptor signaling. Cecal ligation and puncture (CLP) was utilized to induce sepsis 24 h before hemodynamic and renovascular studies were implicated. Renal vasoconstrictions and vasodilatations were induced by cumulative bolus injections of phenylephrine (PE, 0.41-900 ng) and acetylcholine (ACh, 0.01-7.29 nmol), respectively. The data showed that CsA abrogated CLP-evoked hypotension, tachycardia, and impaired renovascular responsiveness. Similarly, the elevation in kidney biomarkers together with the pro-inflammatory cytokines (Tumor necrosis factor-alpha (TNFα) and Interleukin-6 (IL-6)) were also blunted after CsA administration. Likewise, the elevation in nuclear factor kappa-light-chain enhancer of activated B cells (NFκB) and decrease in A2BRs renal tubular expression in sepsis was reversed in CsA-treated rats. These advantageous effects of CsA disappeared upon concurrent exposure to the selective A2BR antagonist, Alloxazine. These results suggest a key role for functional A2BR in CsA counteracting CLP-induced hemodynamic, inflammatory, and renal dysfunction in rats.

Editorial: Purinergic signaling in metabolic diseases and inflammation pharmacology

2025-06-19

Alejandro Sánchez-Melgar , Juana María Sanz , Anna Lisa Giuliani | Frontiers in Pharmacology

ATP6V0A1 protects dopaminergic neurons via the autophagy-lysosomal pathway in Parkinson’s disease

2025-06-19

Yuwan Lin , Zixin Tan , Wenfeng Ye +7 more | Neural Regeneration Research

Abstract Parkinson’s disease is the second most common neurodegenerative disorder. ATPase H + transporting V0 subunit A1 (ATP6V0A1) is a component of vacuolar H + -ATPase (V-ATPase), an ATP-dependent proton … Abstract Parkinson’s disease is the second most common neurodegenerative disorder. ATPase H + transporting V0 subunit A1 (ATP6V0A1) is a component of vacuolar H + -ATPase (V-ATPase), an ATP-dependent proton pump. Our previous research identified an association between the ATP6V0A1 rs601999 variant and Parkinson’s disease; however, the underlying mechanisms of ATP6V0A1 in Parkinson’s disease remain elusive. In this study, we generated ATP6V0A1 knockdown and overexpression models and then examined the degeneration of dopaminergic neurons, lysosomal function, and the autophagy-lysosomal pathway using immunohistochemistry, western blotting, and transmission electron microscopy. We found that ATP6V0A1 protected against lysosomal dysfunction, regulated autophagic flux, and decreased phosphorylated α-synuclein levels in vitro . In vivo , ATP6V0A1 reduced levels of α-synuclein and phosphorylated α-synuclein proteins, mitigated degeneration of dopaminergic neurons, and improved motor dysfunction. Collectively, these findings show that ATP6V0A1 plays a protective role in Parkinson’s disease by modulating the autophagy-lysosomal pathway. A correlation between ATP6V0A1 and Parkinson’s disease susceptibility may serve as a biomarker for Parkinson’s disease, while the protective effects of ATP6V0A1 could represent a potential therapeutic target for the disease.

Tumor‐Associated Glycan Exploits Adenosine Receptor 2A Signaling to Facilitate Immune Evasion

2025-06-18

Jing‐Yan Cheng , Hsiu‐Hui Tsai , Jung‐Tung Hung +7 more | Advanced Science

Abstract Adenosine signaling is a crucial immunosuppressive pathway within the tumor microenvironment, making it a promising target for cancer therapy. In this study, it is demonstrated that Globo H ceramide … Abstract Adenosine signaling is a crucial immunosuppressive pathway within the tumor microenvironment, making it a promising target for cancer therapy. In this study, it is demonstrated that Globo H ceramide (GHCer), the most prevalent tumor‐associated glycosphingolipid, influences the tumor microenvironment by activating adenosine signaling, which results in dual immunosuppressive effects on T cells. It is demonstrated that GHCer interacts with the adenosine receptor 2A (A2AR), triggering cyclic AMP (cAMP) and protein kinase A (PKA) signaling. This interaction leads to a reduction in the proliferation of CD4 + T cells while simultaneously promoting the differentiation of regulatory T cells (Tregs). Furthermore, GHCer enhances the suppressive capacity of Treg cells by upregulating inhibitory molecules such as Lymphocyte‐activation gene 3 (LAG3), cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4), Programmed cell death 1 ligand 1 (PD‐L1), and it stimulates the secretion of the immunosuppressive cytokine Interleukin 35 (IL‐35). Additionally, GHCer‐induced Tregs express CD39 and CD73, which further enhances adenosine production and creates a positive feedback loop in the adenosinergic pathway and A2AR signaling. Mechanistically, it is found that GHCer forms a complex with TRAX (translin‐associated factor‐X) and the C‐terminus of A2AR, which facilitates the activation of A2AR and promotes an immunosuppressive tumor microenvironment.

Pharmacological evaluation of non‐nucleotide purine derivatives as P2X7 antagonists for the treatment of neuroinflammation in traumatic brain injury

2025-06-18

Inés Valencia , Andrea Pastor‐Martínez , Celine Decouty‐Pérez +7 more | British Journal of Pharmacology

Background and purpose Traumatic brain injury (TBI) is considered to be a leading cause of mortality and disability worldwide. After TBI, innate immunity is rapidly activated in response to damage‐associated … Background and purpose Traumatic brain injury (TBI) is considered to be a leading cause of mortality and disability worldwide. After TBI, innate immunity is rapidly activated in response to damage‐associated molecular patterns, such as ATP release, recognised by P2X7 receptors. The P2X7‐NLRP3 inflammasome axis has been identified as one of the main players in neuroinflammation. This study aimed to validate P2X7 receptors as therapeutic target for traumatic brain injury. Experimental approach P2X7 receptors were studied by genetic and pharmacological approaches. Six non‐nucleotide purine derivatives were evaluated as P2X7 antagonists. Compounds that prevented LPS + ATP‐induced IL‐1β release from primary glial cultures were investigated in the closed‐head injury TBI model in vivo in male mice. Finally, we evaluated soluble (s)P2X7 receptor plasmatic levels in a cohort of TBI patients. Key results P2rx7 −/− mice showed an exaggerated inflammatory response 24 h post‐TBI compared to control mice. However, animals treated with the selective P2X7 antagonist JNJ‐47965567 (30 mg kg −1 i.p.) 30 min post‐TBI showed improved neurological and inflammatory parameters. The purine derivative ITH15004 was the most potent compound reducing IL‐1β production in vitro . When administered in vivo 30 min post‐TBI, ITH15004 (1 mg kg −1 i.p.) improved both neuro‐behavioural and inflammatory markers at 24 h. In TBI patients, we showed a tendency towards increase in circulating sP2X7 receptor levels at 24 and 72 h post‐TBI. Conclusions and implications These results highlight the importance of P2X7 receptors in the acute phase of TBI and present ITH15004 as a promising pharmacological tool to counteract P2X7 receptor‐dependent neuroinflammation in vivo .

State of the art indicators for imaging purinergic dynamics in vitro and in vivo

2025-06-17

Yumo Li , L Zhang , Bohan Li +2 more | Purinergic Signalling

Multi-Target Drug Strategies in the Treatment of Diabetic Retinopathy

2025-06-17

Angeline Julius , Suresh Malakondaiah , Ramalakshmi Subbarayalu +2 more | SN Comprehensive Clinical Medicine

ATP‐inducible Receptor Oligomerization Enables Fine‐tuning of T Cell Immunity

2025-06-16

Jianjun He , Haoxiang Li , Feng Liang +5 more | Angewandte Chemie

Receptor oligomerization plays a pivotal role in the regulation of cellular behaviors and functionalities. Stimuli‐responsive artificial circuits are often programmed to rewire cell signaling by fine‐tuning receptor interactions through targeted … Receptor oligomerization plays a pivotal role in the regulation of cellular behaviors and functionalities. Stimuli‐responsive artificial circuits are often programmed to rewire cell signaling by fine‐tuning receptor interactions through targeted stimulation. Notably, the integration of tumor microenvironment (TME)‐responsive systems capable of locally altering cancer cell phenotypes or transforming anti‐tumor responses of immune cells offers a novel and promising strategy for enhancing the therapeutic effectiveness and safety of cancer treatments. In this paper, we introduce an adenosine triphosphate (ATP)‐inducible receptor oligomerization (ATIRO) approach to facilitate the clustering of CD3 and CD8 of T cell and the engagement of CD3 and tumor marker of T cells and targeted cancer cells, to prompt anti‐tumor immunity. ATIRO enables the specific in‐situ activation of T cells by the high‐level ATP presented in TME, thereby effectively suppressing tumor growth in vivo. ATIRO presents a versatile and readily adaptable strategy for the in‐situ reconfiguration of T cell immunity, holding significant potential for advancing cancer therapeutic interventions.

ATP‐inducible Receptor Oligomerization Enables Fine‐tuning of T Cell Immunity

2025-06-16

Jianjun He , Haoxiang Li , Feng Liang +5 more | Angewandte Chemie International Edition

P2Y2 receptors are essential for hepatic clearance of uropathogenic E. coli in a murine sepsis model

2025-06-14

Mette G. Christensen , Nanna Johnsen , Laura V. Sparsoe +2 more | bioRxiv (Cold Spring Harbor Laboratory)

Urosepsis is a life-threatening condition most frequently caused by E. coli expressing important virulence factors, including α-haemolysin (HlyA). The pore-forming exotoxin HlyA releases ATP upon its insertion into cellular membranes, … Urosepsis is a life-threatening condition most frequently caused by E. coli expressing important virulence factors, including α-haemolysin (HlyA). The pore-forming exotoxin HlyA releases ATP upon its insertion into cellular membranes, and the majority of the biological effects of HlyA are mediated through ATP-dependent P2-receptor activation, including the HlyA-mediated thrombocyte activation. We have recently shown that uropathogenic E. coli (UPEC) bind to thrombocytes immediately after entering the blood, and the following hepatic clearance leads to early thrombocytopenia during bacteraemia. Here, we demonstrate that P2Y 2 -deficient mice had markedly shorter survival (LD50 of 185 minutes) compared to wildtype (340 minutes), a response paralleled in mice infused with the P2Y 2 receptor antagonist AR-C118925XX. The P2Y 2 -/- mice exhibited a blunted sepsis-induced thrombocytopenia compared to wildtype and sepsis-induced reduction in mature neutrophils in the blood. Strikingly, the P2Y 2 -deficient mice had inadequate hepatic clearance of UPEC, resulting in the accumulation of bacteria in the lungs, while thrombocytes were mainly sequestered in the kidneys. Hence, it is likely that the insufficient hepatic elimination of UPEC is responsible for the reduced survival in the P2Y 2 -/- mice. Taken together, we show that the lack of functional P2Y 2 receptors is essential for fast and proper hepatic clearance of UPEC and the survival time during urosepsis. Moreover, the data support the notion that an early reduction in circulating thrombocytes is important for a relevant host response to acute bacteraemia.

Depletion of P2X4 receptor alleviates prostate cancer bone metastasis through reduced cancer cell invasiveness and enhanced cell adhesion activities

2025-06-14

Jiepei He , Yuhan Zhou , Hector M. Arredondo Carrera +3 more | Purinergic Signalling

Abstract Prostate cancer (PCa) preferentially metastasizes to bone, which remains incurable and contributes significantly to mortality and morbidity. The P2X4 receptor (P2X4R) is a receptor for ATP that is highly … Abstract Prostate cancer (PCa) preferentially metastasizes to bone, which remains incurable and contributes significantly to mortality and morbidity. The P2X4 receptor (P2X4R) is a receptor for ATP that is highly expressed in many cancer types including PCa and is positively associated with tumorigenesis. To understand the role of P2X4R in PCa biology, particularly in PCa bone metastasis, P2X4R ( P2RX4 ) was knocked out in human PCa cell line PC3 cells using the CRISPR/Cas9 system. Cell proliferation, apoptosis, migration, and invasion were examined using CyQUANT, Cell Meter Caspase 3/7, scratch and transwell assays. Results showed that depleting P2X4R significantly reduced cell proliferation and invasion and increased apoptosis compared to PC3 wildtype (WT) controls in vitro. To test their metastatic potential in vivo, PC3 WT and knock-out (KO) cells were intracardiacally injected into male BALB/c immunocompromised mice. Twenty-five days post-injection, there were no detectable tumours and associated bone destruction in the tibias of mice injected with KO cells, whereas tibias of over 50% mice injected with WT cells were occupied by tumour cells, with significant bone destruction observed ex vivo using micro-CT. Furthermore, RNA-seq and bioinformatic analysis of P2X4R KO cells demonstrated links between P2X4R and PCa cell adhesion, and other key signalling such as Wnt signalling. These findings suggest that P2X4R is a potential therapeutic target for PCa metastasis, particularly bone metastasis.

The Anti-Parkinsonian A2A Receptor Antagonist Istradefylline (KW-6002) Attenuates Behavioral Abnormalities, Neuroinflammation, and Neurodegeneration in Cerebral Ischemia: An Adenosinergic Signaling Link Between Stroke and Parkinson’s Disease

2025-06-13

Michael G. Zaki , Elisabet Jakova , Mahboubeh Pordeli +3 more | International Journal of Molecular Sciences

Stroke, the third leading cause of death worldwide, is a major cause of functional disability. Cerebral ischemia causes a rapid elevation of adenosine, the main neuromodulator in the brain. The … Stroke, the third leading cause of death worldwide, is a major cause of functional disability. Cerebral ischemia causes a rapid elevation of adenosine, the main neuromodulator in the brain. The inhibition of adenosine A2A receptors (A2ARs) has been introduced as a potential target in neurodegenerative disorders involving extracellular adenosine elevation. Istradefylline, a selective A2AR antagonist, has been approved for Parkinson’s disease (PD) adjunctive therapy and showed neuroprotective effects in PD and Alzheimer’s disease. However, the role of A2ARs in post-stroke neuronal damage and behavioral deficits remains unclear. We recently showed that A2AR antagonism prevented the adenosine-induced post-hypoxia synaptic potentiation of glutamatergic neurotransmission following the hypoxia/reperfusion of hippocampal slices. Here, we investigated the potential neuroprotective effects of istradefylline in male Sprague-Dawley rats subjected to pial vessel disruption (PVD) used to model a small-vessel stroke. Rats were treated with either a vehicle control or istradefylline (3 mg/kg i.p.) following PVD surgery for three days. Istradefylline administration prevented anxiety and depressive-like behaviors caused by PVD stroke. In addition, istradefylline significantly attenuated ischemia-induced cognitive impairment and motor deficits. Moreover, istradefylline markedly reduced hippocampal neurodegeneration, as well as GFAP/Iba-1, TNF-α, nNOS, and iNOS levels after PVD, but prevented the downregulation of anti-inflammatory markers TGF-β1 and IL-4. Together, these results suggest a molecular link between stroke and PD and that the anti-PD drug istradefylline displays translational potential for drug repurposing as a neuroprotective agent for cerebral ischemic damage.

906-P: The Predictive Value of Sirtuins and miRNAs in Cardiac Recovery after Acute Myocardial Infarction Treated with Empagliflozin

2025-06-13

ANNA NOWAK-SZWED , Ceren Eyileten , Zofia Wicik +5 more | Diabetes

Introduction and Objective: SGLT2 inhibitors, primarily used for type 2 diabetes, exhibit cardioprotective effects by improving myocardial metabolism, reducing oxidative stress, and modulating inflammation and fibrosis—key factors in acute myocardial … Introduction and Objective: SGLT2 inhibitors, primarily used for type 2 diabetes, exhibit cardioprotective effects by improving myocardial metabolism, reducing oxidative stress, and modulating inflammation and fibrosis—key factors in acute myocardial infarction (AMI). This study explores the molecular mechanisms of SGLT2 inhibitors, focusing on non-coding RNAs and sirtuin pathways, to identify biomarkers and strategies for preventing heart failure post-AMI. Methods: We analyzed microRNAs (miRNAs) involved in sirtuin pathways and validated miRNA and sirtuin gene expressions (SIRT1-7) in 243 patients at baseline and after 26 weeks of treatment (486 samples) using qRT-PCR. We also conducted SHAP analysis, miRNA target predictions, and enrichment analyses. Results: Bioinformatics identified interaction networks for SGLT2 (5225 nodes) and SIRT1-7 (top 100 interactors), annotated for oxidative stress, inflammation, fibrosis, and hypoxia-ischemia. Combining miRNA predictions with these networks identified 13 key miRNAs, including hsa-miR-34a-5p, miR-182-5p, and hsa-miR-302a-3p, targeting SIRT and SGLT2-related pathways. Empagliflozin treatment significantly increased SIRT6, SIRT7, and miR-34a while reducing SIRT4 (p&lt;0.05). Baseline SIRT2 and SIRT4 levels independently predicted unfavorable LVEF outcomes (AUC: 0.655, p=0.002), with improved prediction using a panel including miR-182-5p (AUC: 0.786, p=0.0003). NT-proBNP levels were higher in the unfavorable group after 26 weeks, but no baseline differences were observed. Conclusion: Empagliflozin modulates sirtuin and miRNA expression, suggesting cardioprotective effects in AMI. Baseline SIRT2, SIRT4, and miR-182-5p levels predict unfavorable LVEF outcomes, with a combined biomarker panel offering high accuracy. These findings highlight the potential of integrating sirtuins, miRNAs, and clinical biomarkers for improved cardiac outcome management post-AMI. Disclosure A. Nowak-Szwed: Speaker's Bureau; Novo Nordisk. C. Eyileten: None. Z. Wicik: None. S. Ahmadova: None. J. Siller-Matula: None. D. von Lewinski: Speaker's Bureau; Daiichi Sankyo, Novo Nordisk. Research Support; Merck Sharp & Dohme Corp. H. Sourij: Advisory Panel; Eli Lilly and Company. Speaker's Bureau; Eli Lilly and Company. Research Support; Eli Lilly and Company. Advisory Panel; Boehringer-Ingelheim. Speaker's Bureau; Daiichi Sankyo. Advisory Panel; Novo Nordisk A/S. Speaker's Bureau; Novo Nordisk A/S. Advisory Panel; Novartis AG, Amarin Corporation, Amgen Inc. M. Postula: None. Funding EMPATHYtrial 2019/ABM/01/00037NCN 2022/45/N/NZ7/0246

2137-LB: ATP-P2X7 Mediated T-Cell Activation in Obesity-Induced Type 2 Diabetes

2025-06-13

Yunmeng Liu , Sicheng Deng , P Rogers +4 more | Diabetes

Introduction and Objective: Obesity-induced type 2 diabetes (ObT2D) is known as a key driver of chronic inflammation, underscored by sustained T-cell activation. However, the link between this metabolic disorder and … Introduction and Objective: Obesity-induced type 2 diabetes (ObT2D) is known as a key driver of chronic inflammation, underscored by sustained T-cell activation. However, the link between this metabolic disorder and T-cell responses remains unclear. We propose that in ObT2D, excess extracellular ATP stimulates purinergic receptors on T cells, triggering metabolic shifts that fuel chronic inflammation. Methods: ObT2D was induced in mice by feeding a high-fat diet for 12 weeks. Blood, mesenteric adipose tissue, and spleens were harvested to assess extracellular ATP and the expression of P2X7 receptors on T cells. In vitro, freshly isolated T cells from wild-type and P2X7 knockout (P2X7KO) mice were treated with ATP to evaluate cytokine production and intracellular calcium influx. Additionally, metabolic/glycolytic activity was measured using Seahorse analyzer, and glucose uptake was quantified by flow cytometry using 2-NBDG in T cells treated with ATP. Results: Compared with lean controls, ObT2D mice exhibited markedly increased body weight, elevated blood glucose, and higher circulating ATP levels, along with enhanced ATP production in both mesenteric adipose tissue and splenic CD8T cells. In vitro, ATP stimulation upregulated activation markers and cytokine production in CD8Ts, indicating antigen-independent activation mediated by elevated extracellular ATP. Moreover, the ATP-induced cytokine production and calcium influx were abolished in P2X7KO-CD8Ts, confirming that ATP exerts its effects via the P2X7 receptor. Seahorse assays revealed that ATP-treated CD8Ts displayed enhanced glycolysis, and flow cytometry analysis showed a significant increase in glucose uptake capacity following ATP treatment. Conclusion: Our findings highlight a critical role for ATP-P2X7 signaling in T-cell activation and enhanced production of pro-inflammatory cytokines, thereby contributing to chronic inflammation in ObT2D. Targeting P2X7 may represent a promising therapeutic strategy to mitigate aberrant immune responses and improve metabolic health in ObT2D. Disclosure Y. Liu: None. S. Deng: None. P. Rogers: None. K. Deck: None. C. Mora: None. T. Rafferty: None. S. Mu: None. Funding NIH

Positive allosteric modulators of purinergic P2X4 receptors: design, synthesis and therapeutic potential of “click” ligands for multiple sclerosis

2025-06-12

Paloma Mata , Maialen Sagartzazu‐Aizpurua , Nataliia Berehova +7 more | European Journal of Medicinal Chemistry

Compound muscle action potential as an early functional in vivo measure of Sarm1 inhibition after sciatic nerve transection

2025-06-06

Seong Kwon Hur , Rebecca Leahey , Mitchell Geringer +7 more | Journal of Neuropathology & Experimental Neurology

Abstract The NADase sterile alpha and TIR motif containing 1 (Sarm1) protein drives axon degeneration after injury. Loss or inhibition of Sarm1 structurally protects axons after sciatic nerve transection (SNT) … Abstract The NADase sterile alpha and TIR motif containing 1 (Sarm1) protein drives axon degeneration after injury. Loss or inhibition of Sarm1 structurally protects axons after sciatic nerve transection (SNT) in vivo but whether Sarm1 also drives functional loss after nerve injury is less clear. We established compound muscle action potential (CMAP) as a novel functional correlate of Sarm1 activation in a SNT mouse model and evaluated its relationship with biochemical and a novel Cellpose-based histological axon detection measure. CMAP amplitudes were elicited 8 h post-SNT but reached near-floor levels by 24 h. Decreases in CMAP amplitude are delayed in a gene dose-dependent manner in Sarm1 knockout mice or by pharmacological Sarm1 inhibition. Myelinated axon density, the NAD hydrolysis product cyclic adenosine diphosphate ribose (cADPR), and the axon degeneration plasma biomarker neurofilament light (NfL) were all altered in a Sarm1-dependent manner. In wild type mice, axon density and NfL were altered at time points after that of cADPR and functional loss, indicating that functional deficits preceded structural deficits. We conclude that functional and structural declines after injury are delayed by Sarm1 inhibition and that CMAP measures after SNT can serve as a novel, preclinical, functional, pharmacodynamic readout for Sarm1 inhibition.

Purinergic receptor activation rectifies autism-associated endothelial dysfunction

2025-06-03

Julie Ouellette , Sarah Warsi , Phinea Z. Romero +7 more | bioRxiv (Cold Spring Harbor Laboratory)

Early cerebrovascular alterations affect brain maturation by impacting trophic support and energy supply. Recent evidence in a 16p11.2 deletion mouse model of autism spectrum disorder (ASD) revealed brain endothelial abnormalities … Early cerebrovascular alterations affect brain maturation by impacting trophic support and energy supply. Recent evidence in a 16p11.2 deletion mouse model of autism spectrum disorder (ASD) revealed brain endothelial abnormalities postnatally. Yet, the endothelial alterations eliciting these changes remain unknown. Isolation of brain endothelial cells (ECs) from 14-day old male 16p11.2-deficient and wild-type mice revealed that 16p11.2 deletion-induced endothelial dysfunction is linked to a bioenergetic failure, with reduced intracellular ATP. Intra- or extra-cellular ATP supplementation rescued the function of 16p11.2-deficient ECs in vitro via P2 purinergic receptor activation, specifically P2Y2 receptors. Activating P2Y2 receptors restored cerebrovascular reactivity in 16p11.2-deficient parenchymal arterioles ex vivo and rescued 16p11.2 deletion-associated mouse behaviors. Taken together, this study demonstrates that metabolic reprogramming of brain ECs via purinergic receptor engagement represents a possible therapeutic avenue for ASD.

The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs

2025-06-01

Zimeng Zhuang , Chunshan Han , Meilian Cai +7 more | Journal of Advanced Research

Comparative analysis of the antiglioblastoma activity of adenosine A2A receptor and CK1δ blockers

2025-06-01

Akshaya Murugesan , Anxo Vila Alonso , Konda Mani Saravanan +7 more | European Journal of Pharmacology

Targeting Degradation of the Transcription Factor C/EBPβ Reduces Lung Fibrosis by Restoring Activity of the Ubiquitin-Editing Enzyme A20 in Macrophages

2025-06-01

Shan-shan Liu , Xiao-Xi Lv , Chang Liu +7 more | Immunity

Beneficial and detrimental consequences of AHR activation in intestinal infection

2025-06-01

Oscar E. Diaz , Liang zhou , Christopher Barrington +4 more | bioRxiv (Cold Spring Harbor Laboratory)

The ligand dependent transcription factor aryl hydrocarbon receptor (AHR) is an environmental sensor whose activation can have physiologically beneficial or detrimental consequences for host immune responses depending on the ligand. … The ligand dependent transcription factor aryl hydrocarbon receptor (AHR) is an environmental sensor whose activation can have physiologically beneficial or detrimental consequences for host immune responses depending on the ligand. Here we investigated the hypothesis that prolonged AHR activation either due to inefficient ligand metabolism or due to genetic manipulation may underlie the distinction between beneficial and detrimental effects. Our data indicate that prolonged AHR activation caused toxic endpoints for liver and thymus but was not per se interfering with the host response to infection with the intestinal pathogen C.rodentium. Genetically driven constitutive AHR activation improved resistance to infection, whereas prolonged AHR activation by the pollutant TCDD resulted in delayed clearance of C.rodentium associated with a suppression in antibody production. Combined single cell RNAseq and ATAC-seq analysis provided evidence that TCDD, but not genetic AHR activation, negatively affected dendritic cell functions such as activation, maturation and antigen presentation. Thus, the detrimental impact of environmental pollutants such as TCDD on immune responses cannot solely be attributed to aberrantly prolonged activation of AHR.