P2X currents in peritoneal macrophages of wild type and P2X4−/− mice

Brône, Bert; Moechars, Diederik; Marrannes, Roger; Mercken, Marc; Meert, Theo

doi:10.1016/j.imlet.2007.07.015

Cited by 33 publications

(40 citation statements)

References 41 publications

(59 reference statements)

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“…These results are consistent with the absence of functional P2X 7 receptors in Leydig cells under our experimental conditions. Ivermectin is known to act only on P2X 4 receptors by binding to two distinct sites. The higher-affinity binding site (EC 50 =0.25 µM) is able to increase the maximal ATPactivated currents without changing the ATP concentrationresponse relationship.…”

Section: Discussionmentioning

confidence: 99%

“…The expression of purinergic receptors P2X [1][2][3][4][5][6][7] in Leydig cells was detected by Western blot analysis. Since a large number of high purity Leydig cells are needed for these experiments, we used 20 testes in each preparation.…”

Section: Western Blotsmentioning

confidence: 99%

“…Proteins were then transferred to nitrocellulose membranes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) by Bio-Rad Trans-Blot SD semidry transfer cell (Bio-Rad Laboratories Inc., Richmond, CA, USA). Membranes were blocked with 5% BSA in TBST (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 h, and then incubated for 1 h with anti-P2X [1][2][3][4][5][6][7] (1:400) polyclonal antibodies (Alomone Labs Ltd., Jerusalem, Israel) diluted in 5% BSA in TBST. After washing, membranes were incubated for 1 h with goat anti-rabbit IGg conjugated to horseradish peroxidase, diluted in TBST.…”

Section: Western Blotsmentioning

confidence: 99%

“…The coverslips were incubated 120 min with the same primary antibodies anti-P2X [1][2][3][4][5][6][7] (1:200; rabbit) and anti-calreticulin (Chemicon International, Temecula, California, USA; 1:200; chicken) diluted with 0.01 M PBS, 0.5% Triton X-100, 1% BSA. Then, the cells were incubated with secondary antibodies Alexa Fluor 488 (goat, anti-rabbit) and 594 (goat, anti-chicken; Molecular Probes Inc., Eugene, USA) for 60 min, both in a dilution of 1:800.…”

Section: Immunofluorescencementioning

confidence: 99%

“…Seven P2X receptor subunits (P2X 1-7 ) were cloned and characterized in vertebrates, and each of them can form two type of assemblies, homomeric, P2X 1 [50], P2X 2 [3], P2X 3 [8], P2X 4 [2], P2X 5 [18], P2X 7 [46], and P2X 6 [23], or heterotrimeric P2X 2/3 [31], P2X 1/5 [48], P2X 4/6 [30], P2X 2/6 [27], P2X 1/2 [5], P2X 1/4 [38], and P2X 4/7 [19]. Each subunit is composed by two transmem-brane regions (TM1 and TM2) forming the channel pore, a large extracellular domain, which contains the ATP binding site and both N and C termini located intracellularly [14,48,22].…”

Section: Introductionmentioning

confidence: 99%

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Mouse Leydig cells express multiple P2X receptor subunits

Antonio

Costa

Gomes

et al. 2008

Purinergic Signalling

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ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X 1 -P2X 7 ) and seven heteromeric receptors (P2X 1/2 , P2X 1/4 , P2X 1/5 , P2X 2/3 , P2X 2/6 , P2X 4/6 , P2X 4/7 ) have been described. ATP treatment of Leydig cells leads to an increase in [Ca 2+ ] i and testosterone secretion, supporting the hypothesis that Ca 2+ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X 2 . In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X 2 , P2X 4 , P2X 6 , and P2X 7 subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 μM ivermectin induced an increase (131.2±5.9%) and 3 μM ivermectin a decrease (64.2±4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X 4 subunits. P2X 7 receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X 2/4/6 , is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X 2 subunit.

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Section: Discussionmentioning

confidence: 99%

Section: Western Blotsmentioning

confidence: 99%

Section: Western Blotsmentioning

confidence: 99%

Section: Immunofluorescencementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mouse Leydig cells express multiple P2X receptor subunits

Antonio

Costa

Gomes

et al. 2008

Purinergic Signalling

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NTPDase1 governs P2X₇‐dependent functions in murine macrophages

et al. 2010

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P2X 7 receptor is an adenosine triphosphate (ATP)-gated ion channel within the multiprotein inflammasome complex. Until now, little is known about regulation of P2X 7 effector functions in macrophages. In this study, we show that nucleoside triphosphate diphosphohydrolase 1 (NTPDase1)/CD39 is the dominant ectonucleotidase expressed by murine peritoneal macrophages and that it regulates P2X 7 -dependent responses in these cells. Macrophages isolated from NTPDase1-null mice (Entpd1) were devoid of all ADPase and most ATPase activities when compared with WT macrophages (Entpd1 1/1 ). Entpd1À/À macrophages exposed to millimolar concentrations of ATP were more susceptible to cell death, released more IL-1b and IL-18 after TLR2 or TLR4 priming, and incorporated the fluorescent dye Yo-Pro-1 more efficiently (suggestive of increased pore formation) than Entpd1 1/1 cells. Consistent with these observations, NTPDase1 regulated P2X 7 -associated IL-1b release after synthesis, and this process occurred independently of, and prior to, cytokine maturation by caspase-1. NTPDase1 also inhibited IL-1b release in vivo in the air pouch inflammatory model. Exudates of LPS-injected Entpd1 À/À mice had significantly higher IL-1b levels when compared with Entpd1 1/1 mice. Altogether, our studies suggest that NTPDase1/CD39 plays a key role in the control of P2X 7 -dependent macrophage responses.Key words: ATP-induced death . CD39 . IL-1b . Macrophage . Nucleoside triphosphate diphosphohydrolase 1Supporting Information available online IntroductionMonocytes/macrophages are myeloid inflammatory cells that play important roles in innate host defense and immune regulation [1]. For example, these cells represent the major source of IL-1 during inflammation, a cytokine playing a pivotal role in chronic inflammatory diseases such as rheumatoid arthritis and several neurodegenerative disorders [2,3]. Monocytes/macrophages express functional P2 receptors specific for extracellular nucleotides, which serve as ''danger'' signals and initiate the immune response [4]. At the mRNA level, primary monocytes express the ion-channel P2X 1,4,5,7 receptors and the G-protein-coupled P2Y 1,2,4,6,11-13 receptors, and macrophages express the same receptor subtypes except P2Y 13 [5]. P2Y 1 , P2Y 2 , [6][7][8]. P2X 7 receptor (formerly P2Z) plays a key role in inflammation [9,10]. P2X 7 receptor activation by extracellular adenosine triphosphate (ATP), released under inflammatory conditions, is a co-stimulus for a massive release of mature and bioactive cytokines of the IL-1 family, such as IL-1b and IL-18, from LPS-primed macrophages and other cell types [11]. The activation of P2X 7 in leucocytes also induces cell death, intracellular pathogen clearance and membrane trafficking [12][13][14]. While nano to low micromolar nucleotide concentrations are sufficient for the activation of other P2 receptors, P2X 7 is activated by far higher concentrations of ATP (in the order of the millimolar (mM)) and/ or by mono adenosine diphosphate (ADP) ribosylation [15].The ...

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Physiologic roles of P2 receptors in leukocytes

Alberto

Ferreira

Bonavita

et al. 2022

Journal of Leukocyte Biology

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Since their discovery in the 1970s, purinergic receptors have been shown to play key roles in a wide variety of biologic systems and cell types. In the immune system, purinergic receptors participate in innate immunity and in the modulation of the adaptive immune response. In particular, P2 receptors, which respond to extracellular nucleotides, are widely expressed on leukocytes, causing the release of cytokines and chemokines and the formation of inflammatory mediators, and inducing phagocytosis, degranulation, and cell death. The activity of these receptors is regulated by ectonucleotidases—expressed in these same cell types—which regulate the availability of nucleotides in the extracellular environment. In this article, we review the characteristics of the main purinergic receptor subtypes present in the immune system, focusing on the P2 family. In addition, we describe the physiologic roles of the P2 receptors already identified in leukocytes and how they can positively or negatively modulate the development of infectious diseases, inflammation, and pain.

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P2X currents in peritoneal macrophages of wild type and P2X4−/− mice

Cited by 33 publications

References 41 publications

Mouse Leydig cells express multiple P2X receptor subunits

Mouse Leydig cells express multiple P2X receptor subunits

NTPDase1 governs P2X₇‐dependent functions in murine macrophages

Physiologic roles of P2 receptors in leukocytes

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P2X currents in peritoneal macrophages of wild type and P2X4−/− mice

Cited by 33 publications

References 41 publications

Mouse Leydig cells express multiple P2X receptor subunits

Mouse Leydig cells express multiple P2X receptor subunits

NTPDase1 governs P2X7‐dependent functions in murine macrophages

Physiologic roles of P2 receptors in leukocytes

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NTPDase1 governs P2X₇‐dependent functions in murine macrophages