Journal: Molecular medicine (Cambridge, Mass.)

Article Title: The vasoconstrictor adenosine 5'-tetraphosphate is a danger signal that induces IL-1β.

doi: 10.1186/s10020-025-01116-6

Figure Lengend Snippet: Fig. 3 Effects of Ap4 on P2X7 receptor. A and B HEK293 cells were transfected with P2X7 receptor (pUNO1-hP2RX7) or control plasmid (pUNO-mcs). A After transfection, HEK293 cells were loaded with Fluo-4 AM and probenecid. Ap4 (5 mM), BzATP (300 µM) or calcimycin (10 µM) were added after 20 s. Calcium influx was determined by fluorescence intensity measurement. The graph shows the mean values related to the basal level (n = 3). B Cells were loaded with YO-PRO-1 (2 µM) and stimulated for 1 h without or with BzATP (300 µM) or Ap4 (5 mM). Flow cytometry was used to quantify the change in YO-PRO-1 uptake, as indicated by fluorescence intensity in the FITC channel, after stimulation with either Ap4 or BzATP (depicted by colored curves), in comparison to the untreated control (represented by light grey curves). Heat-killed cells were used as positive control. The dark grey curve shows unstained cells. The histograms are representative of three independent experiments. Mean + SEM (n = 3–6). C THP-1 macrophages were primed with Pam3CSK4 (1 µg/ml) for 3 h and then stimulated with BzATP (300 µM) for 3 h. Ap4 (5 mM) was added 1 h before stimulation. IL-1β release in cell culture supernatants was determined by ELISA. IL-1β release induced by BzATP was set to 100%. All other values were calculated accordingly. Bar graphs show mean ± SEM (n = 3). One-sample t-test against 100%, *P ≤ 0.05

Article Snippet: In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 (1 μM; 4473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxidized ATP (oxATP) (300 μM; 505758, Merck, Darmstadt, Germany), P2X4 receptor antagonist 5-BDBD [5-(3-Bromophenyl)1,3-dihydro-2 H-Benzofuro[3,2-e]-1,4-diazepin-2-one] (25 μM; SML0450-5MG, Sigma-Aldrich, Taufkirchen, Germany), P2 receptor antagonist PPADS (100 μM; 0625, Tocris Bioscience, Bristol, United Kingdom), Gq/11 inhibitor YM-254,890 (1 μM; AG-CN2-0509, Biomol, Hamburg, Germany), NLRP3 inhibitor MCC950 (10 μM; 5479, Tocris Bioscience, Bristol, United Kingdom) or NF-κB inhibitor Bay 11-7082 (20 μM; B5556, SigmaAldrich, Taufkirchen, Germany), Caspase-1 inhibitor Ac-YVAD-cmk (40 μM; 10014, Biomol, Hamburg, Germany), pan-caspase inhibitor Z-VAD-fmk (40 μM; tlrl-vad, Invivogen, Toulouse, France), serine protease inhibitor AEBSF (300 μM; 50985.100, Biomol, Hamburg, Germany), cysteine protease inhibitor E64 (10 μM; 324890, Merck, Darmstadt, Germany), aspartic protease inhibitor pepstatin A (50 μM; 2936, Carl Roth, Karlsruhe, Germany), l-homocysteine (100 μM; Cay30852-5, Biomol, Hamburg, Germany), or ion chelator TPEN [N, N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine] (10 μM; Cay13340-10, Biomol, Hamburg, Germany) for 1 h before stimulation.

Techniques: Transfection, Control, Plasmid Preparation, Fluorescence, Flow Cytometry, Comparison, Positive Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: eNeuro

Article Title: Preconditioning-Induced Facilitation of Lactate Release from Astrocytes Is Essential for Brain Ischemic Tolerance

doi: 10.1523/ENEURO.0494-23.2024

Figure Lengend Snippet: P2X7 receptor-mediated increase in extracellular lactate levels during severe ischemia and the neuroprotective effects of lactate on cerebral ischemic injury. a , b , In the in vivo microdialysis experiment, a probe was inserted into the ipsilateral striatum. Artificial cerebrospinal fluid was perfused and collected to measure lactate. The extracellular lactate levels in the dialysate samples were determined using a lactate assay kit. Control samples were collected from naive WT mice. The extracellular lactate levels during severe middle cerebral artery occlusion (MCAO; for 1 h) were increased compared with the control. In mice who received preconditioning (PC; 15 min of MCAO) 3 d before severe MCAO, extracellular lactate levels during severe MCAO had a greater increase compared with mice who underwent severe MCAO without PC. When P2X7 receptor knock-out mice received PC 3 d before severe MCAO, their extracellular lactate levels during severe MCAO were lower than those in WT mice. Data are presented as the fold increase over control (naive WT) mice. Values are shown as mean ± SEM; ** p < 0.01, one-way ANOVA followed by Tukey's post hoc multiple-comparisons test; n = 4 ( a ); * p < 0.05, unpaired two-tailed Student's t test; n = 3–4 ( b ). Additional data are presented in Extended Data . c , Mice received a lateral ventricle injection of 1 µl of 100 mM lactate 1 d before severe MCAO. Brain damage was assessed by TTC staining of coronal brain sections. Lactate injection protected against severe MCAO-induced ischemic injury. Scale bar, 2 mm. The results are summarized in d . Values are shown as mean ± SEM; ** p < 0.01, unpaired two-tailed Student's t test; n = 5–6.

Article Snippet: To investigate the role of P2X7 receptors in lactate release during severe ischemia, the P2X7 receptor antagonist JNJ-47965567 (30 mg/kg; Selleck Chemicals) dissolved in 30% sulfobutylether-β-cyclodextrin solution was injected intraperitoneally into mice 1 d before severe MCAO (i.e., 1 h of occlusion) was induced.

Techniques: In Vivo, Lactate Assay, Control, Knock-Out, Two Tailed Test, Injection, Staining

Journal: eNeuro

Article Title: Preconditioning-Induced Facilitation of Lactate Release from Astrocytes Is Essential for Brain Ischemic Tolerance

doi: 10.1523/ENEURO.0494-23.2024

Figure Lengend Snippet: P2X7 receptor agonist treatment upregulated CD147 in cultured astrocytes. Primary astrocyte cultures were treated with various concentrations of the P2X7 receptor agonist BzATP for 24 h at the indicated concentrations. Western blotting was then performed. Protein levels of HIF-1α and its target gene CD147 were significantly increased by BzATP treatment. In contrast, monocarboxylate transporter (MCT) 1 and MCT4 protein levels were unchanged by BzATP treatment. Data are representative of four independent experiments. The levels of these proteins were normalized to those of β-actin. Data show the fold increase over controls (no treatment). Values are shown as mean ± SEM; * p < 0.05, ** p < 0.01 versus control, one-way ANOVA followed by Dunnett's post hoc multiple-comparisons test; n = 4.

Article Snippet: To investigate the role of P2X7 receptors in lactate release during severe ischemia, the P2X7 receptor antagonist JNJ-47965567 (30 mg/kg; Selleck Chemicals) dissolved in 30% sulfobutylether-β-cyclodextrin solution was injected intraperitoneally into mice 1 d before severe MCAO (i.e., 1 h of occlusion) was induced.

Techniques: Cell Culture, Western Blot, Control

Journal: eNeuro

Article Title: Preconditioning-Induced Facilitation of Lactate Release from Astrocytes Is Essential for Brain Ischemic Tolerance

doi: 10.1523/ENEURO.0494-23.2024

Figure Lengend Snippet: Schematic diagram of the mechanisms underlying preconditioning (PC)-induced ischemic tolerance. PC-evoked astrocytic activation induces CD147 expression via the P2X7 receptor/HIF-1α signaling pathway. This CD147 expression assists with the translocation of monocarboxylate transporter (MCT) 1 and MCT4 to the cell membrane, thereby promoting lactate release from astrocytes during severe ischemia; this effect likely plays a role in ischemic tolerance.

Article Snippet: To investigate the role of P2X7 receptors in lactate release during severe ischemia, the P2X7 receptor antagonist JNJ-47965567 (30 mg/kg; Selleck Chemicals) dissolved in 30% sulfobutylether-β-cyclodextrin solution was injected intraperitoneally into mice 1 d before severe MCAO (i.e., 1 h of occlusion) was induced.

Techniques: Activation Assay, Expressing, Translocation Assay, Membrane

Journal: eNeuro

Article Title: Preconditioning-Induced Facilitation of Lactate Release from Astrocytes Is Essential for Brain Ischemic Tolerance

doi: 10.1523/ENEURO.0494-23.2024

Figure Lengend Snippet: P2X7 receptor agonist evoked the membrane translocation of monocarboxylate transporter (MCT) 1 and MCT4 via CD147 in cultured astrocytes. a , Primary astrocyte cultures were treated with 10 µM of the P2X7 receptor agonist BzATP for 24 h. Immunocytochemistry was then performed. Representative immunocytochemical images of the cultured astrocytes stained with anti-MCT1 (red) or anti-MCT4 (red) antibodies are shown. BzATP treatment induced the translocation of MCT1 and MCT4 from the perinuclear region to the cell surface in cultured astrocytes. Scale bar, 20 µm. b , Primary astrocyte cultures were treated with 10 µM BzATP and 0.1 µg/ml anti-CD147 or its isotype control for 24 h. Membrane protein extraction and Western blotting were then performed. In cultured astrocytes, the membrane expression levels of MCT1 and MCT4 were significantly increased by BzATP treatment; however, these increases were suppressed by anti-CD147. Data are representative of four independent experiments. The levels of these proteins were normalized to those of Na + /K + -ATPase. Data show the fold increase over control (control IgG treatment). Values are shown as mean ± SEM; * p < 0.05, ** p < 0.01, two-way ANOVA followed by Tukey's post hoc multiple-comparisons test; n = 4.

Article Snippet: To investigate the role of P2X7 receptors in lactate release during severe ischemia, the P2X7 receptor antagonist JNJ-47965567 (30 mg/kg; Selleck Chemicals) dissolved in 30% sulfobutylether-β-cyclodextrin solution was injected intraperitoneally into mice 1 d before severe MCAO (i.e., 1 h of occlusion) was induced.

Techniques: Membrane, Translocation Assay, Cell Culture, Immunocytochemistry, Staining, Control, Protein Extraction, Western Blot, Expressing