Journal: bioRxiv

Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

doi: 10.1101/2021.09.14.460103

Figure Lengend Snippet: (A-C) P2X7 expression analysed by western blotting of macrophages isolated from control, EROS -/- (A) and gp91 phox -/- mice (B) and of control PLB985 cells and an EROS knock-out clone (C). (D-E) P2X7 expression in RAW264.7 cells overexpressing a FLAG-tagged EROS vector (D) and in HEK293 cells transiently expressing the specified constructs (E). (F-G) Interaction between EROS and P2X7 probed by immunoprecipitation of EROS from RAW264.7 EROS-FLAG macrophages followed by immunoblot for P2X7 (F) and by NanoBIT assay in live HEK293 cells expressing the LgBIT-fused EROS vector with a SmBIT-fused P2X7 vector (G). (H) P2X1 expression in macrophages isolated from EROS -/- mice compared to control. n= 5 biological replicates. (I) P2X1 abundance upon co-transfection with EROS construct in HEK293 cells. n= representative of 3 independent experiments. See also Figure S3.

Article Snippet: The following plasmids were obtained from Origene: mouse gp91 phox /nox2/cybb (MC204867), mouse GFP-tagged gp91 phox /nox2/cybb (MG208975), mouse EROS/cybc1 (MC201263), human EROS/CYBC1 (SC324452), human gp91 phox /CYBB (SC122091), human p22 phox /CYBA (SC101113), human NOX4 (SC322623), mouse GFP-tagged P2X7 (MR227216), human P2X1 (SC118594) and human P2X4 (SC122124).

Techniques: Expressing, Western Blot, Isolation, Knock-Out, Plasmid Preparation, Construct, Immunoprecipitation, Cotransfection

Journal: International Journal of Molecular Sciences

Article Title: Spotlight on P2X7 Receptor PET Imaging: A Bright Target or a Failing Star?

doi: 10.3390/ijms24021374

Figure Lengend Snippet: ( A ) Overview of different P2X7R antagonists divided by targeting either the CNS or the peripheral nervous system. ( B ) Common representatives of different structural classes of imaging compounds. Radioisotopes are highlighted with different colors.

Article Snippet: The first P2X7R imaging ligand that went into in vivo testing was [ 11 C]A740003, developed by Abbott Laboratories, after showing promising results in initial in vitro assays.

Techniques: Imaging

Journal: International Journal of Molecular Sciences

Article Title: Spotlight on P2X7 Receptor PET Imaging: A Bright Target or a Failing Star?

doi: 10.3390/ijms24021374

Figure Lengend Snippet: P2X7R PET tracers.

Article Snippet: The first P2X7R imaging ligand that went into in vivo testing was [ 11 C]A740003, developed by Abbott Laboratories, after showing promising results in initial in vitro assays.

Techniques: In Vitro, Binding Assay, In Vivo, Autoradiography, Imaging

Journal: Pharmaceuticals

Article Title: Notopterol Ameliorates Hyperuricemia-Induced Cardiac Dysfunction in Mice

doi: 10.3390/ph16030361

Figure Lengend Snippet: NLRP3 and P2X7R were upregulated in heart tissue of hyperuricemic mice, notopterol suppressed P2X7R and NLRP3 inflammasome signals. ( A ) The expression level of P2X7R, NLRP3, Caspase-1 and Cleaved caspase-1 p20 in heart tissues were analyzed by Western blot. ( B ) The quantitative statistical diagrams of P2X7R, NLRP3, Caspase1, and Cleaved caspase-1 p20 detected by Western blot (n = 5 mice/group). (C) The mRNA level of P2X7R in heart tissues were analyzed by RT-PCR. ( D ) Representative immunohistochemical images of NLRP3 (brown) in mice heart tissues. Scale bar: 100 µm. Data represent means ± S.E.M. The symbol “∗” represents p < 0.05 vs. control. # p < 0.05 vs. hyperuricemia group.

Article Snippet: For siRNA transfection, H9c2 cells were cultured in 6-well culture plate for 12 h. Then the cells were transfected with control siRNA or P2X7R siRNA (RiboBio, Guangzhou, China) using Lipofectamine 2000 ® (11668019; Invitrogen, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Control

Journal: Pharmaceuticals

Article Title: Notopterol Ameliorates Hyperuricemia-Induced Cardiac Dysfunction in Mice

doi: 10.3390/ph16030361

Figure Lengend Snippet: P2X7R was upregulated in uric acid induced H9C2 cells, notopterol suppressed P2X7R signals. ( A ) The expression level of P2X7R was determined by western blot analysis after treatment of various concentrations of uric acid (0, 100, 200, and 400 mg/L) for 24 h in H9c2 cells. ( B ) The quantitative statistical diagrams of P2X7R detected by Western blot (n = 3/group). ( C ) H9c2 cells were treated with uric acid (200 mg/L) with/without notopterol (25 μM) administration for 24 h. The expression level of P2X7R was determined by western blot. ( D ) The quantitative statistical diagrams of P2X7R detected by Western blot (n = 3/group). ( E ) After H9c2 cells were treated with uric acid (200 mg/L) with/without notopterol (25 μM) administration for 24 h, P2X7R was detected by fluorescence analysis in H9c2 cells. Green: P2X7R. Blue, DAPI. ( F ) Fluorescence intensity quantification by image J software (n = 3/group). Scale bar = 50 μm. Data represent means ± S.E.M. ∗ p < 0.05 vs. control; # p < 0.05 vs. uric acid treatment. UA, Uric acid. NOT, notopterol.

Article Snippet: For siRNA transfection, H9c2 cells were cultured in 6-well culture plate for 12 h. Then the cells were transfected with control siRNA or P2X7R siRNA (RiboBio, Guangzhou, China) using Lipofectamine 2000 ® (11668019; Invitrogen, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Fluorescence, Software, Control

Journal: Pharmaceuticals

Article Title: Notopterol Ameliorates Hyperuricemia-Induced Cardiac Dysfunction in Mice

doi: 10.3390/ph16030361

Figure Lengend Snippet: P2X7R regulated NLRP3 inflammasome signals and inflammatory cytokines in H9C2 cells. ( A ) H9c2 cells were treated with uric acid (200 mg/L) with/without BBG (10 μM) administration for 24 h. The expression level of P2X7R, NLRP3, Cleaved caspase-1 p20, IL-18 and IL-1β were determined by western blot. ( B , C ) The quantitative statistical diagrams of P2X7R, NLRP3, Caspase-1 and Cleaved caspase-1 p20 detected by Western blot (n = 3/group). ( D ) H9c2 cells were transfected with control siRNA and siP2X7R, and then treated with/without uric acid (200 mg/L) for 24 h. The expression level of P2X7R, NLRP3, Cleaved caspase-1 p20, IL-18 and IL-1β were determined by western blot. ( E – I ) The quantitative statistical diagrams of P2X7R, NLRP3, Caspase-1 and Cleaved caspase-1 p20 detected by Western blot (n = 3/group). Data represent means ± S.E.M. ∗ p < 0.05 vs. control; # p < 0.05 vs. uric acid treatment. UA, Uric acid. NOT, notopterol.

Article Snippet: For siRNA transfection, H9c2 cells were cultured in 6-well culture plate for 12 h. Then the cells were transfected with control siRNA or P2X7R siRNA (RiboBio, Guangzhou, China) using Lipofectamine 2000 ® (11668019; Invitrogen, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Transfection, Control

Journal: Pharmaceuticals

Article Title: Notopterol Ameliorates Hyperuricemia-Induced Cardiac Dysfunction in Mice

doi: 10.3390/ph16030361

Figure Lengend Snippet: Notopterol alleviated uric acid induced pyroptosis partially via regulating P2X7R/NLRP3 axis. ( A ) H9c2 cells were transfected with control plasmid or oeP2X7R, and then treated with uric acid (200 mg/L) with or without notopterol (25 μM) treatment for 24 h. The expression levels of P2X7R, NLRP3, Cleaved caspase-1 p20, IL-18 and IL-1β were determined by western blot. ( B – F ) The quantitative statistical diagrams of P2X7R, NLRP3, Cleaved caspase-1 p20, IL-18 and IL-1β detected by Western blot (n = 3/group). Data represent means ± S.E.M. ∗ p < 0.05 vs. control; # p < 0.05 vs. uric acid treatment. UA, Uric acid. NOT, notopterol.

Article Snippet: For siRNA transfection, H9c2 cells were cultured in 6-well culture plate for 12 h. Then the cells were transfected with control siRNA or P2X7R siRNA (RiboBio, Guangzhou, China) using Lipofectamine 2000 ® (11668019; Invitrogen, Waltham, MA, USA).

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Western Blot

Journal: Pharmaceuticals

Article Title: Notopterol Ameliorates Hyperuricemia-Induced Cardiac Dysfunction in Mice

doi: 10.3390/ph16030361

Figure Lengend Snippet: Summary of the findings of this study and schematic of mechanisms of Notopterol alleviate pyroptosis and inflammation in cardiac tissue of hyperuricemic mice. P2X7R played an important role in NLRP3 inflammasome activation and IL-18 and IL-18 production induced by uric acid. Notopterol inhibited P2X7R expression and suppressed NLRP3 inflammasome activation, inhibting the cleavage of caspase1, attenuated pyroptosis and inflammation in cardiac tissue of hyperuricemic mice. Black arrows indicate activation or promotion, red arrows represent upregulation. red T-shaped lines indicate inhibition.

Article Snippet: For siRNA transfection, H9c2 cells were cultured in 6-well culture plate for 12 h. Then the cells were transfected with control siRNA or P2X7R siRNA (RiboBio, Guangzhou, China) using Lipofectamine 2000 ® (11668019; Invitrogen, Waltham, MA, USA).

Techniques: Activation Assay, Expressing, Inhibition

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Gene Expression, Expressing, Standard Deviation

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Generated, CRISPR, Injection, In Vivo Imaging, RNA Sequencing, Immunohistochemical staining, Staining, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Suspension, Expressing, Negative Control, Injection, Flow Cytometry, Staining, Immunofluorescence, In Vivo Imaging, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Injection, Flow Cytometry, Isolation, RNA Sequencing, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Injection, Suspension, Purification, RNA Sequencing, Expressing, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: RNA Sequencing, Staining, Flow Cytometry, Confocal Microscopy, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Suspension, Flow Cytometry, Expressing, Isolation, Binding Assay, Cell Culture, Staining, Transfection, Plasmid Preparation, Standard Deviation, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

doi: 10.1523/JNEUROSCI.0852-17.2017

Figure Lengend Snippet: Spinal microglial P2X7R expression and activity are increased in morphine tolerant rats. A, B, Primary rat spinal cord cultured cells. A, Representative dot plot of primary spinal cord cultures imaged using flow cytometry labeled for CD11b (BL2 area, x-axis) and P2X7R (RL1 area, y-axis). B, Histogram of P2X7R (RL1) intensity in CD11b-positive and CD11b-negative populations. C, P2X7R protein levels as measured by Western blot in spinal cord homogenates isolated from rats treated with MS (n = 13) or CTR (n = 13) for 7 d. Unpaired two-tailed t test (t = 3.3, df = 24, p = 0.0027). D–K, Spinal cord cells were acutely isolated from adult rats that had received 7 d of systemic MS or CTR treatment. D, Quantification of P2X7R (RL1) mean intensity in CD11b-negative and-positive populations from control and morphine-treated animals using flow cytometry. Two-way ANOVA (interaction: F(1,16) = 7.7; CD11b +ve/−ve: F(1,16) = 366.9; treatment: F(1,16) = 7.2), Sidak's post hoc test, main effect of population p < 0.0001; CD11b-negative CTR vs MS: p = 0.9969; CD11b-positive CTR vs MS: p = 0.0027. E, Representative dot plot of acutely isolated adult spinal cord cells from control-treated animals imaged using flow cytometry and labeled for CD11b (BL2 area, x-axis) and P2X7R (RL1 area, y-axis). CD11b-negative and CD11b-positive populations used for analysis are circled in black and red, respectively. F, Histogram of CD11b (BL2) single stained control overlaid with unstained cells in adult spinal cord cells. G, Histogram of P2X7R (RL1) single stained control overlaid with unstained cells in adult spinal cord cells. H, Dot plot of primary spinal cultures labeled with IgG-PE and anti-rabbit IgG-647 as controls overlaid with unstained cells. I, Representative image of adult spinal cord cells labeled with α-rat CD11b antibody to identify microglia. Representative images of microglia loaded with the Ca2+ indicator dye fura-2 AM in the 340 nm Ca2+ bound channel (40×). Scale bar, 20 μm. J, Representative Ca2+ tracings from microglia loaded with the Ca2+ indicator dye fura-2 AM. K, Peak rise in intracellular [Ca2+] evoked by BzATP. BzATP-evoked (100 μm) rise in intracellular [Ca2+] was greater in microglia isolated from MS- (n = 42 cells) compared with CTR- (n = 38 cells) treated rats. Unpaired two-tailed t test (t = 5.3, df = 20, p < 0.0001). All data represent mean ± SEM. ****p < 0.0001, **p < 0.01.

Article Snippet: P2X7R 356–371 (NTYASTCCRSRVYPSC, rat), P2X7R 356–371 (NTYSSAFCRSGVYPYC, mouse), P2X7R 379–389 (VNEYYYRKKCE, rat/mouse), inactive P2X7R Y379–389F (VNEFFFRKKCE, rat/mouse), P2X7R 546–556 (RHCAYRSYATW, rat), P2X7R 546–556 (RHRAYRCYATW, mouse), and P2X7R 586–595 (GQYSGFKYPY, rat/mouse) were synthesized by Genemed Synthesis.

Techniques: Expressing, Activity Assay, Cell Culture, Flow Cytometry, Labeling, Western Blot, Isolation, Two Tailed Test, Control, Staining

Journal: The Journal of Neuroscience

Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

doi: 10.1523/JNEUROSCI.0852-17.2017

Figure Lengend Snippet: P2X7R expression and function are increased with repeated morphine in primary and BV2 microglia cultures. A, P2X7Rs and μ-receptors (μR) are expressed on BV2 microglial cell line, primary microglia cultures isolated from P1–P3 rat brain and acutely isolated adult spinal microglia. B, PCR products in whole mouse brain and BV2 microglia amplified with primers targeting μ-receptor mRNA. C, Fluorescent-activated cell sorted microglia from adult rat spinal cord. PCR primers targeting CD11b, GFAP, MAP2, and μ-receptor mRNA amplifying 100 bp bands were used to amplify cDNA and products were run on a DNA gel. D, Western blot analysis of P2X7R protein levels in BV2 cells after one, three, and five daily treatments with morphine (MS, n = 5). Two-way ANOVA (interaction: F(2,24) = 3.3; treatment: F(1,12) = 9.4; days: F(2,24) = 4.2; D5 CTR vs MS: p = 0.0074), Sidak's post hoc test. E, Five days of morphine (MS) produced a dose-dependent increase in P2X7R protein in primary and BV2 microglia (Primary; n = 4; BV2, n = 6). One-way ANOVA (Primary: F(3,12) = 12.8; BV2: F(3,19) = 10.9), Dunnett's post hoc test (Primary: CTR vs 1 μm MS: p = 0.0076; CTR vs 10 μm MS: p = 0.0012; BV2: CTR vs 1 μm MS: p = 0.0229; CTR vs 10 μm MS: p = 0.0001). ***p < 0.001, **p < 0.01, *p < 0.05 compared with CTR. F, Peak rise in intracellular [Ca2+] evoked by BzATP (100 μm; n = 18, 22, 30, 42). Unpaired two-tailed t tests (Primary: t = 2.8, df = 38, p = 0.0085; BV2: t = 3.8, df = 70, p = 0.0012). **p < 0.01 compared with CTR. G, H, A740003, a P2X7R antagonist, (10 μm) was applied to the recording solution 10 min before and throughout Ca2+ imaging or whole-cell patch-clamp recordings. G, A740003 blocked BzATP-evoked rise in intracellular [Ca2+] (CTR, n = 37; MS, n = 109; MS/A740003, n = 39 cells). One-way ANOVA (F(2,182) = 263.7), Sidak's post hoc test (CTR vs MS: p = 0.0229; MS vs MS/A740003: p < 0.0001). H, A740003 reduced BzATP-evoked inward current (charge) and decreased peak amplitude. CTR and MS groups same as shown in Figures 6E, ​,77D, and ​and88C. MS/A740003 experimental group represent n = 4 cells. One-way ANOVA (F(2,17) = 42; F(2,20) = 20.9), Sidak's post hoc test (Charge: CTR vs MS: p < 0.0001; MS vs MS/A740003: p < 0.0001; Peak: CTR vs MS: p = 0.0010; MS vs MS/A740003: p < 0.0001). Quantification of P2X7R and μR protein levels were normalized to actin and represent change from the control group. All data represent mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 compared with CTR; ####p < 0.0001, ###p < 0.001, ##p < 0.01 compared with MS.

Article Snippet: P2X7R 356–371 (NTYASTCCRSRVYPSC, rat), P2X7R 356–371 (NTYSSAFCRSGVYPYC, mouse), P2X7R 379–389 (VNEYYYRKKCE, rat/mouse), inactive P2X7R Y379–389F (VNEFFFRKKCE, rat/mouse), P2X7R 546–556 (RHCAYRSYATW, rat), P2X7R 546–556 (RHRAYRCYATW, mouse), and P2X7R 586–595 (GQYSGFKYPY, rat/mouse) were synthesized by Genemed Synthesis.

Techniques: Expressing, Isolation, Amplification, Western Blot, Produced, Two Tailed Test, Imaging, Patch Clamp, Control

Journal: The Journal of Neuroscience

Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

doi: 10.1523/JNEUROSCI.0852-17.2017

Figure Lengend Snippet: Morphine upregulation of P2X7R expression and function is mediated by μ-receptors (μRs). A–E, BV2 cultured microglia were treated for 5 d with morphine (MS; 1 μm), PBS as a control (CTR), DAMGO (0.1 or 1 μm) a selective μR agonist, or selective δ and κ agonists. Antagonists were coadministered daily with morphine. A, Morphine upregulation of P2X7R protein levels was blocked by CTAP (5 μm), a selective μR antagonist (n = 4). One-way ANOVA (F(2,9) = 12.2), Sidak's post hoc test (CTR vs MS: p = 0.0046; MS vs MS/CTAP: p = 0.0091)b]. B, Total μR protein expression in BV2 microglia was unchanged following MS or CTR treatment (n = 4). Unpaired two-tailed t test (t = 0.6, df = 6), not significant. C, P2X7R protein expression as assessed by Western blotting following treatment with DAMGO (μR selective agonist), DPDPE (δ-receptor selective agonist), or U69593 (κ-receptor selective agonist; n = 5). One-way ANOVA (F(6,21) = 6.5), Dunnett's post hoc test. (CTR vs 1 μm DAMGO: p = 0.0009). D, Representative BzATP-evoked currents from whole-cell patch-clamp recordings of BV2 microglia. E, Quantification of total charge (area under the curve) and peak amplitude for BzATP-evoked intracellular currents in BV2 microglia (n = 9, 8, 7, 8). CTR and MS groups same as shown in Figures 5H, ​,77D, and ​and88C. One-way ANOVA (F(3,27) = 14.9; F(3,30) = 10.5), Sidak's post hoc test (Charge: CTR vs MS: p < 0.0001; MS vs MS/CTAP: p < 0.0001; CTR vs DAMGO: p = 0.0051; Peak: CTR vs MS: p = 0.0003; MS vs MS/CTAP: p = 0.0005; CTR vs DAMGO: p = 0.0133). F, G, BV2 microglia were treated with CTR or MS (1 μm) for 5 d. LPS-RS (10 or 100 ng/ml) was coapplied with MS. F, LPS-RS did not block the morphine-induced potentiation of BzATP-evoked calcium responses (n∼250–350 cells; 10 plates). One-way ANOVA (F(4,1306) = 50.8), Sidak's post hoc test (p < 0.0001). G, LPS-RS did not block morphine-induced increase in total P2X7R protein expression in BV2 microglia (n = 11, 11, 6,6). One-way ANOVA (F(3,30) = 6.0), Sidak's post hoc test (CTR vs MS: p = 0.0296; CTR vs MS/LPS-RS 10 ng/ml: p = 0.0034; CTR vs MS/LPS-RS 100 ng/ml: p = 0.0089). Quantification of P2X7R and μR protein levels were normalized to actin and represent change from the control group. All data represent mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 compared with CTR; ####p < 0.0001, ###p < 0.001, ##p < 0.01 compared with MS.

Article Snippet: P2X7R 356–371 (NTYASTCCRSRVYPSC, rat), P2X7R 356–371 (NTYSSAFCRSGVYPYC, mouse), P2X7R 379–389 (VNEYYYRKKCE, rat/mouse), inactive P2X7R Y379–389F (VNEFFFRKKCE, rat/mouse), P2X7R 546–556 (RHCAYRSYATW, rat), P2X7R 546–556 (RHRAYRCYATW, mouse), and P2X7R 586–595 (GQYSGFKYPY, rat/mouse) were synthesized by Genemed Synthesis.

Techniques: Expressing, Cell Culture, Control, Two Tailed Test, Western Blot, Patch Clamp, Blocking Assay

Journal: The Journal of Neuroscience

Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

doi: 10.1523/JNEUROSCI.0852-17.2017

Figure Lengend Snippet: Morphine potentiation of P2X7R activity depends on Src kinase. A, Average ΔF/F of single-cell BzATP-evoked responses in BV2 microglia expressed relative to CTR. Cells were treated with MS (1 μm) and genistein or genistin (10 μm), PP2, or PP3 (10 μm) for 5 d before imaging (n = 30–42 cells/group). One-way ANOVA (F(5,214) = 52.4), Sidak's post hoc test (p < 0.0001). B, Average of five BzATP-evoked [Ca2+] responses for CTR, MS, and MS/PP2. C, Representative tracings of the BzATP-evoked inward current in BV2 cells treated with MS (1 μm), MS + PP2 (10 μm), or MS + the src antagonist, KBSrc4 (5 μm), or CTR. D, Average charge and peak amplitude for BzATP-evoked currents from BV2 microglia (n = 9, 8, 6, 8, 9). CTR and MS groups same as shown in Figures 5H, ​,66E, and ​and88C. One-way ANOVA (F(4,34) = 30.2; F(4,37) = 13.6), Sidak's post hoc test (Charge: CTR vs MS: p < 0.0001; MS vs MS/PP2: p < 0.0001; CTR vs MS/PP3: p < 0.0001; MS vs MS/KBSrc4: p < 0.0001; Peak: CTR vs MS: p = 0.0006; MS vs MS/PP2: p < 0.0001; CTR vs MS/PP3: p < 0.0171; MS vs MS/KBSrc4: p < 0.0001). E, F, Total src (c-src) was IP from BV2 microglia treated with MS (E) or DAMGO (F). The IP fraction was probed for phosphorylation of the active site of src kinase (pY416). pY416 src is normalized to levels of total src and expressed relative to CTR. E, (n = 8) Unpaired two-tailed t test (t = 3.4, df = 14, p = 0.0072). F, (n = 9, 6) Unpaired two-tailed t test (t = 2.5, df = 13, p = 0.0283). All data represent mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01 *p < 0.05 compared with CTR; ####p < 0.0001 compared with MS.

Article Snippet: P2X7R 356–371 (NTYASTCCRSRVYPSC, rat), P2X7R 356–371 (NTYSSAFCRSGVYPYC, mouse), P2X7R 379–389 (VNEYYYRKKCE, rat/mouse), inactive P2X7R Y379–389F (VNEFFFRKKCE, rat/mouse), P2X7R 546–556 (RHCAYRSYATW, rat), P2X7R 546–556 (RHRAYRCYATW, mouse), and P2X7R 586–595 (GQYSGFKYPY, rat/mouse) were synthesized by Genemed Synthesis.

Techniques: Activity Assay, Imaging, Phospho-proteomics, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

doi: 10.1523/JNEUROSCI.0852-17.2017

Figure Lengend Snippet: Morphine potentiation of P2X7R is modulated by C-terminal amino acid residues Y382–384. A, Palmitoylated peptides designed to mimic short regions of the P2X7R C-terminal domain or intracellular domains were administered (10 μm) to BV2 cells daily with MS to interfere with phosphorylation at these sites. Average ΔF/F of single-cell BzATP-evoked responses in BV2 microglia expressed relative to CTR. Palmitoylated peptide mimicking amino acids 379–389 of the C-terminal domain interfered with morphine-induced potentiation of BzATP-evoked P2X7R calcium responses. Cells were treated with MS (1 μm) and mimetic peptides (n∼150 cells, n = 6–12 plates). One-way ANOVA (F(7,1471) = 43.9), Dunnett's post hoc test (p < 0.0001). B–E, BV2 microglia were treated with the palmitoylated peptide containing amino acids spanning 379–389 of the P2X7R C-terminal domain, with an inactive control peptide with Y382–384F mutation. Peptides (10 μm) were coadministered in culture daily with morphine. B, Representative tracings of the BzATP-evoked intracellular currents from cells treated with CTR, MS and P2X7R379–389 or inactive peptide iP2X7R379–389. C, Average charge and peak amplitude for BzATP-evoked currents (n = 9, 8, 6, 11). CTR and MS groups same as shown in Figures 5H, ​,66E, and ​and77D. One-way ANOVA (F(2,38) = 15.4; F(3,31) = 13.2), Sidak's post hoc test (Charge: CTR vs MS: p = 0.0008; MS vs MS/P2X7R379–389: p < 0.0001; CTR vs MS/iP2X7R379–389: p = 0.0053; Peak: CTR vs MS: p = 0.0021; MS vs MS/P2X7R379–389: p < 0.0001; CTR vs MS/iP2X7R379–389: p = 0.0093). D, Total P2X7R protein expression is increased in BV2 microglia treated with MS (1 μm; n = 32) and KBSrc4 (5 μm; n = 14), P2X7R379–389 (10 μm; n = 27), or iP2X7R379–389 (10 μm; n = 13) for 5 d compared with CTR (n = 31). One-way ANOVA (F(4,112) = 12.9), Dunnett's post hoc test (CTR vs MS: p = 0.417; CTR vs MS/KBSrc4: p = 0.0049; CTR vs MS/P2X7R379–389: p < 0.0001; MS vs MS/iP2X7R379–389: p < 0.0001). E, Cell-surface expression of P2X7R is increased in BV2 microglia treated with MS (1 μm; n = 20) and KBSrc4 (5 μm; n = 12), P2X7R379–389 (10 μm; n = 16) or iP2X7R379–389 (10 μm; n = 15) for 5 d compared with CTR (n = 21). One-way ANOVA (F(4,79) = 11.6), Dunnett's post hoc test (CTR vs MS: p = 0.0001; CTR vs MS/KBSrc4: p < 0.0001; CTR vs MS/P2X7R379–389: p < 0.0001; MS vs MS/iP2X7R379–389: p < 0.0001). F, P2X7R WT and mutant (Y382–384F) constructs were expressed in an astrocytoma cell line (1321) that does not express endogenous P2X7Rs. Transfected cells were treated with morphine (MS; 1 μm) or CTR five times over 3 d before imaging. Average ΔF/F of BzATP-evoked responses in transfected 1321 cells expressed relative to WT CTR (n = 27–32 cells). Two-way ANOVA (interaction: F(1,111) = 6.9; P2X7R WT/mut: F(1,111) = 3.3; treatment: F(1,111) = 4.1), Tukey's post hoc test (WT/CTR vs WT/MS: p = 0.0064; Mutant/CTR vs Mutant/MS: p = 0.9740; WT/MS vs Mutant/MS: p = 0.0127). All data represent mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 compared with CTR; ####p < 0.0001, #p < 0.05 compared with MS.

Article Snippet: P2X7R 356–371 (NTYASTCCRSRVYPSC, rat), P2X7R 356–371 (NTYSSAFCRSGVYPYC, mouse), P2X7R 379–389 (VNEYYYRKKCE, rat/mouse), inactive P2X7R Y379–389F (VNEFFFRKKCE, rat/mouse), P2X7R 546–556 (RHCAYRSYATW, rat), P2X7R 546–556 (RHRAYRCYATW, mouse), and P2X7R 586–595 (GQYSGFKYPY, rat/mouse) were synthesized by Genemed Synthesis.

Techniques: Phospho-proteomics, Control, Mutagenesis, Expressing, Construct, Transfection, Imaging

Journal: The Journal of Neuroscience

Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

doi: 10.1523/JNEUROSCI.0852-17.2017

Figure Lengend Snippet: Spinal P2X7Rs are critically involved in the development of morphine tolerance. A–D, Effects of intrathecal injections of A740003 (0.1 nmol), a selective P2X7R antagonist, on the development of morphine tolerance. A, Thermal and (B) mechanical nociceptive threshold in CTR- (n = 7), MS- (n = 7), MS/A740003- (n = 7), and A740003- (n = 6) treated rats. Latency to withdraw from stimulus, tail-flick latency (TFL) and paw withdrawal thresholds (PWTs), are reported as a percentage of the maximum possible effect (MPE). ****p < 0.0001, ***p < 0.001, *p < 0.05, repeated-measures two-way ANOVA (A: interaction: F(18,161) = 8.7; time: F(6,161) = 15.8; treatment: F(3,161) = 201.7; B, interaction: F(18,161) = 5.4; time: F(6,161) = 10.6; treatment: F(3,161) = 155.3), Dunnett's post hoc test. C, D, Morphine ED50 values following 7 d of treatment. ****p < 0.001 compared with CTR; ####p < 0.0001 compared with MS, one-way ANOVA (C: F(3,23) = 72.5; D: F(3,23) = 22.5), Sidak's post hoc test. C, CTR: 5.0 ± 0.7 mg/kg; MS: 28.4 ± 1.7 mg/kg; MS/A740003: 8.2 ± 1.6 mg/kg; A740003: 6.0 ± 0.8 mg/kg (CTR vs MS: p < 0.0001; MS vs MS/A740003: p < 0.0001). D, CTR: 6.0 ± 0.6 mg/kg; MS: 25.0 ± 3.4 mg/kg; MS/A740003: 10.0 ± 1.2 mg/kg; A740003: 5.7 ± 0.8 mg/kg (CTR vs MS: p < 0.0001; MS vs MS/A740003: p < 0.0001). E, F, Effects of intrathecal injections of A740003 (0.1 nmol) on MS antinociception in rats with established tolerance. E, Thermal and (F) mechanical nociceptive threshold was measured and is reported as a percentage of the MPE. A740003 was administered on days 6–10 with MS (MS, n = 5; MS/A740003, n = 5). All data represent mean ± SEM.

Article Snippet: P2X7R 356–371 (NTYASTCCRSRVYPSC, rat), P2X7R 356–371 (NTYSSAFCRSGVYPYC, mouse), P2X7R 379–389 (VNEYYYRKKCE, rat/mouse), inactive P2X7R Y379–389F (VNEFFFRKKCE, rat/mouse), P2X7R 546–556 (RHCAYRSYATW, rat), P2X7R 546–556 (RHRAYRCYATW, mouse), and P2X7R 586–595 (GQYSGFKYPY, rat/mouse) were synthesized by Genemed Synthesis.

Techniques: Tail Flick Test, Tandem Mass Spectroscopy

Journal: The Journal of Neuroscience

Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

doi: 10.1523/JNEUROSCI.0852-17.2017

Figure Lengend Snippet: P2X7R Y382–384 gates the development of morphine tolerance in male rats. A–D, Male Sprague-Dawley rats were treated with morphine (MS; 15 mg/kg i.p.) or saline as a control (CTR). Mimetic peptide P2X7R379–389 (20 nm), inactive peptide (20 nm), or saline control (CTR) were injected into the intrathecal space. Morphine antinociception was assessed using thermal tail-flick (10 s cutoff). Latency to withdraw from stimulus [tail-flick latency (TFL)] is reported as a percentage of the maximum possible effect (MPE). A, Acute anti-nociceptive response to first MS injection (15 mg/kg) in male rats. Repeated-measures two-way ANOVA, (interaction: F(20,195) = 16.18; time: F(5,195) = 91.52; treatment: F(4,39) = 60.14; subjects: F(39,195) = 2.0), Dunnett's post hoc test. ****p < 0.0001 compared with CTR at 30 min postinjection. B, Daily raw thermal TFL baseline values in seconds before morphine injection. No change in baseline thresholds is observed with peptide treatment. C, Daily thermal TFL in rats treated with MS (15 mg/kg; n = 15) with mimetic peptide P2X7R379–389 (20 nm; n = 9) and inactive peptide (20 nm; n = 8) or CTR (n = 5) and CTR with mimetic peptide P2X7R379–389 (20 nm; n = 7). Repeated-measures two-way ANOVA (interaction: F(24,273) = 13.73; time: F(6,273) = 59.38; treatment: F(4,273) = 351.8), Dunnett's post hoc test. ****p < 0.0001, ***p < 0.001, *p < 0.05 compared with CTR on the same days. D, Morphine dose–response curve and median effective dose (ED50) of thermal TFL as measured on day 8 following 7 d treatment in male rats. CTR: 6.3 ± 0.4 mg/kg; MS: 24.2 ± 2.5 mg/kg; MS/ P2X7R379–389:13.9 ± 2.1 mg/kg; MS/iP2X7R379–389: 25.6 ± 2.4 mg/kg; CTR/P2X7R379–389: 6.3 ± 0.9 mg/kg. One-way ANOVA (F(4,24) = 23.4), Sidak's post hoc test (CTR vs MS: p < 0.0001; MS vs MS/P2X7R379–389: p = 0.0006; CTR vs MS/iP2X7R379–389: p < 0.0001). ****p < 0.0001 compared with CTR; ###p < 0.001 compared with MS. All data represent mean ± SEM.

Article Snippet: P2X7R 356–371 (NTYASTCCRSRVYPSC, rat), P2X7R 356–371 (NTYSSAFCRSGVYPYC, mouse), P2X7R 379–389 (VNEYYYRKKCE, rat/mouse), inactive P2X7R Y379–389F (VNEFFFRKKCE, rat/mouse), P2X7R 546–556 (RHCAYRSYATW, rat), P2X7R 546–556 (RHRAYRCYATW, mouse), and P2X7R 586–595 (GQYSGFKYPY, rat/mouse) were synthesized by Genemed Synthesis.

Techniques: Saline, Control, Injection, Tail Flick Test

Journal: The Journal of Neuroscience

Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

doi: 10.1523/JNEUROSCI.0852-17.2017

Figure Lengend Snippet: P2X7R Y382–384 modulates microglial reactivity in response to morphine. A, Representative images of Iba1 expression in the spinal lumbar dorsal horn following 7 d of CTR or MS treatment with intrathecal injection of the P2X7R mimetic peptide (P2X7R379–389; 20 nm) or its inactive control iP2X7R379–389. Images were acquired at 60×. Scale bar, 50 μm. Yellow boxes represent area magnified three times to show representative microglial morphology. Scale bar, 25 μm. B, Representative images of CD11b (green) and proliferation marker Ki67 (magenta) expression in the spinal lumbar dorsal horn following 7 d of CTR or MS treatment with intrathecal injection of the P2X7R mimetic peptide (P2X7R379–389; 20 nm) or its inactive control iP2X7R379–389. Images were acquired at 20×. Scale bar, 50 μm. C, Morphology classification of Iba1-positive microglia in the lumbar dorsal horn. Microglia are qualitatively assessed for process length, process thickness, number of processes, and cell body size. Individual cells are then classified as resting, intermediate, or activated. The percentage of each cell classification is then calculated per section and averaged across all sections (n = 36, 22, 22, 21). One-way ANOVA (F(3,85) = 12.9), Sidak's post hoc test (CTR vs MS: p = 0.0057; MS vs MS/P2X7R379–389: p = 0.0157; CTR vs MS/iP2X7R379–389: p < 0.0001). D, CD11b mean intensity from dorsal horn sections of the lumbar spinal cord (n = 9, 12, 16, 13). One-way ANOVA (F(3,46) = 9.6), Sidak's post hoc test (CTR vs MS: p = 0.0004; MS vs MS/P2X7R379–389: p = 0.0003; CTR vs MS/iP2X7R379–389: p < 0.0190). E, Percentage of microglia expressing the proliferation marker Ki67 out of all CD11b-positive cells in the field-of-view in the spinal dorsal horn from lumbar sections (n = 9, 11, 14, 12) taken from CTR-, MS-, MS/P2X7R379–389-, and MS/iP2X7R379–389-treated animals. One-way ANOVA (F(3,42) = 13.4), Sidak's post hoc test (CTR vs MS: p = 0.0329; MS vs MS/P2X7R379–389: p = 0.0358; CTR vs MS/iP2X7R379–389: p < 0.0001). All data represent mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 compared with CTR; ###p < 0.001, ##p < 0.01, #p < 0.05 compared with MS. F, Summary: potentiation of P2X7R activity in microglia is required for morphine analgesic tolerance. Morphine signaling through μ-receptors activates Src kinase in microglia. The activation of Src is a key intracellular substrate for morphine-induced enhancement of P2X7R function in microglia. This potentiated function depends on tyrosine residues Y382–384 located within the P2X7R intracellular C-terminal domain. Site-specific P2X7R phosphorylation is critically required for morphine-induced microglial activation, and it is a novel mechanism in the development of morphine analgesic tolerance.

Article Snippet: P2X7R 356–371 (NTYASTCCRSRVYPSC, rat), P2X7R 356–371 (NTYSSAFCRSGVYPYC, mouse), P2X7R 379–389 (VNEYYYRKKCE, rat/mouse), inactive P2X7R Y379–389F (VNEFFFRKKCE, rat/mouse), P2X7R 546–556 (RHCAYRSYATW, rat), P2X7R 546–556 (RHRAYRCYATW, mouse), and P2X7R 586–595 (GQYSGFKYPY, rat/mouse) were synthesized by Genemed Synthesis.

Techniques: Expressing, Injection, Control, Marker, Tandem Mass Spectroscopy, Activity Assay, Activation Assay, Phospho-proteomics