Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 2 Effect of UTP and suramin on IA of small-diameter and FG-labeled TG neurons, with diameters ranging from 18 to 39 μm in control rats. (A) Fluorescence microscopic view of TG neurons from control rats. (a) Retrograde labeling of TG neurons (blue) innervating whisker pad skin. (b) P2Y2 receptor-positive (green) TG neurons were seen in the section of TG. (c) The merged images (purple) of retrograde labeling of TG neurons and P2Y2 receptor-positive TG neurons from the same section, indicating co-localization. (d) Retrograde labeling of TG neurons (blue) innervating whisker pad skin in cultured TG neurons. (B) Electrophysiology recording for small-diameter and FG-labeled TG neurons in control rats. (a) Representative traces showing that the application of 30 μM UTP reduced IA. Suppression of the mean peak amplitudes of IA seen after UTP application was antagonized by suramin 100 μM. (b) Current–voltage relationship for the effects of UTP and suramin on IA. Each value represents the mean ± SEM (con: 0.14 ± 0.01 nA, n = 9; UTP: 0.09 ± 0.01 nA, n = 20, p < 0.05 vs control; suramin: 0.13 ± 0.01 nA, n = 9). IA was initiated via a prepulse (100 ms) of −120 mV and test pulses (400 ms) from −60 to +60 mV in a 10 mV step.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Labeling, Control, Fluorescence, Whisker Assay, Cell Culture

Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 4 Effect of UTP on the expression levels of IA subunits in control TG neurons. (A) Double-immunostaining revealed the expression of Kv1.4, Kv3.4, Kv4.2 and Kv4.3 subunits in P2Y2 receptor-positive neurons in the ION-CCI TG sections. The P2Y2 receptor-positive TG neurons also expressed Kv1.4, Kv3.4, Kv4.2 and Kv4.3, respectively, n = 5 rats. (B) Reduction in the mRNA levels of IA subunits by UTP in cultured TG neurons from control rats. Treatment with suramin (100 μM) in the UTP-incubated (30 μM) TG neurons for 16 h in control rats reversed the decrease in the mRNA levels of Kv1.4, Kv3.4, Kv4.2, and Kv4.3 subunits. n = 10 samples in each group, *, p < 0.05, **, p < 0.01 vs control; ##, p < 0.01 vs UTP.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Expressing, Control, Double Immunostaining, Cell Culture, Incubation

Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 5 The role of P2Y2 receptors in mechanical hyperalgesia in ION-CCI rats. (A) The peripheral target injection to TG of suramin (0.3- 3 μg/μl) reduced mechanical allodynia in the whisker pad. n = 6-8, **, p < 0.01 compared with injection of saline, ##, p < 0.01 compared with injection of high-dose suramin. Suramin led to a time- and dose-dependent increase in PWT, this anti-allodynia effect started 10 min after the suramin injection and remained for at least 45 min. (B) The peripheral target injection to TG of P2Y2 antisense oligodeoxynucleotides significantly alleviated mechanical allodynia of the whisker pad. n = 5, *, p < 0.05, **, p < 0.01 compared with injection of saline. The effect started at 6 h and persisted for at least 120 h. (C) Western blots showed successful suppression of P2Y2 receptor expression in TG by P2Y2 receptor antisense oligodeoxynucleotides treatment n = 4 for each group, **, p < 0.01.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Injection, Whisker Assay, Saline, Western Blot, Expressing

Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 6 Difference of IA channel expression in TG between sham and ION-CCI rats. (A) Double-immunostaining for P2Y2 receptors and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3 on TG neurons in sham and ION-CCI sections, respectively. (B) Percentages of numbers of Kv1.4, Kv3.4, Kv4.2 and Kv4.3 subunits in P2Y2 receptor-positive neurons are significantly decreased in TG from ION-CCI rats compared with sham rats. n = 4 rats, **, p < 0.01). (C) Changes in the mRNA levels of IA subunits in TG after P2Y2 receptor antisense oligodeoxynucleotides treatment. The mRNA levels of Kv1.4, Kv3.4 and Kv4.2 were significantly decreased in the saline group of ION-CCI rats compared with the sham rats. They were reversed after P2Y2 receptor antisense oligodeoxynucleotides treatment. n = 5-9 rats, *, p < 0.05, **p < 0.01 compared with saline groups. There was no difference in the levels of Kv4.3 mRNA among the groups. n = 6-8 rats, p > 0.05.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Expressing, Double Immunostaining, Saline

Journal: Molecular pain

Article Title: Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Figure 7 Role of ERK pathway in activation of P2Y2 receptors mediates an inhibition of IA channels on small-diameter TG neurons in control rats. (A) Comparison of the phosphorylation of ERK1/2 in TG from sham and ION-CCI rats. Western blot results showed that the level of ERK1/2 phosphorylation was significantly increased in the ipsilateral TG after ION-CCI, compared with that from the sham group. n = 5 for each group *, p < 0.05. (B) In TG from ION-CCI rats, treatment with P2Y2 receptor antisense oligodeoxynucleotides (15 μg/50 μl) significantly decreased the expression of ERK protein in TG. n = 5 for each group, **, p < 0.01.

Article Snippet: After transfer, the membranes were blocked with 5% (mass/vol) non-fat dried milk in Tribuffered saline containing 0.05% Tween 20 (TBST) for 1 hour, then incubated with the primary antibodies: P2Y2 (rabbit anti-rat polyclonal, IgG 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or ERK (rabbit anti-rat polyclonal, IgG 1:1000, Cell Signaling) and β-actin (mouse monoclonal, IgG 1:8000, Sigma, USA).

Techniques: Activation Assay, Inhibition, Control, Comparison, Phospho-proteomics, Western Blot, Expressing

Journal: Journal of pharmacological sciences

Article Title: P2Y 2 receptor mediates dying cell removal via inflammatory activated microglia.

doi: 10.1016/j.jphs.2023.06.004

Figure Lengend Snippet: Fig. 4. Effects of AR-C118925, a selective P2Y2 receptor antagonist, on the removal of dying cells by neighboring microglia. (A, B) Time-lapse observation of spontaneous (A) and LPS- induced microglial death (B) and removal of dying cells in the presence of AR-C118925. Microglia were treated with AR-C118925 (30 mM) and images captured every 10 min for 18 h. Images are representative of five independent experiments. Dying cells (white arrowhead); phagocytotic cells (black arrowhead). Time count, hr:min; scale bar, 50 mm, bottom. Movies 7 and 8 show representative live images. (C) Quantification of the removal of dying cells by control and AR-C118925-treated microglia. Fifty cells were randomly selected and manually divided into four groups based on their behavior. Group I, cells that underwent death; Group II, cells that did not make contact with any dying cells; Group III, cells that

Article Snippet: For livecell immunostaining, microglia were incubated with a FITCconjugated antibody against the extracellular domain of the P2Y2 receptor (1:50, ARP-102-F; Alomone) for 30 min on ice and then fixed with 4% paraformaldehyde for 15 min. Immunofluorescence staining of the P2Y2 receptor on the plasma membrane was observed with a fluorescence microscope (BIOREVO Bz-9000; Keyence).

Techniques: Control

Journal: Journal of pharmacological sciences

Article Title: P2Y 2 receptor mediates dying cell removal via inflammatory activated microglia.

doi: 10.1016/j.jphs.2023.06.004

Figure Lengend Snippet: Fig. 5. LPS stimulation caused a change in the intracellular localization of P2Y2 receptors in microglia. (A) Confocal micrographs of the P2Y2 receptor. Microglia were stimulated with LPS (10 ng/mL) for 3 h. After fixation and permeabilization, the cells were stained with an anti-P2Y2 receptor antibody, which recognizes the intracellular domain of the P2Y2 receptor, followed by Alexa Fluor 488-conjugated rabbit IgG as a secondary antibody, and fluorescence micrographs captured by using a confocal laser scanning microscope. White arrows indicate accumulation of the P2Y2 receptor along the edge of the plasma membrane. Experiments were repeated three times. Images are representative of three independent experiments. Scale bar, 50 mm. (B) Live cell images of the P2Y2 receptor on the plasma membrane of microglia. Microglia were stimulated with LPS for 3 h and stained with a FITC- conjugated antibody against the extracellular domain of the P2Y2 receptor for 30 min on ice. White arrowheads indicate the edge of the plasma membrane. Experiments were repeated three times. Images are representative of three independent experiments. Scale bar, 50 mm.

Article Snippet: For livecell immunostaining, microglia were incubated with a FITCconjugated antibody against the extracellular domain of the P2Y2 receptor (1:50, ARP-102-F; Alomone) for 30 min on ice and then fixed with 4% paraformaldehyde for 15 min. Immunofluorescence staining of the P2Y2 receptor on the plasma membrane was observed with a fluorescence microscope (BIOREVO Bz-9000; Keyence).

Techniques: Staining, Laser-Scanning Microscopy, Clinical Proteomics, Membrane

Journal: Journal of pharmacological sciences

Article Title: P2Y 2 receptor mediates dying cell removal via inflammatory activated microglia.

doi: 10.1016/j.jphs.2023.06.004

Figure Lengend Snippet: Fig. 6. Change in mRNA expression of the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12 receptors in microglia after LPS stimulation. Microglia were stimulated with LPS (10 ng/mL) for the indicated time. The mRNA expression level of each P2Y receptor was measured using real-time PCR and normalized to that of b-actin. Data are presented as the means ± SEM of independent experiments. n ¼ 10 for P2Y2; n ¼ 4 for P2Y1, P2Y4, P2Y6, and P2Y12.

Article Snippet: For livecell immunostaining, microglia were incubated with a FITCconjugated antibody against the extracellular domain of the P2Y2 receptor (1:50, ARP-102-F; Alomone) for 30 min on ice and then fixed with 4% paraformaldehyde for 15 min. Immunofluorescence staining of the P2Y2 receptor on the plasma membrane was observed with a fluorescence microscope (BIOREVO Bz-9000; Keyence).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of pharmacological sciences

Article Title: P2Y 2 receptor mediates dying cell removal via inflammatory activated microglia.

doi: 10.1016/j.jphs.2023.06.004

Figure Lengend Snippet: Fig. 7. LPS induces Axl upregulation in a P2Y2 receptor-dependent manner in microglia. (A) LPS stimulation enhanced Axl mRNA expression (left) and suppressed that of Mer (right) in microglia. Microglia were stimulated with LPS (30 ng/mL) for the indicated time, and mRNA expression measured using real-time PCR. Values are expressed as the ratio of Mer or Axl to b-actin. Data are presented as the mean ± SEM of three independent experiments. (B) Effects of the selective P2Y2 receptor antagonist, AR-C118925, and the Pyk2 inhibitor, PF431396, on LPS-induced Axl mRNA upregulation in microglia. Microglia were treated with AR-C118925 (10, 30 mM) for 5 min and PF431396 (0.3, 1 mM) for 15 min and then stimulated with LPS (30 ng/mL) for 6 h. Data are presented as the means ± SEM (%) of LPS-induced Axl mRNA expression (vs. b-actin). n ¼ 5 for AR-C118925; n ¼ 3 for PF431396. C. Effects of AR-C118925 on LPS-induced Pyk2 upregulation in microglia. The mRNA expression levels were measured using real-time PCR and normalized to that of b-actin. Data are presented as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS alone.

Article Snippet: For livecell immunostaining, microglia were incubated with a FITCconjugated antibody against the extracellular domain of the P2Y2 receptor (1:50, ARP-102-F; Alomone) for 30 min on ice and then fixed with 4% paraformaldehyde for 15 min. Immunofluorescence staining of the P2Y2 receptor on the plasma membrane was observed with a fluorescence microscope (BIOREVO Bz-9000; Keyence).

Techniques: Expressing, Real-time Polymerase Chain Reaction