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: Science signaling

Article Title: Spatially-structured cell populations process multiple sensory signals in parallel in intact vascular endothelium

doi: 10.1126/scisignal.aar4411

Figure Lengend Snippet: (A) Representative experiment showing the effect of the P2Y1 antagonist MRS2179 (10 μM) and P2Y2 antagonist ARC118925 (1 μM) on the maximal response to ATP (100 μM). The traces show the average responses from all cells in the field. The P2Y1 antagonists (MRS2179, 10 μM) had only a small effect on the response. The responses are almost fully blocked by the P2Y2 receptor blocker (ARC118925; 1 μM). The small residual response is inhibited by the P2Y1 antagonist. At the end of the experiment CCh (100 μM) was applied to confirm cell viability. (B) Composite Ca2+ images showing the cells that respond to ATP (100 μM) in presence of MRS2179 or ARC118925 (red) or both. The right panel shows the cells that response to CCh (100 μM; green) at the end of the experiment. All images are from the same field of endothelium. (C) Ca2+ signals from the activated cells in B, 30s baseline and 60s activation are shown. (D) Peak Ca2+ (F/F0) response from each individual cell (circle) matched for each treatment (grey line) from a single experiment. Average response indicated by white circle and matched across each treatment by bold line. The Plotting Density (right-hand axis) indicates the distribution of Peak F/F0 values (red high, blue low occurrence of the same values). (E) Paired summary data illustrating changes in Peak F/F0 response values averaged across all cells. Each color represents a different animal and is maintained across the different treatments (n=5). Scale bars, 50μm. p < 0.05.

Article Snippet: The arteries then were incubated with a rabbit anti-P2Y2 receptor antibody (1:100, sc-20124, Santa Cruz, Dallas, TX, USA) and CD31 (1:200, AF3628, R&D Systems,) in PBS containing 1% BSA and 2% Donkey Serum for 12 hours at 4°C.

Techniques: Activation Assay

Journal: Science signaling

Article Title: Spatially-structured cell populations process multiple sensory signals in parallel in intact vascular endothelium

doi: 10.1126/scisignal.aar4411

Figure Lengend Snippet: (A) Fluorescence localization of purinergic P2Y2 receptors and muscarinic M3 receptors in the endothelium. Representative images (from left) show the endothelial cell boundaries as revealed by PECAM-1 labelling (green; anti- CD31/PECAM-1), P2Y2 receptor (red; anti-P2Y2) distribution, M3 receptor distribution (green; fluorescent M3 receptor antagonist, 100 nM) and overlay of all three. The receptors distribution was not uniform across the endothelium and there was relatively little overlap of purinergic and muscarinic receptor staining. (B) Expanded view of the endothelial images shown in A. The expanded region is shown by the red box in A left-panel. (C) Negative control. The left panel is again PECAM-1 labeled to show cell boundary positions. The negative control for P2Y2 staining was obtained by omitting the primary antibody against P2Y2. The control for M3 staining was obtained by labelling with the fluorescent M3 receptor ligand in the presence of the M3 receptor antagonist 4-DAMP (10 μM). The right panel shows an overlay of all three. (D) Summary data showing the levels of M3 staining in the presence and absence of the antagonist 4-DAMP (green), the extent of labelling in the presence and absence of the P2Y2 primary antibody (red); and the extent of overlap of specific M3 and P2Y2 staining (grey). (E) Summary data showing the percentage of cells with M3 staining (green), P2Y2 receptor staining (red) and the percentage of cells expressing both M3 and P2Y2 receptor staining (grey) (n = 3, *p < 0.05, unpaired t-test). Scale bars, 50 μm.

Article Snippet: The arteries then were incubated with a rabbit anti-P2Y2 receptor antibody (1:100, sc-20124, Santa Cruz, Dallas, TX, USA) and CD31 (1:200, AF3628, R&D Systems,) in PBS containing 1% BSA and 2% Donkey Serum for 12 hours at 4°C.

Techniques: Fluorescence, Staining, Negative Control, Labeling, Control, Expressing