Journal: Acta neuropathologica communications

Article Title: Adenosine triphosphate drives head and neck cancer pain through P2X2/3 heterotrimers.

doi: 10.1186/2051-5960-2-62

Figure Lengend Snippet: Figure 1 ATP, P2X2, and P2X3 in human HNSCC microenvironment and pain. a. Representative HPLC chromatograms showing ATP peaks from tumor and matched normal tissue harvested from the same patient. b. Tumor tissues (Ipsi) had higher levels of ATP compared to matched normal sites (Contra) (n = 10, Student’s t-test). c. Pain scores of functional sharpness and intensity were significantly higher than spontaneous sharpness and intensity, respectively (n = 13, Student’s t-test). d. Mean scores of functional pain (Q2, 4, 6, 7, 8) were significantly higher than spontaneous pain (Q1, 3, 5) (Student’s t-test). e. ATP concentration in extracted cancer tissue correlated positively with mean pain scores (linear regression). f. Representative H&E and immunofluorescence staining (P2X2, P2X3, merged) of a human tongue SCC. Sections were taken from adjacent sections of the SCC. Scale bar: 100 μm.

Article Snippet: Control experiments were performed by incubation in secondary antibody alone and by applying P2X2 blocking peptides (Santa Cruz Biotechnology), and P2X3 blocking peptides (Alomone Labs).

Techniques: Functional Assay, Concentration Assay, Immunofluorescence, Staining

Journal: Acta neuropathologica communications

Article Title: Adenosine triphosphate drives head and neck cancer pain through P2X2/3 heterotrimers.

doi: 10.1186/2051-5960-2-62

Figure Lengend Snippet: Figure 3 HNSCC induces neuronal P2X2/3 plasticity that is reversed by anti-NGF. a. Sustained ATP current (top panel) is enhanced and prolonged following SCC co-culture; anti-NGF added into the co-culture reduced the sustained ATP current. Transient ATP current (lower panel) is not affected by either co-culture or anti-NGF. b. Sustained ATP current density is increased by co-culture (One-way ANOVA), and is reversed by anti-NGF. c. Dot plot of sustained ATP current in different diameter neurons. Co-culture increased current in medium-sized TG neurons; this increased current was reversed by anti-NGF. d. Representative immunofluorescence images of P2X2 and P2X3 expression in TG neurons. e. HNSCC co-culture increased the percentage of neurons expressing P2X2 and P2X3 subunits. Anti-NGF treatment significantly reduced percentage of neurons expressing P2X3 but not P2X2 subunits. The significant increase in the percentage of neurons that express both subunits following co-culture was reversed by anti-NGF application (One-way ANOVA). f. P2X2 immunofluorescence intensity was not changed after co-culture or anti-NGF treatment. P2X3 immunofluorescence intensity was significantly increased following co-culture, and was reduced by anti-NGF (one-way ANOVA). g. In mice with tongue HNSCC, mRNA expression for P2X2 was increased, while P2X3 expression was unchanged in TG neurons (Student’s t-test).

Article Snippet: Control experiments were performed by incubation in secondary antibody alone and by applying P2X2 blocking peptides (Santa Cruz Biotechnology), and P2X3 blocking peptides (Alomone Labs).

Techniques: Co-Culture Assay, Immunofluorescence, Expressing

Journal: British Journal of Pharmacology

Article Title: Activation of P2Y 6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

doi: 10.1111/bph.12711

Figure Lengend Snippet: Primary and secondary antibodies used in immunohistochemistry experiments

Article Snippet: Observations were performed and analysed with a laser-scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code Host Dilution Supplier Primary antibodies P2Y 1 APR-009 Rabbit 1:75 Alomone P2Y 6 APR-011 Rabbit 1:100 Alomone P2Y 6 ABIN1386282 Rabbit 1:150 Antibodies Online P2X1 APR-001 Rabbit 1:50 Alomone P2X3 APR-016 Rabbit 1:100 Alomone NTPDase2 BZ1-5F Rabbit 1:400 J. Sevigny Cytokeratin 20 (CK20) M7019 Mouse 1:100 Dako Vimentin M0725 Mouse 1:150 Dako NF160 Ab7794 Mouse 1:600 Abcam VAChT AB1578 Goat 1:750 Chemicon Secondary antibodies Alexa Fluor 488 anti-rb A-21206 Donkey 1:1500 Mol.

Techniques: Immunohistochemistry

Journal: British Journal of Pharmacology

Article Title: Activation of P2Y 6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

doi: 10.1111/bph.12711

Figure Lengend Snippet: Confocal micrographs showing P2Y6, P2Y1 and NTPDase2 immunoreactivity in transverse sections of the detrusor smooth layer of rat urinary bladder. To facilitate visualization of small cholinergic nerve terminals staining for VAChT (red) images correspond to the intensity projections over Z axis of five to six confocal microscopy stacks taken at the smooth muscle layer. No co-localization was found between P2Y6 receptor (green) and VAChT (red) immunoreactivity. Conversely, VAChT-positive cholinergic nerve terminals (red) stained positively with antibodies against the P2Y1 receptor and E-NTPDase2 (green); yellow staining denotes co-localization. Nucleic DNA is stained with DAPI (blue). Scale bars = 50 μm.

Article Snippet: Observations were performed and analysed with a laser-scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code Host Dilution Supplier Primary antibodies P2Y 1 APR-009 Rabbit 1:75 Alomone P2Y 6 APR-011 Rabbit 1:100 Alomone P2Y 6 ABIN1386282 Rabbit 1:150 Antibodies Online P2X1 APR-001 Rabbit 1:50 Alomone P2X3 APR-016 Rabbit 1:100 Alomone NTPDase2 BZ1-5F Rabbit 1:400 J. Sevigny Cytokeratin 20 (CK20) M7019 Mouse 1:100 Dako Vimentin M0725 Mouse 1:150 Dako NF160 Ab7794 Mouse 1:600 Abcam VAChT AB1578 Goat 1:750 Chemicon Secondary antibodies Alexa Fluor 488 anti-rb A-21206 Donkey 1:1500 Mol.

Techniques: Staining, Confocal Microscopy

Journal: British Journal of Pharmacology

Article Title: Activation of P2Y 6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

doi: 10.1111/bph.12711

Figure Lengend Snippet: Schematic representation of the putative mechanisms underlying the control of the voiding frequency by urothelial UDP-sensitive P2Y6 receptors in the anaesthetized rat. Activation of P2Y6 receptors on distended umbrella cells during bladder filling increased, by threefold, the release of ATP from the urothelium. Released ATP, acting via multiple urothelial P2 purinoceptors, triggers a self-regenerating purinergic wave propagating to sensory nerve afferents endowed with P2X3 receptors to initiate the voiding reflex. Bladder activity may be partly reversed by the hydrolysis of ATP into ADP by E-NTPDases, namely E-NTPDase2 located in the lamina propria (probably on interstitial cells) and on cholinergic nerve efferents. ADP accumulation at the neuromuscular synapse decreases ACh release and smooth muscle contraction through the activation of prejunctional inhibitory P2Y1 receptors. The diagram also shows the locus of action of the main drugs used in this study.

Article Snippet: Observations were performed and analysed with a laser-scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code Host Dilution Supplier Primary antibodies P2Y 1 APR-009 Rabbit 1:75 Alomone P2Y 6 APR-011 Rabbit 1:100 Alomone P2Y 6 ABIN1386282 Rabbit 1:150 Antibodies Online P2X1 APR-001 Rabbit 1:50 Alomone P2X3 APR-016 Rabbit 1:100 Alomone NTPDase2 BZ1-5F Rabbit 1:400 J. Sevigny Cytokeratin 20 (CK20) M7019 Mouse 1:100 Dako Vimentin M0725 Mouse 1:150 Dako NF160 Ab7794 Mouse 1:600 Abcam VAChT AB1578 Goat 1:750 Chemicon Secondary antibodies Alexa Fluor 488 anti-rb A-21206 Donkey 1:1500 Mol.

Techniques: Activation Assay, Activity Assay