Journal: Journal of Neuroscience

Article Title: Inhibition of N-Type Voltage-Activated Calcium Channels in Rat Dorsal Root Ganglion Neurons by P2Y Receptors Is a Possible Mechanism of ADP-Induced Analgesia

doi: 10.1523/jneurosci.4019-03.2004

Figure Lengend Snippet: Figure 3. Functional and immunhistochemical evidence for the coexistence of P2X3, VR1, and P2Y1 receptors on the same DRG cell population. Aa, Calcium currents and TTX-insensitive sodium currents were recorded in rat DRGs using the whole-cell configuration of the patch- clamp technique. A NaCl- and TTX-containing extracellular solution (see Materials and Meth- ods)wasused.Voltagestepsweremadefromaholdingpotentialof90to10mVevery20 sectoinduceinwardcurrents.Afterthreecontrolcurrents(c),Ca 2-freeextracellularsolution (noaddedCa 2plus1mMEGTA)wassuperfusedfor2min(0Ca)andthenwaswashedout(w). Subsequently, ATP (30 M) induced an inward current that recovered within seconds to base- line but continued to inhibit the depolarization-evoked currents. This effect was washed out within 1 min (w). Ab, When ICa was calculated by subtraction of the currents recorded in the absenceofCa 2fromthecontrolcurrentsaswellasfromthoserecordedinthepresenceofATP (30M),ATPwasfoundtocauseinhibition.NotethedifferentcurrentcalibrationsinAaandAb. B, Triple immunofluorescence for P2X3, VR1, and P2Y1 receptors of rat DRG neurons investi- gated by means of a laser scanning confocal microscope. Images are of fluorescence for P2X3 (greenCy2immunofluorescence),VR1(redCY3-immunofluorescence),andP2Y1(blueCy5im- munofluorescence) receptor subtypes. Colocalization of P2X3, VR1, and P2Y1 receptors is shown.

Article Snippet: After fixation with ice-cold methanol for 10 min at 4°C, washing with HBSS for 5 min, and blocking with 5% FCS (Seromed, Berlin, Germany) and 0.1% Triton X-100 in TBS (0.05 M; pH 7.6), the cell cultures were incubated in a first step with an antibody mixture of rabbit anti-P2Y1 (1:400; Alomone, Jerusalem, Israel) and guinea pig anti-P2X3 receptor antibodies (1:1000; Neuromics, Minneapolis, MN) in TBS containing 0.1% Triton X-100 and 5% FCS overnight at 4°C.

Techniques: Functional Assay, Patch Clamp, Immunofluorescence, Microscopy, Fluorescence

Journal: Journal of Neuroscience

Article Title: Inhibition of N-Type Voltage-Activated Calcium Channels in Rat Dorsal Root Ganglion Neurons by P2Y Receptors Is a Possible Mechanism of ADP-Induced Analgesia

doi: 10.1523/jneurosci.4019-03.2004

Figure Lengend Snippet: Figure 7. Schematic drawing demonstrating the multiple modulatory effects of ATP on intracellularCa 2inDRGneurons.ATPmayactivateP2X3receptorchannels,therebyfacilitat- ingtheentryofCa 2viathesechannelsintothecell.Moreover,theactivationofP2Y1receptors mayleadviatheGsubunittothegenerationofIP3andthesubsequentreleaseofCa 2from theendoplasmaticreticulum.Finally,P2Y1receptorsmayalsoviaGclosevoltage-operated Ca 2 channels and thereby inhibit the passage of Ca 2 through the VACCs. Hence, the intra- cellular Ca 2 concentration may be modified by three simultaneous and partly opposing ef- fects of ATP, two of which are algogenic (P2X3, P2Y1/G) and one antinociceptive (P2Y1/ G).PLC,phospholipaseC;PIP2,phosphatidylinositol4,5-bisphosphate;DAG,diacylglycerol; ER, endoplasmatic reticulum.

Article Snippet: After fixation with ice-cold methanol for 10 min at 4°C, washing with HBSS for 5 min, and blocking with 5% FCS (Seromed, Berlin, Germany) and 0.1% Triton X-100 in TBS (0.05 M; pH 7.6), the cell cultures were incubated in a first step with an antibody mixture of rabbit anti-P2Y1 (1:400; Alomone, Jerusalem, Israel) and guinea pig anti-P2X3 receptor antibodies (1:1000; Neuromics, Minneapolis, MN) in TBS containing 0.1% Triton X-100 and 5% FCS overnight at 4°C.

Techniques: Concentration Assay, Modification

Journal: Translational Andrology and Urology

Article Title: Evaluation of transurethral GreenLight laser-selective vaporization for refractory overactive bladder in women

doi: 10.21037/tau-24-67

Figure Lengend Snippet: Expressions and comparisons of inflammatory factors and sensory receptors in bladder mucosa tissues before and after treatment. (A) The expressions of TRPV1, P2X3, TNF-α, and IL-6 in patients’ bladder mucosal tissues were stained by immunohistochemical staining before and at 12 weeks after treatment. Representative histological images are shown at 400× magnification. (B) Average optical density value of inflammatory factors and sensory receptors before and after treatment were presented in the bar chart. TNF, tumor necrosis factor; IL-6, interleukin 6; AOD, average optical density.

Article Snippet: Immunohistochemical staining was performed using the avidin-biotin-peroxidase method (ab64212, Abcam, Cambridge, MA, USA) with TRPV1 antibody (CSB-PA822774LA01HU, CUSABIO, Houston, TX, USA), P2X3 antibody (17843-1-AP, Proteintech, Rosemont, IL, USA), tumor necrosis factor alpha (TNF-α) antibody (17590-1-AP, Proteintech, Rosemont, IL, USA), and interleukin 6 (IL-6) antibody (21865-1-AP, Proteintech, Rosemont, IL, USA).

Techniques: Staining, Immunohistochemical 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 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: International Journal of Molecular Sciences

Article Title: Adipose-Derived Stem Cells and Their Derived Microvesicles Ameliorate Detrusor Overactivity Secondary to Bilateral Partial Iliac Arterial Occlusion-Induced Bladder Ischemia

doi: 10.3390/ijms22137000

Figure Lengend Snippet: Purinergic and muscarinic signaling associated molecules changes on western blot study in separated group. showed the expression of muscarinic cholinergic (M2 and M3) and purinergic receptor proteins (P2X2 and P2X3) by Western blot. In the top panel, ( a ) showed the expression of M2 receptor is significantly decreased in BPAO + ADSCs group compared to BAPO group ( # p < 0.05). The expression of M2 receptor had trend of decreasing in BPAO + ADSC-derived MVs group compared to BAPO group without significant differences ( p > 0.05). In the middle panel, ( b ) showed that the expression of M3 receptor was significantly increased in BPAO group compared to sham group (* p < 0.05). The expression of M3 receptor is significantly decreased in BPAO + ADSC-derived MVs group compared to BAPO group ( # p < 0.05). ( c ) showed the expression of P2X2 receptors was significantly increased in BPAO group compared to sham group (* p < 0.05). The expression of P2X2 receptor is significantly decreased in BPAO + ADSC-derived MVs and BAPO + MVs group compared to BAPO group ( # p < 0.05). In the bottom panel, ( d ) showed the expression of P2X3 receptor is of no significant differences between groups.

Article Snippet: Antibodies raised against P2X2 receptors (ab10266, Cambridge, UK), P2X3 receptors (Neuromics, RA141399, Northfield, MN, USA), M2 receptors (Novus bio, nb120-2805, Novus Biologicals, LLC, Colorado, USA), M3 receptors (Abcam, ab87199, Cambridge, UK), and NGF (Abcam, ab6199, Cambridge, UK) were used.

Techniques: Western Blot, Expressing, Derivative Assay