Journal: Cell Communication and Signaling : CCS

Article Title: Bradykinin-induced Ca 2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y 12 receptors activation

doi: 10.1186/1478-811X-11-70

Figure Lengend Snippet: Bradykinin-induced [Ca 2+ ] i mobilization is partially dependent on the activation of P2Y 12 purinoceptors. Panel A shows the effect of bradykinin (BK, 30 μM) after pretreating human subcutaneous fibroblasts with apyrase (2 U/mL, Ai ), which catabolizes ATP/ADP into AMP, and after inhibition of ectonucleotidases, with POM-1 (20 μM, Aii ) or after removing Mg 2+ from the incubation fluid (Aiii) . Panel C shows the effects of BK (30 μM) in the absence or presence of selective P2Y 1 , P2Y 12 and P2Y 13 receptor antagonists, respectively MRS 2179 (0.3 μM, Ci ), AR-C 66096 (0.1 μM, Cii ) and MRS 2211 (10 μM, Ciii ). Cells were pre-incubated with the cell-permeant fluorescent calcium indicator, Fluo-4 NW (2.5 μM, see Methods). [Ca 2+ ] i transients were calibrated to the maximal calcium load produced by ionomycin (5 μM, 100% response). Black arrows indicate the time of drugs application. No changes in baseline fluorescence were observed after application of the modulators. Each point represents pooled data from an n number of experiments. The vertical bars represent S.E.M.. * p < 0.05 represent significant differences from BK (30 μM) alone. Panel B illustrates the time course of the extracellular catabolism of adenine nucleotides in human subcutaneous fibroblasts grown in culture for 11 days. ATP, ADP or AMP (3 μM) were added to the culture medium at time zero; samples (75 μl) were collected at indicated times. Each sample was analyzed by HPLC to separate and quantify ATP (white), ADP (black), AMP (grey), adenosine (ADO, red), inosine (INO, orange) and hypoxanthine (HX, green). Each point represents pooled data from two individuals; 2 replicas were performed for each individual. The calculated half-life time (t ½ ) for each initial substrate is shown for comparison. Panel D shows immunoreactivity of human subcutaneous fibroblasts against the P2Y 12 receptor; shown image is representative of three independent experiments. Image scale bar is 30 μm.

Article Snippet: Cultured cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 minutes, washed 3 times in PBS (10 minutes each) and, subsequently, incubated with blocking buffer I (10% FBS, 1% bovine serum albumin (BSA), 0.1% Triton-X, 0.05% NaN 3 ) for 1 h. Primary antibodies, diluted in blocking buffer II (5% FBS, 1% BSA, 0.1% Triton-X, 0.05% NaN 3 ), were applied [mouse anti-porcine vimentin 1:75 (DAKO); rabbit anti-human collagen I 1:50 (AbDSerotec); mouse anti-human α-smooth muscle actin-FITC 1:250 (Sigma-Aldrich); rabbit anti-human P2Y 12 1:100 (Alomone); rabbit anti-human Cx43 1:600 and rabbit anti-human Panx1 1:1000 (Abcam)] and the slides incubated overnight at 4°C.

Techniques: Activation Assay, Inhibition, Incubation, Produced, Fluorescence

Journal: Blood Transfusion

Article Title: Expression and function of purinergic receptors in platelets from apheresis-derived platelet concentrates

doi: 10.2450/2015.0073-15

Figure Lengend Snippet: Unchanged P2Y12 receptor function during storage of APC.

Article Snippet: Rabbit polyclonal anti-P2Y1, anti-P2Y12 and anti-P2X1 antibodies were from Alomone Labs (Jerusalem, Israel).

Techniques:

Journal: PLoS ONE

Article Title: Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y 12/13 receptors among BV-2 microglia

doi: 10.1371/journal.pone.0183114

Figure Lengend Snippet: MS indicates the mechanical stimulation event. Pentagram indicates the stimulated cell. (A) Application of ADP prior to mechanical stimulation blocks ICWs. (B-D) Application of 10 μM UTP (B) , 10 μM UDP (C) and 10 μM UDP-glucose (D) have no inhibitory effects on ICW communication. (E) Pretreatment with 10 μM for 15 min MRS2395 partially inhibits ICWs. (F) Preincubation with 10 μM MRS2211 for 15 min suppresses ICW propagation. (G) Summary inhibitory effects of nucleotides on ICWs within 75 μm (n ≥ 88 cells for each condition from at least three independent experiments). All values are expressed as mean ± SD. * P < 0.05 and *** P < 0.001, one-way ANOVA, Scheffé post hoc test. N.S., no significant difference. (H) Gene expression of P2Y receptors in BV-2 microglia at mRNA level detected by RT-PCR. The ten lanes in the gel are as follows: P2Y 1,2,4,6,12,13,14 (experimental group with primers directed towards the P2YR mRNA); nuclease-free water (negative control); GAPDH (positive control) and marker (with a list of standardized DNA sequences from 100 bp to 800 bp). (I) Quantitative statistical results of RT-PCR normalized to GAPDH (n = 3). All values are expressed as mean ± SD. (J) Western-blot assay indicates expression of P2Y 12 receptor in BV-2 microglia. (K) Immunolocalization of P2Y 12 receptor on the membrane of BV-2 cells. Scale bar = 30 μm.

Article Snippet: BV-2 microglial cells were prepared as described above and seeded on a glass cover slip in a 6-well plate (Corning, USA) for 24 h. For living cell immunostaining, cells were incubated with anti-P2Y 12 (extracellular) rabbit antibody (1:500; Alomone, Israel) resolved in DMEM at 37°C for 1h, followed by washing with HBSS for three times.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Marker, Western Blot

Journal: PLoS ONE

Article Title: Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y 12/13 receptors among BV-2 microglia

doi: 10.1371/journal.pone.0183114

Figure Lengend Snippet: ① A range of nucleotides, including ATP, ADP and UDP etc., are released into extracellular space in response to mechanical stimulus. ② P2Y 12/13 receptors localized on the membrane of neighboring cells can sense ATP/ADP released from the stimulated cell, then activate PLC/IP 3 /Ca 2+ signaling. ③ Upon sensing and responding to released ATP/ADP, Ca 2+ mobilization sequentially occur in neighboring cells, thus perform as intercellular calcium waves. ④ ATP cannot activate P2Y 12/13 receptors directly. Ecto-NTPase located on the plasma membrane could catalyze hydrolysis of ATP and generates ADP that predominantly activates P2Y 12/13 receptors.

Article Snippet: BV-2 microglial cells were prepared as described above and seeded on a glass cover slip in a 6-well plate (Corning, USA) for 24 h. For living cell immunostaining, cells were incubated with anti-P2Y 12 (extracellular) rabbit antibody (1:500; Alomone, Israel) resolved in DMEM at 37°C for 1h, followed by washing with HBSS for three times.

Techniques:

Journal: Purinergic Signalling

Article Title: Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

doi: 10.1007/s11302-008-9108-0

Figure Lengend Snippet: Expression of P2Y 1 and P2Y 2 receptors in human anagen hair follicles. a P2Y 1 receptors were found in the outer root sheath ( ORS ) and bulb of anagen hair follicles in longitudinal section but not in the inner root sheath ( IRS ). Scale bar = 40 μm. b In transverse section, P2Y 1 receptors were only seen in the outer root sheath ( ORS ). Scale bar = 40 μm. c P2Y 2 receptors were found in the cortex ( CTX ). Scale bar = 40 μm. d Transverse section of anagen hair follicle: P2Y 2 receptors were seen in the cortex ( CTX ) but not in the central medulla ( M ), inner ( IRS ) or outer root sheaths ( ORS ), or in the surrounding adventitial layer ( A ). Scale bar = 40 μm. e , f Controls: the immunoreaction was abolished after preabsorption of the e P2Y 1 and f P2Y 2 receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. Scale bars = 100 μm

Article Snippet: Polyclonal anti-P2Y 1 and P2Y 2 antibodies were obtained from Alomone Labs (Jerusalem, Israel) and corresponded to the third extracellular loop of the P2Y 1 (AA 242–258) and P2Y 2 receptor (AA 227–244).

Techniques: Expressing

Journal: Purinergic Signalling

Article Title: Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

doi: 10.1007/s11302-008-9108-0

Figure Lengend Snippet: Double labelling of P2Y 1 and P2Y 2 receptors with markers for cellular proliferation, and double labelling of P2X 5 receptors with markers for keratinocyte differentiation in anagen hair follicles. a Double labelling of P2Y 1 receptors ( red ) with Ki-67, a nuclear marker for proliferating cells ( green ), to show that P2Y 1 receptors are found in proliferating basal cells in the outer root sheath ( ORS ) and bulb region of the hair follicle in longitudinal section. Scale bar = 50 μm. b Transverse section: double labelling of P2Y 1 receptors ( red ) with Ki-67, a nuclear marker ( green ), to show that P2Y 1 receptors are found in proliferating cells in the outer root sheath ( ORS ) of the hair follicle. Scale bar = 50 μm. c Longitudinal section of anagen hair follicle through the dermal papilla ( DP ): double labelling of P2Y 2 receptors ( red ) with proliferating cell nuclear antigen (PCNA), a marker for proliferating cells ( green ), showed that P2Y 2 receptors were found in the cortex ( CTX ) and at the edge of the medulla ( M ) but not in the central medulla. P2Y 2 receptors were not found in the matrix ( Ma ), where cells were positive for PCNA. Scale bar = 75 μm. d Longitudinal section of anagen hair follicle: P2Y 2 receptors were absent from the keratinised cuticle ( Cu ) of the hair shaft. PCNA was also found in cells of the outer root sheath ( ORS ). Scale bar = 75 μm. e Double labelling of P2X 5 receptors ( red-brown ) with involucrin, a marker for differentiating cells ( green ). Involucrin was expressed both in the inner root sheath ( IRS ), cortex ( CTX ) and in the outermost edge of the medulla ( M ). P2X 5 receptors were expressed in the inner ( IRS ) and outer root sheaths ( ORS ) and in the medulla ( M ) and matrix cells ( Ma ). There was yellow colocalisation with P2X 5 receptors in the inner root sheath and in cells at the outermost edge of the medulla ( arrow ). The cortex only stained positive for involucrin, not P2X 5 receptors. Scale bar = 50 μm. f Transverse section: double labelling of P2X 5 receptors ( red ) with involucrin ( green ). Involucrin was expressed in the inner root sheath ( IRS ) and cortex ( CTX ) and colocalised ( yellow ) with P2X 5 receptor staining in the inner root sheath ( IRS ). Scale bar = 50 μm

Article Snippet: Polyclonal anti-P2Y 1 and P2Y 2 antibodies were obtained from Alomone Labs (Jerusalem, Israel) and corresponded to the third extracellular loop of the P2Y 1 (AA 242–258) and P2Y 2 receptor (AA 227–244).

Techniques: Marker, Staining