Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: List of primary and secondary antibodies used in immunohistochemistry experiments.

Article Snippet: Confocal micrographs shown in demonstrate that P2X4 receptor protein is expressed in the plasma membrane of cardiomyocytes of all assayed regions of the rat heart; in these experiments we used a knock-out validated antibody targeting the amino acid residues 370–388 of the C-terminus of the rat P2X4 receptor (Apr-002 from Alomone).

Techniques: Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p < 0.05, **p < 0.01 (Student’s t -test for paired samples) represent significant differences when compared to the effects of ATP or CTP alone, respectively.

Article Snippet: Confocal micrographs shown in demonstrate that P2X4 receptor protein is expressed in the plasma membrane of cardiomyocytes of all assayed regions of the rat heart; in these experiments we used a knock-out validated antibody targeting the amino acid residues 370–388 of the C-terminus of the rat P2X4 receptor (Apr-002 from Alomone).

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p < 0.05 (Student’s t -test for paired samples) represent significant differences when compared to the effect of ATP alone.

Article Snippet: Confocal micrographs shown in demonstrate that P2X4 receptor protein is expressed in the plasma membrane of cardiomyocytes of all assayed regions of the rat heart; in these experiments we used a knock-out validated antibody targeting the amino acid residues 370–388 of the C-terminus of the rat P2X4 receptor (Apr-002 from Alomone).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.

Article Snippet: Confocal micrographs shown in demonstrate that P2X4 receptor protein is expressed in the plasma membrane of cardiomyocytes of all assayed regions of the rat heart; in these experiments we used a knock-out validated antibody targeting the amino acid residues 370–388 of the C-terminus of the rat P2X4 receptor (Apr-002 from Alomone).

Techniques: Expressing, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .

Article Snippet: Confocal micrographs shown in demonstrate that P2X4 receptor protein is expressed in the plasma membrane of cardiomyocytes of all assayed regions of the rat heart; in these experiments we used a knock-out validated antibody targeting the amino acid residues 370–388 of the C-terminus of the rat P2X4 receptor (Apr-002 from Alomone).

Techniques: Activity Assay, Inhibition

Journal: Brain, Behavior, & Immunity - Health

Article Title: Exercise-induced changes in gene expression do not mediate post exertional malaise in Gulf War illness

doi: 10.1016/j.bbih.2023.100612

Figure Lengend Snippet: Visual representation of genes showing significant main effect for group Note. Pre-exercise gene expression was relativized to a value of 1.0 (gray bar). A significant main effect of group was observed for ACTB (Main Effect: partial η 2 = 0.09, 95% CI = 0.01, 1.00; 30 min post: p = 0.02; 24 h post: p = 0.04), COMT (Main Effect: partial η 2 = 0.10, 95% CI = 0.01, 1.00; 30 min post: p = 0.02; 24 h post: p = 0.04), TLR4 (Main Effect: partial η 2 = 0.10, 95% CI = 0.01, 1.00; 30 min post: p = 0.01; 24 h post: p = 0.03) at both 30 min and 24 h post-exercise as detected by RM-ANOVA.

Article Snippet: 10 ng of cDNA single-plex reactions was performed in triplicate for 13 inventoried gene expression assays (β-actin: ACTB - Hs01060665_g1; β-2 adrenergic receptor: ADRB2 - Hs00240532_s1; acid sensing ion channel 3: ASIC3 - Hs00245097_m1; cluster of differentiation 14: CD14 - Hs02621496_s1; catechol-O-methyltransferase: COMT - Hs00241349_m1; interleukin 6: IL6 - Hs00985639_m1; interleukin 10: IL10 - Hs00961662_m1; lymphotoxin-α: LTA - Hs04188773_g1; nuclear receptor subfamily 3, group C member 1: NR3C1 - Hs00353740_m1; purinoceptor 4: P2RX4 - Hs00602442_m1; purinoceptor 5: P2RX5 - Hs01112471_m1; toll-like receptor 4: TLR4 - Hs00152939_m1; transient receptor potential cation channel subfamily V member 1: TRPV1 - Hs00218912_m1) and 3 housekeeping genes (Extracellular glucosultransferase: GTFB2 - Hs00155321_m1; Proteasome subunit beta 6: PSMB6 - Hs00382586_m1; Importin 8: IPO8 - Hs00183533_m1) on a 384 well plate.

Techniques: Gene Expression

Journal: Brain, Behavior, & Immunity - Health

Article Title: Exercise-induced changes in gene expression do not mediate post exertional malaise in Gulf War illness

doi: 10.1016/j.bbih.2023.100612

Figure Lengend Snippet: Group comparisons of gene expression between Veterans with Gulf War illness (GWI; n = 37) and healthy control Veterans (CON; n = 25).

Article Snippet: 10 ng of cDNA single-plex reactions was performed in triplicate for 13 inventoried gene expression assays (β-actin: ACTB - Hs01060665_g1; β-2 adrenergic receptor: ADRB2 - Hs00240532_s1; acid sensing ion channel 3: ASIC3 - Hs00245097_m1; cluster of differentiation 14: CD14 - Hs02621496_s1; catechol-O-methyltransferase: COMT - Hs00241349_m1; interleukin 6: IL6 - Hs00985639_m1; interleukin 10: IL10 - Hs00961662_m1; lymphotoxin-α: LTA - Hs04188773_g1; nuclear receptor subfamily 3, group C member 1: NR3C1 - Hs00353740_m1; purinoceptor 4: P2RX4 - Hs00602442_m1; purinoceptor 5: P2RX5 - Hs01112471_m1; toll-like receptor 4: TLR4 - Hs00152939_m1; transient receptor potential cation channel subfamily V member 1: TRPV1 - Hs00218912_m1) and 3 housekeeping genes (Extracellular glucosultransferase: GTFB2 - Hs00155321_m1; Proteasome subunit beta 6: PSMB6 - Hs00382586_m1; Importin 8: IPO8 - Hs00183533_m1) on a 384 well plate.

Techniques: Gene Expression, Control

Journal: PLoS ONE

Article Title: ATP Induced Brain-Derived Neurotrophic Factor Expression and Release from Osteoarthritis Synovial Fibroblasts Is Mediated by Purinergic Receptor P2X4

doi: 10.1371/journal.pone.0036693

Figure Lengend Snippet: Representative photomicrographs showing immunohistochemical double staining of OA and RA synovial tissues with P2X4 (brown) and fibroblast marker (green). A . original magnification 400×, box shows the Isotype control, magnification 200×. B . original magnification 630×.

Article Snippet: After blocking, the membranes were probed with antibodies against P2X4 receptor (Alomone Labs, Jerusalem, Israel; 1∶200), p38 MAPK (Cell Signaling, Allschwil, Switzerland; 1∶1000), phospho-p38 (p-p38) MAPK (Cell Signaling; 1∶1000), p44/42 MAPK (Cell Signaling; 1∶1000), phospho-p44/42 (p-p44/42) MAPK (Cell Signaling; 1∶1000), and α-tubulin (Abcam, Cambridge, UK; 1∶5000), respectively.

Techniques: Immunohistochemical staining, Double Staining, Marker, Control

Journal: PLoS ONE

Article Title: ATP Induced Brain-Derived Neurotrophic Factor Expression and Release from Osteoarthritis Synovial Fibroblasts Is Mediated by Purinergic Receptor P2X4

doi: 10.1371/journal.pone.0036693

Figure Lengend Snippet: Endogenous P2X4 expression in OASF was reduced by siRNA transfection before ATP (100 µM) stimulation for 2 h. A . P2X4 mRNA expression was reduced compared to scrambled controls. The ATP-induced BDNF mRNA expression in P2X4 siRNA transfected cells was reduced compared to scrambled controls. B . P2X4 protein levels were reduced by P2X4 siRNA transfection compared to scrambled controls. α-tubulin levels in P2X4 siRNA transfected cells were slightly decreased compared to controls due to experimental conditions. C . Densitometric analysis of Western blot results. Data are shown as means ± standard deviations. N.s., not significant; *, p<0.05, ***, p<0.001.

Article Snippet: After blocking, the membranes were probed with antibodies against P2X4 receptor (Alomone Labs, Jerusalem, Israel; 1∶200), p38 MAPK (Cell Signaling, Allschwil, Switzerland; 1∶1000), phospho-p38 (p-p38) MAPK (Cell Signaling; 1∶1000), p44/42 MAPK (Cell Signaling; 1∶1000), phospho-p44/42 (p-p44/42) MAPK (Cell Signaling; 1∶1000), and α-tubulin (Abcam, Cambridge, UK; 1∶5000), respectively.

Techniques: Expressing, Transfection, Western Blot