kcnk3  (Alomone Labs)

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

Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

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

Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

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: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

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: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

Techniques: Activity Assay, Inhibition

Journal: Cell Reports

Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

doi: 10.1016/j.celrep.2019.03.011

Figure Lengend Snippet: Macrophage Polarization Status Affects Calcium Signal Propagation (A) The surface expression of P2X4R (top) and P2X7R (bottom) was analyzed by flow cytometry in resting, IFNγ-treated (10 ng/mL, 24 h), or IL4-treated (20 ng/mL, 24 h) macrophages. Histograms show the quantification 3 independent biological replicates. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used (ns, non-significant; ∗∗ p

Article Snippet: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

Techniques: Expressing, Flow Cytometry

Journal: Cell Reports

Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

doi: 10.1016/j.celrep.2019.03.011

Figure Lengend Snippet: Extracellular ATP Is Required for Efficient Phagocytosis (A) Primary BMDMs were incubated with PhRodo E. coli fluorescent bioparticles in the presence or absence of 5 mM EGTA to chelate extracellular calcium. Phagocytosis was monitored at 15 or 30 min by flow cytometry (see Figure S4 ). Macrophages incubated with 20 μM cytochalasin D were used as negative reference. The phagocytic index was calculated as the percentage of fluorescent macrophages multiplied by their mean of fluorescence (MFI) and normalized on the cytochalasin-treated samples. (B) Primary BMDMs were loaded with the intracellular calcium chelator BAPTA-AM or its vehicle (loading solution) before performing the phagocytosis assay. (C) Primary BMDMs were incubated with PhRodo E. coli , PhRodo Zymosan, or PhRodo S. aureus fluorescent bioparticles, in the presence or absence of apyrase (5 U/mL). (D) Primary BMDMs were pretreated with the P2X4R inhibitor 5BDBD (100 μM), the P2X7R inhibitor A740003 (100 μM), or their vehicle (DMSO), or were left untreated, before performing the phagocytosis assay. (E) Phagocytosis was performed for 30 min in the presence or absence of MSC-derived EVs, pre-incubated or not with ARL-67516 (30 min, 200 μM). The graphs are representative of at least 3 independent biological replicates, each performed in technical triplicate. Error bars represent SEM. For data analysis, a two-way ANOVA followed by Tukey’s multiple comparisons test was used ( ∗ p

Article Snippet: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

Techniques: Incubation, Flow Cytometry, Fluorescence, Phagocytosis Assay, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Two‐Pore K+ Channel TREK‐1 Regulates Sinoatrial Node Membrane Excitability

doi: 10.1161/JAHA.115.002865

Figure Lengend Snippet: TREK ‐1 deficiency does not affect activity or expression of other channels important for SAN function. A and B, Representative current traces of pacemaker current ( I f ) and summary current‐voltage data in WT and Kcnk2 −/− SAN cells (n=5 from 4 preparations for WT , n=5 from 3 preparations for Kcnk2 −/− , P = NS ). Voltage ramp protocol is shown in inset. C, Immunoblots showing expression of voltage‐gated K + channels important for SAN cell repolarization (K v 2.1, K v 4.2, K v 4.3), HCN 4, and K 2P family member TASK ‐1 in detergent‐soluble lysates from WT and α MHC ‐Kcnk2 f/f ( Kcnk2 −/− ) SAN (3 pooled samples from 3 preparations). D, Isolated WT and Kcnk2 −/− adult SAN myocytes immunostained for HCN 4, TREK ‐1, and TASK ‐1. Scale bar=5 μm. SAN indicates sinoatrial node; WT, wild type.

Article Snippet: The following antibodies were used for immunoblotting, immunoprecipitation, or immunostaining: TREK‐1 (C‐terminal: sc50412, N‐terminal: sc11554; Santa Cruz Biotechnology), βIV ‐spectrin (N‐terminal, HPA043370, lot A96519; Sigma‐Aldrich), Kir 2.1 (Alomone), Kv 2.1 (NeuroMab), Kv 4.2 (Alomone), Kv4.3, TASK‐1 (APC024; Alomone), HCN4 (32675, lot GR153269‐7; Abcam), N‐cadherin (333900, lot 1397763A; Invitrogen), Connexin43 (MAB3067, lot 23070602; Invitrogen), and GAPDH (10R‐G109A; Fitzgerald).

Techniques: Activity Assay, Expressing, Western Blot, Isolation

Journal: Frontiers in Physiology

Article Title: Leptin Signaling in the Carotid Body Regulates a Hypoxic Ventilatory Response Through Altering TASK Channel Expression

doi: 10.3389/fphys.2018.00249

Figure Lengend Snippet: Stimulatory effect of leptin on pSTAT3/STAT3 and TASK-1,-2,-3 channel protein expression in CBs. Gel images and bar charts showing quantitative analysis of leptin-stimulated expression of ob-Rb (A) pSTAT3/STAT3 (B) and TASK-1,-2,-3 (C–E) in the CB ( n = 8) of each group ( n = 4). ** P

Article Snippet: The membranes were blocked with bovine serum albumin and incubated at 4°C overnight with primary antibodies anti-ob-R (1:2000, #ab5593, Abcam, USA), anti-TASK-1 (1:200, #APC024, Alomone labs, Israel), anti-TASK-2 (1:200, #APC037, Alomone labs, Israel), anti-TASK-3 (1:200, #APC044, Alomone labs, Israel), anti-pSTAT3 (1:2000, #9145, Cell Signaling Technology, USA), anti-STAT3 (1:1000, #9139, Cell Signaling Technology, USA), and anti-β-actin (1:3,000, #T0022, Affinity Biosciences, USA).

Techniques: Expressing