psalmotoxin 1  (Alomone Labs)


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    Alomone Labs psalmotoxin 1
    D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM <t>PcTx1</t> (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.
    Psalmotoxin 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    psalmotoxin 1 - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction"

    Article Title: Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

    Journal: Neuroscience

    doi: 10.1016/j.neuroscience.2021.01.036

    D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM PcTx1 (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.
    Figure Legend Snippet: D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM PcTx1 (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.

    Techniques Used: Incubation

    High concentrations of the ASIC inhibitors Benzamil and PcTx1 reduce mEPP and EPP amplitude. MEPPs, EPPs and Quantal Content are plotted from NMJs incubated in either control saline (n=75, same data show in 1A), 100 μM benzamil (n=15, 3 mice), and 100 nM PcTx1 (n=25, 5 mice). Data are plotted as in Fig. 1. P values are indicated and were calculated from Student’s t-test or ordinary one-way ANOVA.
    Figure Legend Snippet: High concentrations of the ASIC inhibitors Benzamil and PcTx1 reduce mEPP and EPP amplitude. MEPPs, EPPs and Quantal Content are plotted from NMJs incubated in either control saline (n=75, same data show in 1A), 100 μM benzamil (n=15, 3 mice), and 100 nM PcTx1 (n=25, 5 mice). Data are plotted as in Fig. 1. P values are indicated and were calculated from Student’s t-test or ordinary one-way ANOVA.

    Techniques Used: Incubation

    Lowering extracellular pH underlies the upregulation of QC triggered by d-TC. A. mEPPs and EPPs were measured and QC estimated before and after a 10 min incubation in pH 7.2 saline with 100nM PcTx1 present throughout (n=25, 5 mice). The data for mEPPs, EPPs and QC are presented as violin plots. The plot on the far right includes the data from Fig. 3B to depicts the effect of pH 7.2 saline under control conditions vs. in the presence of 100 nM PcTx1. The Mean differences are plotted as a bootstrap sampling distribution. Means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA. B. mEPPs and EPPs were measured and QC was estimated in pH 7.4 saline, after a 7 min incubation in pH 7.2 saline, and then after a 15 min incubation with d-TC, still in pH 7.2 saline (n = 5 mice). Although dTC had its normal effects on mEPP and EPP amplitude (compare to Fig. 1A), dTC did not alter QC when compared to pH 7.2 saline. The data is plotted as in panel A. C. The ratio of QC after application of dTC compared to before dTC was applied (post/pre dTC) is shown for pH 7.4 saline (this is the same data presented in Fig 1B, Control) and for pH 7.2 saline (the same data presented in Fig. 4B).
    Figure Legend Snippet: Lowering extracellular pH underlies the upregulation of QC triggered by d-TC. A. mEPPs and EPPs were measured and QC estimated before and after a 10 min incubation in pH 7.2 saline with 100nM PcTx1 present throughout (n=25, 5 mice). The data for mEPPs, EPPs and QC are presented as violin plots. The plot on the far right includes the data from Fig. 3B to depicts the effect of pH 7.2 saline under control conditions vs. in the presence of 100 nM PcTx1. The Mean differences are plotted as a bootstrap sampling distribution. Means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA. B. mEPPs and EPPs were measured and QC was estimated in pH 7.4 saline, after a 7 min incubation in pH 7.2 saline, and then after a 15 min incubation with d-TC, still in pH 7.2 saline (n = 5 mice). Although dTC had its normal effects on mEPP and EPP amplitude (compare to Fig. 1A), dTC did not alter QC when compared to pH 7.2 saline. The data is plotted as in panel A. C. The ratio of QC after application of dTC compared to before dTC was applied (post/pre dTC) is shown for pH 7.4 saline (this is the same data presented in Fig 1B, Control) and for pH 7.2 saline (the same data presented in Fig. 4B).

    Techniques Used: Incubation, Sampling

    psalmotoxin 1  (Alomone Labs)


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    Structured Review

    Alomone Labs psalmotoxin 1
    D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM <t>PcTx1</t> (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.
    Psalmotoxin 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/psalmotoxin 1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    psalmotoxin 1 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction"

    Article Title: Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

    Journal: Neuroscience

    doi: 10.1016/j.neuroscience.2021.01.036

    D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM PcTx1 (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.
    Figure Legend Snippet: D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM PcTx1 (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.

    Techniques Used: Incubation

    High concentrations of the ASIC inhibitors Benzamil and PcTx1 reduce mEPP and EPP amplitude. MEPPs, EPPs and Quantal Content are plotted from NMJs incubated in either control saline (n=75, same data show in 1A), 100 μM benzamil (n=15, 3 mice), and 100 nM PcTx1 (n=25, 5 mice). Data are plotted as in Fig. 1. P values are indicated and were calculated from Student’s t-test or ordinary one-way ANOVA.
    Figure Legend Snippet: High concentrations of the ASIC inhibitors Benzamil and PcTx1 reduce mEPP and EPP amplitude. MEPPs, EPPs and Quantal Content are plotted from NMJs incubated in either control saline (n=75, same data show in 1A), 100 μM benzamil (n=15, 3 mice), and 100 nM PcTx1 (n=25, 5 mice). Data are plotted as in Fig. 1. P values are indicated and were calculated from Student’s t-test or ordinary one-way ANOVA.

    Techniques Used: Incubation

    Lowering extracellular pH underlies the upregulation of QC triggered by d-TC. A. mEPPs and EPPs were measured and QC estimated before and after a 10 min incubation in pH 7.2 saline with 100nM PcTx1 present throughout (n=25, 5 mice). The data for mEPPs, EPPs and QC are presented as violin plots. The plot on the far right includes the data from Fig. 3B to depicts the effect of pH 7.2 saline under control conditions vs. in the presence of 100 nM PcTx1. The Mean differences are plotted as a bootstrap sampling distribution. Means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA. B. mEPPs and EPPs were measured and QC was estimated in pH 7.4 saline, after a 7 min incubation in pH 7.2 saline, and then after a 15 min incubation with d-TC, still in pH 7.2 saline (n = 5 mice). Although dTC had its normal effects on mEPP and EPP amplitude (compare to Fig. 1A), dTC did not alter QC when compared to pH 7.2 saline. The data is plotted as in panel A. C. The ratio of QC after application of dTC compared to before dTC was applied (post/pre dTC) is shown for pH 7.4 saline (this is the same data presented in Fig 1B, Control) and for pH 7.2 saline (the same data presented in Fig. 4B).
    Figure Legend Snippet: Lowering extracellular pH underlies the upregulation of QC triggered by d-TC. A. mEPPs and EPPs were measured and QC estimated before and after a 10 min incubation in pH 7.2 saline with 100nM PcTx1 present throughout (n=25, 5 mice). The data for mEPPs, EPPs and QC are presented as violin plots. The plot on the far right includes the data from Fig. 3B to depicts the effect of pH 7.2 saline under control conditions vs. in the presence of 100 nM PcTx1. The Mean differences are plotted as a bootstrap sampling distribution. Means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA. B. mEPPs and EPPs were measured and QC was estimated in pH 7.4 saline, after a 7 min incubation in pH 7.2 saline, and then after a 15 min incubation with d-TC, still in pH 7.2 saline (n = 5 mice). Although dTC had its normal effects on mEPP and EPP amplitude (compare to Fig. 1A), dTC did not alter QC when compared to pH 7.2 saline. The data is plotted as in panel A. C. The ratio of QC after application of dTC compared to before dTC was applied (post/pre dTC) is shown for pH 7.4 saline (this is the same data presented in Fig 1B, Control) and for pH 7.2 saline (the same data presented in Fig. 4B).

    Techniques Used: Incubation, Sampling

    pctx1  (Alomone Labs)


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    Alomone Labs pctx1
    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTx1</t> (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "ASIC1a senses lactate uptake to regulate metabolism in neurons"

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    Journal: Redox Biology

    doi: 10.1016/j.redox.2022.102253

    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.
    Figure Legend Snippet: Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.

    Techniques Used: Cell Culture, Fluorescence

    ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
    Figure Legend Snippet: ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

    Techniques Used: Fluorescence

    pctx 1  (Alomone Labs)


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    Alomone Labs pctx 1
    Proton and non-proton activation of ASIC1a in HEK cells. (A) Representative membranes of lysates of HEK cells treated with pH6 or MitTx for 2 or 10 min or preincubated with <t>Pctx-1</t> compared to untreated cells (control, Ctr) and detected with phosphoERK (pERK), total ERK (tERK), and ASIC1 antibodies. (B) Representative membrane of the same lysates to detect pCaMKII levels. (C) Plots showing detected levels of pERK (top panel) or pCaMKII (lower panel). Notice that the increase in kinase levels goes further at a later time point (2 vs. 10 min) in MitTx treated cultures compared to pH6 treated ones that show an increase at 2 min followed by a reversal to control levels consistent with the proton-activated desensitizing mechanism. (A) Notice that plots are the result of the signal intensity of the bands—with tERK and tubulin used as loading controls between loaded samples—and expressed relative to control samples. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments against the control were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; *** p 0.0001–0.001; ns: no significant differences. Mean values expressed relative to control ( Ctr ) levels ± SEM are as follows: for pERK/tERK: pH6 2’ 6.10 ± 0.13; pH6 10’ 1.23 ± 0.11; pH6 2’ Pctx 1.39 ± 0.08; MitTx 2’ 7.98 ± 0.10; Mittx10 ’ 10.90 ± 0.17; MitTx 2’ Pctx 1.18 ± 0.05 ; MitTx 10’ Pctx 1.42 ± 0.05. For pCaMKII/Tub: pH6 2’ 7.31 ± 0.21; pH6 10’ 1.16 ± 0.06; pH6 2’ Pctx 1.14±0.07; Mittx 2’ 8.88 ± 0.15; Mittx 10’ 10.76 ± 0.32; Mittx 2’ Pctx 1.17 ± 0.07.
    Pctx 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Signaling Pathways in Proton and Non-proton ASIC1a Activation"

    Article Title: Signaling Pathways in Proton and Non-proton ASIC1a Activation

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2021.735414

    Proton and non-proton activation of ASIC1a in HEK cells. (A) Representative membranes of lysates of HEK cells treated with pH6 or MitTx for 2 or 10 min or preincubated with Pctx-1 compared to untreated cells (control, Ctr) and detected with phosphoERK (pERK), total ERK (tERK), and ASIC1 antibodies. (B) Representative membrane of the same lysates to detect pCaMKII levels. (C) Plots showing detected levels of pERK (top panel) or pCaMKII (lower panel). Notice that the increase in kinase levels goes further at a later time point (2 vs. 10 min) in MitTx treated cultures compared to pH6 treated ones that show an increase at 2 min followed by a reversal to control levels consistent with the proton-activated desensitizing mechanism. (A) Notice that plots are the result of the signal intensity of the bands—with tERK and tubulin used as loading controls between loaded samples—and expressed relative to control samples. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments against the control were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; *** p 0.0001–0.001; ns: no significant differences. Mean values expressed relative to control ( Ctr ) levels ± SEM are as follows: for pERK/tERK: pH6 2’ 6.10 ± 0.13; pH6 10’ 1.23 ± 0.11; pH6 2’ Pctx 1.39 ± 0.08; MitTx 2’ 7.98 ± 0.10; Mittx10 ’ 10.90 ± 0.17; MitTx 2’ Pctx 1.18 ± 0.05 ; MitTx 10’ Pctx 1.42 ± 0.05. For pCaMKII/Tub: pH6 2’ 7.31 ± 0.21; pH6 10’ 1.16 ± 0.06; pH6 2’ Pctx 1.14±0.07; Mittx 2’ 8.88 ± 0.15; Mittx 10’ 10.76 ± 0.32; Mittx 2’ Pctx 1.17 ± 0.07.
    Figure Legend Snippet: Proton and non-proton activation of ASIC1a in HEK cells. (A) Representative membranes of lysates of HEK cells treated with pH6 or MitTx for 2 or 10 min or preincubated with Pctx-1 compared to untreated cells (control, Ctr) and detected with phosphoERK (pERK), total ERK (tERK), and ASIC1 antibodies. (B) Representative membrane of the same lysates to detect pCaMKII levels. (C) Plots showing detected levels of pERK (top panel) or pCaMKII (lower panel). Notice that the increase in kinase levels goes further at a later time point (2 vs. 10 min) in MitTx treated cultures compared to pH6 treated ones that show an increase at 2 min followed by a reversal to control levels consistent with the proton-activated desensitizing mechanism. (A) Notice that plots are the result of the signal intensity of the bands—with tERK and tubulin used as loading controls between loaded samples—and expressed relative to control samples. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments against the control were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; *** p 0.0001–0.001; ns: no significant differences. Mean values expressed relative to control ( Ctr ) levels ± SEM are as follows: for pERK/tERK: pH6 2’ 6.10 ± 0.13; pH6 10’ 1.23 ± 0.11; pH6 2’ Pctx 1.39 ± 0.08; MitTx 2’ 7.98 ± 0.10; Mittx10 ’ 10.90 ± 0.17; MitTx 2’ Pctx 1.18 ± 0.05 ; MitTx 10’ Pctx 1.42 ± 0.05. For pCaMKII/Tub: pH6 2’ 7.31 ± 0.21; pH6 10’ 1.16 ± 0.06; pH6 2’ Pctx 1.14±0.07; Mittx 2’ 8.88 ± 0.15; Mittx 10’ 10.76 ± 0.32; Mittx 2’ Pctx 1.17 ± 0.07.

    Techniques Used: Activation Assay

    Proton and non-proton activation of overexpressed ASIC1a channels. (A) Representative membranes of lysates of cells control (ctr) or transfected with eGFP-ASIC1a (eASIC) at two levels (1x or x3) to obtained different levels of expression of the protein (“eASICx1 or eASICx3”), and treated with pH6 or MitTx with or without pre-incubation of Pctx-1 or untreated. (B) Plots showing the increase in pERK and pCaMKII levels calculated from membranes as that shown in (A) , consistent with the increase in eASIC expressed. Notice the level of increase achievable via MitTx incubation at the highest overexpressed level of eASIC, higher than that obtained via pH6. (C) Representative membrane showing the different levels of eASIC in cells overexpressing the channel (1x or 3x), detected with an ASIC1 antibody. (D) Comparison between the different ASIC1 proteins expressed (the endogenous human ASIC1a; of approx. 67 kDa) and the overexpressed eASIC (approx. 110 kDa, and expressed at different levels; x1 or x3). (A) Notice that plots are the result of the signal intensity of the band detected,—tERK and tubulin are used as loading controls between loaded samples—and expressed relative to eASICx1 levels. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments and conditions were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; ns: no significant differences. Mean values expressed relative to eASICx1 levels ± SEM are as follows: eASICx3 3.16 ± 0.06; eASIC MitTx 3.42 ± 0.15; eASICx3 MitTx 9.96 ± 0.12; eASIC MitTx Pctx 1.10 ± 0.05; eASIC pH6 2’ 1.85 ± 0.08; eASIC pH6 2’ Pctx 1.08 ± 0.05; eASICx3 pH6 2’ 4.00 ± 0.12.
    Figure Legend Snippet: Proton and non-proton activation of overexpressed ASIC1a channels. (A) Representative membranes of lysates of cells control (ctr) or transfected with eGFP-ASIC1a (eASIC) at two levels (1x or x3) to obtained different levels of expression of the protein (“eASICx1 or eASICx3”), and treated with pH6 or MitTx with or without pre-incubation of Pctx-1 or untreated. (B) Plots showing the increase in pERK and pCaMKII levels calculated from membranes as that shown in (A) , consistent with the increase in eASIC expressed. Notice the level of increase achievable via MitTx incubation at the highest overexpressed level of eASIC, higher than that obtained via pH6. (C) Representative membrane showing the different levels of eASIC in cells overexpressing the channel (1x or 3x), detected with an ASIC1 antibody. (D) Comparison between the different ASIC1 proteins expressed (the endogenous human ASIC1a; of approx. 67 kDa) and the overexpressed eASIC (approx. 110 kDa, and expressed at different levels; x1 or x3). (A) Notice that plots are the result of the signal intensity of the band detected,—tERK and tubulin are used as loading controls between loaded samples—and expressed relative to eASICx1 levels. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments and conditions were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; ns: no significant differences. Mean values expressed relative to eASICx1 levels ± SEM are as follows: eASICx3 3.16 ± 0.06; eASIC MitTx 3.42 ± 0.15; eASICx3 MitTx 9.96 ± 0.12; eASIC MitTx Pctx 1.10 ± 0.05; eASIC pH6 2’ 1.85 ± 0.08; eASIC pH6 2’ Pctx 1.08 ± 0.05; eASICx3 pH6 2’ 4.00 ± 0.12.

    Techniques Used: Activation Assay, Transfection, Expressing, Incubation

    pctx 1  (Alomone Labs)


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    Pctx 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    psalmotoxin 1  (Alomone Labs)


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    Psalmotoxin 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    psalmotoxin 1 pctx1  (Alomone Labs)


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    pctx1  (Alomone Labs)


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    (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTX1</t> (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.
    Figure Legend Snippet: (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.

    Techniques Used: Fluorescence, Concentration Assay, Cell Culture

    (A) Representative [Na+]m fluorescence traces in digitonin-permeabilized HEK293-T cells overexpressing ASIC1a (See Materials and Methods). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion.
    Figure Legend Snippet: (A) Representative [Na+]m fluorescence traces in digitonin-permeabilized HEK293-T cells overexpressing ASIC1a (See Materials and Methods). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion.

    Techniques Used: Fluorescence

    A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca2+ concentration ([Ca2+]c) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion.
    Figure Legend Snippet: A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca2+ concentration ([Ca2+]c) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion.

    Techniques Used: Fluorescence, Concentration Assay

    pctx1  (Alomone Labs)


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    Alomone Labs pctx1
    (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTX1</t> (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.
    Figure Legend Snippet: (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.

    Techniques Used: Fluorescence, Concentration Assay, Cell Culture

    (A) Representative [Na+]m fluorescence traces in digitonin-permeabilized HEK293-T cells overexpressing ASIC1a (See Materials and Methods). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion.
    Figure Legend Snippet: (A) Representative [Na+]m fluorescence traces in digitonin-permeabilized HEK293-T cells overexpressing ASIC1a (See Materials and Methods). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion.

    Techniques Used: Fluorescence

    A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca2+ concentration ([Ca2+]c) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion.
    Figure Legend Snippet: A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca2+ concentration ([Ca2+]c) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion.

    Techniques Used: Fluorescence, Concentration Assay

    psalmotoxin 1 pctx1  (Alomone Labs)


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    Alomone Labs psalmotoxin 1 pctx1
    A role for ASIC1b in acid-induced mechanical hyperalgesia. (A) Mechanical hyperalgesia of wild-type ( Asic1b + / + , N = 7) and ASIC1b-knockout ( Asic1b – /– , N = 8) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 1 and evaluated by 0.20-mN von Frey filament. Data were analyzed by two-way ANOVA (Interaction F ( 10 , 143 ) = 5.34, P < 0.0001; Time F ( 10 , 143 ) = 14.5, P < 0.0001; Genotype F ( 1 , 143 ) = 23.11, P < 0.0001), followed by Sidak post hoc test. ∗∗∗ P < 0.001 for genotype difference at specific times. (B) Mechanical hyperalgesia of Asic1b + / + ( N = 10) and Asic1b – /– ( N = 7) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 5. Data were analyzed by two-way ANOVA [Interaction F ( 12 , 195 ) = 5.933, P < 0.0001; Time F ( 12 , 195 ) = 21.28, P < 0.0001; Genotype F ( 1 , 195 ) = 59.07, P < 0.0001], followed by Sidak post hoc test. **** P < 0.0001; ∗∗∗ P < 0.001; ∗∗ P < 0.01; ∗ P < 0.05 Asic1b + / + vs. Asic1b – /– . For comparison, the effect of mambalgin-1 on acid-induced hyperalgesia in Asic1b – /– mice was plotted. (C) Effect of mambalgin-1 (MB-1) on acid-induced mechanical hyperalgesia (vehicle, N = 7; MB-1 [3 pmol], N = 6; MB-1 [15 pmol], N = 7; MB-1 [30 pmol], N = 7). Data were analyzed by two-way ANOVA [Interaction F ( 21 , 168 ) = 4.014, P < 0.0001; Time F ( 7 , 168 ) = 26.61, P < 0.0001; Drug dose F ( 3 , 168 ) = 29.32, P < 0.0001], followed by Sidak post hoc test. ### P < 0.001, #### P < 0.0001, MB-1 (30 pmol) vs. vehicle; $$$$ P < 0.0001, MB-1 (15 pmol) vs. vehicle. (D) Cumulative withdrawal response after second acid injection 5 days before acid + MB-1 injection is shown as the area under the receiver operating characteristic curve (AUC) calculated by the trapezoidal method. Data were analyzed by one-way ANOVA [ F ( 3 , 21 ) = 4.719, P = 0.0114], followed by Dunnett post hoc test. ∗∗ P < 0.01, ∗ P < 0.05 vs. vehicle. (D’) While 30 pmole mambalgin-1 was applied in the second acid injection, it blocked the development of the acid-induced chronic hyperalgesia. (E) Effect of <t>PcTx1</t> on acid-induced mechanical hyperalgesia. Data were analyzed by two-way ANOVA (vehicle, N = 4; PcTx1 [120 nmol], N = 6) Interaction F ( 7 , 64 ) = 0.2935, P = 0.9541; Time F ( 7 , 64 ) = 31.29, P < 0.0001; Drug dose F ( 1 , 64 ) = 0.054, P = 0.8169). (F) Cumulative withdrawal response after a second acid injection 5 days before acid + PcTx1 injection is shown as the AUC calculated by the trapezoidal method. Data were analyzed by unpaired t -test [ F ( 3 , 5 ) = 2.191, P = 0.4149). Black arrows, the time mice received intramuscular injection of pH 4.0 saline. Red arrows, the time mice received intramuscular injection of pH-4.0 saline mixed with mambalgin-1 or PcTx1. B, baseline. Data are mean ± SEM.
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Involvement of Acid-Sensing Ion Channel 1b in the Development of Acid-Induced Chronic Muscle Pain"

    Article Title: Involvement of Acid-Sensing Ion Channel 1b in the Development of Acid-Induced Chronic Muscle Pain

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2019.01247

    A role for ASIC1b in acid-induced mechanical hyperalgesia. (A) Mechanical hyperalgesia of wild-type ( Asic1b + / + , N = 7) and ASIC1b-knockout ( Asic1b – /– , N = 8) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 1 and evaluated by 0.20-mN von Frey filament. Data were analyzed by two-way ANOVA (Interaction F ( 10 , 143 ) = 5.34, P < 0.0001; Time F ( 10 , 143 ) = 14.5, P < 0.0001; Genotype F ( 1 , 143 ) = 23.11, P < 0.0001), followed by Sidak post hoc test. ∗∗∗ P < 0.001 for genotype difference at specific times. (B) Mechanical hyperalgesia of Asic1b + / + ( N = 10) and Asic1b – /– ( N = 7) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 5. Data were analyzed by two-way ANOVA [Interaction F ( 12 , 195 ) = 5.933, P < 0.0001; Time F ( 12 , 195 ) = 21.28, P < 0.0001; Genotype F ( 1 , 195 ) = 59.07, P < 0.0001], followed by Sidak post hoc test. **** P < 0.0001; ∗∗∗ P < 0.001; ∗∗ P < 0.01; ∗ P < 0.05 Asic1b + / + vs. Asic1b – /– . For comparison, the effect of mambalgin-1 on acid-induced hyperalgesia in Asic1b – /– mice was plotted. (C) Effect of mambalgin-1 (MB-1) on acid-induced mechanical hyperalgesia (vehicle, N = 7; MB-1 [3 pmol], N = 6; MB-1 [15 pmol], N = 7; MB-1 [30 pmol], N = 7). Data were analyzed by two-way ANOVA [Interaction F ( 21 , 168 ) = 4.014, P < 0.0001; Time F ( 7 , 168 ) = 26.61, P < 0.0001; Drug dose F ( 3 , 168 ) = 29.32, P < 0.0001], followed by Sidak post hoc test. ### P < 0.001, #### P < 0.0001, MB-1 (30 pmol) vs. vehicle; $$$$ P < 0.0001, MB-1 (15 pmol) vs. vehicle. (D) Cumulative withdrawal response after second acid injection 5 days before acid + MB-1 injection is shown as the area under the receiver operating characteristic curve (AUC) calculated by the trapezoidal method. Data were analyzed by one-way ANOVA [ F ( 3 , 21 ) = 4.719, P = 0.0114], followed by Dunnett post hoc test. ∗∗ P < 0.01, ∗ P < 0.05 vs. vehicle. (D’) While 30 pmole mambalgin-1 was applied in the second acid injection, it blocked the development of the acid-induced chronic hyperalgesia. (E) Effect of PcTx1 on acid-induced mechanical hyperalgesia. Data were analyzed by two-way ANOVA (vehicle, N = 4; PcTx1 [120 nmol], N = 6) Interaction F ( 7 , 64 ) = 0.2935, P = 0.9541; Time F ( 7 , 64 ) = 31.29, P < 0.0001; Drug dose F ( 1 , 64 ) = 0.054, P = 0.8169). (F) Cumulative withdrawal response after a second acid injection 5 days before acid + PcTx1 injection is shown as the AUC calculated by the trapezoidal method. Data were analyzed by unpaired t -test [ F ( 3 , 5 ) = 2.191, P = 0.4149). Black arrows, the time mice received intramuscular injection of pH 4.0 saline. Red arrows, the time mice received intramuscular injection of pH-4.0 saline mixed with mambalgin-1 or PcTx1. B, baseline. Data are mean ± SEM.
    Figure Legend Snippet: A role for ASIC1b in acid-induced mechanical hyperalgesia. (A) Mechanical hyperalgesia of wild-type ( Asic1b + / + , N = 7) and ASIC1b-knockout ( Asic1b – /– , N = 8) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 1 and evaluated by 0.20-mN von Frey filament. Data were analyzed by two-way ANOVA (Interaction F ( 10 , 143 ) = 5.34, P < 0.0001; Time F ( 10 , 143 ) = 14.5, P < 0.0001; Genotype F ( 1 , 143 ) = 23.11, P < 0.0001), followed by Sidak post hoc test. ∗∗∗ P < 0.001 for genotype difference at specific times. (B) Mechanical hyperalgesia of Asic1b + / + ( N = 10) and Asic1b – /– ( N = 7) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 5. Data were analyzed by two-way ANOVA [Interaction F ( 12 , 195 ) = 5.933, P < 0.0001; Time F ( 12 , 195 ) = 21.28, P < 0.0001; Genotype F ( 1 , 195 ) = 59.07, P < 0.0001], followed by Sidak post hoc test. **** P < 0.0001; ∗∗∗ P < 0.001; ∗∗ P < 0.01; ∗ P < 0.05 Asic1b + / + vs. Asic1b – /– . For comparison, the effect of mambalgin-1 on acid-induced hyperalgesia in Asic1b – /– mice was plotted. (C) Effect of mambalgin-1 (MB-1) on acid-induced mechanical hyperalgesia (vehicle, N = 7; MB-1 [3 pmol], N = 6; MB-1 [15 pmol], N = 7; MB-1 [30 pmol], N = 7). Data were analyzed by two-way ANOVA [Interaction F ( 21 , 168 ) = 4.014, P < 0.0001; Time F ( 7 , 168 ) = 26.61, P < 0.0001; Drug dose F ( 3 , 168 ) = 29.32, P < 0.0001], followed by Sidak post hoc test. ### P < 0.001, #### P < 0.0001, MB-1 (30 pmol) vs. vehicle; $$$$ P < 0.0001, MB-1 (15 pmol) vs. vehicle. (D) Cumulative withdrawal response after second acid injection 5 days before acid + MB-1 injection is shown as the area under the receiver operating characteristic curve (AUC) calculated by the trapezoidal method. Data were analyzed by one-way ANOVA [ F ( 3 , 21 ) = 4.719, P = 0.0114], followed by Dunnett post hoc test. ∗∗ P < 0.01, ∗ P < 0.05 vs. vehicle. (D’) While 30 pmole mambalgin-1 was applied in the second acid injection, it blocked the development of the acid-induced chronic hyperalgesia. (E) Effect of PcTx1 on acid-induced mechanical hyperalgesia. Data were analyzed by two-way ANOVA (vehicle, N = 4; PcTx1 [120 nmol], N = 6) Interaction F ( 7 , 64 ) = 0.2935, P = 0.9541; Time F ( 7 , 64 ) = 31.29, P < 0.0001; Drug dose F ( 1 , 64 ) = 0.054, P = 0.8169). (F) Cumulative withdrawal response after a second acid injection 5 days before acid + PcTx1 injection is shown as the AUC calculated by the trapezoidal method. Data were analyzed by unpaired t -test [ F ( 3 , 5 ) = 2.191, P = 0.4149). Black arrows, the time mice received intramuscular injection of pH 4.0 saline. Red arrows, the time mice received intramuscular injection of pH-4.0 saline mixed with mambalgin-1 or PcTx1. B, baseline. Data are mean ± SEM.

    Techniques Used: Knock-Out, Injection

    Electrophysiological properties of three types of ASIC1b-TdTomato(+) DRGs with different desensitization rates.
    Figure Legend Snippet: Electrophysiological properties of three types of ASIC1b-TdTomato(+) DRGs with different desensitization rates.

    Techniques Used: Inhibition

    Effect of amiloride, mambalgin-1, APETx2 and PcTx1 on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.
    Figure Legend Snippet: Effect of amiloride, mambalgin-1, APETx2 and PcTx1 on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.

    Techniques Used: Expressing, Patch Clamp, Labeling

    psalmotoxin 1  (Alomone Labs)


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    Alomone Labs psalmotoxin 1
    Psalmotoxin 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs psalmotoxin 1
    D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM <t>PcTx1</t> (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.
    Psalmotoxin 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/psalmotoxin 1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    psalmotoxin 1 - by Bioz Stars, 2023-02
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    94
    Alomone Labs pctx1
    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTx1</t> (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs pctx 1
    Proton and non-proton activation of ASIC1a in HEK cells. (A) Representative membranes of lysates of HEK cells treated with pH6 or MitTx for 2 or 10 min or preincubated with <t>Pctx-1</t> compared to untreated cells (control, Ctr) and detected with phosphoERK (pERK), total ERK (tERK), and ASIC1 antibodies. (B) Representative membrane of the same lysates to detect pCaMKII levels. (C) Plots showing detected levels of pERK (top panel) or pCaMKII (lower panel). Notice that the increase in kinase levels goes further at a later time point (2 vs. 10 min) in MitTx treated cultures compared to pH6 treated ones that show an increase at 2 min followed by a reversal to control levels consistent with the proton-activated desensitizing mechanism. (A) Notice that plots are the result of the signal intensity of the bands—with tERK and tubulin used as loading controls between loaded samples—and expressed relative to control samples. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments against the control were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; *** p 0.0001–0.001; ns: no significant differences. Mean values expressed relative to control ( Ctr ) levels ± SEM are as follows: for pERK/tERK: pH6 2’ 6.10 ± 0.13; pH6 10’ 1.23 ± 0.11; pH6 2’ Pctx 1.39 ± 0.08; MitTx 2’ 7.98 ± 0.10; Mittx10 ’ 10.90 ± 0.17; MitTx 2’ Pctx 1.18 ± 0.05 ; MitTx 10’ Pctx 1.42 ± 0.05. For pCaMKII/Tub: pH6 2’ 7.31 ± 0.21; pH6 10’ 1.16 ± 0.06; pH6 2’ Pctx 1.14±0.07; Mittx 2’ 8.88 ± 0.15; Mittx 10’ 10.76 ± 0.32; Mittx 2’ Pctx 1.17 ± 0.07.
    Pctx 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs psalmotoxin 1 pctx1
    Proton and non-proton activation of ASIC1a in HEK cells. (A) Representative membranes of lysates of HEK cells treated with pH6 or MitTx for 2 or 10 min or preincubated with <t>Pctx-1</t> compared to untreated cells (control, Ctr) and detected with phosphoERK (pERK), total ERK (tERK), and ASIC1 antibodies. (B) Representative membrane of the same lysates to detect pCaMKII levels. (C) Plots showing detected levels of pERK (top panel) or pCaMKII (lower panel). Notice that the increase in kinase levels goes further at a later time point (2 vs. 10 min) in MitTx treated cultures compared to pH6 treated ones that show an increase at 2 min followed by a reversal to control levels consistent with the proton-activated desensitizing mechanism. (A) Notice that plots are the result of the signal intensity of the bands—with tERK and tubulin used as loading controls between loaded samples—and expressed relative to control samples. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments against the control were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; *** p 0.0001–0.001; ns: no significant differences. Mean values expressed relative to control ( Ctr ) levels ± SEM are as follows: for pERK/tERK: pH6 2’ 6.10 ± 0.13; pH6 10’ 1.23 ± 0.11; pH6 2’ Pctx 1.39 ± 0.08; MitTx 2’ 7.98 ± 0.10; Mittx10 ’ 10.90 ± 0.17; MitTx 2’ Pctx 1.18 ± 0.05 ; MitTx 10’ Pctx 1.42 ± 0.05. For pCaMKII/Tub: pH6 2’ 7.31 ± 0.21; pH6 10’ 1.16 ± 0.06; pH6 2’ Pctx 1.14±0.07; Mittx 2’ 8.88 ± 0.15; Mittx 10’ 10.76 ± 0.32; Mittx 2’ Pctx 1.17 ± 0.07.
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM PcTx1 (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.

    Journal: Neuroscience

    Article Title: Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

    doi: 10.1016/j.neuroscience.2021.01.036

    Figure Lengend Snippet: D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM PcTx1 (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.

    Article Snippet: In and , benzamil hydrochloride hydrate (Sigma-Aldrich, St. Louis, MO), psalmotoxin-1 (PcTx1; Alomone Labs), or mambalgin-3 (Mamb3; Alomone Labs), were applied (in the presence of μ-Ctx) for 30 minutes prior to recording.

    Techniques: Incubation

    High concentrations of the ASIC inhibitors Benzamil and PcTx1 reduce mEPP and EPP amplitude. MEPPs, EPPs and Quantal Content are plotted from NMJs incubated in either control saline (n=75, same data show in 1A), 100 μM benzamil (n=15, 3 mice), and 100 nM PcTx1 (n=25, 5 mice). Data are plotted as in Fig. 1. P values are indicated and were calculated from Student’s t-test or ordinary one-way ANOVA.

    Journal: Neuroscience

    Article Title: Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

    doi: 10.1016/j.neuroscience.2021.01.036

    Figure Lengend Snippet: High concentrations of the ASIC inhibitors Benzamil and PcTx1 reduce mEPP and EPP amplitude. MEPPs, EPPs and Quantal Content are plotted from NMJs incubated in either control saline (n=75, same data show in 1A), 100 μM benzamil (n=15, 3 mice), and 100 nM PcTx1 (n=25, 5 mice). Data are plotted as in Fig. 1. P values are indicated and were calculated from Student’s t-test or ordinary one-way ANOVA.

    Article Snippet: In and , benzamil hydrochloride hydrate (Sigma-Aldrich, St. Louis, MO), psalmotoxin-1 (PcTx1; Alomone Labs), or mambalgin-3 (Mamb3; Alomone Labs), were applied (in the presence of μ-Ctx) for 30 minutes prior to recording.

    Techniques: Incubation

    Lowering extracellular pH underlies the upregulation of QC triggered by d-TC. A. mEPPs and EPPs were measured and QC estimated before and after a 10 min incubation in pH 7.2 saline with 100nM PcTx1 present throughout (n=25, 5 mice). The data for mEPPs, EPPs and QC are presented as violin plots. The plot on the far right includes the data from Fig. 3B to depicts the effect of pH 7.2 saline under control conditions vs. in the presence of 100 nM PcTx1. The Mean differences are plotted as a bootstrap sampling distribution. Means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA. B. mEPPs and EPPs were measured and QC was estimated in pH 7.4 saline, after a 7 min incubation in pH 7.2 saline, and then after a 15 min incubation with d-TC, still in pH 7.2 saline (n = 5 mice). Although dTC had its normal effects on mEPP and EPP amplitude (compare to Fig. 1A), dTC did not alter QC when compared to pH 7.2 saline. The data is plotted as in panel A. C. The ratio of QC after application of dTC compared to before dTC was applied (post/pre dTC) is shown for pH 7.4 saline (this is the same data presented in Fig 1B, Control) and for pH 7.2 saline (the same data presented in Fig. 4B).

    Journal: Neuroscience

    Article Title: Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

    doi: 10.1016/j.neuroscience.2021.01.036

    Figure Lengend Snippet: Lowering extracellular pH underlies the upregulation of QC triggered by d-TC. A. mEPPs and EPPs were measured and QC estimated before and after a 10 min incubation in pH 7.2 saline with 100nM PcTx1 present throughout (n=25, 5 mice). The data for mEPPs, EPPs and QC are presented as violin plots. The plot on the far right includes the data from Fig. 3B to depicts the effect of pH 7.2 saline under control conditions vs. in the presence of 100 nM PcTx1. The Mean differences are plotted as a bootstrap sampling distribution. Means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA. B. mEPPs and EPPs were measured and QC was estimated in pH 7.4 saline, after a 7 min incubation in pH 7.2 saline, and then after a 15 min incubation with d-TC, still in pH 7.2 saline (n = 5 mice). Although dTC had its normal effects on mEPP and EPP amplitude (compare to Fig. 1A), dTC did not alter QC when compared to pH 7.2 saline. The data is plotted as in panel A. C. The ratio of QC after application of dTC compared to before dTC was applied (post/pre dTC) is shown for pH 7.4 saline (this is the same data presented in Fig 1B, Control) and for pH 7.2 saline (the same data presented in Fig. 4B).

    Article Snippet: In and , benzamil hydrochloride hydrate (Sigma-Aldrich, St. Louis, MO), psalmotoxin-1 (PcTx1; Alomone Labs), or mambalgin-3 (Mamb3; Alomone Labs), were applied (in the presence of μ-Ctx) for 30 minutes prior to recording.

    Techniques: Incubation, Sampling

    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.

    Journal: Redox Biology

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    doi: 10.1016/j.redox.2022.102253

    Figure Lengend Snippet: Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.

    Article Snippet: Experiments done on WT neurons, untreated or treated with PcTX1 (Alomone labs, #STP200), were performed using the imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany).

    Techniques: Cell Culture, Fluorescence

    ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

    Journal: Redox Biology

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    doi: 10.1016/j.redox.2022.102253

    Figure Lengend Snippet: ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

    Article Snippet: Experiments done on WT neurons, untreated or treated with PcTX1 (Alomone labs, #STP200), were performed using the imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany).

    Techniques: Fluorescence

    Proton and non-proton activation of ASIC1a in HEK cells. (A) Representative membranes of lysates of HEK cells treated with pH6 or MitTx for 2 or 10 min or preincubated with Pctx-1 compared to untreated cells (control, Ctr) and detected with phosphoERK (pERK), total ERK (tERK), and ASIC1 antibodies. (B) Representative membrane of the same lysates to detect pCaMKII levels. (C) Plots showing detected levels of pERK (top panel) or pCaMKII (lower panel). Notice that the increase in kinase levels goes further at a later time point (2 vs. 10 min) in MitTx treated cultures compared to pH6 treated ones that show an increase at 2 min followed by a reversal to control levels consistent with the proton-activated desensitizing mechanism. (A) Notice that plots are the result of the signal intensity of the bands—with tERK and tubulin used as loading controls between loaded samples—and expressed relative to control samples. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments against the control were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; *** p 0.0001–0.001; ns: no significant differences. Mean values expressed relative to control ( Ctr ) levels ± SEM are as follows: for pERK/tERK: pH6 2’ 6.10 ± 0.13; pH6 10’ 1.23 ± 0.11; pH6 2’ Pctx 1.39 ± 0.08; MitTx 2’ 7.98 ± 0.10; Mittx10 ’ 10.90 ± 0.17; MitTx 2’ Pctx 1.18 ± 0.05 ; MitTx 10’ Pctx 1.42 ± 0.05. For pCaMKII/Tub: pH6 2’ 7.31 ± 0.21; pH6 10’ 1.16 ± 0.06; pH6 2’ Pctx 1.14±0.07; Mittx 2’ 8.88 ± 0.15; Mittx 10’ 10.76 ± 0.32; Mittx 2’ Pctx 1.17 ± 0.07.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Signaling Pathways in Proton and Non-proton ASIC1a Activation

    doi: 10.3389/fncel.2021.735414

    Figure Lengend Snippet: Proton and non-proton activation of ASIC1a in HEK cells. (A) Representative membranes of lysates of HEK cells treated with pH6 or MitTx for 2 or 10 min or preincubated with Pctx-1 compared to untreated cells (control, Ctr) and detected with phosphoERK (pERK), total ERK (tERK), and ASIC1 antibodies. (B) Representative membrane of the same lysates to detect pCaMKII levels. (C) Plots showing detected levels of pERK (top panel) or pCaMKII (lower panel). Notice that the increase in kinase levels goes further at a later time point (2 vs. 10 min) in MitTx treated cultures compared to pH6 treated ones that show an increase at 2 min followed by a reversal to control levels consistent with the proton-activated desensitizing mechanism. (A) Notice that plots are the result of the signal intensity of the bands—with tERK and tubulin used as loading controls between loaded samples—and expressed relative to control samples. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments against the control were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; *** p 0.0001–0.001; ns: no significant differences. Mean values expressed relative to control ( Ctr ) levels ± SEM are as follows: for pERK/tERK: pH6 2’ 6.10 ± 0.13; pH6 10’ 1.23 ± 0.11; pH6 2’ Pctx 1.39 ± 0.08; MitTx 2’ 7.98 ± 0.10; Mittx10 ’ 10.90 ± 0.17; MitTx 2’ Pctx 1.18 ± 0.05 ; MitTx 10’ Pctx 1.42 ± 0.05. For pCaMKII/Tub: pH6 2’ 7.31 ± 0.21; pH6 10’ 1.16 ± 0.06; pH6 2’ Pctx 1.14±0.07; Mittx 2’ 8.88 ± 0.15; Mittx 10’ 10.76 ± 0.32; Mittx 2’ Pctx 1.17 ± 0.07.

    Article Snippet: Incubation of cells: ASIC inhibitors were used at the following concentrations before incubation with other reagents: Pctx-1 (Alomone, STP-200), 20 nM, 30 min before; as previously used in Salinas et al. ( ).

    Techniques: Activation Assay

    Proton and non-proton activation of overexpressed ASIC1a channels. (A) Representative membranes of lysates of cells control (ctr) or transfected with eGFP-ASIC1a (eASIC) at two levels (1x or x3) to obtained different levels of expression of the protein (“eASICx1 or eASICx3”), and treated with pH6 or MitTx with or without pre-incubation of Pctx-1 or untreated. (B) Plots showing the increase in pERK and pCaMKII levels calculated from membranes as that shown in (A) , consistent with the increase in eASIC expressed. Notice the level of increase achievable via MitTx incubation at the highest overexpressed level of eASIC, higher than that obtained via pH6. (C) Representative membrane showing the different levels of eASIC in cells overexpressing the channel (1x or 3x), detected with an ASIC1 antibody. (D) Comparison between the different ASIC1 proteins expressed (the endogenous human ASIC1a; of approx. 67 kDa) and the overexpressed eASIC (approx. 110 kDa, and expressed at different levels; x1 or x3). (A) Notice that plots are the result of the signal intensity of the band detected,—tERK and tubulin are used as loading controls between loaded samples—and expressed relative to eASICx1 levels. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments and conditions were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; ns: no significant differences. Mean values expressed relative to eASICx1 levels ± SEM are as follows: eASICx3 3.16 ± 0.06; eASIC MitTx 3.42 ± 0.15; eASICx3 MitTx 9.96 ± 0.12; eASIC MitTx Pctx 1.10 ± 0.05; eASIC pH6 2’ 1.85 ± 0.08; eASIC pH6 2’ Pctx 1.08 ± 0.05; eASICx3 pH6 2’ 4.00 ± 0.12.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Signaling Pathways in Proton and Non-proton ASIC1a Activation

    doi: 10.3389/fncel.2021.735414

    Figure Lengend Snippet: Proton and non-proton activation of overexpressed ASIC1a channels. (A) Representative membranes of lysates of cells control (ctr) or transfected with eGFP-ASIC1a (eASIC) at two levels (1x or x3) to obtained different levels of expression of the protein (“eASICx1 or eASICx3”), and treated with pH6 or MitTx with or without pre-incubation of Pctx-1 or untreated. (B) Plots showing the increase in pERK and pCaMKII levels calculated from membranes as that shown in (A) , consistent with the increase in eASIC expressed. Notice the level of increase achievable via MitTx incubation at the highest overexpressed level of eASIC, higher than that obtained via pH6. (C) Representative membrane showing the different levels of eASIC in cells overexpressing the channel (1x or 3x), detected with an ASIC1 antibody. (D) Comparison between the different ASIC1 proteins expressed (the endogenous human ASIC1a; of approx. 67 kDa) and the overexpressed eASIC (approx. 110 kDa, and expressed at different levels; x1 or x3). (A) Notice that plots are the result of the signal intensity of the band detected,—tERK and tubulin are used as loading controls between loaded samples—and expressed relative to eASICx1 levels. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments and conditions were performed, mean values above bars; n = 3 membranes, **** p < 0.0001; ns: no significant differences. Mean values expressed relative to eASICx1 levels ± SEM are as follows: eASICx3 3.16 ± 0.06; eASIC MitTx 3.42 ± 0.15; eASICx3 MitTx 9.96 ± 0.12; eASIC MitTx Pctx 1.10 ± 0.05; eASIC pH6 2’ 1.85 ± 0.08; eASIC pH6 2’ Pctx 1.08 ± 0.05; eASICx3 pH6 2’ 4.00 ± 0.12.

    Article Snippet: Incubation of cells: ASIC inhibitors were used at the following concentrations before incubation with other reagents: Pctx-1 (Alomone, STP-200), 20 nM, 30 min before; as previously used in Salinas et al. ( ).

    Techniques: Activation Assay, Transfection, Expressing, Incubation