pctx1  (Alomone Labs)


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    Structured Review

    Alomone Labs pctx1
    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 <t>PcTX1;</t> (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
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 19 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

    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
    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

    Techniques Used: Fluorescence

    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
    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

    Techniques Used: Cell Culture, Fluorescence

    2) 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

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (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. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (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. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). 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. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). 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. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] 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. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] 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. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    3) 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

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (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. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (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. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). 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. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). 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. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] 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. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] 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. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    4) 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

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (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. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (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. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). 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. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). 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. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] 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. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] 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. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    5) 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

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (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. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (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. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). 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. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). 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. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] 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. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] 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. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

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    Alomone Labs pctx1
    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 <t>PcTX1;</t> (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
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    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

    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

    Article Snippet: 4.4 Fluorescence imaging 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

    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

    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

    Article Snippet: 4.4 Fluorescence imaging 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