Journal: The Journal of Neuroscience

Article Title: Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

doi: 10.1523/JNEUROSCI.2087-20.2021

Figure Lengend Snippet: Citrate potentiates ASIC-dependent pain behavior. A–C , Each mouse was injected with either PBS, pH 7.4 ( A, C ), or citrate, pH 7.4 ( B ) in one hindpaw and then PBS, pH 5.5 ( A ); or citrate, pH 5.5 ( B ); or citrate, pH 7.4 ( C ), in the other hindpaw, or vice versa. The presence (+) or absence (o) of a withdrawal reflex was recorded; n = 4–6 per group, n.s. p > 0.05, * p < 0.05, two-tailed Mann–Whitney U test, in which (+) was ranked as 1 and (o) was ranked as 0. D–F , Each mouse was injected in either hindpaw with PBS, pH 7.4 or 5.5, as described for A–C , with or without amiloride (100 μ m ). The presence (+) or absence (o) of a withdrawal reflex was recorded; n = 4–14 per group, n.s. p > 0.05, *** p < 0.001, two-tailed Mann–Whitney U test, in which (+) was ranked as 1 and (o) was ranked as 0. G , Each mouse was injected with PBS, pH 5.5, in one hindpaw and then citrate, pH 5.5 (25 m m ), in the other hindpaw, or vice versa. The relative withdrawal intensity between the two hindpaws was compared and recorded: (+) for greater, (=) for equivalent, and (−) for lesser withdrawal response; n = 7 per group, * p < 0.05, two-tailed Mann–Whitney U test, in which (+) was ranked as +1, (=) was ranked as 0, and (−) was ranked as −1. H , Summary of the results from . n.s., Not significant.

Article Snippet: Sodium citrate tribasic dihydrate and capsaicin were purchased from Sigma-Aldrich (catalog #S4641 and #M2028, respectively), amiloride was purchased from Alomone Labs (catalog #A-140), 10% neutral buffered formalin was purchased from TONYAR BIOTECH.

Techniques: Injection, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

doi: 10.1523/JNEUROSCI.2087-20.2021

Figure Lengend Snippet: Citrate also potentiates ASIC lacking ASIC1 subunit in mouse primary sensory neurons. A , Primary sensory neurons isolated from the mouse dorsal root ganglia were perfused in a bath solution, pH 7.4, containing no citrate, and each stimulus consisted of a 3 s exposure (at 30 s intervals) to a test solution, pH 5.5, with or without citrate (25 m m ) or amiloride (100 μ m ). B , Summarized result from A . C , D , Similar to B , except the primary sensory neurons were from mice lacking ASIC1 ( ASIC1 −/− ; C ) and ASIC2 ( ASIC2 −/− ; D ), respectively. In B–D , error bars indicate the mean ± SEM; n = 5 per group, ** p < 0.01, *** p < 0.001, one-way ANOVA with a post hoc Tukey's test.

Article Snippet: Sodium citrate tribasic dihydrate and capsaicin were purchased from Sigma-Aldrich (catalog #S4641 and #M2028, respectively), amiloride was purchased from Alomone Labs (catalog #A-140), 10% neutral buffered formalin was purchased from TONYAR BIOTECH.

Techniques: Isolation

Journal: The Journal of Neuroscience

Article Title: Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

doi: 10.1523/JNEUROSCI.2087-20.2021

Figure Lengend Snippet: Concentrated citric acid at pH 3.5 but not at pH 5.5 induces paw-licking behavior that is ASIC1 independent. A , Each mouse was injected with either 5% formalin; 30 m m citrate, pH 5.5; or 100 m m citrate, pH 5.5 or 3.5, into one hindpaw, and the duration of paw licking over a period of 30 min was recorded; n = 6 per group, n.s. p > 0.05, *** p < 0.001, one-way ANOVA with a post hoc Tukey's test. B , C , Each mouse was injected with either 5% formalin, with or without amiloride (100 μ m ; B ) or 100 m m citrate, pH 3.5, with or without amiloride (100 μ m ; C ) into one hindpaw, and the duration of paw licking over a period of 30 min was recorded; n = 6 per group, n.s. p > 0.05, two-tailed Student's t test. D , wild-type mice (WT) or mice with homozygous ASIC1 deletion ( ASIC1 −/− ) were injected with 100 m m citrate, pH 3.5, into one hindpaw, and the duration of paw licking over a period of 30 min was recorded; n = 6 per group, n.s. p > 0.05, two-tailed Student's t test. n.s., Not significant.

Article Snippet: Sodium citrate tribasic dihydrate and capsaicin were purchased from Sigma-Aldrich (catalog #S4641 and #M2028, respectively), amiloride was purchased from Alomone Labs (catalog #A-140), 10% neutral buffered formalin was purchased from TONYAR BIOTECH.

Techniques: Injection, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

doi: 10.1523/JNEUROSCI.2087-20.2021

Figure Lengend Snippet: Acetic acid but not citric acid induces writhing behavior that is ASIC1 independent. A , B , Each mouse received intraperitoneal injection of either 1% acetic acid, pH 3.5; 30 m m citric acid, pH 5.5; or 100 m m citric acid, pH 3.5; and the numbers of abdominal contractions ( A ) and body contortions ( B ) over a period of 10 min were recorded; n = 5 per group. n.s. p > 0.05, *** p < 0.001, one-way ANOVA with a post hoc Tukey's test. C , D , Each mouse received intraperitoneal injection of 1% acetic acid, pH 3.5, with or without amiloride (100 μ m ), and the numbers of abdominal contractions ( C ) and body contortions ( D ) over a period of 10 min were recorded; n = 5 per group, n.s. p > 0.05, two-tailed Student's t test. E , F , wild-type mice (WT) or mice with homozygous ASIC1 deletion ( ASIC1 −/− ) received intraperitoneal injection of 1% acetic acid, pH 3.5, and the numbers of abdominal contractions ( E ) and body contortions ( F ) over a period of 10 min were recorded; n = 6 per group, n.s. p > 0.05, two-tailed Student's t test. n.s., Not significant.

Article Snippet: Sodium citrate tribasic dihydrate and capsaicin were purchased from Sigma-Aldrich (catalog #S4641 and #M2028, respectively), amiloride was purchased from Alomone Labs (catalog #A-140), 10% neutral buffered formalin was purchased from TONYAR BIOTECH.

Techniques: Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Systemic administration of a reported extracellular vesicle inhibitor, dimethyl amiloride, induces preterm birth and fetal growth restriction in pregnant mice

doi: 10.1101/2025.08.13.670125

Figure Lengend Snippet: The amount of maternal weight gained in Control (blue) or DMA-treated (red) pregnant mice. Control (saline) or 7.5 mg/kg DMA were injected on E15.5, E16.5 and E17.5. Maternal weights were recorded 24hrs later - on E16.5, E17.5, and E18.5 - for each pregnancy (2A). The cumulative amount of weight gained throughout the study period in Control (blue) and DMA-treated (red) pregnant mice (2B).

Article Snippet: At E15.5, pregnant mice were intraperitoneally (i.p.) injected with 7.5 mg/kg DMA (Alomone Labs, #D-165) or 0.9% saline (vehicle Control) for three consecutive days.

Techniques: Control, Saline, Injection

Journal: Journal of veterinary internal medicine

Article Title: Effects of the potassium-sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells.

doi: 10.1111/jvim.15382

Figure Lengend Snippet: FIGURE 2 Combination treatment with calculations of the combination index (CI) in D17, Abrams, and Dharma treated with amiloride and doxorubicin, or amiloride and carboplatin. Decreased cell viability in each cell line after treatment with increasing doses of (A) amiloride and doxorubicin or (B) amiloride and carboplatin for 72 hours. X-axis shows dose pairings for amiloride (Am, μM), doxorubicin (Dox, nM) and carboplatin (Carb, μM), which were chosen from IC50 curves. Relative absorbance values at 590 nm (A590) were presented with median range (N = 9). (C) Mean CI values from 3 independent experiments, stratified by dose pairing. CI < 1 was synergistic, C = 1 additive, and C > 1 antagonistic. Significance was recorded at *P < .05. (D) Mean CI values stratified by cell line independent of drug pairings. N = 15 with N = 3/drug pairing. IC50, half-maximal inhibitory concentration

Article Snippet: Amiloride hydrochloride was purchased from Tocris Bioscience (Bristol, United Kingdom) and resuspended at a stock concentration of 100 mM with dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri).

Techniques: Concentration Assay

Journal: Journal of veterinary internal medicine

Article Title: Effects of the potassium-sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells.

doi: 10.1111/jvim.15382

Figure Lengend Snippet: FIGURE 3 Apoptosis analysis of canine OSA cells treated with amiloride and doxorubicin alone or in combination. (A) Density plots obtained after compensation for spillover by the BD Accuri C6 flow cytometer. Combination treatments were compared to the 100 nM doxorubicin-only control, D100, and high-dose etoposide-positive control (50 μM). Cells were treated with increasing doses of amiloride, from A10 to A150 (μM), or treated with increasing doses of amiloride and doxorubicin in combination, D + A. (B) Combined analysis of early and late apoptotic fractions, compared to the vehicle control with median range shown from 3 independent experiments. Significance was reported at *P < .05, **P < .01, ***P < .001, ****P < .0001. OSA, osteosarcoma; PI, propidium iodide

Article Snippet: Amiloride hydrochloride was purchased from Tocris Bioscience (Bristol, United Kingdom) and resuspended at a stock concentration of 100 mM with dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri).

Techniques: Flow Cytometry, Control, Positive Control

Journal: Journal of veterinary internal medicine

Article Title: Effects of the potassium-sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells.

doi: 10.1111/jvim.15382

Figure Lengend Snippet: FIGURE 4 Western blot of proteins involved in p53 and Akt signaling in (A) D17, (B) Abrams, and (C) Dharma cells. (D) Viability after pharmacological inhibition of p53 translocation to the mitochondria. Cells were treated with pifithrin-μ (3 nM) alone or in combination with 300 μM amiloride for 24 hours. Cell viability was measured with the crystal violet assay. Means were presented with SD, and significance reported at *P < .05

Article Snippet: Amiloride hydrochloride was purchased from Tocris Bioscience (Bristol, United Kingdom) and resuspended at a stock concentration of 100 mM with dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri).

Techniques: Western Blot, Inhibition, Translocation Assay, Crystal Violet Assay

Journal: Journal of veterinary internal medicine

Article Title: Effects of the potassium-sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells.

doi: 10.1111/jvim.15382

Figure Lengend Snippet: FIGURE 5 Immunofluorescence of p53 localization in canine OSA cells after amiloride treatment. (A) Representative images of p53 localization in D17 cells after treatment with high-dose amiloride (300 μM) or vehicle for 24 hours before p53 staining. Cells were imaged with the Leica DMLB fluorescent microscope. Scale bar 10 μm. (B) Mean fluorescence intensity (MFI) of p53 staining in D17, Abrams, and Dharma cells as calculated by ImageJ/Fiji image analysis. Native images were taken at 10x/0.7 aperture, the regions of interest (ROI) were defined and quantified using ImageJ. N = 3 per group, with mean SD. Significance was reported at *P < .05 and **P < .01. OSA, osteosarcoma

Article Snippet: Amiloride hydrochloride was purchased from Tocris Bioscience (Bristol, United Kingdom) and resuspended at a stock concentration of 100 mM with dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri).

Techniques: Immunofluorescence, Staining, Microscopy, Fluorescence

Journal: Journal of veterinary internal medicine

Article Title: Effects of the potassium-sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells.

doi: 10.1111/jvim.15382

Figure Lengend Snippet: FIGURE 7 Combined energy phenotype analysis reveals a dose-dependent effect of amiloride on ECAR and OCR in D17 cells. (A) Linear regression model of ECAR during the first 50 minutes, representing readings after amiloride injection. (B) Fold changes in ECAR expressed across all treatment groups tested after amiloride injection. Coupling efficiency (C) and maximal respiration (D) as measured using the mitochondrial stress test. (E-F) Energy phenotype analysis after a 24-hour pretreatment with amiloride (100 μM). ECAR and OCR were normalized to cell density (4 x 10−4 cells), and vehicle-treated cells were compared to amiloride-treated cells after oligomycin injection. A metabolic switch is indicated by the black arrow between the phenotype markers. Median range values were presented in box plots. Mean SD values were presented in energy phenograms. Significance was recorded at *P < .05, **P < .01, ****P < .0001. ECAR, extracellular acidification rates; OCR, oxygen consumption rates

Article Snippet: Amiloride hydrochloride was purchased from Tocris Bioscience (Bristol, United Kingdom) and resuspended at a stock concentration of 100 mM with dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri).

Techniques: Injection

Journal: bioRxiv

Article Title: Dynamic modulation of auditory hair cell stereocilia membrane mechanics by the scrambling mechanotransduction complex

doi: 10.1101/2024.09.27.615395

Figure Lengend Snippet: A) FLIM images of P10 Tmc1 −/− ; Tmc2 −/− and Tmc1 −/ ; Tmc2 −/ HC bundles (top rows) and soma (bottom row). B) Example fluorescence decay curves comparing the HC bundles and PC soma of double knockout mice and the littermate controls. C) Quantification of the mean lifetime from Tmc1 −/− ; Tmc2 −/− and littermate controls. D, E) Intensity images of BODIPY 1c in P10 rat mid-apical turn of control, 1mM curare and 1mM amiloride treated organ of Corti with focus plane at the hair bundles (top row) and soma (bottom panel). F, G) Quantification of BODIPY 1c intensity in the F) soma of the hair cells and G) the hair bundles. Boxes in C, F and G represent the SD, and the star symbol indicates the mean. Each data point corresponds to a hair bundle or a cell (for soma). * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar = 10 μm.

Article Snippet: MET channel blockers: In a subset of experiments, during and post BODIPY 1c staining, the tissue was incubated in 1 mM tubocurarine (93750, Sigma) or 1mM amiloride (0890, TOCRIS) to block MET channel.

Techniques: Fluorescence, Double Knockout, Control

Journal: bioRxiv

Article Title: Dynamic modulation of auditory hair cell stereocilia membrane mechanics by the scrambling mechanotransduction complex

doi: 10.1101/2024.09.27.615395

Figure Lengend Snippet: A) FLIM images of a 30-minute time course where amiloride was continuously applied. B) Quantification of the mean lifetime across samples from hair bundles during amiloride treatment. C) Example lifetime decay curves for control, curare (1 mM) and amiloride (1 mM) from early time point (∼10 mins). D) Early (∼10 mins) and late (∼30 mins) FLIM images of hair bundles for each compound. E) Summary of mean lifetime from hair bundles across preparations and cells for early and late time points from control, curare and amiloride treatments. Boxes in E represent the SD, and the star symbol indicates the mean and the line indicates the median. *** p < 0.001. Scale bar = 10 μm.

Article Snippet: MET channel blockers: In a subset of experiments, during and post BODIPY 1c staining, the tissue was incubated in 1 mM tubocurarine (93750, Sigma) or 1mM amiloride (0890, TOCRIS) to block MET channel.

Techniques: Control

Journal: bioRxiv

Article Title: Dynamic modulation of auditory hair cell stereocilia membrane mechanics by the scrambling mechanotransduction complex

doi: 10.1101/2024.09.27.615395

Figure Lengend Snippet: A) FLIM images from P10 rat mid-apical organ of Corti with different monovalent ions (Na + vs TRIS + ), Ca 2+ concentrations (2 mM vs 0.02 mM) with presence or absence of 1 mM amiloride. The manipulations are represented schematically above each representative FLIM image. B) Summary plot showing the mean lifetime measured from the hair bundles from each manipulation. Each manipulation was performed in at least 4 animals. Boxes in B represent the SD, and the star symbol indicates the mean and the line indicates the median. *** p < 0.001. Scale bar = 10 μm.

Article Snippet: MET channel blockers: In a subset of experiments, during and post BODIPY 1c staining, the tissue was incubated in 1 mM tubocurarine (93750, Sigma) or 1mM amiloride (0890, TOCRIS) to block MET channel.

Techniques:

Journal: Cell calcium

Article Title: Native and recombinant ASIC1a receptors conduct negligible Ca2+ entry.

doi: 10.1016/j.ceca.2008.12.002

Figure Lengend Snippet: Fig. 1. Negligible Ca2+ entry through native ASIC1a. (A and B) stimulation of chick DRG neurons or HEK-293 cells with acid (pH 6) evoked inward currents inhibited by amiloride (30 M) and PcTx1 (30 nM). (C–E) Representative traces showing whole cell current (top), fura-2 fluorescence (gray, bottom), and the integrated current (dashed, bottom). Stimulation of chick DRGs (C) or HEK-293 cells (D) with acid evoked an inward current, but no measurable change in fura-2 fluorescence. (E) HEK-293-P2X3 cells responded to ATP (10 M) with a transient inward current and a deflection in F380 in that followed the time course of the integrated current.

Article Snippet: This current was transient in nature and inhibited by both the broad-spectrum ASIC channel blocker, amiloride (30 M) and the selective ASIC1a receptor antagonist psalmotoxin 1 (PcTx1; 30 nM; Peptides International, Louisville KY) (Fig. 1A) [25].

Techniques: