Journal: Pflugers Archiv

Article Title: Functional expression of the proton sensors ASIC1a, TMEM206, and OGR1 together with BK Ca channels is associated with cell volume changes and cell death under strongly acidic conditions in DAOY medulloblastoma cells

doi: 10.1007/s00424-024-02964-7

Figure Lengend Snippet: DAOY cells and UW228 cells express functional BK Ca channels. (a) Left, representative traces of a DAOY cell before (ctrl), during (iono) and after (wash) treatment with 1 μM ionomycin. Recordings were made with a control (ctrl) or pipette solution containing Cs + . Middle, IV curves (mean ± SD). Right, mean current amplitudes at + 120 mV (n ≥ 4). (b) As in a, but with UW228 cells ( n = 6). (c) mRNA expression of KCNMA1 in DAOY and UW228 cells cultured with a pH 7.4 medium (ctrl) and in DAOY cells cultured as MS for 7 days. qPCR data was normalized to HPRT1 expression levels (mean ± SD; n = 3). (d) Left, representative traces of DAOY cells treated with the following 3 conditions at + 50 mV: 1 μM ionomycin, 1 μM ionomycin plus 20 μM bepridil (bep), 1 μM ionomycin plus 100 nM iberiotoxin (IbTx). Right, summary of current amplitudes (mean ± SD; n ≥ 4). (e) Left, representative trace of a DAOY cell treated with 10 μM NS-11021 (NS) and 100 nM IbTx. Right, mean membrane potentials ( n = 5). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (paired Student’s t-tests)

Article Snippet: PcTx1 and IbTx (Smartox Biotechnology, Sainte-Égrève, France), YM-254890 (Biozol, Eching, Germany), DIDS (Sigma-Aldrich, St. Louis, MI, USA), bepridil hydrochloride (MedChemExpress, Monmouth Junction, New Jersey, United States), NS-11021 (Alomone Labs, Jerusalem, Israel), and pregnenolone sulfate (Sigma-Aldrich) were purchased in research quality.

Techniques: Functional Assay, Control, Transferring, Expressing, Cell Culture, Membrane

Journal: Cardiovascular Research

Article Title: Contractility measurements for cardiotoxicity screening with ventricular myocardial slices of pigs

doi: 10.1093/cvr/cvad141

Figure Lengend Snippet: Screening test with drugs from CiPA list with known interactions and risks performed in three different laboratories ( N = 3–5, n = 6–10). The effects of bepridil, cisapride, disopyramide, ibutilide, and risperidone (red) as well as negative controls DMSO and acetylsalicylic acid (black) were compared to time-matched controls treated only with PBS (grey). ( A ) Kaplan–Meier curve displaying the pacing capability of slices. Statistical analysis by Fisher’s exact test. ( B–H ) Mean and SEM of effects compared to PBS with a two-way ANOVA with Dunnett’s multiple comparison test on the normalized pacing threshold ( B ), normalized force ( C ), FFR ( D ), normalized contraction duration ( E ), CDFR ( F ), normalized ERP ( G ), and changes in diastolic force ( H ). Absolute values of each slice were normalized to values before adding the drug or solvent. Exact n and P values for each condition are given in , .

Article Snippet: Drugs from the CiPA list were added at five different concentration steps into 3–4 mL from the following stocks: bepridil hydrochloride (Tocris Bioscience, UK) 10 mM dissolved in DMSO; ibutilide hemifumarate (Sigma-Aldrich, USA) 10 μM dissolved in H 2 O; disopyramide phosphate (Sigma-Aldrich, USA) 10 mM dissolved in H 2 O; cisapride monohydrate 100 μM dissolved in DMSO; risperidone (Sigma-Aldrich, USA) 1 mM dissolved in DMSO; and acetylsalicylic acid (Sigma-Aldrich, USA) 100 μM dissolved in H 2 O.

Techniques: Comparison, Solvent

Journal: Nature Communications

Article Title: MicroRNA-27a controls the intracellular survival of Mycobacterium tuberculosis by regulating calcium-associated autophagy

doi: 10.1038/s41467-018-06836-4

Figure Lengend Snippet: Cacna2d3 regulates survival of Mtb via Ca 2+ -mediated autophagy. a , b Bacterial survival of Mtb in ( a ) primary macrophages treated with Cacna2d3 siRNA; ( b ) macrophages from WT and Cacna2d3 mutant ( Cac3 PB/PB ) mice, after infection with Mtb (MOI = 5) for 24 h, compared to 2 h. c , d WT or Cac3 PB/PB macrophages were infected with Mtb , and then subjected to ( c ) TEM analysis; red arrow indicated the bacteria. ( d ) IB of lysates. e–h Confocal detection of mRFP-GFP-LC3B spots in Cacna2d3 -siRNA-treated macrophages ( e , f ) or Cac3 PB/PB macrophages, after infection with Mtb (MOI = 5) for 6 h ( g , h ); Bar, 5 μM. i Calcium assay in WT or Cac3 PB/PB macrophages infected with Mtb (MOI = 5) for the duration of 1 min. j , k CFU assay ( j ) and intracellular survival of Mtb ( k ) in WT or Cac3 PB/PB macrophages pretreated with Bepridil (BPD) (10 μM), and then infected with Mtb (MOI = 5) for 2 h or 24 h. l , m Confocal detection of mRFP-GFP-LC3B spots in WT or Cac3 PB/PB macrophages pretreated with DMSO or BPD infected with Mtb at MOI 5 for 6 h. Bar, 5 μM. n , o IB of lysates for phosphorylated-CaMKKβ ( n ) and phosphorylated-ULK1 ( o ) from WT or Cac3 PB/PB macrophages infected with Mtb for indicated times. NS, not significant ( p > 0.05), * p < 0.05 and ** p < 0.01 by the unpaired t -test ( f , h ) or Mann-Whitney U test ( a , b , j ). Data are from three independent experiments with biological duplicates in each ( a , b , f , h , j , k , m ; mean ± s.e.m. of n = 3 duplicates) or representative of three independent experiments ( c–e , g , i , l , n , o )

Article Snippet: The VGCC inhibitor Bepridil (sc-202974) was purchased from Santa Cruz Biotechnology (Santa Cruz, USA).The vacuolar ATPase inhibitor Bafilomycin A1 (S1413) and CQ (S4157) was purchased from Selleck Chem. (Shanghai, China).

Techniques: Mutagenesis, Infection, Bacteria, Calcium Assay, Colony-forming Unit Assay, MANN-WHITNEY

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Recording and blockade of the Na + /Ca 2+ exchanger (NCX) currents in glioblastoma cells. (a) Human glioblastoma U87 cells were held at −50 mV; the membrane currents associated with NCX were recorded by a voltage ramp from +60 to −120 mV. Ni 2+ (10 mM) was used to block the NCX currents. (b, c) NCX currents recorded before and after application of bepridil (0.1–100 μM) or KB‐R7943 (0.1–100 μM). (d–f) The currents recorded before and after application of the reverse NCX blockers SEA0400 (0.1–100 μM), SN‐6 (0.1–30 μM), and YM244769 (0.1–30 μM). The reverse NCX current was measured at +50 mV, and forward NCX current was measured at −110 mV

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Membrane, Blocking Assay

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Effect of the Na + /Ca 2+ exchanger (NCX) blockers on viability of human glioblastoma cell lines. (a, b) Viability of adult glioblastoma cell lines (U87 and U251) after exposure to NCX blockers bepridil, KB‐R7943, SEA0400, SN‐6, or YM244769 at 0–100 μM for 72 hr. n = 5 independent tests per group. (c) Viability of paediatric glioblastoma cell line SF188 after 72‐hr incubation with the same NCX blockers at 0–100 μM. n = 5 independent tests per group. (d) For U87, U251, and SF188 cell lines, concentration of the NCX blockers required to produce half maximal inhibition (IC 50 : μM, mean ± SEM) of cell viability. IC 50 was obtained by fitting the normalized concentration–response relationship to the equation: V / V 0 = 1/{1 + [C/IC 50 ] n } , where V 0 and V are the cell viability measured in control and in the presence of a blocker, C is the concentration of the blocker, and n is the Hill coefficient. n = 5 independent cultures in each group

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Incubation, Concentration Assay, Inhibition, Control

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Effect of bepridil and KB‐R7943 on the level of [Ca 2+ ] i measured by Ca 2+ imaging. (a) Flua‐4 AM‐loaded U87 cells were imaged and perfused with bepridil (25 or 50 μM) in the HEPES‐buffered solution containing 2‐mM Ca 2+ . (b) Ca 2+ imaging of U87 cells before and after perfusion with KB‐R7943 (25 or 50 μM). (c) In the Ca 2+ ‐free solution, Ca 2+ imaging of U87 cells before and after perfusion with bepridil or KB‐R7943. (d) Ca 2+ imaging before and after perfusion with bepridil or KB‐R7943, followed by application of 5‐μM thapsigargin. (e) U87 cells were pretreated with a Ca 2+ chelator BAPTA‐AM (20 μM) for 2 hr and then imaged and perfused with bepridil or KB‐R7943. The traces are representative of three separate experiments, and n = 12–18 cells were imaged per experiment

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Imaging

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Impact of bepridil on human astrocytes (HA) and the Na + /Ca 2+ exchanger (NCX) isoforms in each cell line. (a) Viability of U87, U251, and SF188 cells and HA after exposure to bepridil (25 μM, 48 hr); n = 5 independent tests in each group. (b) The bands of NCX1, NCX2, and NCX3 isoforms detected by Western blot analysis in the sample of HA and glioblastoma cell lines U87, U118, A172, U251, and SF188. (c) Representative recording of the NCX currents in HA and glioblastoma cell lines. (d) Viability of U87, U251, and SF188 cells and HA after exposure to KB‐R7943 (25 μM, 48 hr). n = 5 independent tests in each group

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Western Blot

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Concentration of compounds required to produce half‐maximal inhibition (IC 50 : μM) of the currents related to the reverse and forward NCX operation

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Concentration Assay, Inhibition, Blocking Assay