Bumetanide

Manufactured by Merck Group

Sourced in United States, United Kingdom, Germany, Denmark

Bumetanide is a pharmaceutical compound used as a loop diuretic. It functions by inhibiting the Na-K-2Cl cotransporter in the thick ascending limb of the loop of Henle, leading to increased excretion of sodium, chloride, and water.

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Lab products found in correlation

Amiloride, by Merck Group (12 mentions) Forskolin, by Merck Group (9 mentions) Dimethyl sulfoxide (dmso), by Merck Group (9 mentions) Ouabain, by Merck Group (8 mentions) Furosemide, by Merck Group (7 mentions) Isoguvacine, by Merck Group (6 mentions) Glibenclamide, by Merck Group (4 mentions) Hepes, by Merck Group (4 mentions) Gabazine, by Merck Group (4 mentions) D glucose, by Merck Group (4 mentions) Nifedipine, by Merck Group (4 mentions) Picrotoxin, by Bio-Techne (4 mentions) Acetazolamide, by Merck Group (4 mentions) Trypsin edta, by Thermo Fisher Scientific (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Benzamil, by Merck Group (3 mentions) Gabazine, by Bio-Techne (3 mentions) Picrotoxin, by Merck Group (3 mentions) Mannitol, by Merck Group (3 mentions) 5 nitro 2 3 phenylpropylamino benzoic acid nppb, by Merck Group (3 mentions)

100 protocols using bumetanide

Ion Transport Inhibition Assay

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The following chemicals and solutions were used in the experiment:

RH—Ringer solution, a basic solution with iso-osmotic properties and pH 7.4. Composition: Cl⁻ 160.8 mM; Na⁺ 147.2 mM; K⁺ 4.0 mM; Mg²⁺ 2.6 mM; Ca²⁺ 2.2 mM; HEPES 10.0 mM (4-(2-hydroxyethyl)piperazine-1-ethanosulfonic acid, 238.30 g/mol);

Amiloride (A)—used as an inhibitor of transepithelial transport of sodium ions, in a concentration in 0.1 mM solution of amidynoamide acid, 3,5-diamino-6-chloro-2-carboxylic acid (266.09 g/mol), dissolved and diluted in RH.

Bumetanide (B)—used as an inhibitor of transepithelial transport of chloride ions, in a concentration in 0.1 mM solution of 3-butylamino-4-phenoxy-5-sulfamoylbenzoic acid (364.42 g/mol), dissolved in 0.1% DMSO (dimethyl sulfoxide) and diluted in RH.

Reagents: Amiloride, Bumetanide, DMSO and HEPES were purchased from Sigma-Aldrich (USA). Mineral compounds: KCl, NaCl, CaCl₂, MgCl₂ were purchased from POCH (Poland).

Hołyńska-Iwan I, & Szewczyk-Golec K. (2020). Analysis of changes in sodium and chloride ion transport in the skin. Scientific Reports, 10, 18094.

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SIET Recordings with Ion Inhibitors

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The ROMK inhibitor (VU591 and BaCl₂), NKCC inhibitor (bumetanide), NKA inhibitor (ouabain) and tight junction inhibitor (2,4,6-triaminopyrimidine, TAP) were obtained from Sigma-Aldrich. A stock solution of VU591 was prepared by dissolving it into dimethyl sulfoxide (DMSO, Sigma-Aldrich). The final concentration of DMSO in the working solutions (including the control group) was 0.1%. BaCl₂ stock solution was prepared by dissolving it in redistilled water. Stock solutions of bumetanide were prepared by dissolving it in ethanol (Sigma-Aldrich). The final concentration of ethanol in the working solutions (including the control group) was 0.1%. TAP was directly dissolved with NW to the final concentrations. Before SIET recordings, the larvae were incubated in 1 ml of NW medium with different concentrations of inhibitors for 30 min. 0.1% DMSO or ethanol was added to the control group. After incubation, the larvae were transferred to the recording medium that contained no inhibitor. The inhibitor was not added to the recording medium to prevent alteration of the selectivity of the microelectrodes. In preliminary tests, VU591 (100 μM) was found to increase the background voltage of recording medium by 2.6% and decrease the Nernstian slope to 54.5 ± 0.5 (n = 5). Other inhibitors used in this study did not significantly alter the voltage or slope of calibration curve.

Horng J.L., Yu L.L., Liu S.T., Chen P.Y, & Lin L.Y. (2017). Potassium Regulation in Medaka (Oryzias latipes) Larvae Acclimated to Fresh Water: Passive Uptake and Active Secretion by the Skin Cells. Scientific Reports, 7, 16215.

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Bumetanide Dosage for Neonatal Seizures

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The effective systemic dose of Bumetanide on neonatal seizures in rats is 0.1–0.5 mg/kg (21 (link), 25 (link)). We administered 0.2 mg/kg of Bumetanide twice a day in repeated doses as previously described (26 (link)). Bumetanide (B3023; Sigma-Aldrich) was dissolved in 100% ethyl alcohol and then diluted with 0.9% saline to a final concentration of 0.05 mg/mL. Bumetanide solution (0.2 mg/kg) or equal volumes of 0.9% saline (4 mL/kg) was injected twice daily from P12 to P20 or P26. MEMRI was performed on P21 or P27 for each rat.

Tahara M., Higurashi N., Hata J., Nishikawa M., Ito K., Hirose S., Kaneko T., Mashimo T., Sakuma T., Yamamoto T, & Okano H.J. (2023). Developmental changes in brain activity of heterozygous Scn1a knockout rats. Frontiers in Neurology, 14, 1125089.

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Epithelial Cell Culture Protocol

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T84 (#CCL-248) and Caco2 (#HTB-37) human epithelial cell lines were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and were cultured in 95% air with 5% CO₂ at 37°C according to instructions provided by ATCC. Where indicated, cells were cultured on 5.0 μM or 0.4 μm pore polyester transwell inserts for 10–14 days to obtain confluent cell monolayers as measured by transepithelial resistance (CoStar, Cambridge, MA). Bumetanide (10 μM), S3226 (5 μM), indole (1 mM), myricetin (10 μM), and 4-aminobenzoic hydrazide (4-ABAH) (500 μM) obtained from Sigma–Aldrich (St. Louis, MA) were added to sterile filtered HBSS (indole and 4-ABAH) or DMSO (Bumetanide and S3226). Anti-CD11b (M1/70) and IgG1 rat isotype control monoclonal antibodies (10 μg/ml) were obtained from BioLegend (San Diego, CA). Anti-CD47 (B6H12) and IgG1 mouse isotype control monoclonal antibody (10 μg/ml) was obtained from Fisher Scientific (Hampton, NH). Anti-CD55 (clone OE-1) mouse monoclonal antibody (10 μg/ml) was produced as previously described.^{14 (link)} All inhibitors were used at established, noncytotoxic concentrations.

Cartwright I.M., Dowdell A.S., Hanson C., Kostelecky R.E., Welch N., Steiner C.A, & Colgan S.P. (2022). Contact-dependent, polarized acidification response during neutrophil–epithelial interactions. Journal of leukocyte biology, 112(6), 1543-1553.

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Whole-cell and Perforated Patch-Clamp Recordings

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The bathing solution consisted of (in mM) 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₅, 1 MgCl₂, 2 CaCl₂, 2.5 KCl, 10 glucose and was equilibrated with 95% O₂/5% CO₂ at least 1 h before use (pH 7.4, osmolarity 306 mOsm). Two pipette solutions for whole-cell recordings were used: for a pipette Cl^- concentration ([Cl^-]_p) of 10 mM the solution was composed of (in mM) 128 K-gluconate, 2 KCl, 4 NaCl, 1 CaCl₂, 11 EGTA, 10 K-HEPES, 2 Mg²-ATP, 0.5 Na-GTP, and 2 lidocaine-N-ethyl chloride (pH adjusted to 7.4 with KOH and osmolarity to 306 mOsm with sucrose). For a [Cl^-]_p of 50 mM the solution contained (in mM) 86 K-gluconate, 44 KCl, 4 NaCl, 1 CaCl₂, 11 EGTA, 10 K-HEPES, 2 Mg2-ATP, 0.5 Na-GTP. Pipette solution for gramicidin-perforated patch experiments consisted of (in mM) 10 Na-gluconate, 120 KCl, 1 CaCl₂, 2 MgCl₂, 11 EGTA, and 10 K-HEPES. For gramicidin-perforated patch-clamp recordings 10 μg/ml Gramicidin D (Sigma, St. Louis, MO, United States) was added from a stock solution (2 mg/ml in DMSO) on the day of experiment. Gramicidin D, bumetanide, and lidocaine-N-ethyl chloride was obtained from Sigma-Aldrich and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) was obtained from Biotrend (Cologne, Germany). CNQX and bumetanide were used from a dimethylsulfoxide (DMSO, Sigma-Aldrich) stock solution. The DMSO concentration of the final solution never exceeded 0.1%.

Lombardi A., Jedlicka P., Luhmann H.J, & Kilb W. (2018). Giant Depolarizing Potentials Trigger Transient Changes in the Intracellular Cl- Concentration in CA3 Pyramidal Neurons of the Immature Mouse Hippocampus. Frontiers in Cellular Neuroscience, 12, 420.

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Systemic and Topical Ototoxicity Induction

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For systemic assays, animals were injected sub-cutaneously with a solution of gentamicin sulphate or kanamycin sulphate in normal saline (400–500 mg/kg; Sigma, Gillingham, U.K.) followed 20–30 min later by an intra-peritoneal injection of bumetanide (50 mg/kg; Sigma) or furosemide solution (100 mg/kg; Sigma). For topical application to the cochlea, anaesthesia was induced with isoflurane, the auditory bulla was accessed through a retro-auricular incision and windowed using a scalpel blade. Aminoglycoside and diuretic solutions (as above) were applied directly to the round window; in some cases, animals were implanted with a gelatin sponge (Cutanplast, Sheffield, U.K.) pre-soaked in an aminoglycoside/diuretic mixture. The bulla was sealed using a plug of fascia held in place with Vetbond adhesive (3 M, Bracknell, U.K.). The muscle layer and wound were closed with absorbable Vicryl suture material (Ethicon, Norderstedt, Germany) and the animal allowed to recover.

Abbas L, & Rivolta M.N. (2015). Aminoglycoside ototoxicity and hair cell ablation in the adult gerbil: A simple model to study hair cell loss and regeneration. Hearing Research, 325, 12-26.

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Ion Transporter Inhibitor Effects

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Polyclonal anti-KCC1 antibody was purchased from Alomone Labs, Israel. Bumetanide, amiloride, benzamil, barium, glibenclamide, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), CFTR(inh)-172, and forskolin were obtained from Sigma while GlyH 101 was from Calbiochem. All stock solutions were prepared in DMSO. Normal salt diet and low salt diet were purchased from Harlan Teklad. Ringer solution contains (in mM) 140.8 Na⁺, 5.2 K⁺, 123.5 Cl^-, 25 HCO₃^-, 1.25 Ca²⁺, 1.25 Mg²⁺ and 5 glucose, pH 7.4. Cl^--free Ringer solution contains HCO₃^- (25 mM) with no Cl^-. HCO₃^--free Ringer solution contains Cl^- with no HCO₃^-. Na⁺-free Ringer solution contains no Na⁺. In these solutions, isethionate was used in place of anion Cl^- and HCO₃^- and N-Methyl-D-glucamine (NMDG) was used as a substitute for Na⁺. HCO₃^--containing solutions were gassed with 95% O₂-5% CO₂; HCO₃^--free solution was gassed with 100% O₂.

Tang L., Fang X., Winesett S.P., Cheng C.Y., Binder H.J., Rivkees S.A, & Cheng S.X. (2017). Bumetanide increases Cl--dependent short-circuit current in late distal colon: Evidence for the presence of active electrogenic Cl- absorption. PLoS ONE, 12(2), e0171045.

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Pharmacological Reagents for Ion Channel Modulation

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The following reagents were obtained from Sigma-Aldrich (Gillingham, UK); amiloride, atropine, bumetanide, capsaicin, carbachol, forskolin, veratrine, veratridine.
Tetrodotoxin came from Tocris Bioscience and 4-chlorobenzo(F)isoquinoline from Ubichem plc. Note veratrine is predominantly veratridine together with other similarly acting alkaloids of natural origin.

Cuthbert A.W., Murthy M, & Darlington A.P. (2015). Neural control of submucosal gland and apical membrane secretions in airways. Physiological Reports, 3(6), e12398.

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Modeling Fat Embolism Pathogenesis in Mice

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One hundred twenty C57BL/6J male mice weighing 22–24 g were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. and used in the present study. Ninety-six mice were randomly divided into control and fat embolism groups 1–5 (n = 16 per group). Injectable fat was gathered from perinephric fat from allogeneic mice and homogenized to rupture the cell membrane, then centrifuged. Mice in the fat embolism groups were intravenously injected with 2 μL/g fat. Lung tissue was obtained 4, 6, 12, 24 and 48 h after fat injection. Sixteen mice were intravenously injected with the same volume of sterile saline as the control group. Pentobarbital at a dosage of 70 mg/kg was intraperitoneally injected as anesthesia. For protein function experiments, the AQP1 inhibitor bumetanide (Sigma, St. Louis, MO, USA) was dissolved in a NaOH liquor at a concentration of 2.25 g/L and intraperitoneally injected at a dose of 0.1 mg/g before fat injection, another AQP1 inhibitor Acetazolamide (Sigma) was intragastrically administered at a volume of 0.1 mL per mice (40 mg·kg⁻¹·d⁻¹), according to the manufacturer’s instructions.

Zhang Y., Tian K., Wang Y., Zhang R., Shang J., Jiang W, & Wang A. (2016). The Effects of Aquaporin-1 in Pulmonary Edema Induced by Fat Embolism Syndrome. International Journal of Molecular Sciences, 17(7), 1183.

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Bumetanide Transport Assay Protocol

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Bumetanide and all of the other compounds (with the exception of AR-C155858) were purchased from Sigma-Aldrich (St. Louis, MO). AR-C155858 was purchased from Tocris Bioscience (Bristol, U.K.). [³H]Bumetanide (15–30 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). GIBCO Leibovitz’s L-15 medium with glutamine (Cat. No. 41300-039) and all Western blotting materials were supplied by Thermo Fisher Scientific (Rockford, IL). The pCR2.1-TOPO, TOPO TA cloning kit, TRIzol Reagent, and mMESSAGE mMACHINE T7 transcription kit were purchased from Thermo Fisher Scientific (Rockford, IL). pGH19 vector was kindly provided by Dr. Walter F. Boron (Case Western Reserve University, Department of Physiology and Biophysics, Cleveland, Ohio).^{24 (link)} The FlashGel System was purchased from Lonza (Portsmouth, NH). All enzymes were purchased from New England Biotechnology (Ipswich, MA). The gel extraction and PCR purification kits were purchased from Qiagen (Valencia, CA). DNA purity and concentration were verified using a NanoDrop 1000 instrument (Thermo Fisher Scientific, Rockford, IL). A ZOE fluorescent cell imager made by Bio-Rad (Hercules, CA) was used for fluorescent microscopy. The mouse anti-Egfp antibody (JL-8, Cat. No. 632381) was purchased from Clontech (Mountain View, CA).

Jones R.S., Parker M.D, & Morris M.E. (2017). Quercetin, Luteolin, Phloretin, and Morin Are Dietary Flavonoid Inhibitors of Monocarboxylate Transporter 6. Molecular pharmaceutics, 14(9), 2930-2936.

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