Fpl 64176

Manufactured by Merck Group

Sourced in United States

The FPL 64176 is a laboratory equipment product manufactured by Merck Group. It is designed for performing specific laboratory procedures, but a detailed and unbiased description of its core function cannot be provided without the risk of making interpretations or extrapolations.

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7 protocols using fpl 64176

Whole-Cell Patch-Clamp Recording of L-type Ca2+ Current

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The L-type Ca²⁺ current was recorded at room temperature using the whole-cell patch-clamp technique with the Axopatch 200B (Axon Instruments), as we previously described.⁹⁰ (link) Extracellular solution contained 140 mM NaCl, 2.7 mM KCl, 10 mM HEPES, and 10 mM EGTA (pH7.4). Glass pipettes were filled with solution containing 100 mM CsCl, 30 mM CsF, 4 mM ATP-Mg, 2 mM MgCl₂, 10 mM HEPES, and 10 mM EGTA (pH 7.2 adjusted with CsOH). After the membrane was ruptured, cells were voltage-clamped at a holding potential of −70 mV, and inward currents were evoked by a 240-ms test pulse to +40 mV in 10-mV increments. After a further 30-min interval, the effects of 300 nM FPL64176 and 3 μM nifedipine (all from Sigma-Aldrich) were tested. Data collection and analysis were performed using pClamp 9.0 software (Axon Instruments).

Jiang L., Liang J., Huang W., Ma J., Park K.H., Wu Z., Chen P., Zhu H., Ma J.J., Cai W., Paul C., Niu L., Fan G.C., Wang H.S., Kanisicak O., Xu M, & Wang Y. (2021). CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy. Molecular Therapy, 30(1), 54-74.

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Aortic Ring Isometric Contraction Assay

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DTAs from wild-type, smLRP1^−/−, and smaLRP1^−/− mice were dissected and placed in a tissue culture dish containing Krebs solution (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, 25 mM NaHCO₃, pH 7.4). Aortic rings were prepared and equilibrated as detailed in the online-only Data Supplement. The following stimuli were tested: 120 mM KCl (Sigma-Aldrich), phenylephrine (PE; Sigma-Aldrich), 1 μM U-46619 (Sigma-Aldrich), 1 μM FPL 64176 (Sigma-Aldrich), 0.3 μM calyculin A (Sigma-Aldrich), and 1 mM 4-chloro-m-cresol (4-CmC; Sigma-Aldrich). After each stimulus, aortic rings were washed and re-equilibrated for 15–30 minutes before application of the next stimulus. Force measurements were acquired and recorded using LabChart Pro (ADInstruments). For each stimulus, aortic rings were normalized to their respective baseline force measurement recorded immediately before addition of the stimulus. Aortic ring isometric contraction assays using PE are presented as means ± SEM with 95% confidence intervals indicated.

Au D.T., Ying Z., Hernández-Ochoa E.O., Fondrie W.E., Hampton B., Migliorini M., Galisteo R., Schneider M.F., Daugherty A., Rateri D.L., Strickland D.K, & Muratoglu S.C. (2018). LRP1 regulates smooth muscle contractility by modulating Ca2+ signaling and expression of cytoskeletal related proteins.. Arteriosclerosis, thrombosis, and vascular biology, 38(11), 2651-2664.

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Insulin Release Assay Protocols

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Drugs employed for insulin release studies were purchased from Tocris Bioscience (nifedipine, FPL64176, oxotremorine-M, exendin-4, veratridine), Sigma (GLP-1) or Millipore (AIP2). The Drosophila homeoprotein Antennapaedia leader peptide (RQIKIWFQNRRMKWKK) was obtained from GenScript.

Zhu L., Almaça J., Dadi P.K., Hong H., Sakamoto W., Rossi M., Lee R.J., Vierra N.C., Lu H., Cui Y., McMillin S.M., Perry N.A., Gurevich V.V., Lee A., Kuo B., Leapman R.D., Matschinsky F.M., Doliba N.M., Urs N.M., Caron M.G., Jacobson D.A., Caicedo A, & Wess J. (2017). β-arrestin-2 is an essential regulator of pancreatic β-cell function under physiological and pathophysiological conditions. Nature Communications, 8, 14295.

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Emetic Dose Response Evaluation

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Apomorphine HCl, McN-A-343, quinpirole HCl, serotonin HCl and 2-methyl-serotonin maleate salt (2-Me-5-HT) were obtained from Sigma/RBI (St. Louis, MO). FPL 64176, GR 73632 and Resiniferatoxin, were purchased from Tocris (Minneapolis, MN). Resiniferatoxin was dissolved in ethanol: Tween-80: water in a 1:1:18 ratio. 5-HT, 2-Me-5-HT, GR 73632, apomorphine HCL and quinpirole HCL, pilocarpine HCL, and McN-A-343 were dissolved in water. FPL 64176 was dissolved in DMSO (Sigma) and then diluted with three volumes of distilled water to a final DMSO concentration of 25%, and thapsigargin in 10% DMSO in water. Shrews were divided into groups (n = 6–10 per group) and each shrew received a 1-h pretreatment with varying doses of RTX (μg/kg body weight, s.c.) or the corresponding vehicle (s.c.). Following the 1-h- pretreatment, each treated animal was injected with a fully effective emetic dose of either a nonselective or receptor selective emetogen.

Darmani N.A., Henry D.A., Zhong W, & Chebolu S. (2020). Ultra-low doses of the transient Receptor Potential Vanilloid 1 Agonist, resiniferatoxin, prevents vomiting evoked by diverse emetogens in the least shrew (Cryptotis Parva). Behavioural pharmacology, 31(1), 3-14.

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Ion Channel Modulator Preparation

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Isradipine, nifedipine, verapamil, diltiazem, FPL-64176, Iso and ACh were purchased from Sigma-Aldrich. Drugs were prepared as 10 mM stock solutions in DMSO and then diluted in the external solution. TTX (Wako Chemical Co.) was dissolved in distilled water at a concentration of 10 mM and then diluted to the final concentration of 10 µM in the experimental solution.

Toyoda F., Mesirca P., Dubel S., Ding W.G., Striessnig J., Mangoni M.E, & Matsuura H. (2017). CaV1.3 L-type Ca2+ channel contributes to the heartbeat by generating a dihydropyridine-sensitive persistent Na+ current. Scientific Reports, 7, 7869.

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RT-qPCR Analysis of BDNF Expression

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RNA was isolated using the RNeasy kit (Qiagen) and genomic DNA was removed with the DNA-Free kit (Ambion). cDNA was synthesised using 0.5 µg total RNA with the DyNAmo™ cDNA Synthesis Kit (Thermo). Technical replicates as well as no template and no RT negative controls were included and at least three biological replicates were studied in each case. Real-time quantitative PCR reactions were set up with DyNAmo ColorFlash SYBR Green qPCR kit (Thermo) or TaqMan Universal PCR Master Mix (Applied Biosystems) and run on a CFX96 System (BioRad) or 7300 Real Time PCR System (Applied Biosystems). The data were analysed using the iCycler software (BioRad) or the MxPro QPCR analysis software (Stratagene) and the qbase PLUS software (Biogazelle) for statistical comparisons. Primer sequences are provided in Table S1. Human foetal brain total RNA (21 weeks old) sample was purchased from Stratagene. BDNF induction was assessed by qRT-PCR after membrane depolarization of five week old neurons with 25 mM KCl and 5 µM FPL 64176 (Sigma) treatment in full media for four hours. Primer sequences are given in Table S1.

Bilican B., Livesey M.R., Haghi G., Qiu J., Burr K., Siller R., Hardingham G.E., Wyllie D.J, & Chandran S. (2014). Physiological Normoxia and Absence of EGF Is Required for the Long-Term Propagation of Anterior Neural Precursors from Human Pluripotent Cells. PLoS ONE, 9(1), e85932.

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Compound 1 Evaluation in Aorta and SHR

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Compound 1 was obtained previously [4] . Carbamoylcholine chloride (carbachol), ( ± )-noradrenaline bitartrate hydrate (NA), serotonin chloride (5-HT), nifedipine, 1H-[1, 2, 4]oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), glibenclamide, tetraethylammonium chloride (TEA), FPL 64176, components of physiological solution, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). For ex vivo and in vivo experiments, compound 1 was dissolved in distilled water and DMSO (1 % and 10 % v/v, respectively). Preliminary experiments showed that DMSO had no effect on tension development on isolated aorta and on the blood pressure of SHR.

Estrada-Soto S., Rendón-Vallejo P., Villalobos-Molina R., Millán-Pacheco C., Vázquez M., Hernández-Borja F, & Hernández-Núñez E. (2022). 6-Amino-3-Methyl-4-(2-nitrophenyl)-1,4-Dihydropyrano[2,3-c]Pyrazole-5-Carbonitrile Shows Antihypertensive and Vasorelaxant Action via Calcium Channel Blockade. Drug research, 72(1).

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