The stock solutions of the reagents used included A-317491, a P2X3 receptor antagonist (Sigma-Aldrich); 10PANX, a selective PANX-1 blocker; and GsMTx-4, a selective Piezo1 channel blocker (R&D Systems, Inc, Minneapolis, United States) were prepared in sterilized PBS (Life Technologies, Ltd, Paisley, United Kingdom) and diluted with physiological saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) to the appropriate concentration for administration. Stock solutions of the TRPA1 channel antagonist, HC-030031 (Abcam), and TRPC5 channel antagonist, HC-070 (MedChemExpress, Monmouth Junction, NJ, United States), were prepared in DMSO, and diluted with physiological saline to the appropriate concentration for administration. Mefloquine, a non-selective PANX-1 blocker, was purchased from Hisamitsu Pharmaceutical Co., Inc, and Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Because this reagent is water-insoluble, it was administered orally with physiological saline. Anesthesia was induced with 3% isoflurane (FUJIFILM Wako Pure Chemical Co., Osaka, Japan).
A 317491
Manufactured by Merck Group
Sourced in United States
A-317491 is a chemical compound used in laboratory research and analysis. It functions as a selective antagonist of the P2X3 and P2X2/3 receptors. The core function of this product is to facilitate the study of the physiological and pharmacological roles of these receptor subtypes in various biological systems.
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Lab products found in correlation
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Hc 030031, by Abcam (1 mentions)
Hc 070, by MedChemExpress (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Human tnf α, by Merck Group (1 mentions)
F12 medium, by Merck Group (1 mentions)
Apetx2, by Peptide Institute (1 mentions)
Naloxone, by Bio-Techne (1 mentions)
Escherichia coli lps, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
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Octoxynol 9, by Merck Group (1 mentions)
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Sumatriptan, by Merck Group (1 mentions)
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13 protocols using a 317491
1
Antagonists of P2X3, PANX-1, and Piezo1 Channels
2
Intrathecal AOAA and A317491 Behavioral Study
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AOAA (an inhibitor of CBS), A317491 (an antagonist of the P2X3 receptor), and ATP were purchased from Sigma-Aldrich and were freshly prepared in 0.9% normal saline. AOAA was intrathecally injected once daily for 7 consecutive days for molecular expression experiments and behavioral tests. A317491 was administered by a single intrathecal injection for behavioral tests.
3
Empathic Pain Hypersensitivity Modulation
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Whether A317491 (0.2 mg/10 μl, Sigma, USA), a selective P2X3 receptor antagonist, has blocking effect on the development of empathic mechanical pain hypersensitivity in the CO rats was examined by s.c. injection of the drug into the plantar surface of one hindpaw randomly (left or right) 15 min prior to the dyadic social interactions.
To determine whether peripheral postganglionic sympathetic innervations exists and if exists, which adrenergic receptor subtypes are involved in the development of empathic mechanical pain hypersensitivity, s.c. administration of prazosin (40 μg/25 μl), a selective α1 adrenergic receptor antagonist, yohimbine (30 μg/25 μl), a potent α2 adrenergic receptor antagonist, and propranolol (5 μg/25 μl), a β adrenergic receptor antagonist, was performed, respectively, in the CO rats 15 min prior to the dyadic priming social interaction. The dose selection of the pharmacological agents was based on, but slightly different from, the following studies: A317491 (Hsieh et al., 2012 (link)), clonidine (Jørum, 1988 (link)), DSP-4 (Ross and Stenfors, 2015 (link)), guanethidine (Chen et al., 2010 (link)), prazosin (Lee et al., 2000 (link)), yohimbine (Silva et al., 2015 (link)) and propranolol (Wang et al., 2013 (link)). The CC rats and vehicles (0.9% physiological saline) of the above drugs served as controls.
To determine whether peripheral postganglionic sympathetic innervations exists and if exists, which adrenergic receptor subtypes are involved in the development of empathic mechanical pain hypersensitivity, s.c. administration of prazosin (40 μg/25 μl), a selective α1 adrenergic receptor antagonist, yohimbine (30 μg/25 μl), a potent α2 adrenergic receptor antagonist, and propranolol (5 μg/25 μl), a β adrenergic receptor antagonist, was performed, respectively, in the CO rats 15 min prior to the dyadic priming social interaction. The dose selection of the pharmacological agents was based on, but slightly different from, the following studies: A317491 (Hsieh et al., 2012 (link)), clonidine (Jørum, 1988 (link)), DSP-4 (Ross and Stenfors, 2015 (link)), guanethidine (Chen et al., 2010 (link)), prazosin (Lee et al., 2000 (link)), yohimbine (Silva et al., 2015 (link)) and propranolol (Wang et al., 2013 (link)). The CC rats and vehicles (0.9% physiological saline) of the above drugs served as controls.
4
Cell Culture with ATP Stimulation
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F12 medium, Fetal Bovine Serum (FBS), L-glutamine, penicillin, streptomycin, ATP, A-317491, human TNF-α, and all salts were purchased from Sigma-Aldrich (Toluca, MX). ATP stock solution (100 mM) was made using deionized distiller water and stored frozen; these were diluted to obtain the desired final concentration in external solution prior to use, and the pH was adjusted to 7.4 with NaOH.
5
P2X3-Mediated Nociception Modulation
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α β-me ATP (P2X3 agonist) and A317491 (P2X3 antagonist) were purchased from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, USA), and dissolved in sterile 0.9 % saline solution to prepare stock solution (stored at –20 °C). They were diluted to the requested concentrations before each experiment. For i.pl, α β-me ATP (600 nmol,10 μL) and A317491 (300 nmol,10 μL) were injected subcutaneously into the dorsal surface of the right hindpaw of rats. For i.t., α β-me ATP (300 nmol, 25 μL) and A317491 (100 nmol, 25 μL) were administered once on days 3 after CFA injection.
6
Pharmacological Inhibition of Nociceptive Signaling
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The TRPV1 antagonist N-(3-methoxyphenyl)-4-chlorocinnamide (SB366791; BIOMOL International; Plymouth Meeting, PA, USA), a selective blocker to the ASIC3 channel APETx2 (PEPTIDE Institute, Inc., Osaka, Japan), and the selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors A317491 (Sigma-Aldrich Japan, Yokohama, Japan) were purchased as shown. SB366791 was administered intraperitoneally to OVX mice at a dose of 1.0 mg/kg16 (link). APETx2 was administered intramuscularly at a dose of 20 µM into the left gastrocnemius muscle17 (link). A-317491 was administered subcutaneously at a dose of 10 mg/kg18 . Mice were given a single injection at 6 weeks after OVX or the sham operation, and the femur and serum were isolated at 6 hours after administration.
7
Intrathecal Receptor Blockade in Rats
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Twelve days after inoculation, the rats were catheterized by placing a PE-10 polyethylene catheter into the subarachnoid space between vertebrae L5 and L6. Three days later, rats (n = 6 for each group) were randomized to receive via the catheter P2X3 receptor antagonist A-317491 (30 nM; Sigma-Aldrich, St. Louis, MO, USA), LPAR1 receptor blocker VPC32183 (2 mM in 30 µL 3% bovine serum albumin; Avanti Ploar Lipids, Alabaster, AL, USA), Rho-inhibitor botulinum toxin C3 (BoTXC3, 300 pg in 30 µL normal saline; Calbiochem San Diego, CA, USA), ROCK blocker Y27632 (48 µg in 12 µL normal saline; Calbiochem), or normal saline.
8
Tongue SCC Mouse Model Evaluation
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The tongue SCC model was produced in athymic BALB/c nu/nu mice as previously described [22 (link)]. The anatomic and functional features of this mouse cancer model parallel those found in HNSCC patients [22 (link)]. After baseline Dolognawmeter gnaw times were established, BALB/c nude mice were inoculated with 50 μl total volume (of 106) HSC-3 cells in DMEM and Matrigel™ into the tongue through a transoral approach. Control mice received 50 μl of vehicle injection. 20 μl of an ATP-hydrolyzing enzyme apyrase (100 μM, Sigma-Aldrich), A-317491 (3 mg/kg, Sigma-Aldrich), AF-353 (3 mg/kg, Afferent Pharmaceuticals) or normal saline solution was injected into the tongue of mice on post-inoculation day 14.
9
P2X Receptor Agonist and Antagonist Study
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In this study we used α,β-meATP (0.75–12.5 mM), a selective P2X receptor including P2X1, P2X2/3, and P2X3 subtypes agonist12 ,20 (link),54 (link); A-317491 (10 mM), a selective P2X2/3 and P2X3 receptor antagonist55 (link); and ADP, a selective P2Y receptor agonist26 (link). Naloxone (100 mM), specific opioids receptor antagonist33 (link),38 (link) and 2% procaine, local anesthetic drug3 ,78 (link) also were used.
Each drug was dissolved in phosphate buffer solution (PBS, pH 7.35) to a stock concentration. The pH of working solution of each drug was adjusted with 1 M of NaHCO3 [8.4% (wt/vol)] to a final value of 7.35. Procaine, A-317491 and α,β-meATP were purchased from Sigma-Aldrich (St. Louis, MO). Naloxone was purchased from Tocris (Minneapolis, MN). The stock solution was prepared weekly and stored in a −20 °C freezer and the working solution was prepared daily.
Each drug was dissolved in phosphate buffer solution (PBS, pH 7.35) to a stock concentration. The pH of working solution of each drug was adjusted with 1 M of NaHCO3 [8.4% (wt/vol)] to a final value of 7.35. Procaine, A-317491 and α,β-meATP were purchased from Sigma-Aldrich (St. Louis, MO). Naloxone was purchased from Tocris (Minneapolis, MN). The stock solution was prepared weekly and stored in a −20 °C freezer and the working solution was prepared daily.
10
Chronic Visceral Hypersensitivity Rat Model
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The neonatal rats were randomly divided into normal group (NG) and model group (MG), rats in the NG did not receive any intervention, and the model rats were given CRD stimulation to establish the chronic visceral hypersensitivity rat model. After modeling, rats in the MG were randomly divided into MG, EA, P2X3 receptor antagonist (A-317491), and P2X3 receptor agonist (α, β-meATP) groups, 6 rats in each group. Rats in the NG and MG groups received the same fixation as the EA group. Rats in the EA group received EA intervention on bilateral ST25 and ST37 using the Han's meridian stimulator (2/100 Hz, 1 mA) for 30 min, and the depth of acupuncture was 5 mm, once a day for 7 days; rats in the A-317491 group received an intrathecal injection of A-317491 (10 μl, 10 μmol/μl, Sigma) [16 (link)] every three days (day 1, day 3, day 7); rats in the α, β-meATP group received an intrathecal injection of α, β-meATP (10 μl, 10 nmol/μl, Sigma) [17 (link)] every three days as the A-317491 group.
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