Pyridoxalphosphate 6 azophenyl 2 4 disulfonic acid ppads

Manufactured by Merck Group

Sourced in United States

Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) is a chemical compound used in laboratory research. It serves as a selective antagonist for P2X receptors, which are involved in various physiological processes. PPADS is utilized as a tool to study the role of these receptors in biological systems.

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5 protocols using pyridoxalphosphate 6 azophenyl 2 4 disulfonic acid ppads

Pharmacological Evaluation of Vascular Mediators

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Acetylcholine, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), indomethacin, MRS2159, N-[2-(Cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398), (E)-3-[4-(Imidazol-1-ylmethyl)phenyl]propenoic acid hydrochloride hydrate (ozagrel), phenylephrine, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole (SC560) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Up₄A was obtained from Biolog Life Science (Bremen, Germany). [1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-xabicyclo[2.2.1] hept-2-yl]-5-heptenoic acid (SQ29548) and thromboxane B2 enzyme immunoassay (EIK) kit were purchased from Cayman Chemical (Ann Arbor, MI, USA). indomethacin, DPCPX, NS398, SC560 and SQ29548 were firstly dissolved in DMSO. All subsequent dilutions (at least 1000 fold) and other drugs were obtained with distilled water. PPADS and MRS2159 were protected from light.

Zhou Z., Sun C., Tilley S.L, & Mustafa J. (2015). Mechanisms underlying uridine adenosine tetraphosphate-induced vascular contraction in mouse aorta: role of thromboxane and purinergic receptors. Vascular pharmacology, 73, 78-85.

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Extraction and Analysis of Bioactive Compounds from P. giganteus

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Nerve growth factor (NGF), U0126, PD98059, LY294002, suramin, pyridoxal phosphate-6-azophenyl-2'4'-disulfonic acid (PPADS) were purchased from Sigma (St. Louis, MO, USA). Ethanol and methanol were purchased from Merck (Germany). Linoleic acid, oleic acid, cinnamic acid, caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine were previously detected in the basidiocarps of P. giganteus using GCMS and LCMS/MS (Fig 1) [12 ]. All the compounds were purchased from Sigma (St. Louis, MO, USA).

Phan C.W., David P., Wong K.H., Naidu M, & Sabaratnam V. (2015). Uridine from Pleurotus giganteus and Its Neurite Outgrowth Stimulatory Effects with Underlying Mechanism. PLoS ONE, 10(11), e0143004.

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Cytokine and Peptide Preparations

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Recombinant mouse TNF and human TNF were produced in Escherichia coli and purified (>95%) in the VIB Protein Core (IRC, Ghent, Belgium). The biological activity of murine TNF (in vivo) and human TNF (in vitro) was 7.94 × 10⁹ IU/mg and 6.8 × 10⁷ IU/mg respectively, and was determined using an MTT [3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide] assay. Peptides (purity > 95%) were purchased from Pepnome (China) and Genosphere Biotechnologies (Paris, France). Endotoxin contamination was checked by a Limulus Amebocyte Lysate assay and was <1.0 EU/mg. Gap27 (SRPTEKTIFII), TAT-Gap19 (YGRKKRRQRRR-KQIEIKKFK), Gap19 (KQIEIKKFK), TAT-CT9 (YGRKKRRQRRR-RPRPDDLEI), CT9 (RPRPDDLEI) and TAT (YGRKKRRQRRR) were diluted in endotoxin-free phosphate-buffered saline (PBS) or pipette solution for in vivo or in vitro use respectively. BAPTA was purchased from Invitrogen (B1212). BAPTA-AM (Invitrogen, B6769) was dissolved in dimethylsulfoxide (DMSO) (+0.01% pluronic® (Invitrogen)) and further diluted in endotoxin-free PBS for in vivo use. Pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, Sigma-Aldrich), and KN-93 (Calbiochem) were dissolved in DMSO and further diluted in endotoxin-free PBS for in vivo use. Fluorescein isothiocyanate (FITC)-dextran (4kDa; Sigma-Aldrich) was used in vascular permeability studies.

Delvaeye T., De Smet M.A., Verwaerde S., Decrock E., Czekaj A., Vandenbroucke R.E., Lemeire K., Gonçalves A., Declercq W., Vandenabeele P., Krysko D.V, & Leybaert L. (2019). Blocking connexin43 hemichannels protects mice against tumour necrosis factor-induced inflammatory shock. Scientific Reports, 9, 16623.

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Krebs' Solution Preparation for Tissue Experiments

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During experiments, tissues were maintained in Krebs’ solution consisting of (mM): NaCl 118.4, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25 and glucose 11.7. ATP, tetraethylammonium chloride (TEA), tetrodotoxin, atropine sulfate monohydrate and carbachol were obtained from FUJIFILM-Wako (Osaka, Japan). D-tubocurarine, suramin, methoctramine hydrate, 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (PPADS), cibacron blue F3GA (CBF3GA; synonym reactive blue 2) and 4-aminopyridine (4-AP) were obtained from Sigma-Aldrich (St Louis, MO, USA). Glibenclamide and nicorandil were obtained from Tokyo Chemical Industry (Tokyo, Japan). GSK1016790A was obtained from Cayman Chemical (Ann Arbor, MI, USA). Apamin was obtained from Peptide Institute (Osaka, Japan). tetrodotoxin was dissolved in citrate solution. Glibenclamide, nicorandil and GSK1016790A were dissolved in DMSO. Other drugs were dissolved in distilled water. The highest concentration of vehicles (0.1%) for the drugs alone had no effect on the basal tone and contractile responses at the concentrations used. The concentrations of drugs given were final concentrations in the bath solution.

Shiina T., Suzuki Y., Horii K., Sawamura T., Yuki N., Horii Y, & Shimizu Y. (2024). Purinergic inhibitory regulation of esophageal smooth muscle is mediated by P2Y receptors and ATP-dependent potassium channels in rats. The Journal of Physiological Sciences : JPS, 74, 26.

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Selective Receptor Blockade in Neurotransmission

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The following neurotransmitter/neuropeptide receptor blockers were used: cholecystonkinin-A receptor antagonist devazepide ([2mg/kg] in 5% DMSO PBS; Sigma)^{14 ,55}; cocktail of the ionotropic glutamate receptor antagonist kynurenic acid (KA) ([150μg/kg] in PBS, stock made in 1M NaOH then diluted, pH 7.4; Sigma)^{14 ,56} and the metabotropic glutamate receptor antagonist DL-2-Amino-3-phosphonopropionic acid (AP-3) ([1mg/kg] in PBS, stock made in 1M NaOH diluted, pH 7.4; Sigma)^{14 ,57}; and non-selective P2-purinoreceptor antagonist pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid (PPADs) ([25mg/kg] in PBS; Sigma)^{33 (link),58 –60}. Following recording of a pre-inhibitor response, one inhibitor was delivered over one minute (devazepide and PPADS were delivered 10μL/g; KA/AP3 cocktail was delivered 20μL/g). Infusion of the selected sugar ligand was repeated for post-inhibitor recording after an incubation period: 5-8 minutes for devazepide and 3-5 minutes for KA/AP3 and PPADs.

Buchanan K.L., Rupprecht L.E., Kaelberer M.M., Sahasrabudhe A., Klein M.E., Villalobos J.A., Liu W.W., Yang A., Gelman J., Park S., Anikeeva P, & Bohórquez D.V. (2022). The preference for sugar over sweetener depends on a gut sensor cell. Nature Neuroscience, 25(2), 191-200.

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