We used (±)-norepinephrine (+)-bitartrate salt (Sigma-Aldrich, St Louis, MO), isoproterenol hydrochloride (Millipore, Billerica, MA), propranolol hydrochloride (Wako, Osaka, Japan), oxymetazoline hydrochloride (Wako), p38 inhibitor, SB203580 (Wako), MEK/ERK inhibitor, PD98059 (Millipore), STAT3 inhibitor, S31-201 (Sigma-Aldrich), recombinant human TGFβ1 (rTGFβ) (R&D systems, Minneapolis, MN), ET-1 (Sigma-Aldrich) and soluble IL-6 receptor (sIL-6R) (Peprotech, Rocky Hill, NJ).
Norepinephrine bitartrate salt
Manufactured by Merck Group
Sourced in Germany
(±)-norepinephrine (+)-bitartrate salt is a chemical compound used in research and laboratory settings. It is a salt of the racemic mixture of the neurotransmitter norepinephrine and the organic acid bitartaric acid. This product is utilized by researchers and scientists for various experimental and analytical purposes.
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Lab products found in correlation
Propranolol hydrochloride, by Fujifilm (1 mentions)
S31 201, by Merck Group (1 mentions)
Pd98059, by Merck Group (1 mentions)
Sil 6r, by Thermo Fisher Scientific (1 mentions)
Sb203580, by Fujifilm (1 mentions)
Sodium sulfite, by Merck Group (1 mentions)
Sulfonamide, by Merck Group (1 mentions)
Acetylthiocholine iodide, by Merck Group (1 mentions)
N 1 naphthyl ethylenediamine dihydrochloride, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Thiobarbituric acid, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Phosphoric acid, by Merck Group (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (1 mentions)
Perchloric acid, by Merck Group (1 mentions)
Isoproterenol hydrochloride, by Merck Group (1 mentions)
Propranolol hydrochloride, by Merck Group (1 mentions)
2 2 dithiodipyridine dtdp, by Merck Group (1 mentions)
7 protocols using norepinephrine bitartrate salt
1
Norepinephrine Signaling Pathway Modulation
2
Reserpine and Cerebrolysin Neuroprotective Protocol
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Reserpine (Mallinckrodlt. Inc, Paris-Kantucky) was dissolved in glacial acetic acid (1 µg/µl) and completed with distilled water to reach 25 ml. Cerebrolysin was purchased from EVER Neuropharma GmbH A-4866 Unterach, Austria. Lithium chloride was obtained from BDH (England). Ethylene diaminetetraacetic acid (EDTA), thiobarbituric acid (TBA), trichloroacetic acid (TCA), acetylthiocholine iodide, 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), and 96% ethanol, N-(1-Naphthyl) ethylenediaminedihydrochloride (NED), phosphoric acid, sulfonamide, and perchloric acid were supplied by Sigma Aldrich. Norepinephrine bitartrate salt (Sigma, Taufkirchen – Germany), dopamine hydrochloride and serotonin (Fluka – Sigma – Aldrich, Taufkirchen – Germany) were used to prepare the standards. n-heptane (Labscan Ltd., Dublin, Ireland), 1-butanol (POCH SA, Gliwice, Poland), hydrochloric acid, acetic acid, ethyl alcohol (EDWIC, El-Nasr Pharmaceutical Chemicals Co., Egypt), sodium acetate (Fluka, Buchs, Switzerland), Iodine (Panreac, Barcelona Spain), ethylenediaminetetraacetic acid (EDTA) (S.D. fine – Chem Ltd. Mumbai, India), sodium sulfite and O-phthalaldehyde (OPT) (Merck, Schuchardt, Germany) were used for the quantitative determination of monoamines in the striatum and midbrain.
3
Skin Secretion Extraction from Frogs
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To promote release of skin secretions, frogs were stimulated by injecting 40 nmol/grams body weight norepinephrine bitartrate salt (Sigma-Aldrich, St. Louis, MO, USA) dissolved in sterile 200 µL water at two sites into dorsal lymph sacs. Skin secretions were collected by placing the stimulated frog in a covered glass beaker with 40 mL of collection buffer, 25 mM NaCl and 25 mM ammonium acetate, pH 7.0, [43 ,44 (link)] for 15 min. After removing the frog, collected skin secretions were acidified with hydrochloric acid (1% v/v) and centrifuged 30 min at 5000× g. Supernatants were immediately desalted and concentrated using Sep-Pak C-18 cartridges (Waters Associates, Milford, MA, USA) as previously described [18 (link)]. Concentrated and desalted protein solution was named the skin secretions extract (SSE). Protein concentration was determined using Microplate BCATM Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). After norepinephrine stimulation and recovery of SSE, none of the frogs tested showed any distress related to this process. This protocol was reviewed and approved by the Institutional Committee for the Use and Care of Lab Animals of Tecnologico de Monterrey (Comité Institucional para el Cuidado y Uso de Animales de Laboratorio (CICUAL) del Tecnologico de Monterrey) under the protocol number 2015-007 (8 May 2015).
4
Pharmacological Agents for Cell Signaling Research
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We obtained (−)-isoproterenol hydrochloride (ISO, I6504), (±)-norepinephrine (+)-bitartrate salt (NE, A0937), 2,2′-dithiodipyridine (DTDP, D5767), prazosin hydrochloride (P7791), (±)-propranolol hydrochloride (P0884), methoxamine hydrochloride (M6524) and streptozotocin (STZ, S0130) from Sigma Aldrich (Darmstadt, Germany). Fluo4-AM (F311) and N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 343-05401) were purchased from Dojindo (Kumamoto, Japan). FluoZin™−3 AM (F24195) was from Thermo Fisher (Hampton, New Hampshire, USA). We purchased 1-hydrazinophthalazine hydrochloride (hydralazine hydrochloride, H0409) from Tokyo Chemical Industry (Tokyo, Japan). The antibodies used in this study are listed in Supplementary Table 9 .
5
Pharmacological Modulation of Zebrafish Cardiac Function
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Tg(kdrl:GFP)s843;Tg(gata1:DsRED)sd2 embryos at 96 hpf were arrayed in 96-well plates as previously described and maintained in E3 medium until drug treatment. The cardiac rate of each individual embryo was recorded immediately before the addition of each drug. Tricaine (MS-222, A5040, Sigma) or 2,3-BDM (2,3-butanedione monoxime, 31550, Sigma) was added in E3 medium at final concentrations of 500 μM and 20 μM, respectively. DL-Isoproterenol (isoprenaline hydrochloride, I5627, Sigma) and norepinephrine ((±)-Norepinephrine (+)-bitartrate salt, A0937, Sigma) were used at 50 μM and 1 mM, respectively.
For the analysis of the effects of pharmacological treatments on cardiac rate, embryos were treated with the following drugs: NS-398 (N194 Sigma) at 30 μM, L-NAME (L-NG-Nitroarginine methyl ester, Sigma) at 500 μM, SNAP (S-Nitroso-N-Acetyl-D,L-Penicillamine) at 100 μM or vehicle alone from 48 to 72 hpf. For PDE inhibition, 100 μM IBMX (I5879, Sigma), 100 μM caffeine (C0750, Sigma), 100 μM Ro-201724 (4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone, 557502, Calbiochem), 50 μM Cilostamide (N-Cyclohexyl-N-methyl-4-(1,2-dihydro-2-oxo-6-quinolyloxy)butyramide (0915, Tocris) or vehicle was added from 48 to 72 hpf. After 24 hours of treatment, embryos were placed in 96-well plates for imaging and re-incubated with fresh drugs/chemicals for the duration of the experiments.
For the analysis of the effects of pharmacological treatments on cardiac rate, embryos were treated with the following drugs: NS-398 (N194 Sigma) at 30 μM, L-NAME (L-NG-Nitroarginine methyl ester, Sigma) at 500 μM, SNAP (S-Nitroso-N-Acetyl-D,L-Penicillamine) at 100 μM or vehicle alone from 48 to 72 hpf. For PDE inhibition, 100 μM IBMX (I5879, Sigma), 100 μM caffeine (C0750, Sigma), 100 μM Ro-201724 (4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone, 557502, Calbiochem), 50 μM Cilostamide (N-Cyclohexyl-N-methyl-4-(1,2-dihydro-2-oxo-6-quinolyloxy)butyramide (0915, Tocris) or vehicle was added from 48 to 72 hpf. After 24 hours of treatment, embryos were placed in 96-well plates for imaging and re-incubated with fresh drugs/chemicals for the duration of the experiments.
6
Pharmacological Modulation of Neuronal Activity
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Angiotensin II (Ang II; Tocris Cat# 1158), Ang-(1–7) (Tocris Cat# 1562), AT2R-Agonist CGP42112 (Tocris Cat# 2569), Losartan potassium (Tocris Cat# 3798) and (+-)-Norepinephrine (+)-bitartrate salt (Sigma Cat# A0937, Cas # 3414-63-9) were dissolved in sterile H2O. Ang II and CGP 42112 were used at a final working concentration of 500 nM, Ang-(1–7) at 10 nM, losartan at 5 μM and norepinephrine at 10 μM.
Regarding all pharmacological LFP experiments with Ang II, Ang-(1–7), AT2R-Agonist and losartan, rhythmic activity was recorded for 15 min under control condition. Treatment was applied for 20 min followed by a 40 min washout (if performed), with one exception. In one experiment losartan was applied for 10 min after which Ang II was co-applied for another 20 min. Regarding the calcium imaging experiments, calcium activity was recorded under control condition for 30–60 s after which Ang II was applied and recorded for another ∼7 min. Immediately after this, a new recording was started in which the calcium activity under the previously applied Ang II was recorded again for 30–60 s, then norepinephrine was co-applied.
Regarding all pharmacological LFP experiments with Ang II, Ang-(1–7), AT2R-Agonist and losartan, rhythmic activity was recorded for 15 min under control condition. Treatment was applied for 20 min followed by a 40 min washout (if performed), with one exception. In one experiment losartan was applied for 10 min after which Ang II was co-applied for another 20 min. Regarding the calcium imaging experiments, calcium activity was recorded under control condition for 30–60 s after which Ang II was applied and recorded for another ∼7 min. Immediately after this, a new recording was started in which the calcium activity under the previously applied Ang II was recorded again for 30–60 s, then norepinephrine was co-applied.
7
Synthesis and Evaluation of Novel Quinoxaline Derivatives
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Thirty-three new test compounds were synthesized. Derivatives of 2-phenyl-5,6,7,8-tetrahydroquinoxaline with different functional groups on the phenyl ring were synthesized. See Table 1 for detailed information on all test compounds which were synthesized and tested.
100-mM stock solutions of the test compounds were made up in 100% dimethyl sulfoxide (DMSO). Serial dilutions were performed on the day of experimentation using MilliQ water and DMSO as required to reach the desired concentration. The volumes of MilliQ water and DMSO used were dependent on the solubility of each individual test compound, and vehicle solutions were made up containing the same ratio of the two solvents as used to dissolve and dilute each test compound.
αß-Methylene ATP, prazosin, and acetylcholine were dissolved and diluted in MilliQ water, while noradrenaline was dissolved and diluted in a catecholamine diluent (NaCl, 154 mM; NaH2PO4·H2O, 1.2 mM; ascorbic acid, 0.2 mM).
Acetylcholine chloride, dimethyl sulfoxide (DMSO), αß-methyleneadenosine 5′-triphosphate lithium salt, norepinephrine bitartrate salt, and prazosin hydrochloride were purchased from Sigma-Aldrich.
100-mM stock solutions of the test compounds were made up in 100% dimethyl sulfoxide (DMSO). Serial dilutions were performed on the day of experimentation using MilliQ water and DMSO as required to reach the desired concentration. The volumes of MilliQ water and DMSO used were dependent on the solubility of each individual test compound, and vehicle solutions were made up containing the same ratio of the two solvents as used to dissolve and dilute each test compound.
αß-Methylene ATP, prazosin, and acetylcholine were dissolved and diluted in MilliQ water, while noradrenaline was dissolved and diluted in a catecholamine diluent (NaCl, 154 mM; NaH2PO4·H2O, 1.2 mM; ascorbic acid, 0.2 mM).
Acetylcholine chloride, dimethyl sulfoxide (DMSO), αß-methyleneadenosine 5′-triphosphate lithium salt, norepinephrine bitartrate salt, and prazosin hydrochloride were purchased from Sigma-Aldrich.
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