2‐Methylamino‐1‐(p‐tolyl)propan‐1‐one hydrochloride (mephedrone, MW: 213.70), 2‐amino‐1‐(p‐tolyl)propan‐1‐one hydrochloride (nor‐mephedrone, MW: 199.68) and 1‐(4‐(hydroxymethyl)phenyl)‐2‐(methylamino)propan‐1‐one hydrochloride (4‐OH‐mephedrone, MW: 229.70) were synthesized as racemic mixtures. In the case of 2‐(methylamino)‐1‐(p‐tolyl)propan‐1‐ol hydrochloride (dihydromephedrone, MW: 215.72), all four stereoisomers [syn‐(1R,2R), syn‐(1S,2S), anti‐(1R,2S) and anti‐(1S,2R)] were synthesized in their enantiopure form (99%) and tested as 1:1:1:1 mixture. Synthetic procedures and chemical characterization data are given in detail in the Supporting Information . Reagents used in the experiments for uptake inhibition and release in HEK293 cells were used as mentioned in Hofmaier et al., 2014 . Plasmids encoding human SERT were a generous gift of Dr Randy D. Blakely. For uptake and release experiments in HEK293‐cells and rat brain synaptosomes, [3H]‐1‐methyl‐4‐phenylpyridinium ([3H]‐MPP+; 80–85 μCi mmol−1) and [3H]‐5‐HT (28.3 μCi mmol−1) were purchased from American Radiolabeled Chemicals (St. Louis, MO, USA) and Perkin Elmer (Boston, MA, USA) respectively. All other chemicals and cell culture supplies were from Sigma‐Aldrich (St. Louis, MO, USA) with the exception of cell culture dishes, which were obtained from Sarstedt (Nuembrecht, Germany).
3h mpp
Manufactured by American Radiolabeled Chemicals
Sourced in United States
3H-MPP is a radiolabeled compound containing the tritium isotope of hydrogen. It is used as a research tool to study relevant biological processes in experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Cell culture dishes, by Sarstedt (2 mentions)
14c metformin, by American Radiolabeled Chemicals (2 mentions)
3h 1 methyl 4 phenylpyridinium, by American Radiolabeled Chemicals (1 mentions)
3h 5 ht, by PerkinElmer (1 mentions)
Cell culture supplies, by Merck Group (1 mentions)
Beta scintillation counter, by PerkinElmer (1 mentions)
Non tritiated mpp, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mycoplasma detection kit, by Minerva Biolabs (1 mentions)
8 protocols using 3h mpp
1
Synthesis and Characterization of Cathinone Derivatives
2
Radiolabeled Transporter Assay Protocol
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3H-MPP (1-Methyl-4-phenylpyridinium iodide) and 14C-metformin were purchased from American Radiolabeled Chemicals Saint louis; Missouri, USA. All non labelled chemicals were obtained from Sigma-Aldrich, Darmstadt, Germany. For transfection, the following cDNAs were used: hOCT1 (GeneBank: accession number: NM_003057.2), mOct1 (NM_009202.5), rOct1 (NM_012697.1), hMATE1 (NM_018242.2), and hOCT2 (NM_003058.3). The hOCT1 cloned has the genotype Ser14, Arg61, Cys88, Phe160, Gly401,Met408, Met420 and Gly465, which corresponds to the OCT1*1B allele according to the nomenclature suggested by Seitz et al. [29 (link)].
3
Synthesis and Characterization of Fluorinated Morpholines
4
Radiotracer Uptake Assay for MPP+ in Cells
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On the day of the experiment, cells were incubated with 50 µL Krebs-HEPES-buffer (KHB; 10 mM HEPES, 120 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgSO4 and 20 mM D-glucose, pH adjusted to 7.3) containing increasing concentrations of 1-methyl-4-phenylpyridinium (MPP+; Sigma-Aldrich, St. Louis, MO, USA) together with 50 nM [3H]-MPP+ (80–85 μCi mmol−1; American Radiolabeled Chemicals, St. Louis, USA) for 10 min. Time-dependent uptake was determined using 50 nM [3H]-MPP+ for the time indicated. Unspecific uptake was determined in the presence of 100 µM decynium-22. Cells were washed with KHB and then lysed with 100 µL 1% sodium dodecyl sulfate (SDS). Lysate was transferred to a counting vial, containing 2 mL of scintillation cocktail. Uptake of tritiated substrate was assessed with a beta scintillation counter (Perkin Elmer, Waltham, USA).
5
Radioligand Uptake Assays for Transporter Characterization
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14C-metformin (115 Ci/mmol) was purchased from Moravek, and 3H-MPP+ (80 Ci/mmol) was purchased from American Radiolabeled Chemicals. Uptake assays were performed similarly to a previous report58 . Injections of 30 ng cRNA were performed, with 2–4 day expression at 17°C. Specific radioactivities of 0.06 and 5 Ci/mmol were used for 3H-MPP+ and 14C-metformin for Kt measurements shown in Fig. 1a and Fig. 1d , respectively. Full specific radioactivities were used for mutant uptake assessments in Fig. 1f , Fig. 2c , Fig. 2e , Fig. 4d . For IC50 experiments, specific radioactivities of 80 Ci/mmol were used for all constructs, except for OCT1CS for which a specific radioactivity of 8 Ci/mmol was used. Water injected oocytes were used for background correction.
6
Kinetic Characterization of Metformin and MPP+ Uptake
7
MPP+ Uptake Kinetics in Cultured Cells
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Prior to the experiment, cultures were washed with 800 µL buffer consisting of (in mM): NaCl (140), glucose (20), HEPES (10), CaCl2 (2.5), MgCl2(2), KOH (3), NaOH (2). Cells were then preincubated in buffer containing either β-estradiol (E2), corticosterone, decynium-22 (D22), cocaine or desipramine for 10 min. To compensate for effects of solvent, all solutions contained 0.25% DMSO (Sigma-Aldrich, Vienna, Austria). Thereafter, preincubation solutions were removed and uptake was initiated by addition of 100 µl of buffer containing 50 nM [3H]MPP+ (specific activity: 80 Ci·mmol−1, American Radiolabeled Chemicals, St. Louis, MO, United States) plus 10 µM non-tritiated MPP+ (Sigma-Aldrich, Vienna, Austria). After 5 min, uptake was terminated by the addition of 800 µl of ice cold buffer to each well and putting the plates on ice. Cells were washed with 800 µl of ice cold buffer once and lysed by addition of 1% sodium dodecyl sulfate. Radioactivity contained in these samples was assessed by liquid scintillation counting (Packard Tri-Carb 2800 TR, PerkinElmer, Brunn am Gebirge, Austria).
8
Radiometric Assay for Organic Cation Transporters
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14C-metformin (1,1-Dimethylbiguanide hydrochloride) and 3H-MPP (1-Methyl-4-phenylpyridinium iodide) were purchased from American Radiolabeled Chemicals, St. Louis, MO, USA. All non labelled chemicals were obtained from Sigma-Aldrich, Darmstadt, Germany.
With cDNA of hOCT2 (GeneBank: accession number: NM_003058.3) or mOct2 (NM_013667.2), stably overexpressing HEK293 cells were used for the experiments and empty vector-transfected HEK cells were used as control-HEK cells.
All HEK-293 cell lines (hOCT2-, mOct2 and the vector-HEK-cells) were routinely tested for mycoplasma using Mycoplasma Detection Kit (Venor®GeM, Minerva biolabs, Berlin, Germany) for conventional PCR and used if free from mycoplasma.
With cDNA of hOCT2 (GeneBank: accession number: NM_003058.3) or mOct2 (NM_013667.2), stably overexpressing HEK293 cells were used for the experiments and empty vector-transfected HEK cells were used as control-HEK cells.
All HEK-293 cell lines (hOCT2-, mOct2 and the vector-HEK-cells) were routinely tested for mycoplasma using Mycoplasma Detection Kit (Venor®GeM, Minerva biolabs, Berlin, Germany) for conventional PCR and used if free from mycoplasma.
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