Non tritiated mpp

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).

Bulk chemicals were purchased from Sigma-Aldrich (Vienna, Austria). [³H] 1-methyl-4-phenylpyridinium (MPP⁺) was purchased from American Radiolabeled Chemicals (St. Louis, MO, United States) and non-tritiated MPP⁺ from Sigma-Aldrich (Vienna, Austria). Corticosterone, β-estradiol, and D22 were obtained from Sigma-Aldrich (Vienna, Austria), THP (Vienna, Austria) and Synthon Chemicals (Bitterfeld-Wolfen, Germany) respectively. Tetrodotoxin (TTX) was purchased from Latoxan (Valence, France).