4.2. Monoamine Uptake Transporter Inhibition

The monoamine uptake transporter inhibition was examined in accordance with previously described methods by [56,57] for each substance of interest.

Human embryonic kidney 293 (HEK293) cells (Invitrogen, Zug, Switzerland) stably transfected with the human norepinephrine (hNET), dopamine (hDAT), or serotonin (hSERT) uptake transporters were briefly cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, Zug, Switzerland) containing 10% fetal bovine serum and 250 μg/mL geneticin (Gibco, Life Technologies, Zug, Switzerland). Next, the cells were detached at a confluency of 70−90% and resuspended in Krebs–Ringer Bicarbonate Buffer (Sigma-Aldrich, Buchs, Switzerland) at a concentration of 3 × 10⁶ cells per ml. The uptake buffer was additionally supplemented with 0.2 mg/mL of ascorbic acid (Sigma–Aldrich, Buchs, Switzerland) for the [³H]-DA uptake experiments.

In summary, using round bottom 96-well plates, 100 μL of cell suspension was incubated in 25 μL of buffer containing test substances, vehicle control (0.7% dimethyl sulfoxide, DMSO), or 10 μM of the respective monoamine uptake inhibitor, mainly fluoxetine (SERT), nisoxetine (NET), or mazindol (DAT) for 10 min at room temperature while on a rotary shaker at 450 rpm (Thermomixer Comfort, Eppendorf, Hamburg, Germany). Thereafter, the monoamine uptake transporter was initiated by the addition of 50 μL of each respective radiolabelled neurotransmitter ([³H]-5-HT, [³H]-DA or [³H]-NE) dissolved in the uptake buffer to a final concentration of 5 nM for an additional 10 min. Then, 100 μL of cell suspension mixture was transferred to microcentrifuge tubes containing 50 μL of 3 M potassium hydroxide (KOH, Sigma–Aldrich, Buchs, Switzerland) and 200 μL silicon oil (1:1 mixture of silicon oil type AR20 and AR200; Sigma–Aldrich, Buchs, Switzerland). Immediately after, the tubes were centrifugated (3 min, 13200 rpm) to terminate the uptake reaction by allowing the cells to move through the silicon oil into the KOH, which then lysed the cells. Quickly after, the tubes were immediately frozen with liquid nitrogen. Afterwards, the frozen cell pellet was cut off into 6 mL scintillation vials (Perkin–Elmer, Schwerzenbach, Switzerland) filled with 500 μL of lysis buffer (5 mM EDTA, 0.05 M TRIS-HCl, 50 mM NaCl and 1% NP-40 in water). Directly after the vials were shaken for 1 h at 700 rpm and then each vial was filled with 3 mL of scintillation fluid (Ultimagold, Perkin–Elmer, Schwerzenbach, Switzerland). The uptake of the monoamines was measured using a liquid scintillation counter (Packard Tri-Carb Liquid Scintillation Counter 1900 TR). Specific monoamine uptake was examined by subtracting the nonspecific uptake in the presence of selective inhibitor from the total counts measured.

The data were analysed using Prism software (version 8, GraphPad, San Diego, CA, USA) and was fitted by a nonlinear regression to variable-slope sigmoidal dose-response curve. The IC₅₀ values were extracted in order to determine each drug’s inhibition potency at the various monoamine transporters. Furthermore, the DAT/SERT ratio expressed as 1/DAT IC₅₀: 1/SERT IC₅₀ was calculated to assess whether a substance exhibited stronger serotonergic effects (ratio < 1, more entactogenic and similar to MDMA) or stronger dopaminergic effects (ratio > 1, more psychostimulant and similar to cocaine).