4.3. Monoamine Efflux Mediated by the Transporters

Monoamine efflux was assessed using human embryonic kidney 293 (HEK293) cells (Invitrogen, Zug, Switzerland) stably transfected with the human norepinephrine (hNET), dopamine (hDAT), or serotonin (hSERT) uptake transporters. In summary, the cells were seeded in poly-D-lysine coated XF24 cell culture microplates (Seahorse Biosciences, North Billerica, MA, USA) and cultured overnight at a concentration of 100,000 cells per well.

The cells were then exposed to Krebs–HEPES release buffer (85 μL/well) comprised of 130 mM NaCl, 1.3 mM KCl, 2.2 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 10 mM HEPES, and 10 mM D-glucose at a pH of 7.5, which also contained 10 nM radiolabelled neurotransmitter ([³H]-5-HT for SERT, [³H]-DA for DAT or [³H]-NE for NET), 1 μM unlabelled neurotransmitter (DA or NE, only), 10 μM pargyline, and 0.2 mg/mL of ascorbic acid. This enabled the loading of the cells with their respective neurotransmitters via the uptake transporters. Afterwards, the release buffer was replaced by fresh buffer to wash the cells twice and the cells were then incubated for 15 min (DAT and SERT) or 45 min (NET) to 100 μM of test drugs dissolved in 1 mL of Krebs–HEPES buffer while shaking at 300 rpm and 37 °C. Termination of the release reaction occurred by removing the buffer from the cells and washing them with ice-cold buffer. Next, 50 μL of lysis buffer was added to the cells for 1 h in order to lyse the cells. Thereafter, 40 μL of the lysed cell mixture was transferred into scintillation vials (Perkin–Elmer, Schwerzenbach, Switzerland) containing 3 mL of scintillation fluid (Ultimagold, Perkin–Elmer, Schwerzenbach, Switzerland) and measured using liquid scintillation (as previously described in Section 2.2).

To quantify the “pseudo efflux” caused by the non-transporter mediated monoamine release and successive reuptake inhibition [58], each monoamine efflux experiment included a control where the cells were exposed to each respective transporter blocker (nisoxetine for NET, mazindol for DAT, and citalopram for SERT). The radioactivity inside the cells without any drug was set as 100%. The nonspecific release was subtracted from the total observed release at 100 μM of each examined drug to calculate the specific transporter mediated release. One single concentration of the test drugs was used (100 μM) and release exposure durations were set based on previously evaluated kinetics of the release-over-time curves [56].

A substance which produced significantly (p-value < 0.05) higher monoamine efflux compared to the efflux observed in the presence of a monoamine transporter inhibitor, was identified as a monoamine releaser. An ANOVA followed by a Holm–Sidak test was conducted to compare each drug’s specific transporter-mediated efflux based on at least three independent experiments to the control condition.