2.3.2. Plasmalemmal [3H]DA uptake.
In order to obtain a first evidence of the direct blockade (competitive inhibition) of [3H]DA uptake in the presence of different α-aminovalerophenone derivatives, synaptosomes from rat striatum were prepared as described above. Competitive blockade of [3H]DA uptake was performed as described by (López-Arnau et al., 2012), with some modifications. Briefly, reaction tubes were composed of 0.125 mL of pentedrone, N-ethyl-pentedrone, N,N-diethyl-pentedrone, α-PVP or α-PpVP at different concentrations in HEPES-buffered solution containing pargyline and ascorbic acid. Moreover, 0.025 mL of [3H]DA was also added (final concentration 5 nM). The reaction tubes as well as the synaptosomal suspension were warmed for 10 min at 37°C before the addition of 0.1 mL of synaptosome suspension, after which incubation was carried out for a further 5 min. Then, the uptake reaction was terminated by rapid vacuum filtration through Whatman GF/B glass fibre filters (Whatman Intl Ltd., Maidstone, UK) presoaked in 0.5% polyethyleneimine. Tubes and filters were washed rapidly three times with 4 mL ice-cold 50 mM Tris– HCl. The radioactivity trapped on the filters was measured by liquid scintillation spectrometry. Non-specific uptake was determined at 4°C in parallel samples containing cocaine (300 µM). All determinations were performed per duplicate. All experiments were performed three times (N=3). No [3H]5-HT uptake assays in rat synaptosomes were performed due to the results obtained in inhibition assays in HEK293 cells thus reducing the number of rats used.