2.4.2. Uptake inhibition assays
Before starting the uptake inhibition experiments, the media was removed
from the cell culture 96-well plates and replaced with 200 µl per well
of Krebs-HEPES-Buffer (KHB; 10 mM HEPES, 120 mM NaCl, 3 mM KCl, 2 mM
CaCl2 · 2 H2O, 2mM MgCl2· 6 H2O supplemented with 20 mM D-glucose; pH 7.3).
Afterwards, cells were incubated with different concentrations of the
test drugs diluted in KHB at a final volume of 50 µl per well for some
time to ensure equilibrated conditions (preincubation). The
preincubation time was 5 min in case of the uptake-1 inhibition assays
(hDAT and hSERT) and 10 min for the uptake-2 assays (OCT-2, OCT-3). On
preincubation, the tritiated substrates were added: 0,02 µM
[3H]MPP+ for hDAT, 0,1 µM
[3H]5-HT for hSERT and 0,05 µM
[3H]MPP+ for hOCT-2 and hOCT-3. The uptake
incubation times were 3 min for hDAT, 1 min for hSERT and 10 min for
uptake-2 experiments. The uptake was terminated by removing the
tritiated substrate, washing the cells with ice-cold KHB and lysing them
with sodium dodecyl sulfate (SDS) 1%. The lysate was added to
scintillation fluid and the released radioactivity was quantified with a
beta-scintillation counter (Perkin Elmer, Waltham, MA, USA).
Non-specific uptake was determined in parallel samples containing
cocaine 100 µM for hDAT, paroxetine 30 µM for hSERT and decynium-22
(D22) 100 µM for hOCT-2 andhOCT-3.
The non-specific uptake value was
<10% of total uptake and was subtracted from the data to
yield specific uptake. The uptake in absence of the test compounds was
normalized to 100% and uptake in the presence of different
concentrations of drugs was expressed as a percentage thereof. All
determinations were performed per triplicate. All experiments were
performed three times (N=3).