Dopamine and serotonin transporter measurements

Chinese hamster ovary (CHO) cells were purchased from American Type Culture Collection. CHO cells were cultured in Ham’s F-12 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Amarillo TX, USA), 1mM L-glutamine, 100U/mL penicillin and 100μg/mL streptomycin (Sigma-Aldrich). The human DAT and SERT cDNAs were cloned into the pEYFP-C1 vector and used to transfect CHO cells with Lipofectamine 2000 (Invitrogen, Waltham MA, USA). For stable transfections, DAT- and SERT-expressing single clones (CHO-DAT and CHO-SERT cells, respectively) were selected with G418 (Gibco), verified by DAT and SERT immunoblot, and maintained in Ham’s F-12 media containing 0.5mg/mL G418. DAT and SERT functions were analyzed via DA and 5-HT uptake experiments, which were conducted 24–48 h after seeding cells in 24-well plates coated with Poly-D-Lysine (Sigma-Aldrich). For the treatments, cells were incubated with different drug conditions for 10 min at 37°C. Conditions are described as follows: Areca extract ≤ 1 kDa fraction (supari) tested at 6 mg/ml dry weight, Areca anion-exchange fraction tested at 0.72 mg/ml dry weight, Areca cation-exchange fraction tested at 0.72 mg/ml dry weight, 10 μM {"type":"entrez-protein","attrs":{"text":"GBR12935","term_id":"2281712181","term_text":"GBR12935"}}GBR12935 (DAT blocker, only for CHO-DAT cells, Sigma-Aldrich), and 0.5 μM Fluoxetine (SERT blocker, only for CHO-SERT cells, Sigma Aldrich).

[3H]-DA or [3H]-5HT uptake measurements in CHO cells transfected with human DAT or SERT were performed as previously described (14). Briefly, cells seeded in 24-well plates at 80% confluency were pre-incubated for 5 min at 37°C with 200 μl of uptake buffer (4 mM Tris base, 6.25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, 1.25 mM MgSO4, 0.57 mM ascorbic acid, 5.6 mM glucose, 1 mM tropolone, pH 7.4) containing the tested areca fractions. Uptake assays were conducted in the presence of 60 nM of [³H]-DA (3,4-[7–3H] dihydroxyphenylethylamine, Perkin Elmer, Waltham, MA, USA) or 50 nM of [³H]-5HT (5-Hydroxytryptamine Creatinine Sulfate, Perkin Elmer) at 37°C for 5 min. After loading with [³H]-DA or [³H]-5HT, cells were washed with 1 ml ice-cold uptake buffer. Samples were solubilized in 0.4 ml of 1% SDS and incubated at room temperature for 1 h. Samples were then transferred to scintillation vials and filled with 4 ml scintillation counting fluid (RPI Bio-safe IITM). Counts per min (cpm) were obtained using a LS-6500 liquid scintillation counter (Beckman Coulter, Brea, CA, USA). Experiments were conducted three times with four replicates in each experiment (total n = 12). The values of [³H]-DA or [³H]-5HTwere analyzed using one-way ANOVA with Bonferroni post-hoc test. Minimal significance was set at p < 0.05.