Neurotransmitter Uptake Inhibition Assay

[³H]Dopamine, [³H]norepinephrine, and [³H]5-HT (specific activity ranging from 30–50 Ci/mmol) were purchased from Perkin Elmer (Shelton, CT, USA). All other chemicals and reagents were acquired from Sigma-Aldrich (St. Louis, MO, USA). Rats were euthanized by CO₂ inhalation, and brains were processed to yield synaptosomes as previously described [25 (link)]. Rat caudate tissue was used for DAT assays, whereas rat whole brain minus caudate and cerebellum was used for NET and SERT assays. Transport activity at DAT, NET, and SERT was assessed using 5 nM [³H]dopamine, 10 nM [³H]norepinephrine, and 5 nM [³H]5-HT, respectively. The selectivity of uptake assays was optimized for a single transporter by including unlabeled blockers to prevent uptake of [³H]transmitter by competing transporters. Uptake inhibition assays were initiated by adding 100 µL of tissue suspension to 900 µL Krebs-phosphate buffer (126 mM NaCl, 2.4 mM KCl, 0.83 mM CaCl₂, 0.8 mM MgCl₂, 0.5 mM KH₂PO₄, 0.5 mM Na₂SO₄, 11.1 mM glucose, 0.05 mM pargyline, 1 mg mL⁻¹ bovine serum albumin, and 1 mg mL⁻¹ ascorbic acid, pH 7.4) containing test peptide and [³H]transmitter. Assays were terminated by rapid vacuum filtration through Whatman GF/B filters, and retained radioactivity was quantified by liquid scintillation counting. Statistical analyses were carried out using GraphPad Prism 6.0 (GraphPad Scientific, San Diego, CA, USA). IC₅₀ values for uptake inhibition were calculated based on non-linear regression analysis.