3h granisetron

[³H]Tropisetron was synthesized (Metis Laboratories Inc, NY, USA) from its desmethyl analogue (0.5 mg) in a tritiomethylation reaction by heating at 80 °C for 2 h with 50 mCi [³H]methyl iodide in tetrahydrofuran (THF) (0.4 ml). After evaporation of the THF the residue was subjected to reversed phase chromatography (Kromasil 100 C18, 7 μm, 250 × 10 mm, 2 mL min⁻¹) using 31% ACN in 0.1% TFA as eluent. The eluent was stripped off and the residue was dissolved in 12 mL absolute ethanol. The [³H]tropisetron had a specific activity of 73.5 Ci/mmol and radiochemical purity of >99%. [³H]granisetron (63.5 Ci/mmol) and [³H]epibatidine (55.8 Ci/mmol) were purchased from Perkin Elmer (Waltham, MA) and were >97% chemical purity.

Transfected HEK 293 cells were washed twice with phosphate buffered saline (PBS) at room temperature, scraped into 1 ml of ice-cold HEPES buffer (10 mM, pH 7.4), homogenised and frozen. After thawing, they were washed with HEPES buffer, resuspended, and 50 μg of cell suspension incubated in 0.5 ml HEPES buffer and the relevant concentration of radioligand at 0 °C. Non-specific binding was determined using 2 mM quipazine. Equilibrium reactions were incubated for at least 3 h for [³H] granisetron (63.5 Ci/mmol, PerkinElmer, Boston, Massachusetts, USA) and 48 h for [³H]VUF10166. Incubations were terminated by vacuum filtration onto GF/B filters pre-soaked in 0.3% polyethyleneimine, followed by three rapid washes with 3.5 ml ice cold buffer. Radioactivity was determined by scintillation in Ecoscint A (National Diagnostics, Atlanta, Georgia) using a Beckman LS6000SC (Fullerton, California, USA). Each method was performed on at least three independent cell samples on at least three separate days.