BRL-54443

230, 31

Adult male cats

Distilled water containing 1 equivalent 2, 3-dihydroxybutanedioic acid+0.9% NaCl

BRL 54443 has also measurable affinity for the 5-HT2A receptor. BRL 54443 induces contraction with a logEC50 of 6.52. Contraction caused by BRL 54443 is likely mediated through stimulation of 5-HT5-HT2A receptors. [2]

Membrane preparation and binding assay, The binding affinity of a set of 5-HT receptor agonists and antagonists (samples) at 5-HT1F receptors is tested. To do this h5-HT1F-HEK 293 cells are cultured in Petri dishes till 90% confluency is reached. The cells are scraped from the plates and suspended in 50 mM Tris–HCl buffer (pH 7.4). After centrifugation (23, 500, g) the pellet is suspended in 5 mM Tris–HCl buffer (pH 7.4) and homogenized with an Ultra Turrax homogeniser. The membranes are collected by centrifugation at 18, 000, g for 20 minutes, resuspended in 50 mM Tris–HCl (pH 7.4) and stored at 80 C. At the day of the binding experiment, the membranes are defrosted and homogenized. The protein concentration is determined using the Bradford protein assay and the membranes are diluted in the assay buffer down to 65 ug protein/mL (50 mM Tris–HCl, 10 mM MgCl2, 1 mM EGTA and 10 uM pargyline, pH 7.4). Assay mixtures (0.5 mL) contained 0.4 mL membrane preparation and 2 nM of tritiated 5-HT (118 Ci/mmol), mine the non-specific binding 1 uM sumatriptan is added. Per sample studied 15 mixtures are created containing different concentrations (10 uM–1 pM) of the sample. The mixtures are then incubated for 60 minutes at 25 C (protected from light), incubation is stopped by rapid filtration on a Filtermate 96. Bound radioactivity is determined by liquid scintillation counting. Data are analysed graphically with inhibition curves and IC50 values are derived. Ki values are calculated according to the equation Ki =IC50/(1+(C/Kd)), with C as the concentration of [3H]5-HT (2 nM) and Kd as the equilibrium dissociation constant of [3H]5-HT (2.61 nM).

BRL 54443 is a 5-HT1E and 5-HT1F receptor agonist with pKi of 8.7 and 9.25, respectively, with a weak binding affinity for 5-HT1A, 5-HT1B, 5-HT1D receptors.