Evidence that the atypical 5‐HT3 receptor ligand, [3H]‐BRL46470, labels additional 5‐HT3 binding sites compared to [3H]‐granisetron

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Abstract
1 The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen 2 In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 ± 0.18, 2.49 ± 0.30, 1.84 ± 0.27, 3.46 ± 0.36, respectively; mean ± s.e. mean, n = 3–4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg−1 protein): 102 ± 16, 44 ± 4, 968 ± 32 and 2055 ± 105, respectively; mean ± s.e.mean, n = 3–4) but failed to display specific binding in human putamen homogenates 3 In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 ± 0.61, 2.31 ± 0.44, 1.89 ± 0.36, 2.03 ± 0.42 and 6.46 ± 2.58 respectively; mean ± s.e.mean, n = 3–4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg−1 protein): 39 ± 4, 20±2, 521 ± 47, 870 ± 69 and 18 ± 2, respectively; mean±s.e.mean, n = 3–4) 4 Competition studies with a range of structurally different 5-HT3 receptor ligands indicated that in both rat cerebral cortex/hippocampus and rat ileum homogenates, [3H]-BRL46470 binding exhibited a pharmacological profile consistent with the labelling the 5-HT3 receptor with compounds competing with Hill coefficients close to unity 5 In HEK-5-HT3As cell homogenates, [3H]-BRL46470 and [3H]-granisetron associated rapidly ((3.84 ± 0.4)106 M−1s−1 and (5.85 ± 0.2)106 M−ls−l respectively, mean ± s.e.mean, n = 3–4) in an apparently monophasic manner. Following the establishment of equilibrium, both [3H]-BRL46470 and [3H]-granisetron at a saturating concentration ([3H]-BRL46470 approximately 16 nM; [3H]-granisetron approximately 18 nM) and at a sub-Kd concentration (approximately 1 nM for both radioligands) dissociated biphasically in HEK-5-HT3As cell homogenates (saturating concentration; [3H]-BRL46470 4.05 × 10−3±2.53 × 10−3 s−1 and 5.83 × 10−5 ± 0.91 × 10−5 s−1; [3H]-granisetron 3.20 × 10−3± 1.70 × 10−3 s−1 and 18.58 × 10−5±4.19 × 10−5 s−1: sub-Kd concentration; [3H]-BRL46470 2.47 × 10−3± 1.18 × 10−3 s−1 and 9.30 × 10−5 ± 2.59 × 10−5 s−1; [3H]-granisetron 65.91 × 10−3±22.14 × 10−3 s−1 and 49.96 × 10−5± 12.26 × 10−5 s−1, mean ± s.e.mean, n= 4–8) when induced by a 300 fold dilution in ice-cold Tris/Krebs 6 In conclusion, the present study provides evidence that [3H]-BRL46470 specifically labels the 5-HT3 receptor in rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, but fails to label the 5-HT3 receptor expressed in human putamen. Whilst the pharmacological profile of the site labelled by [3H]-BRL46470 is directly comparable to that labelled by [3H]-granisetron, [3H]-BRL46470 consistently labelled approximately twice the density of sites compared to [3H]-granisetron in the same tissue homogenates prepared from rat cortex/hippocampus, rat ileum, NG108-15 cells and HEK-5-HT3As cells.

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