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Evidence that the atypical 5‐HT 3 receptor ligand, [ 3 H]‐BRL46470, labels additional 5‐HT 3 binding sites compared to [ 3 H]‐granisetron
Author(s) -
Steward Lucinda J.,
Ge Jian,
Bentley Kim R.,
Barber Peter C.,
Hope Anthony G.,
Lambert Jeremy J.,
Peters John A.,
Blackburn Thomas P.
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb16663.x
Subject(s) - radioligand , population , putamen , receptor , medicine , endocrinology , hippocampus , ligand (biochemistry) , biology , microbiology and biotechnology , chemistry , environmental health
1 The radioligand binding characteristics of the 3 H‐derivative of the novel 5‐HT 3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5‐HT 3 receptor radioligand [ 3 H]‐granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108‐15 cells, HEK‐5‐HT 3 As cells and human putamen 2 In rat cerebral cortex/hippocampus, rat ileum, NG108‐15 cell and HEK‐5‐HT 3 As cell homogenates, [ 3 H]‐BRL46470 bound with high affinity ( K d (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 (B max (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‐HT 3 As cells and human putamen as used for the [ 3 H]‐BRL46470 studies, [ 3 H]‐granisetron also bound with high affinity ( K d (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 ( B max (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‐HT 3 receptor ligands indicated that in both rat cerebral cortex/hippocampus and rat ileum homogenates, [ 3 H]‐BRL46470 binding exhibited a pharmacological profile consistent with the labelling the 5‐HT 3 receptor with compounds competing with Hill coefficients close to unity 5 In HEK‐5‐HT 3 As cell homogenates, [ 3 H]‐BRL46470 and [ 3 H]‐granisetron associated rapidly ((3.84 ± 0.4)10 6 M −1 s −1 and (5.85 ± 0.2)10 6 M −l s −l respectively, mean ± s.e.mean, n = 3–4) in an apparently monophasic manner. Following the establishment of equilibrium, both [ 3 H]‐BRL46470 and [ 3 H]‐granisetron at a saturating concentration ([ 3 H]‐BRL46470 approximately 16 nM; [ 3 H]‐granisetron approximately 18 nM) and at a sub‐ K d concentration (approximately 1 nM for both radioligands) dissociated biphasically in HEK‐5‐HT 3 As cell homogenates (saturating concentration; [ 3 H]‐BRL46470 4.05 × 10 −3 ±2.53 × 10 −3 s −1 and 5.83 × 10 −5 ± 0.91 × 10 −5 s −1 ; [ 3 H]‐granisetron 3.20 × 10 −3 ± 1.70 × 10 −3 s −1 and 18.58 × 10 −5 ±4.19 × 10 −5 s −1 : sub‐ K d concentration; [ 3 H]‐BRL46470 2.47 × 10 −3 ± 1.18 × 10 −3 s −1 and 9.30 × 10 −5 ± 2.59 × 10 −5 s −1 ; [ 3 H]‐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 [ 3 H]‐BRL46470 specifically labels the 5‐HT 3 receptor in rat cerebral cortex/hippocampus, rat ileum, NG108‐15 cell and HEK‐5‐HT 3 As cell homogenates, but fails to label the 5‐HT 3 receptor expressed in human putamen. Whilst the pharmacological profile of the site labelled by [ 3 H]‐BRL46470 is directly comparable to that labelled by [ 3 H]‐granisetron, [ 3 H]‐BRL46470 consistently labelled approximately twice the density of sites compared to [ 3 H]‐granisetron in the same tissue homogenates prepared from rat cortex/hippocampus, rat ileum, NG108‐15 cells and HEK‐5‐HT 3 As cells.