Evidence that the atypical 5‐HT 3 receptor ligand, ( 3 H)‐BRL46470, labels additional 5‐HT 3 binding sites compared to ( 3 H)‐granisetron

Evidence that the atypical 5‐HT 3 receptor ligand, ( 3 H)‐BRL46470, labels additional 5‐HT... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Pharmacology Wiley

Evidence that the atypical 5‐HT 3 receptor ligand, ( 3 H)‐BRL46470, labels additional 5‐HT 3 binding sites compared to ( 3 H)‐granisetron

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Publisher
Wiley
Copyright
1995 British Pharmacological Society
ISSN
0007-1188
eISSN
1476-5381
DOI
10.1111/j.1476-5381.1995.tb16663.x
Publisher site
See Article on Publisher Site

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.

Journal

British Journal of PharmacologyWiley

Published: Sep 1, 1995

References

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