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

Lucinda J. Steward, Jian Ge, Kim R. Bentley, Peter C. Barber, Anthony G. Hope, Jeremy J. Lambert, John A. Peters, Thomas P. Blackburn, Nicholas M. Barnes

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

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 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 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) 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 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 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. 1995 British Pharmacological Society

Original languageEnglish
Pages (from-to)1781-1788
Number of pages8
JournalBritish Journal of Pharmacology
Volume116
Issue number2
DOIs
Publication statusPublished - Sep 1995

Fingerprint

Granisetron
Binding Sites
Ligands
Ileum
Hippocampus
Cerebral Cortex
Putamen
Pharmacology
Population

Keywords

  • 5‐HT As receptor subunit
  • 5‐HT receptor
  • human brain
  • NG108‐15 cells
  • Nicholas M. Barnes
  • rat brain
  • [H]‐BRL46470
  • [H]‐granisetron

Cite this

Steward, Lucinda J. ; Ge, Jian ; Bentley, Kim R. ; Barber, Peter C. ; Hope, Anthony G. ; Lambert, Jeremy J. ; Peters, John A. ; Blackburn, Thomas P. ; Barnes, Nicholas M. / Evidence that the atypical 5‐HT3 receptor ligand, [3H]‐BRL46470, labels additional 5‐HT3 binding sites compared to [3H]‐granisetron. In: British Journal of Pharmacology. 1995 ; Vol. 116, No. 2. pp. 1781-1788.
@article{b412a77b43cc418f8c1c8e8e0a8a9a7f,
title = "Evidence that the atypical 5‐HT3 receptor ligand, [3H]‐BRL46470, labels additional 5‐HT3 binding sites compared to [3H]‐granisetron",
abstract = "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 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 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) 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 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 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. 1995 British Pharmacological Society",
keywords = "5‐HT As receptor subunit, 5‐HT receptor, human brain, NG108‐15 cells, Nicholas M. Barnes, rat brain, [H]‐BRL46470, [H]‐granisetron",
author = "Steward, {Lucinda J.} and Jian Ge and Bentley, {Kim R.} and Barber, {Peter C.} and Hope, {Anthony G.} and Lambert, {Jeremy J.} and Peters, {John A.} and Blackburn, {Thomas P.} and Barnes, {Nicholas M.}",
year = "1995",
month = "9",
doi = "10.1111/j.1476-5381.1995.tb16663.x",
language = "English",
volume = "116",
pages = "1781--1788",
journal = "British Journal of Pharmacology",
issn = "0007-1188",
publisher = "Wiley",
number = "2",

}

Evidence that the atypical 5‐HT3 receptor ligand, [3H]‐BRL46470, labels additional 5‐HT3 binding sites compared to [3H]‐granisetron. / Steward, Lucinda J.; Ge, Jian; Bentley, Kim R.; Barber, Peter C.; Hope, Anthony G.; Lambert, Jeremy J.; Peters, John A.; Blackburn, Thomas P.; Barnes, Nicholas M.

In: British Journal of Pharmacology, Vol. 116, No. 2, 09.1995, p. 1781-1788.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Evidence that the atypical 5‐HT3 receptor ligand, [3H]‐BRL46470, labels additional 5‐HT3 binding sites compared to [3H]‐granisetron

AU - Steward, Lucinda J.

AU - Ge, Jian

AU - Bentley, Kim R.

AU - Barber, Peter C.

AU - Hope, Anthony G.

AU - Lambert, Jeremy J.

AU - Peters, John A.

AU - Blackburn, Thomas P.

AU - Barnes, Nicholas M.

PY - 1995/9

Y1 - 1995/9

N2 - 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 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 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) 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 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 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. 1995 British Pharmacological Society

AB - 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 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 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) 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 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 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. 1995 British Pharmacological Society

KW - 5‐HT As receptor subunit

KW - 5‐HT receptor

KW - human brain

KW - NG108‐15 cells

KW - Nicholas M. Barnes

KW - rat brain

KW - [H]‐BRL46470

KW - [H]‐granisetron

UR - http://www.scopus.com/inward/record.url?scp=0028981568&partnerID=8YFLogxK

U2 - 10.1111/j.1476-5381.1995.tb16663.x

DO - 10.1111/j.1476-5381.1995.tb16663.x

M3 - Article

C2 - 8528560

AN - SCOPUS:0028981568

VL - 116

SP - 1781

EP - 1788

JO - British Journal of Pharmacology

JF - British Journal of Pharmacology

SN - 0007-1188

IS - 2

ER -