Characterization of Histaminergic Receptors

Abstract

This unit describes radioligand binding protocols for histamine H1, H2, H3, and H4 receptors. Because these receptors demonstrate low amino acid sequence homology and divergent pharmacological characteristics, assay conditions vary for each. These protocols can be employed in high‐throughput screening programs aimed at identifying selective agonists and antagonists for these sites.

Pharmacological Properties and Procognitive Effects of ABT-288, a Potent and Selective Histamine H3Receptor Antagonist

“…3 H]N-␣-methylhistamine (0.5-1.0 nM) in the presence or absence of increasing concentrations (from 5 to 11 concentrations over a five-log unit range) of competing ligands for 30 min at 25°C in a final volume of 0.5 ml of 50 mM Tris/5 mM EDTA, pH 7.4 buffer as described previously Strakhova and Esbenshade, 2007). Nonspecific binding was defined with 10 M thioperamide.…”

“…Nonspecific binding was defined with 10 M thioperamide. Cloned human histamine H 1 , H 2 , and H 4 receptor radioligand binding assays were performed as described previously Strakhova and Esbenshade, 2007) 3 H]histamine, respectively. All binding reactions were terminated by vacuum filtration onto polyethylenimine (0.3%) presoaked Unifilter plates (PerkinElmer Life and Analytical Sciences) or Whatman GF/B filters (for human cortex H 3 R and human H 4 receptor) followed by three brief washes with 2 ml of ice-cold 50 mM Tris/5 mM EDTA buffer.…”

“…IC 50 values and Hill slopes were determined by Hill transformation for all of the radioligand competition binding data as described previously (Strakhova and Esbenshade, 2007). K i values were determined by the Cheng-Prusoff equation (Cheng and Prusoff, 1973), and the data are presented as the mean K i with 95% confidence intervals.…”

In vitro and in vivo characterization of A‐940894: a potent histamine H4 receptor antagonist with anti‐inflammatory properties

“…For the rat H 4 ‐FLAG cell line B max = 1624 fmol·mg −1 and K d = 109 nmol·L −1 (p K d = 7.70 ± 0.04, data not shown). Radioligand binding assays for cloned human histamine H 1 , H 2 and H 3 receptors were performed as described (Strakhova and Esbenshade, 2006) using [ 3 H]‐mepyramine, [ 3 H]‐tiotidine and [ 3 H]‐N‐α‐methylhistamine respectively. All binding reactions were terminated by filtration under vacuum onto polyethylenimine (0.3%) pre‐soaked Unifilters (Perkin Elmer Life Sciences, Boston, MA, USA) or Whatman GF/B filters (for histamine H 4 receptors) followed by three brief washes with 2 mL of ice‐cold TE buffer.…”

“…For all of the radioligand competition binding assays, IC 50 values and Hill slopes were determined by Hill transformation of the data as previously described (Strakhova and Esbenshade, 2006) and p K i values were determined by the Cheng–Prusoff equation (Cheng and Prusoff, 1973) using K d values derived from saturation studies.…”