Correlation of apparent affinity values from H₃‐receptor binding assays with apparent affinity (pK_app) and intrinsic activity (α) from functional bioassays

Abstract

Background and purpose: Agonist apparent affinities (pK I 0 ) in histamine H 3 -receptor binding assays were higher than expected from apparent affinity values (pK app ) estimated in bioassay. Here, we investigate whether the degree of pK I 0 overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pK I 0 of ligands with varying intrinsic activity. Experimental approach: In the guinea-pig ileum bioassay, intrinsic activity (a) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pK app values were estimated using the method of Furchgott. The pK L of [ 3 H]clobenpropit in guinea-pig cerebral cortex was estimated by saturation analysis in 20 mM HEPES-NaOH buffer (buffer B (0,0,0) ), or buffer B (0,0,0) containing 70 mM CaCl 2 , 100 mM NaCl and 100 mM KCl (buffer B (0.07,0.1,0.1) ). PK I values were determined in competition studies in both buffers. Key results: [ 3 H]clobenpropit saturation isotherms had n H values of unity in both buffers. In buffer B (0.07,0.1,0.1) , agonist pK I 0 values were closer to pK app values than in buffer B (0,0,0) but were associated with n H values o1. A two-site analysis of agonist data in buffer B (0.07, 0.1, 0.1) provided a better fit than a one-site fit and low affinity values (pK IL ) were comparable to pK app . Differences between the pK I 0 in buffer B (0,0,0) and pK IL values in buffer B (0.07,0.1,0.1) (DpK) were correlated with a. Conclusions and implications: H 3 -receptor binding assays conducted in buffer B (0,0,0) and buffer B (0.07,0.1,0.1) can provide a measure of ligand affinity (pK app ) and intrinsic efficacy. The assay predicts that some ligands previously classified as H 3 -receptor antagonists may possess residual intrinsic efficacy.

Histamine H3‐receptor agonists and imidazole‐based H3‐receptor antagonists can be thermodynamically discriminated

“…In buffer A, the mean Δ H °′ values for JB96132, thioperamide and [ 3 H]clobenpropit were negative (Table 4) and mean Δ S °′ values were positive (Table 4). The mean Δ H °′ and Δ S °′ values for the ligands classified as agonists in the guinea‐pig ileum longitudinal muscle myenteric plexus (see Harper et al ., 2007 ) were also positive (Table 4).…”

“…In this study, we have determined the thermodynamic parameters underlying the binding of 10 ligands (seven agonists and three antagonists) at histamine H 3 ‐receptors of guinea‐pig cortex. In previous studies, we have shown that agonist affinity values are overestimated in H 3 ‐receptor radioligand‐binding assays, when buffer does not contain salts, and that the degree of affinity overestimation is correlated with the ligand's intrinsic activity ( α ) (see Harper et al ., 2007 ). In addition, we have shown that when H 3 ‐receptor radioligand‐binding assays are conducted in the presence of salts, p K I estimates are closer to p K app values estimated by the method of Furchgott in the guinea‐pig ileum bioassay (see Harper et al ., 2007 ).…”

“…In an additional series of experiments, we investigated the effect of temperature (4, 12, 21 and 30°C) on the binding of [ 3 H]clobenpropit in the presence of 300 m M CaCl 2 (buffer A Ca ). This was because in previous studies, we found that this buffer was more effective than 100 m M NaCl, 100 m M KCl and 70 m M CaCl 2 at reducing R ‐ α ‐MH p K I values to those equivalent to p K app values estimated in a functional bioassay; see Harper et al ., 2007 ).…”

“…Therefore, the primary aim of this study was to determine whether the binding of ligands, characterised as agonists and antagonists at histamine H 3 ‐receptor in a functional bioassay of the guinea‐pig ileum, could be discriminated thermodynamically. However, in previous studies, we have shown that agonist affinity values are overestimated in H 3 ‐receptor radioligand‐binding assays conducted in the absence of buffer salts by an amount which is related to the ligand's intrinsic activity (see Harper et al ., 2007 ) and, additionally, that when radioligand‐binding assays are conducted in the presence of salts, p K I estimates are closer to p K app values estimated by the method of Furchgott in a guinea‐pig ileum bioassay. Therefore, an additional aim of this study was to establish whether the thermodynamic parameters underlying the binding interaction of agonists paralleled those of the antagonists, when the assay was conducted under conditions where their p K I values were similar to p K app values and where the agonist n H values were not different from unity.…”

“…The agonist ligands that were selected for this study had varying intrinsic activity ( α ) as defined by bioassay on the guinea‐pig ileum myenteric plexus longitudinal muscle (see Figure 1 and Harper et al ., 2007 ) (immepip, α =1.0; imetit, α =0.90; R ‐ α ‐methylhistamine ( R ‐ α ‐MH), α =1.0; proxyfan, α =0.35; chloroproxyfan α =0.45; bromoproxyfan α =0.65; and iodoproxyfan, α =0.90). The antagonist ligands, as defined by bioassay (thioperamide, JB96132 and [ 3 H]clobenpropit), all contained an imidazole moiety (Figure 1 and Harper et al ., 2007 ).…”