Correlation of apparent affinity values from H3-receptor binding assays with apparent affinity (pKapp) and intrinsic activity (alpha) from functional bioassays

Abstract

Background and purpose: Agonist apparent affinities (pK(I)') 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)' overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pK(I)' of ligands with varying intrinsic activity.

Experimental approach: In the guinea-pig ileum bioassay, intrinsic activity (alpha) 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)' values were closer to pK(app) values than in buffer B(0,0,0) but were associated with n (H) values <1. 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)' in buffer B(0,0,0) and pK(IL) values in buffer B(0.07,0.1,0.1) (DeltapK) were correlated with alpha.

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.

Figure 1

Chemical structures of histamine H₃-receptor ligands.

Figure 2

Effect of histamine H₃-receptor ligands on the electrically induced contraction of the guinea-pig ileum. Each point represents the mean±s.e.m. of determinations in at least three separate preparations (see Table 2).

Figure 3

Effect of EEDQ (0.3 μM) on (a) R-α-MH, (b) imetit, (c) S-α-MH and (d) histamine-induced inhibition of the electrically induced contraction of the guinea-pig ileum. Each point is the mean±s.e.m. of determinations in at least six separate preparations.

Figure 4

Representative saturation isotherms of [³H]clobenpropit to sites in guinea-pig cerebral cortex membranes in buffer B_(0,0,0) (a and c) and buffer B_{(0.07,0.1,0.1)} (b and d). The lines shown superimposed on the data points are the saturation isotherm obtained by fitting the Hill equation with n_H constrained to unity to the data. Guinea-pig cortical membranes (1.6 mg) were incubated for 165 min at 21±3°C in a final volume of 0.5 ml with HEPES–NaOH buffer and [³H]clobenpropit. Total and nonspecific binding of [³H]clobenpropit were defined using buffer B_(0,0,0) or buffer B_{(0.07,0.1,0.1)} and 1 μM thioperamide, respectively. All determinations were made in triplicate.

Figure 5

Representative saturation isotherms of [³H]clobenpropit to sites in guinea-pig cerebral cortex membranes in (a) buffer B_(0,0,0), (b) buffer B_(0.03,0,0), (c) buffer B_(0.07,0,0), (d) buffer B_(0.1,0,0), (e) buffer B_(0.2,0,0) and (f) buffer B_(0.3,0,0). The lines shown superimposed on the data points are the saturation isotherm obtained by fitting the Hill equation with n_H constrained to unity to the data. Guinea-pig cortical membranes (1.6 mg) were incubated for 165 min at 21±3°C in a final volume of 0.5 ml with HEPES–NaOH buffer and [³H]clobenpropit. Total and nonspecific binding of [³H]clobenpropit were defined using appropriate buffer and 1 μM thioperamide, respectively. All determinations were made in triplicate.

Figure 6

Effect of increasing concentration of CaCl₂ in buffer B on (a) pK_L and (b) B_max of [³H]clobenpropit at sites in guinea-pig cerebral cortex membranes. The line shown superimposed on the data was obtained by fitting a hyperbolic function.

Figure 7

Competition curves for H₃-receptor agonists and antagonists at sites labelled with [³H]clobenpropit in guinea-pig cerebral cortex. (a) Effect of increasing concentrations of ligands on [³H]clobenpropit binding (dpm). Data were obtained in a single experiment in buffer B_(0,0,0) and buffer B_{(0.07,0.1,0.1)} and errors are the mean±s.e.m. of triplicates. (b) Mean competition curve data for ligands, expressed as percentage specific binding, in buffer B_(0,0,0) or (c) buffer B_{(0.07,0.1,0.1)}. Guinea-pig cortical membranes (1.6 mg) were incubated for 165 min at 21±3°C in a final volume of 0.5 ml with HEPES–NaOH buffer, [³H]clobenpropit (0.2 nM) and increasing concentrations of ligands. Total and nonspecific binding of [³H]clobenpropit were defined using appropriate buffer and 1 μM thioperamide, respectively. Data are the mean±s.e.m. of between four and six experiments (see Table 4). The lines shown superimposed on the data for imetit, chloroproxyfan and S-α-methylhistamine were obtained using a two-site fit. The line shown superimposed on the thioperamide data was obtained using a one-site fit.

Figure 8

Comparison of the apparent affinities (pK_I or pK_I′) of histamine H₃-receptor agonists and antagonists obtained in buffer B_(0,0,0) and in buffer B containing 70 mM CaCl₂, 100 mM KCl and 100 mM NaCl (buffer B_{(0.07,0.1,0.1)}). The broken line represents the line of identity.

Figure 9

Comparison of the affinities of histamine H₃-receptor agonists (pK_app) and antagonists (pA₂ or pK_B) at H₃-receptors in the guinea-pig ileum bioassay (see Table 2) with those estimated in radioligand binding assays (see Table 4). pK_I or pK_I′ values for (a) agonists and antagonists in standard buffer (B_(0,0,0)), (b) agonists and antagonists in B_{(0.07,0.1,0.1)}, (c) agonists in B_(0,0,0), (d) agonists in B_{(0.07,0.1,0.1)}, (e) antagonists in B_{(0.07,0.1,0.1)} and (f) antagonists in B_{(0.07,0.1,0.1).} The broken line represents the line of identity (y=x).

Figure 10

Comparison of (a) pK_I′ and (b) pK_IL values for agonists in buffer B_{(0.07,0.1,0.1)} with ileum pK_app estimates.

Figure 11

Comparison of ligand ΔpK values and α measured in the guinea-pig ileum bioassay.

Figure 12

(a) The extended TCM as described by Samama et al. (1993). (b) The cubic TCM of Weiss et al. (1996). In both models, H is hormone, G is G protein and R is the inactive receptor state and R^* the ‘active' state. To allow simple comparison of the models, the terms used for ‘inactive', R_i and ‘active' receptor states (R_a) in the model of Weiss et al. (1996) have been modified to R and R^*, respectively.