Characterization of the binding of [3H]-clobenpropit to histamine H3-receptors in guinea-pig cerebral cortex membranes

Abstract

1. We have investigated the binding of a novel histamine H3-receptor antagonist radioligand, [3H]-clobenpropit ([3H]-VUF9153), to guinea-pig cerebral cortex membranes. 2. Saturation isotherms for [3H]-clobenpropit appeared biphasic. Scatchard plots were curvilinear and Hill plot slopes were significantly less than unity (0.63 ± 0.03; n = 12 ± s.e.mean). The radioligand appeared to label two sites in guinea-pig cerebral cortex membranes with apparent affinities (pK(D)') of 10.91 ± 0.12 (B(max) = 5.34 ± 0.85 fmol mg-1 original wet weight) and 9.17 ± 0.16 (B(max) = 23.20 ± 6.70 fmol mg-1). 3. In the presence of metyrapone (3 mM) or sodium chloride (100 mM), [3H]-clobenpropit appeared to label a homogeneous receptor population (B(max) = 3.41 ± 0.46 fmol mg-1 and 3.49 ± 0.44 fmol mg-1, pK(D)' = 10.59 ± 0.17 and 10.77 ± 0.02, respectively). Scatchard plots were linear and Hill slopes were not significantly different from unity (0.91 ± 0.04 and 0.99 ± 0.02, respectively). Granisetron (1 μM), rilmenidine (3 μM), idazoxan (0.3 μM), pentazocine (3 μM) and 1,3-di-(2-tolyl)guanidine (0.3 μM) had no effect on the binding of [3H]-clobenpropit. 4. The specific binding of [3H]-clobenpropit appeared to reach equilibrium after 25 min at 21 ± 3°C and remained constant for > 180 min. The estimated pK(D)' (10.27 ± 0.27; n = 3 ± s.e.mean) was not significantly different from that estimated by saturation analysis in the presence of metyrapone. 5. A series of histamine H3-receptor ligands expressed affinity values for sites labelled with [3H]-clobenpropit which were not significantly different from those estimated when [3H]-R-α-MH was used to label histamine H3-receptors in guinea-pig cerebral cortex membranes.