Characterization of the binding of a novel radioligand to CCKB/gastrin receptors in membranes from rat cerebral cortex

We have investigated the binding of a novel radiolabelled CCKB/gastrin receptor ligand, [3H]-JB93182 (5[[[(1S)-[[(3,5-dicarboxyphenyl)amino]carbonyl]-2-phenylethylamino]-carbonyl]-6-[[(1-adamantylmethyl) amino]carbonyl]-indole), to sites in rat cortex membranes. The [3H]-JB93182 was 97% radiochemically pure as assessed by reverse-phase HPLC (RP-HPLC) and was not degraded by incubation (150 min) with rat cortex membranes. Saturation analysis indicated that [3H]-JB93182 labelled a homogeneous population of receptors in rat cortex membranes (pKD=9.48±0.08, Bmax=3.61∼plusmn;0.65 pmol g−1 tissue, nH=0.97±0.02, n=5). The pKD was not significantly different when estimated by association-dissociation analysis (pKD=9.73±0.11; n=10). In competition studies, the low affinity of the CCKA receptor antagonists, L-364,718; SR27897 and 2-NAP, suggest that, under the assay conditions employed, [3H]-JB93182 (0.3 nM) does not label CCKA receptors in the rat cortex. The affinity estimates obtained for reference CCKB/gastrin receptor antagonists were indistinguishable from one of the affinity values obtained when a two site model was used to interpret [125I]-BH-CCK8S competition curves obtained in the same tissue (Harper et al., 1999). This study provides further evidence for the existence of two CCKB/gastrin sites in rat cortex. [3H]-JB93182 appears to label selectively sites previously designated as gastrin-G1 and therefore it may be a useful compound for the further discrimination and characterization of these putative receptor subtypes. British Journal of Pharmacology (1999) 126, 1504–1512; doi:10.1038/sj.bjp.0702447