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

1 We have investigated the binding of a novel radiolabelled CCKB/gastrin re ceptor ligand, [H-3]-JB93182 (5[[[(1S)-[[3, 5-dicarboxyphenyl)amino]carbony l]-2-phenylethylamino]-carbonyl]-6-[[(1-adamantylmethyl) amino]carbonyl]-in dole), to sites in rat cortex membranes, 2 The [H-3]-JB93182 was 97% radiochemically pure as assessed by reverse-pha se HPLC (RP-HPLC) and was not degraded by incubation (15 min) wit-h rat cor tex membranes. 3 Saturation analysis indicated that [H-3]-JB93182 labelled a homogeneous p opulation of receptors in rat cortex membranes (pK(D) = 9.48 +/- 0.08, B-ma x = 3.61 +/- 0.65 pmol g(-1) tissue, n(H) = 0.97 +/- 0.02, n = 5). The pK(D ) was not significantly different when estimated by association-dissociatio n analysis (pK(D) = 9.73 +/- 0.11; n = 10). 4 In competition studies, the low affinity of the CCKA receptor antagonists , L-364,718; SR27897 and 2-NAP, suggest that, under the assay conditions em ployed, [H-3]-JB93182 (0.3 nM) does not label CCKA receptors in the fat cor tex. 5 The affinity estimates obtained for reference CCKB/gastrin receptor antag onists were indistinguishable from one of the affinity values obtained when a two site model was used to interpret [I-125]-BH-CCK8S competition curves obtained in the same tissue (Harper et al,, 1999). 6 This study provides further evidence for the existence of two CCK(B/)gast rin sites in rat cortex. [H-3]-JB93182 appears to label selectively sites p reviously designated as gastrin-G(1) and therefore it may be a useful compo und for thefurther discrimination and characterization of these putative re ceptor subtypes.