Analysis of the behaviour of selected CCK_B/gastrin receptor antagonists in radioligand binding assays performed in mouse and rat cerebral cortex

Abstract

James Black Foundation, 68 Half Moon Lane, Dulwich, London SE24 9JE, England 1 The previously described complex behaviour of the CCK B /gastrin receptor antagonist, L-365,260, in radioligand binding assays could be explained by a variable population of two binding sites. We have investigated whether other CCK B /gastrin receptor ligands (PD134,308, PD140,376, YM022 and JB93182) can distinguish between these sites. 2 In the mouse cortex assay, Hill slopes were not dierent from unity and the ligand pK I values did not dier when either [ I]-BH-CCK-8S or [3 H]-PD140,376 was used as label as expected for a single site (G 2 ). 3 In the rat cortex, where previous analysis of replicate (n=48) L-365,260 data indicated the presence of two CCK B /gastrin sites (G 1 and G 2 ), the competition data for PD134,308, PD140,376, YM022 and JB93182 could be explained by a homogeneous population of CCK B /gastrin sites because the Hill slope estimates were not signi®cantly dierent from unity. However, the estimated anity values for JB93182 and YM022 were signi®cantly higher and that for PD134,308 was signi®cantly lower than those obtained in the mouse cortex when the same radioligand was used. In view of our previous data obtained with L-365,260, the rat cortex data were also interpreted using a two-site model. In this analysis, SR27897 expressed *9 fold, PD134,308 *13 fold and PD140,376 *11 fold selectivity for the G 2 site. In contrast, JB93182 expressed *23 fold and YM022 *4 fold selectivity for the G 1 site. If the two-site interpretation of the data is valid then, because of its reverse selectivity to L-365,260, JB93182 has been identi®ed as a compound which if radiolabelled could provide a test of this receptor subdivision.

Pharmacological evidence for putative CCK1 receptor heterogeneity in human colon smooth muscle

“…The competitors all behaved simply in the gallbladder assay with Hill slopes not significantly different from unity. The corresponding p K I value for 2‐NAP was indistinguishable from previously reported affinity estimates obtained in a guinea‐pig pancreas ([ 125 I]‐BH‐CCK‐8S) binding assay (p K I =5.3; Harper et al ., 1999) and gallbladder smooth muscle functional bioassays (human pA 2 =5.9, guinea‐pig pA 2 =6.4; Hull et al ., 1993). The values obtained for L‐364,718 and SR27897, although still consistent with selective CCK 1 receptor binding, were higher than those previously reported at human CCK 1 receptors (SR27897 p K I =8.7 at the cloned receptor, Gigoux et al ., 1999; L‐364,718 p K I =9.2 in human gallbladder membranes; Xiao et al ., 1999).…”

“…The values obtained for L‐364,718 and SR27897, although still consistent with selective CCK 1 receptor binding, were higher than those previously reported at human CCK 1 receptors (SR27897 p K I =8.7 at the cloned receptor, Gigoux et al ., 1999; L‐364,718 p K I =9.2 in human gallbladder membranes; Xiao et al ., 1999). The lower affinity values estimated for the CCK 2 ‐receptor selective ligands were also consistent with their interaction at the CCK 1 ‐receptor subtype in animal tissue (p K I for YM220=8.1, PD‐134,308=6.1, JB93182=5.3; Harper et al ., 1999), which demonstrated that the selectivity of these compounds was maintained in human tissue. In contrast, in the human colon assay the competition curves for SR27897, 2‐NAP, YM220 and PD‐134,308 were all significantly flat and better described by the two‐site rather than the one‐site model.…”

“…in a final assay volume of 500 μl for 2.5 h at room temperature (21±3°C). Non‐specific binding was defined using 1 μ M SR27897 (p K i ∼9.6 in the CCK 1 guinea‐pig pancreas assay; Harper et al ., 1999). All data points were determined in triplicate.…”

Regulation of Aggression by Obesity-Linked GenesTfAP-2andTwzThrough Octopamine Signaling inDrosophila

“…They were then fed by the capillary feeding assay method for 2-3 days with 1, 3, and 5 mM of the octopamine antagonists phentolamine (Dudai 1982), Sigma-Aldrich) or epinastine (Stevenson et al 2005;Unoki et al 2005) or with 1 mM CCK antagonist (SR27897, Sigma-Aldrich) (Harper et al 1999). Each fly was transferred to a transparent plastic cylindrical vial (9 3 2 cm) (height 3 diameter), containing 1% agarose (5 cm high) to provide moisture and humidity for the flies, and the opening of the vial was covered with paraffin film.…”

Cholecystokinin antagonists: Pharmacological and therapeutic potential

“…18 Competition radioligand binding studies with diverse CCK 2 receptor antagonists have provided evidence for the existence of two binding sites, or binding states, for this receptor subtype in gastric glands and cortex of some animal species. [68][69][70] Furthermore, the distinct behavioral responses elicited by the CCK 2 selective agonists BC 197 (anxiogenic) and CB 264 (nonanxiogenic and increases memory and attention) have been also related with the existence of two different binding states of these receptors in the brain. 71,72 …”