Autoradiographic Localization of Cholecystokinin A and B Receptors in Rat Brain Using [125I]D-Tyr25(NIe28,31)-CCK 25-33S

Abstract

The distribution of receptors for the sulphated octapeptide cholecystokinin 26–33 (CCK-8S) in rat brain was investigated by radioligand binding in conjunction with autoradiography using the novel iodinable, non-oxidizable, amino- and thiolendopeptidase-resistant CCK analogue, d-Tyr²⁵(Nle^28,31)-CCK 25–33S. Labelling of the peptide was achieved by synthesis utilizing Na¹²⁵l and Chloramine-T. [¹²⁵l]D-Tyr²⁵(Nle^26,31)-CCK 25–33S (100 pM) bound rapidly and reversibly to a single population of sites on slide-mounted coronal sections of rat forebrain with a dissociation constant of 34 pM. Specific binding was fully inhibited by CCK-8S, CCK-8, CCK-4, L-365,260 and L-364,718, with inhibition constants 2.7, 9.8, 35, 7.0 and 130 nM, respectively. These inhibition data may indicate that the [¹²⁵l] ligand binds preferentially to a CCK_B subtype of receptor, but may also reflect the relative paucity of CCK_A receptors in the rat forebrain. Optimum conditions for autoradiography combined the preincubation of brain sections in unlabelled 10 pM d-Tyr²⁵(Nle^28,31)-CCK 25–33S with a 60-min wash after incubation with the [¹²⁵l] ligand. Analyses of the autoradiograms obtained from the use of coronal and horizontal brain sections were aided by the high levels of specific binding (80–90%), and revealed that CCK receptors were topographically distributed through the neuroaxis. High densities of receptor-associated silver grains were found in the olfactory bulb (internal plexiform layer), neocortex (layer III), nucleus accumbens, parasubiculum, subbrachial nucleus, parabigeminal nucleus, dorsal vagal complex, area postrema and the A2 region. Moderate labelling was observed in many telencephalic and diencephalic nuclei. The majority of these receptors were of the CCK_B subtype, as shown by the use of subtype-selective antagonists, although CCK_A receptors were present in moderate to high densities in the A2 area, area postrema and nucleus tractus solitarii, and at low density in the interpeduncular nucleus and central amygdala. These findings provide further evidence for the widespread, topographic distribution of CCK receptors and indicate that [¹²⁵l]d-Tyr²⁵(Nle^28,31)-CCK 25–33S is very suitable for autoradiographic investigations because of its low non-specific binding.