A new, highly selective CCK-B receptor radioligand ([³H][N-methyl-Nle^28,31]CCK_26-33): Evidence for CCK-B receptor heterogeneity

[N-methyl-Nle²⁸,³¹]CCK_26-33 (SNF 8702) is a nonsulfated cholecystokinin octapeptide analog that is highly selective for cholecystokinin-B (CCK-B) receptors. Inhibition studies using [¹²⁵I]Bolton-Hunter-labeled CCK-8 show that SNF 8702 has over 4,000-fold greater affinity for CCK receptors in guinea pig cortex relative to those in guinea pig pancreas. SNF 8702 was tritium-labeled to a specific activity of 23.7 Ci/mmol and its binding properties characterized for guinea pig brain membrane preparations. [³H]SNF 8702 binds to a single site with high affinity (K(d) = 0.69-0.90 nM) in guinea pig cortex, cerebellum, hippocampus and pons-medulla. Of these four tissues, the highest receptor density was measured in the cortex (86 fmol/mg of protein) and the lowest in the pons-medulla (22 fmol/mg of protein). In contrast to findings of single-site binding in some brain regions, evidence for CCK-B receptor heterogeneity is observed under other conditions. [³H]SNF 8702 binding to membranes prepared from whole guinea pig brain shows biphasic association kinetics at a concentration of 2.0 nM consistent with the presence of binding site heterogeneity. Binding site heterogeneity is consistently observed for [³H]SNF 8702 binding to guinea pig whole brain membranes in saturation studies where a high-affinity site (K(d) = 0.31 nM) is distinguished from a low-affinity site (K(d) = 3.3 nM). Binding site heterogeneity is also observed for the midbrain-thalamic region. CCK-B receptor heterogeneity is suggested by the effect of the guanyl nucleotide analogue, guanylyl-imidodiphosphate (Gpp(NH)p), on [³H]SNF 8702 binding to CCK-B receptors in the cerebellum. The addition of Gpp(NH)p to cerebellar membranes reveals the presence of a low concentration of high-affinity (Gpp(NH)p insensitive sites and a high concentration of low-affinity (Gpp(NH)p sensitive) sites. Cortical and hippocampal CCK-B receptors have reduced binding affinity for [³H]SNF 8702 in the presence of Gpp(NH)p but this effect is smaller than for cerebellar Gpp(NH)p sensitive sites and additional high affinity binding sites are not observed. The data show that SNF 8702 is a highly selective ligand for CCK-B receptors. Regional variations in the observed binding properties of [³H]SNF 8702 suggest the presence of CCK-B receptor subtypes.