Characterization of type A and type B CCK receptor binding sites in rat vagus nerve

We employed quantitative receptor autoradiography to analyze pharmacological properties of ¹²⁵I-Bolton Hunter cholecystokinin (CCK-8)-labeled binding sites in sections of rat cervical vagus nerve that had been ligated 24 h prior to extraction. Binding densities were detected in segments of nerve proximal and distal to the ligature. Analysis was confined to proximal segments. Saturation and competitive binding studies were carried out using sulphated CCK-8 and two selective CCK receptor antagonists: MK-329, to define type-A (CCK_A) binding sites; and, L-365,260, to define type-B (CCK_B) binding sites. Sulphated CCK-8 was the most potent inhibitor of vagal ¹²⁵I-CCK binding (IC₅₀ = 2 nM). Nonlinear curve fitting analysis of the CCK binding data favored the presence of a single class of vagal CCK receptors KD_i = 1 nM). However, both MK-329 (IC₅₀ = 18 nM) and L-365,260 (IC₅₀ = 45 nM) completed for vagal ¹²⁵I-CCK binding indicating the presence of CCK_A and CCK_B binding sites. Co-analysis of the antagonist binding data suggested that CCK_A and CCK_B receptors were transported in equal concentration within the vagus. MK-329 bound with high affinity to CCK_A sites (K_i = 3 nM) and low-affinity to CCK_B sites (K_i = 462 nM) while L-365,260 bound with high affinity to CCK_B sites (K_i = 10 nM) and low-affinity to CCK_A sites (K_i = 775 nM). These same ligands were used to characterize the specificity of ¹²⁵I-CCK binding in the medial and lateral divisions of the nucleus of the solitary tract (NTS), two regions innervated by primary vagal afferents carrying CCK receptors. Sulphated CCK-8 was the most potent inhibitor of ¹²⁵I-CCK binding in both regions of the NTS (IC₅₀ = 0.6 nM, medial; IC₅₀ = 0.4 nM, lateral). In the medial NTS, MK-329 was a potent inhibitor (IC₅₀ = 21.8 nM) while MK-329 was a weak inhibitor (IC₅₀ = 341 nM) of ¹²⁵I-CCK binding. In contrast, in the lateral NTS, L-365,260 was a potent inhibitor (IC₅₀ = 21.8 nM) while MK-329 was a weak inhibitor (IC> 1,0000 nM) of ¹²⁵I-CCK binding. These results are consistent with the existence of two populations of vagal afferent fibers projecting to different regions of the NTS. One, containing CCK_A receptors, projects principally to the medial NTS, while the other containing CCK_B receptors, projects principally to the lateral NTS.