Characterization of cholecystokinin receptors on bovine gallbladder membranes

Abstract

Cholecystokinin (CCK) binding to its receptors on a muscularis membrane fraction of bovine gallbladder was characterized using a biologically active CCK-33-[125I]Bolton-Hunter conjugate. Receptor binding was localized to the muscularis layer of the gallbladder; no binding was seen on either mucosal or serosal membranes. At 24 degrees C and pH 6.5, binding was maximal after 60-90 min of incubation, remained at a plateau for at least 240 min, and was reversed by the addition of unlabeled CCK-8. Optimal binding was seen at pH of 5.5 and required the presence of magnesium. Gallbladder binding data, best fit by a two-parameter model using a nonlinear least-squares computer program, was consistent with a single order of binding sites with a Kd of 618 +/- 168 pM and a binding capacity of 100.5 +/- 15.7 fmol/mg prot (mean +/- SE, n = 5). CCK-8 and CCK-33 inhibited 125I-CCK binding to gallbladder membranes with similar potencies, whereas desulfated CCK-8, gastrin I and II, and CCK-4 were at least 500 times less potent than CCK-33. The CCK antagonists dibutyryl cGMP and proglumide inhibited 125I-CCK binding with an IC50 of 31 and 600 microM, respectively. The present studies therefore demonstrate the existence of a specific CCK receptor on bovine gallbladder muscularis membranes with a high degree of selectivity for CCK analogues.

Cholecystokinin and Gastrin Receptors

“…CCK induces gallbladder contraction via activation of CCK1R located on smooth muscle cells. Presence of CCK1R on the muscularis layer of bovine gallbladder, where binding sites are absent on both the mucosal and serosal membranes, demonstrated a direct myogenic effect of the hormone for the contraction of the gallbladder (494). This localization was further confirmed by autoradiographic studies in several species including human (14,359,445,529,548).…”

Biochemical and pharmacological characterization of an extremely potent and selective nonpeptide cholecystokinin antagonist.

“…1251I-labeled CCK (1251-CCK) binding assays were similar to those described by others for rat pancreas (7), guinea pig cerebral cortex (8,9), and bovine gallbladder membranes (10), with the modifications noted below.…”

The peripheral cholecystokinin receptors

“…In addition to the pancreas, the gall bladder was the first recognized target organ for CCK, since CCK has been initially discovered on the basis of its cholecystokinetic activity [l]. Ligand binding studies on bovine [245,2461 and human gall bladder muscle membranes [247] and on tissue sections from guineapig [248] confirmed the presence of high-affinity CCK-A receptors. The presence of both CCK-A and CCK-B receptors in the gall bladder was suggested in a study using receptor protection by selective agonists and antagonists [249].…”