Both CCK-A and CCK-B/gastrin receptors mediate pepsinogen release in guinea pig gastric glands.

We evaluated the affinity of cholecystokinin octapeptide (CCK-8), gastrin, and subtype-selective CCK agonists for CCK/gastrin receptors and compared it with the ability of these peptides to stimulate phosphoinositide (PI) hydrolysis and pepsinogen release in guinea pig gastric glands. Competitive binding studies using 125I-labeled Bolton-Hunter-CCK-8 and 125I-gastrin showed the presence of CCK-B/gastrin receptors in gastric glands and dispersed chief cells. In contrast, the potency of peptides in stimulating PI hydrolysis in both gastric glands and dispersed chief cells displayed a profile similar to CCK-A receptors found in pancreatic acini, i.e., CCK-8 = A 71378 greater than A 71623 greater than A 70874 much greater than A 72962 = CCK-8 (desulfated) greater than gastrin II greater than gastrin I. In general, the rank order of potency of peptides for stimulation of PI hydrolysis correlated well with their ability to stimulate pepsinogen release. At concentrations greater than 10 microM, efficacies of gastrin I and II in stimulating pepsinogen release from gastric glands were near 90% of the maximal activity of CCK-8. The inhibitory potency of MK-329, a selective CCK-A receptor antagonist, was similar against either CCK-8 (10 nM) or gastrin I (10 microM), except that a minor portion (approximately 30-40%) of gastrin I-induced pepsinogen release was insensitive to MK-329. The MK-329-insensitive component was inhibited by CI-988, a potent and selective CCK-B/gastrin receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of two novel cholecystokinin tetrapeptide (30-33) analogues, A-71623 and A-70874, that exhibit high potency and selectivity for cholecystokinin-A receptors.

Based on their relative affinities for cholecystokinin octapeptide (26-33) (CCK-8), cholecystokinin tetrapeptide (30-33) (CCK-4), desulfated CCK-8, and gastrin, cholecystokinin (CCK) receptors have been classified as CCK-A (alimentary) and CCK-B (brain). Selective nonpeptide antagonists of CCK-A and CCK-B receptors, as well as highly selective CCK-A and CCK-B peptide agonists, have been described. We report here the characterization of two novel CCK-4-based peptides, A-71623 and A-70874. In radioligand binding assays, the IC50 values for A-71623 and A-70874 were 3.7 and 4.9 nM in guinea pig pancreas (CCK-A) and 4500 and 710 nM in cerebral cortex (CCK-B), respectively. Both were agonists in stimulating pancreatic amylase release, and their stimulatory effects were potently inhibited by the CCK-A antagonist L-364,718. A-71623 was a full agonist and A-70874 was a partial agonist (approximately 80%) in stimulating phosphoinositide breakdown in pancreas. Both peptides also were potent agonists in stimulating CCK-A receptors in the ileum. They were, however, weak and behaved as partial agonists in calcium studies in NCI-H345 cells, which possess CCK-B/gastrin receptors. In guinea pig gastric glands, the affinities of A-71623 and A-70874 for the CCK-B/gastrin receptor were 11 and 1.6 microM, respectively. These results demonstrate that A-71623 and A-70874 are potent and selective agonists at CCK-A receptors. The preferential interaction of these novel CCK-4 analogs with CCK-A receptors is in contrast to other CCK-4-based peptides, which are primarily selective for CCK-B receptors. In addition, A-71623 and A-70874 are the first two examples of potent CCK-A agonists that do not contain a tyrosine residue whose sulfation is required for potent CCK-A agonist activity of larger peptides.

A71378: a CCK agonist with high potency and selectivity for CCK-A receptors.

Receptors for the brain and gut peptide cholecystokinin (CCK) have been classified into two classes, CCK-A and CCK-B. To date, peptide analogues with selectivity for the CCK-B receptors have been identified, and selective antagonists for CCK-A and CCK-B receptors have been reported as well; until now, there have been no reports of highly selective CCK-A agonists. Herein we describe the properties of A71378 [desamino-Try(SO3H)-Nle-Gly-Trp-Nle-(N-methyl)Asp-Phe-NH2], a highly selective CCK-A receptor ligand. Characterization of A71378 was carried out in the guinea pig pancreas, cortex, gastric gland, and ileum, as well as in NCI-H345 cells. The IC50 values of A71378 for the pancreatic CCK-A, cortical CCK-B, and gastrin receptor were 0.4 nM, 300 nM, and 1,200 nM, respectively. A71378 proved to be a potent agonist in eliciting pancreatic amylase secretion (EC50 = 0.16 nM) and ileal muscle contraction (EC50 = 3.7 nM). In contrast, A71378 was relatively weak (EC50 = 600 nM) in mobilizing intracellular calcium from NCI-H345 cells, which express CCK-B/gastrin receptors. The high potency and selectivity of A71378 for the CCK-A over CCK-B and gastrin receptors is unprecedented among CCK peptides. Studies on CCK-7 analogues indicate that N-methylation of the Asp residue is responsible for the observed selectivity for CCK-A receptors. This discovery of a selective CCK-A agonist should prove valuable for studies aimed at understanding the physiological roles of CCK-A receptors in the brain and periphery.

Distinct requirements for activation at CCK-A and CCK-B/gastrin receptors: studies with a C-terminal hydrazide analogue of cholecystokinin tetrapeptide (30-33).

We describe here the properties of tert-butyloxycarbonyl-Trp-Leu-Asp-Phe-NHNH2 (A-57696), a C-terminal hydrazide analogue of tert-butyloxycarbonyl-CCK4 (Boc-Trp-Met-Asp-Phe-NH2), at four cholecystokinin (CCK) receptor-bearing tissues, the guinea pig pancreas and gall bladder (Type A), guinea pig cortex (Type B), and NCI-H345 cells, a human small cell lung cancer cell line that expresses CCK-B/gastrin receptors. Using 125I-Bolton-Hunter-cholecystokinin octapeptide (26-33) (125I-Bolton-Hunter-CCK8) as the radioligand, A-57696 was found to be selective for cortical CCK-B receptors (IC50 = 25 nM), compared with pancreatic CCK-A receptors (IC50 = 15 microM). A-57696 behaved as a competitive antagonist in reversing CCK8-stimulated pancreatic amylase secretion and phosphoinositide breakdown. By Schild analysis, its Kd was determined to be 4.7 and 6.8 microM in amylase and phosphoinositide assays, respectively. A-57696 (100 microM) did not elicit gall bladder contraction, and it inhibited contractions induced by CCK8. The Kd of A-57696 at gall bladder CCK-A receptors was 19 microM. In contrast, A-57696 behaved as a partial agonist (80% of maximal CCK8 response) in stimulating calcium mobilization at CCK-B/gastrin receptors on NCI-H345 cells. A-57696 and CCK8 inhibited each other in calcium mobilization experiments utilizing the fluorescent dye Indo-1. Stimulatory actions of CCK8 and A-57696 were reversed by the CCK-B-selective (R)-L-365,260 (100 nM), whereas at the same concentration, the CCK-A-selective (S)-L-365,260 was ineffective. Binding studies using 125I-Bolton-Hunter-CCK8 and 125I-gastrin indicated that binding sites labeled by these two ligands displayed similar affinities for CCK8, desulfated CCK8, gastrin, A-57696, and both enantiomers of L-365,260. A-57696 represents a new class of CCK-A peptide antagonist at guinea pig pancreas a new class of CCK-A peptide antagonist at guinea pig pancreas and gall bladder. Its contrasting functional activities at guinea pig CCK-A and CCK-B/gastrin receptors in a human tumor cell demonstrate that, in addition to the previously described differences in binding specificity for selective agonists and antagonists, CCK-A receptors and CCK-B/gastrin receptors have different requirements for activation.

Type-A cholecystokinin binding sites in cow brain: characterization using (-)-[3H]L364718 membrane binding assays.

(-)-[3H]L364718 membrane binding assays were employed to localize and characterize cholecystokinin (CCK)-A binding sites in rat and cow brain. Specific binding was detected in all brain areas tested, but in all areas of rat brain and most areas of cow brain the level was too low to allow characterization of the ligand binding specificity of these sites. Membranes prepared from cow nucleus accumbens and striatum contained higher levels of (-)-[3H]L364718 specific binding which represented 55-70% of total binding. Characterization of the ligand binding properties of (-)-[3H]L364718 binding sites in cow nucleus accumbens revealed that these sites are similar to CCK-A sites found in pancreatic membranes. Binding of (-)-[3H]L364718 was saturable and had high affinity (Kd = 45 pm). Sites labeled by (-)-[3H]L364718 displayed stereospecificity for the stereoisomers of CR1409. The competition curve for CCK8 was shallow and was steepened and shifted to the right by the presence of the stable GTP analog guanosine 5'-(beta,delta-imido)triphosphate. The potency of CCK8, but not (-)-L36478, was also affected by the buffer in which the assay was conducted. Future use of (-)-[3H]L364718 membrane binding assays using cow nucleus accumbens and/or striatum will help explore the possibility of differences in ligand recognition among CCK-A sites found in brain and peripheral tissues.

Type-A cholecystokinin receptors in CHP212 neuroblastoma cells: evidence for association with G protein and activation of phosphoinositide hydrolysis.

125I-Bolton Hunter-cholecystokinin octapeptide (BH-CCK8) and (-)-[3H]L-364718 membrane binding assays were used to identify and characterize cholecystokinin (CCK) receptors in CHP212 human neuroblastoma cells. The ligand binding properties of CCK receptors in these cells are similar to those found in pancreas (CCK-A sites) and differ from the predominant type of CCK binding site found in brain (CCK-B sites). The specific binding of 125I-BH-CCK8 but not (-)-[3H]L-364718 was reduced by the metabolically stable GTP analog guanosine 5'-(beta-delta-imido)trisphosphate. A substantial difference in the Bmax for the radiolabeled agonist (125I-BH-CCK8) and antagonist [(-)-[3H]L-364718] was noted. These observations are consistent with CCK receptors existing in guanine nucleotide-binding protein-coupled and -uncoupled states. Similar to its action in pancreatic acinar cells, CCK8(S) stimulated the accumulation of [3H]inositol phosphates in cells prelabeled with [3H]myo-inositol (EC50 = 3.2 +/- 0.4 nM; maximum response = 4.5 +/- 0.4 x basal). The intrinsic activity of CCK analogues in stimulating phosphoinositide hydrolysis was substantially less than their reported intrinsic activity in stimulating phosphoinositide hydrolysis in pancreatic acinar cells. The CHP212 neuroblastoma cell may serve as a useful model for the recently reported CCK-A binding site found in the central nervous system.