title: Distinct requirements for activation at CCK-A and CCK-B/gastrin receptors: studies with a C-terminal hydrazide analogue of cholecystokinin tetrapeptide (30-33).
abstract: 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.
authors.affiliation: Neuroscience Research Division, Abbott Laboratories, Abbott Park, Illinois.
authors.forename: C W
authors.initials: CW
authors.lastname: Lin
authors.forename: M W
authors.initials: MW
authors.lastname: Holladay
authors.forename: R W
authors.initials: RW
authors.lastname: Barrett
authors.forename: C A
authors.initials: CA
authors.lastname: Wolfram
authors.forename: T R
authors.initials: TR
authors.lastname: Miller
authors.forename: D
authors.initials: D
authors.lastname: Witte
authors.forename: J F
authors.initials: JF
authors.lastname: Kerwin
authors.forename: F
authors.initials: F
authors.lastname: Wagenaar
authors.forename: A M
authors.initials: AM
authors.lastname: Nadzan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001570:Benzodiazepinones / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002766:Cholecystokinin / Q000037:antagonists & inhibitors*; D020109:Devazepide; D005755:Gastrins; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D011377:Proglumide / Q000031:analogs & derivatives / Q000494:pharmacology; D011949:Receptors, Cholecystokinin / Q000187:drug effects*; D013758:Tetragastrin / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D001570:Benzodiazepinones; D005755:Gastrins; D010446:Peptide Fragments; D011949:Receptors, Cholecystokinin; D013758:Tetragastrin; C062180:A 57696; D002766:Cholecystokinin; D011377:Proglumide; D020109:Devazepide; C048181:lorglumide; D002118:Calcium