title: Cytosolic Ca2+ evaluation in rabbit parietal cells: a novel method to screen gastrin receptor antagonists.
abstract: We have evaluated the application of the fura-2 method to detect cytosolic Ca2+ increase in gastric cells expressing CCKB/gastrin receptors, in order to screen gastrin receptor antagonists, as an alternative to functional studies. We have characterized the receptors on parietal cell suspension from rabbit gastric mucosa and validated the method using both the CCKB and CCKA receptor agonists and antagonists. Human gastrin I (gastrin) (0.1 nM-4 microM) and sulfated cholecystokinin 26-33 (CCK-8) (0.01 nM-2 microM) dose-dependently augmented cytosolic Ca2+. The efficacies of the two agonists were similar, but the potency of CCK-8 (EC50 1.03 nM) was about 10-fold greater than that of gastrin (11 nM). Response to a submaximal dose of gastrin (50 nM) was dose-dependently blocked by the CCKB-receptor antagonists CAM-1028 (4-[[2-[[3-(1 H-indol-3-yl)-2-methyl-1-oxo-2-[[[1,7,7-trimethylbicyclo[2, 2,1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl]amino-4-oxo -[1 S-1 alpha, 2 beta [S'(S')4 alpha]]-butanoate-N-methyl-D-glucamine) (IC50 1.9 nM), L-365,260 (3 R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1 H-1, 4-benzodiazepin-3-yl)-N'-(3-methylphenyl)urea) (IC50 10 nM) and spiroglumide ((R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4.5]decan -8-yl)-5-oxopentanoic acid) (IC50 2 microM). The results were in agreement with those obtained from binding studies in guinea-pig cortical membranes. The model was employed to optimize the synthesis of a new class of spiroglumide analogues which led to a new molecule, (S)-4-¿(R)-4'-(3,5-dichlorobenzoylamino)-5'-(8-azaspiro[4.5] decan-8-yl)-5'-oxo)-pentanoylamino-5-(1-naphthylamino)-5-oxo pentanoic acid (CR 2622), whose potency was about 100-fold greater than that of spiroglumide. CR 2622, as well as the other CCKB receptor antagonists tested, exhibited no effect on basal [Ca2+]i. The simplicity and the reproducibility of this method suggest that it is a useful model to screen gastrin and antigastrin activity in parallel or as an alternative to binding studies.
authors.affiliation: Rotta Research Laboratorium S.p.A., Monza (MI), Italy.
authors.forename: O
authors.initials: O
authors.lastname: Letari
authors.forename: L
authors.initials: L
authors.lastname: Mennuni
authors.forename: L
authors.initials: L
authors.lastname: Revel
authors.forename: S
authors.initials: S
authors.lastname: Colombo
authors.forename: F
authors.initials: F
authors.lastname: Makovec
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D014151:Anti-Anxiety Agents / Q000494:pharmacology; D001570:Benzodiazepinones / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020109:Devazepide; D004305:Dose-Response Relationship, Drug; D016257:Fura-2 / Q000378:metabolism; D005755:Gastrins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008536:Meglumine / Q000031:analogs & derivatives / Q000494:pharmacology; D010295:Parietal Cells, Gastric / Q000187:drug effects / Q000378:metabolism*; D010671:Phenylurea Compounds; D011817:Rabbits; D011949:Receptors, Cholecystokinin / Q000235:genetics / Q000378:metabolism*; D012844:Sincalide / Q000037:antagonists & inhibitors / Q000494:pharmacology
chemical_list: D014151:Anti-Anxiety Agents; D001570:Benzodiazepinones; D005755:Gastrins; D006727:Hormone Antagonists; D007211:Indoles; D010671:Phenylurea Compounds; D011949:Receptors, Cholecystokinin; C065600:PD 135158; C058121:L 365260; D008536:Meglumine; D020109:Devazepide; D012844:Sincalide; D002118:Calcium; D016257:Fura-2