CYTOSOLIC CA2-CELLS - A NOVEL METHOD TO SCREEN GASTRIN RECEPTOR ANTAGONISTS( EVALUATION IN RABBIT PARIETAL)

We have evaluated the application of the fura-2 method to detect cytos olic 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 c ell suspension from rabbit gastric mucosa and validated the method usi ng both the CCKB and CCKA receptor agonists and antagonists. Human gas trin I (gastrin) (0.1 nM-4 mu M) and sulfated cholecystokinin 26-33 (C CK-8) (0.01 nM-2 mu M) dose-dependently augmented cytosolic Ca2+. The efficacies of the two agonists were similar, but the potency of CCK-8 (EC(50) 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-dependent ly blocked by the CCKB-receptor antagonists CAM-1028 [[2-[13-(1H-indol -3-yl)-2-methyl-1-oxo-2-[[[1,7,7- mino]propyl]amino]-1-phenylethyl]ami no-4-oxo-[1S-1 alpha,2 beta[S'(S')4 alpha]]-butanoate-N-methyl-D-gluca mine) (IC50 1.9 nM), L-365,260 (+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phe nyl-1H-1,4- benzodiazepin-3-yl)-N'-(3-methylphenyl)urea) (IC50 10 nM) and e((R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4,5] decan-8-yl)-5-o xopentanoic acid) (IC50 2 mu M). The results were in agreement with th ose obtained from binding studies in guinea-pig cortical membranes. Th e model was employed to optimize the synthesis of a new class of spiro glumide analogues which led to a new molecule, -pentanoylamino-5-(1-na phthylamino)-5-oxopentanoic acid (CR 2622), whose potency was about 10 0-fold greater than that of spiroglumide. CR 2622, as well as the othe r CCKB receptor antagonists tested, exhibited no effect on basal [Ca2](i). The simplicity and the reproducibility of this method suggest th at it is a useful model to screen gastrin and antigastrin activity in parallel or as an alternative to binding studies.