Cholecystokinin (CCK) has important physiological roles in gallbaldder contraction and release of pancreatic enzymes and potential effects on gastric acid secretion, gastric emptying and food intake. The relative importance of CCK-58, the largest molecular form of CCK isolated so far, is unknown for these actions. Sufficient amounts of canine, rat, and bovine CCK-58 will be isolated by reverse phase and ion-exchange PHLC for their chemical and biological analysis. Chemical analysis will allow accurate estimation of peptide amount, ensure that all components for bioactivity are intact, and allow evaluation of peptide modification during various bioassays. Three increasingly complex biological systems will be used to compare the activity of canine CCK-58 and CCK-8. The simplest system will be binding to brain, pancreatic and gallbladder membrane receptors. More complex interactions will be evaluated by in vitro bioassay using release of amylase from isolated acini and contraction of isolated gallbladder strips. The most complex system will be intact, conscious dogs in which actions on pancreatic function and gallbladder contraction, gastric secretion and gastric emptying will be compared for the different CCK forms. Rat, bovine, and canine CCK-58 bioactivities will be compared in the in vitro systems to determine whether differences in their amino termini cause changes in bioactivity. The structure of canine preproCCK will be determined by cDNA techniques, and synthetic peptides corresponding to regions outside CCK-58 will be used for antibody production. Radioimmunoassays specific for regions of proCCK outside CCK-58 will be used to detect peptides in intestinal extracts. These peptides will purified, sequenced, and synthetic duplicates made. The synthetic duplicates will be tested for specific binding in several tissues. Bioassays will be done on tissues shown to have specific binding. Hypotheses to be tested are: CCK-58 is the major endocrine cholecystokinin form in mammals; the relative molar potency of CCK-58 varies from other CCK molecular forms for many biological activities; the amino terminus of CCK-58 from various species shields the active carboxyl terminus to different extents due to variation in structure; that our understanding of the significance of cholecystokinin in putative actions such as inhibition of gastric acid secretion and gastric emptying will change once the actions of CCK-58 are studied; that gene products other than CCK have biological activity; and that degradation of CCK-58 is important in determining its activity. The major goal of these studies is to determine the physiological significance of CCK-58.
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