A variety of neuropeptides are involved in the central control of gastro- intestinal (GI) functions. Amongst these peptides (which are localized in CNS neurons) are cholecystokinin (CCK; CCK-8; CCK-(26-33)) and gastrin- releasing peptide (GRP-(1-27)). The role which these peptides play is linked to the mechanisms which regulate their biosynthesis and metabolism. Since the biosynthetic rates of peptide precursors are generally determined by cellular contents of their encoding mRNAs, and metabolic rates by the specific peptidases associated with their metabolism, insight into the mechanisms controlling CCK mRNA and GRP mRNA levels and CCK and GRP metabolism in neuronal systems can further elucidate the physiological roles of CCK and GRP ( and related peptides) in the brain with respect to regulation of GI tract function and control. Therefore, the central question of this project is: how are CCK mRNA and GRP mRNA levels and CCK and GRP metabolism regulated in neuronal systems? Our hypothesis is that gene expression and metabolic enzymes associated with CCK- and GRP-producing neurons can be affected by transmitters, neuropeptides and steroid hormones. Transmitters and peptides bind to membrane receptors and activate intracellular messenger systems, while steroid hormones have intracellular soluble receptors.
The specific aims are (1) to characterize which second messenger systems influence levels of CCK mRNA and GRP mRNA, levels of proteolytic enzymes and levels of CCK and GRP (including various forms); (2) to characterize which specific proteolytic enzymes are involved in the metabolism of CCK and GRP in cyto and in vivo; (3) which transmitters and neuropeptides are involved in regulating these effects, and (4) which steroid hormones regulate CCK mRNA and GRP mRNA levels and metabolic enzymes. To meet these aims, the regulation of CCK mRNA and GRP mRNA and metabolic enzymes will be studied in cyto in a clonal human neuroblastoma cell line (derived from autonomic nervous system) which has high levels of CCK and GRP gene expression secretes CCK upon stimulation and in vivo in the rat brain. By combining mRNA measurements, CCK and GRP levels and metabolic enzyme measurements we are very confident that we will be successful in determining how CCK and GRP are regulated in neuronal systems.
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