The specific aim of this proposal is to examine the hypothesis that proteolytic enzymes determine the duration and extent of action of locally released peptides in the stomach. These peptide degrading enzymes are either ectopeptidases (enzymes bound to the external cell surfaces) or intracellular lysosomal enzymes in the target cells for the peptides. These enzymes differentially degrade larger and smaller forms of the peptides. The smaller naturally occurring molecular forms of gastrin releasing peptide (GRP), GRP-10, and of somatostatin, SS-14, are preferentially degraded by neutral endopeptidase (NEP), an ectopeptidase. Degradation of the larger forms (GRP-27 and SS-28) requires receptor binding and internalization, leading to lyosomal degradation. The internalization and degradation rates may differ for receptor subtypes. The physiological importance of NBP in regulating the gastrin-regulating actions of arterially infused large and small forms of GRP and somatostatin will be determined in the perfused stomach. The role of NBP in regulating the relative proportions of these peptides released into gastric venous blood from gastric tissue stores also will be examined. The degradation products of large and small forms of GRP and somatostatin during surface or intracellular degradation and the structural requirements of these peptides for degradation by NEP and by lysosomal enzymes will be studied by biochemical methods. Morphological studies with specific antibodies will reveal the cellular distribution of NEP compared with that of GRP and somatostatin receptor subtypes in the gastric mucosal and muscle layers. Ultrastructural analyses will reveal whether there is a close physical association between NEP and neuropeptide receptors in native and transfected cells. The necessity for both enzyme and receptor to be present on the same cell to result in rapid degradation of the hormonal ligand will be studied in cells transfected with enzyme and/or receptors. Cells expressing receptors but not NBP may require receptor internalization in order to degrade GRP and somatostatin. Manipulation of receptor internalization should alter ligand uptake and degradation in such cells. Finally, inhibition of receptor/ligand internalization may prolong the stimulatory effects of GRP upon Swiss 3T3 cells and proliferative gastric mucosal cells. These studies will provide basic information about mechanisms by which neuropeptide and paracrine peptide signals are terminated within their target tissues.
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