The specific aims of these studies are to examine the mechanisms by which GI peptide producing cells direct the regulated release of peptides to their basolateral and/or apical surfaces. We will be examining the physical and functional compartmentalization of gastrointestinal (GI) peptides that are co-processed within the same cell. Specifically peptide tyrosine-tyrosine (PYY) and oxyntomodulin-(enteroglucagon)-like peptides (GLP) from the GI mucosa, and glucagon and glucagon like peptide-1 6- 37(NH2) from the pancreatic islets will be examined as model systems. We will investigate the control of differential release of these peptides, and whether the release of PYY and GLP or glucagon and GLP1 6-37 (NH2) are regulated at the site of release, such as at the apical or basolateral membranes, or intravesicularly. To allow the study of stimulus secretion coupling events in polarized epithelial cells we have developed a parental cell line from the rat colonic mucosa (ARCE-139) and have selected a thymidine kinase minus mutant which has been transfected with a plasmid conferring hygromycin resistance. With these cells as parental fusion partners we have established an acinar cell line (rCI-253-20), an insulin positive beta cell line (rCI-150) and a glucagon alpha cell line (rCI-120. With ARCE tk hmr-139 and rCI-120 cells we will investigate the dual packaging and polarized release of GI peptide containing secretory vesicles, namely those containing glucagon and GLP (pro-enteroglucagon) fragments and to glucagon and pro glucagon 72-108 amide. With these antibodies, and with our gp3067 anti-PYY and gp 1120 anti-oxyntomodulin antisera, we will also examine the possibility that distinct populations of mature secretory vesicles are released through the basolaterol or apical surfaces. With light and EM autoradiography it will also be determined, in situ, if there are receptors for PYY and GLP on the apical surfaces of the mucosal cells adjacent to the L-cells, suggesting a transcellular mode of action for PYY. Differential release of GLP1 6-37 (NH2)/glucagon/or PYY/enteroglucagon and their propeptide forms will be further examined both in primary cultured cells and cell lines by presentation of various secretagogues such as epinephrine, forskolin, bombesin, and oleate. These secretagogues will also allow us to test the possibility that secretory granule release is dependent on whether the stimulus for release is tat concentration of other GLP molecules besides oxyntomodulin (such as GLP1 6- 37 (NH2), GLP-1 or GLP-2), we will attempt to characterize the mature secretory granule by its vesicular content. Secretory granules will be purified and the prohormone content will be assayed after affinity purification.
