The regulation of intestinal peptide-YY (PYY) synthesis and secretion has not been well studied. We have used fetal rat intestinal cells in culture to examine the intra- and extracellular factors controlling the production of PYY. Immunohistochemical analysis demonstrated a distinct population of cells containing immunoreactive PYY (IR-PYY). When examined by HPLC, the IR-PYY stored and secreted by fetal rat intestinal cell cultures eluted as a single moiety with the same elution time as synthetic rat PYY. Pro-PYY mRNA transcript levels and secretion of IR-PYY into the cell medium were increased by activation of protein kinase-A with either a long-acting cAMP analog or forskolin. In contrast, IR-PYY secretion only was stimulated in a synergistic fashion through calcium- and protein kinase-C-dependent pathways (stimulated with A23187 and phorbol myristate acetate, respectively). The intestinal endocrine peptide, gastric inhibitory peptide, and the neurocrine peptide, gastrin-releasing peptide, were found to stimulate IR-PYY secretion in a dose-dependent fashion, with significant effects observed at concentrations as low as 10(-8) and 10(-12) M, respectively (P less than 0.05-0.001). Cholecystokinin and vasoactive intestinal peptide were without effect on IR-PYY secretion at doses of 10(-12)-10(-6) M. The fatty acid sodium oleate and the cholinergic agonist bethanechol were also found to stimulate IR-PYY secretion, each at a concentration of 10(-4) M (P less than 0.001). The results of the present study indicate that the synthesis and secretion of PYY by the rat intestine is under the regulatory control of a wide variety of extracellular agents, mediated by several intracellular signalling pathways.