We will study cellular controls involved in stimulating secretion of pepsinogen by peptic glands and located in the mucosa of the distal esophagus of Rana catesbeiana. These gland contain no acid-secreting cells and thus provide a model for studying peptic cells independently of acid secretion. We use gland-bearing sheets of esophageal mucosa vectorially mounted in chambers in which the lumen and the sub-mucosa can be separately perfused. Moreover, dispersed peptic cell preparations of high purity, viability and normal responsiveness can be routinely obtained from these glands. Receptors for muscarinic, peptidergic and Beta-adrenergic receptors will be characterized as to density, affinity, desensitization, subunits, messengers and interactions using ligand-binding techniques in parallel with pharmacologic studies measuring cell messengers and secretion. Cellular responses to agonist stimulation which include enhanced phosphatidyl inositol metabolism; N protein activation, Ca2+ fluxes, cyclic nucleotide changes, and protein phosphorylation will be measured. Cell messengers - cAMP, Ca2+ and protein kinase C - each separately activated, have been shown to stimulate secretion independently of receptors and to interact synergistically. Synergism is Ca2+-dependent. We plan to study interactions among messengers and the influence of messengers alone and in combination on receptor function and binding, and on """"""""down stream"""""""" events, such as protein phosphorylation. Experiments are designed to quantitate the effects of each messenger and to study transients of secretion. In winter when the frogs hibernate, pepsinogen secretion is much diminished due to reduction or loss of receptor activity an reduction in cellular pepsinogen content. However, the cells can still be fully stimulated by combinations of messengers and can thus be usefully studied throughout all seasons. We plan to study the receptor modifications associated with seasonal changes and to determine whether these are due to environmental cues or to lack of food or both. Definition of cellular controls at the levels proposed here will provide further understanding of the steps between stimulation and secretion of cell products. Such information should be widely applicable to many cells that secrete hormones, peptides and proteins.
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