The overall hypothesis addressed in this work is that Ca-regulatory hormonal peptides, especially parathyroid hormone (PTH) and calcitonin gene-related peptide (CGRP), play important roles in regulation of gut function. PTH is a highly effective relaxant of GI smooth muscle. This project will examine in detail the nature of the GI PTH receptors and the second messengers involved. Rat GI smooth muscle and mucosa will be studied using standard ligand binding techniques, receptor chemical cross- linking, and autoradiography in order to compare GI receptors to classical PTH receptors in bone and kidney and to localize the receptors to specific GI cells. Second messengers to be examined include cyclic nucleotides, intracellular Ca++, and inositol phosphates. Also addressed is the hypothesis that PTH-related protein (PTHrP), a newly discovered tumor- derived peptide involved in humoral hypercalcemia of malignancy, is present normally in GI mucosal or muscle cells and acts locally as a paracrine or autocrine factor to regulate GI events. Molecular biological technology will be used to search for PTHrP mRNA in the gut and, by in situ hybridization histochemistry, to localize the mRNA to specific GI cells. Immunocytochemistry will be employed concurrently to localize PTHrP itself. Developmental changes in association with gestation, lactation, aging, and wound healing will be examined. The physiological significance of the effect of PTH-like peptides in the gut will be examined by studying nonabsorbable markers in vivo in the rat gut to determine the influence of these peptides on GI motility. CGRP, also known to affect GI function, especially contractility, also will be studied using the approaches outlined for PTHrP. Receptors in rat and pig GI tissue will be characterized, second messengers will be identified, and the localization of mRNA and the peptide itself will be studied using hybridization and immunocytochemical techniques. The studies will help elucidate the physiological importance of PTHrP, a new peptide functionally associated at present only with tumor tissue. The work ultimately should establish new roles for Ca-related peptides in the gut, clarify the mechanisms involved, promote our understanding of their roles in health and disease, and enable us to employ these peptides or their analogs as therapeutic agents.
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