We propose to test the hypotheses that parathormone degradation in the parathyroid is an integral component of its calcium-controlled secretory system, that the rate of cellular hormone destruction is inversely related to its rate of secretion, and that the secretion of peptide fragments of parathormone is a direct result of this physiologic process. We propose to identify the enzymes responsible for the cleavages of the hormone peptide chain which produce major hormone fragments such as the 34-84 and 37-84 carboxy-terminal peptides. We used radioimmunoassay and gel electrophoresis to demonstrate parathormone fragments in bovine parathyroid glands and cells. The peptides will be identified using radioisotope microsequencing methods. The rate of fragment formation as a function of secretion will be studied using dispersed bovine parathyroid cells and primary monolayer cell cultures - a system newly developed by the P.I. Secretion will be altered by varying calcium, and fragment content will be assayed. High performance liquid chromatography will separate fragments derived from cells and those from culture media to determine if they are the same peptide species. We have shown that cathepsin B cleaves parathormone to produce the 37-84 fragment, the 1-30 fragment and others, and that cathepsin D cleaves PTH to form primarily the 35-84 and the 1-34 fragments. Hormone fragments, produced by these enzymatic digestions of parathormone will be further identified, isolated, and their biological and immunological properties studied. Enzymes which produce the same hormone fragments as those found in tissue and cells will be tested for their roles in situ. Parathyroid cells will be maintained in culture in the presence of non-toxic protease inhibitors such as leupeptin and pepstatin and the effect of these agents on general protein turnover and production of parathormone fragments will be assessed. The results will define the importance of parathormone turnover in various secretory states. With the cell culture system, we hope to determine if hormone turnover is abnormally depressed or uncontrolled in cases of chronic stimulation of the parathyroid as occurs in secondary hyperparathyroidism resulting from renal failure. Such information might help to develop effective treatments in such cases.
| MacGregor, R R; McGregor, D H; Lee, S H et al. (1986) Structural analysis of parathormone fragments elaborated by cells cultured from a hyperplastic human parathyroid gland. Bone Miner 1:41-50 |
