Primary hyperparathyroidism is a relatively common endocrine dysfunction with an incidence of 40-45 cases per 100,000 population. Secondary hyperparathyroidism is a manifestation of renal failure. There are over 100,000 patients on dialysis in the United States. Thus, disorders of parathyroid function impose a significant burden on health care management and loss from the labor force each year. The control of parathyroid hormone secretion is almost unique in that calcium ultimately inhibits secretion whereas in most other systems calcium stimulates secretion. This investigation focuses on molecular mechanisms that are involved in the control of calcium-mediated PTH secretion. The role of GTP-binding signal transducing proteins and protein kinase C in normal parathyroid secretion will be studied. Synthetic oligonucleotide probes will be used to select for DNA sequences in a normal bovine parathyroid expression library constructed in phage lambda gtl0. The DNA corresponding to the signal transduction proteins will be subcloned and sequenced by the dideoxy chain-termination method to deduce the types of GTP-binding proteins expressed in the parathyroid cell. This will establish whether the parathyroid cell contains signal transduction proteins common to other secretory tissues, or if there is a unique transducing protein to the parathyroid which can account for the reverse calcium sensitivity of hormone secretion. Based upon the derived amino acid sequences, peptides of specific sequence will be synthesized. These peptides will be used to generate polyclonal antisera specific to the individual signal transduction proteins in order to quantitate these proteins in parathyroid cells. The transducing proteins which serve as substrates for pertussis toxin and cholera toxin, and a toxin-insensitive protein linked to phospholipase C will be characterized. The effect of pertussis toxin and cholera toxin on calcium, magnesium and strontium-induced suppression of PTH secretion and changes in cytosolic calcium will be evaluated. The role of protein kinase C in parathyroid secretion will be evaluated by monitoring enzyme activity, and through the use of anti-peptide antisera specific for the three isoforms of protein kinase C. Sphingoid base metabolism as a function of extracellular calcium, magnesium and strontium will be evaluated for the production of protein kinase C inhibitors. These studies utilizing the parathyroid expression library, DNA sequencing and antipeptide antibodies will form a basis for the future examination of normal and abnormal parathyroid function.
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