This proposal examines two hypotheses concerning the function of the mammalian parathryoid: 1) adaptation to changing conditions of calcium concentration and vitamin D status over periods in excess of 1 week involves an important contribution of a cellular system for the degradation of parathyroid hormone (PTH), in addition to direct effects of the change on the process of excytosis. 2) The degradative system which participates in adaptation is not lysosomal, but is a previously unreported degradative system resident in the Golgi and newly formed secretory vesicles. In order to test these hypotheses, a new tissue model will be used: cultures of parathyroid cell aggregates which maintain calcium regulated PTH secretion for several weeks, called organoids. The first goal is to examine the amount of PTH which is degraded under culture conditions which alter hormone secretion, compared to the amount which is synthesized. The second goal is to characterize the degradative system which responds to changes in secretory activity. Organoids will be cultured under different conditions and then incubated with redioactive amino acids. Changes in the amount of secreted hormone fragments will indicate effects on the putative Goli/vesicle pathway. Responses of the lysosomal degradative system will be examined as well. The second major goal is to separate and characterize the enzymes involved in the newly described system, and to provide experimental evidence of their subcellular location(s). Fractions of parathyroid homogenates will be prepared and incubated with radioactive PTH, and PTH fragments, produced will be indentified using HPLC, peptide mapping, and microsequence analyses. Active fractions will be charactaerized morphologically and enzymatically. Isolation of the cleavage enzymes(s) will be performed, antibodies will be generated, and immunocytochemical analyses will be used to establish the identity of the cellular organelle(s) responsible for cleavage. The results of these studies may provide information relevant to control of secondary hyperparathyroidism in cases of renal failure.
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