This investigation will examine the role of diglyceride and glutathione metabolism in the regulation of parathyroid cell function. Collagenase-dispersed bovine parathyroid cells will be used throughout. The studies will focus initially on the role of calcium-induced changes in diglyceride formation and resultant effects on phosphatidyl-inositol levels, protein phosphorylation and parathyroid hormone secretion. The approach will be to assess the rate of radiolabeled glycerol incorporation into diglyceride and related metabolites. Lipids will be separated by means of thin layer and high performance liquid chromatography and radiolabel content determined by liquid scintillation spectroscopy. The metabolism of the synthetic diglyceride 1-oleoyl-2-acetylglycerol by the parathyroid cells into phosphatidic acid phosphatidylinositol will be determined. Protein phosphorylation/dephosphorylation patterns of cells incubated at various extracellular calcium ion concentrations or cells treated with phorbol myristate acetate, synthetic diglyceride or isoproterenol will be evaluated. The phosphoproteins will be separated by two-dimensional electrophoresis and visualized by radioautography. Proteins in which the phosphorylation/dephosphorylation changes coincide with changes in hormone secretion will be selected for further evaluation and characterization. Multiple phosphorylation sites within secretion-associated proteins will be evaluated by tryptic digestion and separation of the phosphopeptides by thin layer peptide mapping and high performance liquid chromatography. Specific phosphoamino acids will be identified by acid hydrolysis followed by two-dimensional thin layer electrophoresis. The subcellular localization of secretion-associated phosphopeptides will be evaluated. The participation of myosin light chain phosphorylation in the control of hormone secretion will be determined. The involvement of calcium-mediated changes in hexose monophosphate shunt activity and reduced glutathione levels in hormone secretion will be determined. The role of glutathione reductase in hormone secretion will be evaluated by inhibition with BCNU. The contribution of mitochondria (NADPH sensitive) and microsomal (reduced glutathione sensitive) calcium uptake systems to parathyroid cell calcium homeostasis and parathyroid hormone secretion will be measured. The results will be integrated to provide information on the manner in which changes in extracellular calcium influence the participation of diglyceride-activated protein kinase C, cyclic AMP-dependent protein kinase and reduced glutathione in the control of parathyroid hormone secretion.
