REGULATION OF PARATHYROID FUNCTION

本研究将探讨甘油二酯和谷胱甘肽的作用 甲状旁腺细胞功能的调节代谢。 整个过程将使用胶原酶分散的牛甲状旁腺细胞。 这些研究最初将集中在钙诱导的变化在 甘油二酯的形成及其对磷脂酰肌醇的影响 水平、蛋白磷酸化和甲状旁腺激素分泌。 的 方法是评估放射性标记甘油的掺入率 甘油二酯和相关代谢物。 脂质将通过 薄层和高效液相色谱法， 通过液体闪烁光谱法测定放射性标记物含量。 的 合成甘油二酯1-油酰基-2-乙酰基甘油的代谢 将甲状旁腺细胞转化为磷脂酸磷脂酰肌醇， 测定 细胞的蛋白质磷酸化/去磷酸化模式 在不同的细胞外钙离子浓度或细胞中孵育 用佛波醇肉豆蔻酸酯乙酸酯、合成甘油二酯或 将评价异丙肾上腺素。 磷蛋白将通过 二维电泳，并通过放射自显影法可视化。 磷酸化/去磷酸化变化一致的蛋白质 将选择激素分泌变化的患者进行进一步评估 和特征化。 多个磷酸化位点 分泌相关蛋白将通过胰蛋白酶消化进行评价， 用薄层肽图法和高效液相色谱法分离磷酸肽 高效液相色谱法 特定的磷酸氨基酸将是 通过酸水解然后二维薄层鉴定 电泳 分泌相关蛋白的亚细胞定位 将评估磷酸肽。 肌球蛋白轻链的参与 将测定磷酸化在控制激素分泌中的作用。 钙离子介导的磷酸己糖分流的改变 激素分泌中的活性和还原型谷胱甘肽水平将 测定 谷胱甘肽还原酶在激素分泌中的作用将是 用BCNU抑制评价。 线粒体（NADPH）的贡献 敏感）和微粒体（还原型谷胱甘肽敏感）钙摄取 甲状旁腺细胞钙稳态和甲状旁腺激素系统 将测量分泌。 结果将被整合以提供 关于细胞外钙离子变化方式的信息 影响甘油二酯活化蛋白激酶C的参与， 环腺苷酸依赖性蛋白激酶和还原型谷胱甘肽的对照 甲状旁腺激素分泌的影响。

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.