REGULATION OF PARATHYROID FUNCTION

这项研究将检验甘油二酯和谷胱甘肽的作用。 甲状旁腺细胞功能调节中的代谢。 胶原酶分散的牛甲状旁腺细胞将贯穿始终。 这些研究最初将集中在钙诱导的改变在 二甘油三酯的形成及其对磷脂酰肌醇的影响 水平、蛋白质磷酸化和甲状旁腺激素分泌。这个 方法是评估放射性标记甘油的参入率 转化成二甘油酯和相关的代谢物。脂质将通过以下方式分离 薄层和高效液相色谱方法 液体闪烁光谱分析测定放射性标记物含量。这个 合成双甘油酯1-油酰基-2-乙酰甘油的代谢 甲状旁腺细胞转化为磷脂酸将磷脂酰肌醇 下定决心。细胞的蛋白质磷酸化/去磷酸化模式 在不同细胞外钙离子浓度或细胞内孵育 用佛波醇肉豆蔻酸酯、合成双甘油酯或 将对异丙肾上腺素进行评估。磷蛋白将通过以下方式分离 双向电泳法和放射自显影技术。 磷酸化/去磷酸化变化一致的蛋白质 随着激素分泌的变化，将被选作进一步评估 和角色塑造。内含多个磷酸化位点 分泌相关蛋白将通过胰酶消化和 薄层肽谱和高效毛细管电泳法分离磷酸小肽 高效液相色谱。特定的磷酸氨基酸将是 通过酸解和二维薄层进行鉴定 电泳法。分泌相关基因的亚细胞定位 将对磷酸肽进行评估。肌球蛋白轻链的参与 控制激素分泌的磷酸化将被确定。 钙离子参与一磷酸己糖分流的变化 激素分泌的活性和谷胱甘肽水平的降低将是 下定决心。谷胱甘肽还原酶在激素分泌中的作用将是 用BCNU进行抑制评价。线粒体的贡献(NADPH 敏感)和微粒体钙摄取(还原型谷胱甘肽敏感) 甲状旁腺细胞钙稳态和甲状旁腺激素系统 分泌物将被测量。结果将综合起来，以提供 关于细胞外钙变化方式的信息 影响二甘油三酯活化蛋白激酶C的参与， 对照组中cAMP依赖的蛋白激酶和还原型谷胱甘肽 甲状旁腺激素的分泌。

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.