HORMONAL CONTROL OF MITOCHONDRIAL METABOLISM

本研究的总体目标是阐明作用机制 胰高血糖素和肾上腺素能激素的能力， 刺激肝内造血和尿素生成。 测量 分离的肝细胞和灌注的谷氨酸和天冬氨酸浓度 肝脏结合碳通量的估计表明， 谷氨酸-天冬氨酸线粒体转运蛋白的参数可以是 由胰高血糖素和α-肾上腺素能激动剂改变。 这些激素 刺激：1）从乳酸盐中生成尿素，2）尿素生成，和3） 减少了从胞质溶胶到线粒体的等效转运。 所有三 肝脏中的过程涉及天冬氨酸从线粒体的转运 进入细胞质。 因此，注意力将集中在这方面， 线粒体代谢 我们还将评估 α-甘油磷酸穿梭是由激素刺激继发于 细胞质游离Ca++增加。 游离Ca++将在 含有“quin 2”的细胞胞质溶胶，这是一种Ca++指示剂荧光染料，可以 被困在肝细胞里 为了确定 Ca++是由Ca++依赖性蛋白激酶介导的，我们将比较 完整细胞中线粒体代谢对监测的 在存在和不存在以下物质的情况下，游离胞质Ca++水平 三氟拉嗪，钙调素拮抗剂。 苹果酸-天冬氨酸穿梭的细胞代谢物和 将在存在和不存在以下情况下测量α-甘油磷酸穿梭： 激素与不同水平的底物的同源性（即， 乳酸盐）、尿素生成（即NH3）和还原当量转运（即 乙醇）。 通过谷氨酸-天冬氨酸的通量的依赖性 转运蛋白和通过α-甘油磷酸穿梭代谢物 将在广泛的通量范围内进行评估，以确定 激素作用的靶点。 类似的实验将在 灌注的心脏 苹果酸-天冬氨酸穿梭的组分将 在存在和不存在去甲肾上腺素的情况下进行监测， 灌注液乳酸盐浓度和心脏功。 进一步的实验将 旨在更精确地识别激素 刺激靶蛋白，并可能涉及分离特定酶或 载体蛋白

The overall objective of this study is to elucidate the mechanism of action of glucagon and adrenergic hormones with respect to their ability to stimulate gluconeo-genesis and ureagenesis in liver. Measurements of glutamate and aspartate concentrations in isolated hepatocytes and perfused livers combined with estimates of carbon flux suggest that the kinetic parameters of the glutamate-aspartate mitochondrial transporter may be altered by glucagon and Alpha-adrenergic agonists. These hormones stimulate: 1 ) gluconeogenesis from lactate, 2) ureagenesis, and 3) reducing equivalent transport from cytosol to mitochondria. All three processes in the liver involve transport of aspartate from the mitochondria into the cytosol. Thus, attention will be focussed on this aspect of mitochondrial metabolism. We will also evaluate the possibility that the Alpha-glycerophosphate shuttle is stimulated by the hormones secondary to an increase in cytosolic free Ca++. Free Ca++ will be monitored in the cell cytosol with "quin 2", a Ca++ indicator fluorescent dye which can be trapped inside liver cells. In order to determine whether the effects of Ca++ are mediated by Ca++ dependent protein kinases, we will compare the sensitivity of mitochondrial metabolism in intact cells to the monitored levels of free cytosolic Ca++ in the presence and absence of trifluoperazine, a calmodulin antagonist. Cellular metabolites of the malate-aspartate shuttle and the Alpha-glycerophosphate shuttle will be measured in the presence and absence of hormones with varied levels of substrates of gluconeogenesis (i.e. lactate), ureagenesis (i.e. NH3), and reducing equivalent transport (i.e. ethyl alcohol). The dependence of flux through the glutamate-aspartate transporter and through the alpha-glycerophosphate shuttle on metabolites will be evaluated over a wide range of fluxes in order to identify the target of the hormone action. Similar experiments will be carried out in perfused hearts. The components of the malate-aspartate shuttle will be monitored in the presence and absence of norepinephrine while varying perfusate lactate concentration and heart work. Further experiments will be designed to identify more precisely the mechanism by which the hormones stimulate target proteins and may involve isolation of specific enzymes or carrier proteins.