HORMONAL CONTROL OF MITOCHONDRIAL METABOLISM

这项研究的总体目标是阐明其作用机制。 胰高血糖素和肾上腺素能激素的能力 促进肝脏的糖异生和尿失禁。测量 离体肝细胞中谷氨酸和天冬氨酸的浓度 肝脏结合碳通量的估计表明， 谷氨酸-天冬氨酸线粒体转运体的参数可以是 被高血糖素和α-肾上腺素能激动剂改变。这些荷尔蒙 刺激：1)乳酸的糖异生，2)尿失禁，3) 减少从细胞质到线粒体的等量运输。三个都是 肝脏中的过程涉及从线粒体运输天冬氨酸。 进入细胞质。因此，人们的注意力将集中在这方面 线粒体代谢。我们还将评估 α-甘油磷酸盐穿梭受到继发性激素的刺激 细胞内游离钙离子浓度升高。游离Ca++将在 细胞胞浆中加入“Quin 2”，一种钙离子指示剂，可以 困在肝细胞内。以确定是否影响了 Ca~(++)是由Ca~(++)依赖蛋白激酶介导的，我们将比较 完整细胞线粒体代谢对监测的敏感性 细胞内游离Ca~(++)水平在细胞内存在和不存在时的变化 三氟拉嗪，钙调素拮抗剂。 苹果酸-天冬氨酸穿梭的细胞代谢产物和 α-甘油磷酸盐穿梭将在存在和不存在时进行测量 具有不同糖异生底物水平的激素(即 乳酸)、尿失禁(即NH3)和减少等量转运(即 酒精)。通过谷氨酸-天冬氨酸的通量的依赖性 代谢物上的转运蛋白和通过α-甘油磷酸穿梭 将在广泛的焊剂范围内进行评估，以确定 荷尔蒙作用的靶标。类似的实验将在 灌流的心脏。苹果酸-天冬氨酸航天飞机的组件将是 在有无去甲肾上腺素的情况下进行监测 灌流液乳酸浓度和心脏工作。进一步的实验将 被设计成更准确地识别荷尔蒙 刺激靶蛋白，可能涉及分离特定的酶或 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.