ROLE OF CALCIUM IN THE REGULATION OF ENERGY METABOLISM

这个项目是为了评估生理意义的 钙离子在调节能量代谢中的作用。我们有 应用荧光螯合剂方案，这是 为测量细胞内游离钙而开发的，到 线粒体基质游离钙((Ca~(2+)m的测定 使我们能够直接研究(Ca~(2+))m和 基质酶丙酮酸脱氢酶的激活 首次在分离的大鼠心肌线粒体负载的研究中 使用INDO-1。发现激活的KO.5为300 NM钙： 该值不受程序的影响，例如省略镁离子， Na+，它改变了钙浓度梯度的大小 穿过线粒体膜。我们还研究了学位 丙酮酸脱氢酶的激活与速率的关系 电刺激离体豚鼠心脏的实验研究 平行研究中，线粒体被快速地从刺激中分离出来 心脏，使用一种协议来防止丢失或获得 线粒体钙，并允许我们将这个数量与水平联系起来 脱氢酶的激活。作为脱氢酶的意义 激活在于NADH/NAD比率，该比率可维持在 面对上升的工作负荷，我们也测量了这一比例 饱和状态下大鼠心肌线粒体悬液中NADH/NAD的变化 和非饱和浓度的丙酮酸或2-羟基戊二酸 呼吸底物，在存在或不存在钙离子的情况下，以及 这与通过氧化磷酸化的通量相关联。 最后，我们检测了心肌线粒体的能力。 衰老的狗氧化底物，重点是脂肪酸) 作为生活方式的一种功能，即久坐与锻炼。

This project is designed to assess the physiological importance of calcium ions in the regulation of energy metabolism. We have applied the fluorescent chelating-agent protocol, which was developed for the measurement cytosolic free Ca2+, to the measurement of mitochondrial matrix free Ca ((Ca2+))m. This allowed us to study directly the relationship between (Ca2+)m and the activation of the matrix enzyme pyruvate dehydrogenase for the first time in studies with isolated rat cardiac mitochondria loaded with indo-1. The KO.5 for activation was found to be 300 nM Ca2: this value was unaffected by procedures, e.g. omission of Mg2+, Na+, which change the magnitude of the Ca concentration gradient across the mitochondrial membrane. We have also studied the degree of activation of pyruvate dehydrogenase as a function of the rate of electrical stimulation of isolated guinea pig hearts: in a parallel study, mitochondria were rapidly isolated from stimulated hearts, using a protocol which prevents loss or gain of mitochondrial Ca and allows us to relate this quantity to the level of dehydrogenase activation. As the significance of dehydrogenase activation lies in the NADH/NAD ratio which can be maintained in the face of elevated work -loads, we have also measured the ratio NADH/NAD in suspensions of rat heart mitochondria with saturating and non-saturating concentrations of pyruvate or 2-oxoglutarate as respiratory substrate, in the presence or absence of Ca2+, and correlated this with the flux through oxidative phosphorylation. Finally, we have examined the ability of cardiac mitochondria from senescent dogs to oxidize substrates with emphasis on fatty acids) as a function of lifestyle, viz-sedentary versus exercised.