ELECTRON PROBE ANALYSIS OF SUBCELLULAR CALCIUM IN LIVER

本课题的主要目的是研究细胞内钙离子的作用机制， 肝脏的调节。 这些实验的重点将是（a）确定 线粒体Ca浓度的生理范围和 Ca 2+在线粒体中的转运，特别是为了研究这一假设， 它用于调节基质游离Ca 2+和（B）以鉴定主要的 激活剂Ca 2+的细胞内来源，即，Ca存储被动员起来， 用加压素或α肾上腺素能激动剂刺激，导致 胞浆Ca 2+增加和关键酶如糖原的激活 磷酸化酶 这些研究的结果将有助于一个普遍的 了解其他上皮组织中的刺激-分泌偶联， 其中Ca 2+被认为起着重要作用，例如腺泡细胞， 胰腺外分泌和β细胞胰岛素分泌素。 线粒体、内质网（ER）和 将用电子探针微量分析（EPMA）测量核蛋白， 大鼠肝实质细胞，原位快速冷冻保存细胞 形态和离子分布。 亚细胞Ca分布将是 在来自刺激和未刺激的肝脏的细胞中测量，以确定 生理条件下线粒体和内质网中Ca浓度的范围 条件 本研究中使用的组合技术，即，一种新的原地快速 冷冻技术、冷冻切片术和电子探针，构成了一个独特的， 直接测量细胞内Ca分布的强大方法 在原地。

The major aim of this project is to investigate mechanisms of cellular Ca regulation in liver. The focus of these experiments will be (a) to define the physiological range of mitochondrial Ca concentrations and the role of Ca2+ transport in mitochondria, specifically to study the hypothesis that it serves to regulate matrix free Ca2+ and (b) to identify the major intracellular source(s) of activator Ca2+, i.e., Ca stores mobilized upon stimulation with vasopressin or alpha adrenergic agonists, resulting in an increase in cytoplasmic Ca2+ and activation of key enzymes such as glycogen phosphorylase. The results of these studies will contribute to a general understanding of stimulus-secretion coupling in other epithelial tissues, where Ca2+ is thought to play an essential role, such as acinar cells of the exocrine pancreas, and in Beta-cell insulin secretin. Total Ca concentration in mitochondria, endoplasmic reticulum (ER), and nuclein will be measured by electron probe microanalysis (EPMA) in parenchymal cells of rat liver, rapidly frozen in situ to preserve cell morphology and ion distribution. Subcellular Ca distribution will be measured in cells from stimulated and unstimulated liver to determine the range of Ca concentrations in mitochondria and ER under physiological conditions. The combined techniques to be used in this study i.e., a new in situ rapid freezing technique, cryoultramicrotomy and EPMA, constitute a unique and powerful approach for directly measuring the intracellcular Ca distribution in situ.