Studies on Phosphoinositide Compartmentalization in WRK-1 Cells

我们目前对细胞代谢事件调节的了解受到我们在体内复制亚细胞组织成分的能力的限制。在目前的提案中，我们概述了一系列实验，旨在研究大鼠细胞系WRK-1中激素响应的肌醇磷脂周期的组织。我们以前报道过在这些细胞中同时存在对加压素敏感和不敏感的磷脂酰肌醇，最近的数据也表明了周期的物理分隔。我们目前的目标有三个：1)使用两种不同的激动剂，血管加压素和缓激肽，我们将确定共同的或不同的磷脂酰肌醇脂池参与对不同刺激的循环代谢反应。这些实验的结果将表明该受体与激素反应的脂类有多么密切的联系。2)为了确定肌醇磷脂循环在多大程度上是细胞内的封闭代谢现象，我们将进行实验，以测试外源底物中间体是否参与激素刺激的循环。在这种情况下，否定的结果将表明循环中的所有酶都以这样的方式排列，即来自一个反应的产物被直接引导到途径中的下一个酶。3)我们将使用完整细胞和破碎细胞方案相结合的方法来研究在周期过程中刺激磷脂酰肌醇再合成的机制。我们将确定激素诱导的磷脂酰肌醇合成增加是底物浓度增加还是酶激活的结果，还是两者兼而有之。这些研究的综合结果应该可以深入了解肌醇磷脂循环的组织方面，让我们更准确地了解参与细胞调控的生化事件。多年来，人们一直认识到，激素跨膜信号的主要途径之一涉及细胞膜特定磷脂成分的分解。摩纳哥博士之前的工作已经证明，膜中只有一小部分总磷脂容易受到激素刺激的破坏。因此，只有该部分能够在细胞对激素的反应中充当第二信使的来源。这项研究的继续是为了进一步鉴定实际上将细胞膜磷脂划分为激素敏感和不敏感组分的机制。这项研究的结果将有助于我们从根本上理解细胞对细胞外信号做出反应的复杂机制。

Our present understanding of the regulation of cellular metabolic events is limited by our ability to reproduce subcellular organizational components in vivo. In the present proposal, we outline a series of experiments designed to investigate the organization of the hormone- responsive phosphoinositide cycle in the rat cell line, WRK-1. We have previously reported the existence of both vasopressin-sensitive and insensitive phosphoinositides in these cells, and more recent data suggests a physical compartmentation of the cycle, as well. Our present goals are three fold: 1) Using two different agonists, vasopressin and bradykinin, we will determine whether a common or distinct phosphoinoitide lipid pool is involved in the cyclic metabolic response to different stimuli. The results of these experiments will indicates how closely the receptor is associated with hormone-responsive lipid pools. 2) To determine the degree to which the phosphoinositide cycle is a closed metabolic phenomenon within the cell, we will carry out experiments designed to test whether or not exogenous substrate intermediates can participate in hormone-stimulated cycling. A negative result in this instance would suggest that all the enzymes of the cycle are arranged in such a manner that the product from one reaction is channeled directly to the next enzyme in the pathway. 3) We will examine the mechanism by which phosphatidylinositol resynthesis is stimulated during cycling using a combination of whole cell and broken cell protocols. We will determine whether the hormone induced increase in synthesis of phosphatidylinositol is the result of increased substrate concentrations or enzyme activation or both. The combined results of these studies should provide insight into the organizational aspects of phosphoinositide cycling, giving us a more accurate picture of the biochemical events involved in cellular regulation. It has for a number of years been appreciated that one of the major pathways of transmembrane signaling by hormones involves the breakdown of particular phospholipid components of the cell membrane. Dr. Monaco's prior work has demonstrated that only a fraction of the total phospholipid present in the membrane is susceptible to hormone- stimulated breakdown. Thus, only that portion is capable of serving as a source of second messenger in the cellular response to hormone. The continuation of this research is directed toward further identification of the mechanisms that, in effect, compartmentalize the cell membrane phospholipids into hormone-sensitive and insensitive fractions. The results of this research will contribute to our fundamental understanding of the complex mechanisms by which cells respond to extracellular signals.