Studies on Phosphoinositide Compartmentalization in WRK-1 Cells

我们目前对细胞代谢事件调节的理解受到我们在体内复制亚细胞组织成分的能力的限制。在本提案中，我们概述了一系列旨在研究大鼠细胞系WRK-1中激素响应磷酸肌肽周期组织的实验。我们以前曾报道过这些细胞中存在抗利尿激素敏感和不敏感的磷酸肌苷，最近的数据表明该周期也存在物理分隔。我们目前的目标有三个方面：1)使用两种不同的激动剂，抗利尿激素和缓激肽，我们将确定一个共同的或独特的磷脂磷脂脂池是否参与对不同刺激的循环代谢反应。这些实验的结果将表明受体与激素反应性脂质池的关系有多密切。2)为了确定磷酸肌肽循环在细胞内是一种封闭的代谢现象的程度，我们将进行旨在测试外源性底物中间体是否可以参与激素刺激的循环的实验。在这种情况下，阴性结果表明循环的所有酶都以这样一种方式排列，即一个反应的产物直接输送到途径中的下一个酶。3)我们将通过使用全细胞和破细胞方案的组合来研究在循环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的增加是底物浓度增加或酶激活的结果，还是两者兼而有之。这些研究的综合结果应该提供对磷酸肌苷循环的组织方面的见解，使我们更准确地了解细胞调节中涉及的生化事件。多年来，人们已经认识到，激素跨膜信号传导的主要途径之一涉及细胞膜特定磷脂成分的分解。莫纳科博士先前的研究表明，细胞膜中只有一小部分磷脂容易受到激素刺激的破坏。因此，在细胞对激素的反应中，只有这一部分能够作为第二信使的来源。这项研究的继续是为了进一步确定将细胞膜磷脂划分为激素敏感和不敏感部分的机制。这项研究的结果将有助于我们对细胞响应细胞外信号的复杂机制的基本理解。

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.