INTEGRATED VASCULAR SMOOTH MUSCLE METABOLISM

血管平滑肌(VSM)的整合代谢起着中枢作用 在所有细胞功能中的作用，特别是离子的维持 梯度和血管紧张度。我们认为VSM的一个核心问题 生物能量学是研究碳水化合物分解代谢是否 分割的，如果是这样，是什么机制基础 车厢分隔。我们将严格审查一般概念 葡萄糖中间代谢的细胞内区隔和 设计实验，从反应的角度进一步定义概念- 扩散概念。我们提出了一种使用强大技术的新方法 还没有用于研究平滑肌能量代谢的物质。 拟议的项目将产生重要的新信息，将使 对胞质这一重要问题的新水平的研究 车厢分隔。具体目标是专门针对VSM的，但 制定的方法和战略以及定义的概念将是 适用于其他组织。拟议的工作利用了一系列 包括31P和13C核磁共振在内的生化和生理技术 用光谱学研究猪颈动脉。第一个具体目标 严格检查这种明显的隔间是否是 允许独立调节通量的细胞内分隔 两条路中的一条。它有三个组成部分：a)使用差异 13C标记的葡萄糖和糖原，我们将检查 糖酵解和糖原分解混合物的中间体，如预期的那样 在小细胞中扩散的寿命相对较长的中间体；b)使用 定量测量实际糖酵解率和 糖原分解，我们将确定这两条途径的速率 可以独立监管-这在以下情况下尤其重要 通路是被分隔的；c)我们将检查一个细胞 异质性可以解释明显的区隔。第二 特定的目的表征了糖原周转的模式，即 对解释具体目标1的结果很重要，对 确定糖原水平调节的性质：a)确定 最近合成的糖原的糖基单位是第一个 B)确定糖原合成和降解是否可以 同时发生。第三个具体目标允许我们测试这些 分隔的通路可能会与新陈代谢的其他成分相互作用， 具体地说，脂质利用的途径：a)我们将通过 外源性脂质通过何种机制影响糖原分解和 糖酵解；b)我们将测试(通过脂质)改变的葡萄糖和糖原 底物)导致代谢物区隔和途径改变 使用特定目标#1中的方法的流量。因此，所有 具体目标是密切相关的，最后一个目标是提供 获得的关于脂代谢与 糖酵解和糖原分解，将被用来发展进一步的特异性 这些系统的操作将使我们能够严格定义 胞质分隔的性质和限度。

The integrated metabolism of vascular smooth muscle (VSM) plays a central role in all cellular functions especially the maintenance of ionic gradients and of vascular tone. We believe a central issue of VSM bioenergetics is the whether carbohydrate catabolic metabolism is compartmented and if so, what is the mechanistic basis for the compartmentation. We will rigorously examine the general concept of intracellular compartmentation of glucose intermediary metabolism and design experiments that define the concept further in terms of a reaction- diffusion concept. We propose a novel approach using powerful techniques that have not been used in the study of smooth muscle energy metabolism. The proposed project will yield important new information which will allow a new level of investigation into this important issue of cytosolic compartmentation. The specific aims are oriented specifically to VSM, but the methods and strategies developed and concepts defined will be applicable to other tissues. The proposed work utilizes a range of biochemical and physiological techniques including 31P and 13C NMR spectroscopy to study hog carotid arteries. The first specific aim rigorously examines whether this apparent compartmentation is an intracellular compartmentation which allows independent regulation of flux of the two pathways. It has three components: a) Using differentially 13C-labelled glucose and glycogen, we will examine whether the intermediates of glycolysis and glycogenolysis mix, as expected for relatively long lived intermediates diffusing in small cells; b) Using quantitative measurements of the actual rates of glycolysis and glycogenolysis, we will determine whether the rates of these two pathways can be independently regulated - which is especially important if the pathways are compartmented; c) We will examine whether a cellular heterogeneity can account for the apparent compartmentation. The second specific aim characterizes the pattern of glycogen turnover which is important both to the interpretation of results in specific aim 1, also to define the nature of regulation of glycogen levels: a) Determine whether the most recently synthesized glucosyl units of glycogen are the first to be utilized; b) Determine whether glycogen synthesis and degradation can occur simultaneously. The third specific aim permits us to test how these compartmented pathways might interact with other components of metabolism, specifically the pathways for lipid utilization: a) We will determine by what class of mechanism exogenous lipids affect glycogenolysis and glycolysis; b) We will test whether altered glucose and glycogen (by lipid substrates) results in altered metabolite compartmentation and pathways fluxes using the approaches in specific aim #1. Hence the all of the specific aims are closely inter-related, and the last, providing information obtained about the interaction of lipid metabolism with glycolysis and glycogenolysis, will be used to develop further specific manipulations of those systems which will allow us to rigorously define the nature of and limits to cytosolic compartmentation.