INTEGRATED VASCULAR SMOOTH MUSCLE METABOLISM

血管平滑肌（VSM）的综合代谢起着中心作用， 在所有细胞功能中的作用，特别是维持离子 梯度和血管张力。 我们认为VSM的一个核心问题是 生物能量学是碳水化合物分解代谢是否 如果是这样的话，什么是机械基础， 分隔 我们将严格审查的一般概念， 葡萄糖中间代谢的细胞内区室化， 设计实验，进一步定义反应的概念- 扩散概念 我们提出了一种使用强大技术的新方法 还没有用于平滑肌能量代谢的研究。 拟议的项目将产生重要的新信息， 对细胞溶质这一重要问题的研究达到了一个新的水平， 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 适用于其他组织。 拟议的工作利用了一系列 生物化学和生理学技术，包括31 P和13 C NMR 光谱学来研究猪的颈动脉。 第一个具体目标 严格审查是否这种明显的划分是一个 允许独立调节流量的细胞内区室化 这两条路。 它有三个组成部分：（a）区别使用 13 C标记的葡萄糖和糖原，我们将检查是否 糖酵解和糖原分解的中间体混合，正如预期的那样， 相对长寿命的中间体在小细胞中扩散; B）使用 糖酵解的实际速率的定量测量， 糖原分解，我们将确定这两种途径的速率是否 可以独立监管--如果 c）我们将检查细胞是否存在 异质性可以解释明显的区室化。 第二 具体目标的特点是糖原周转的模式， 对具体目标1的结果的解释， 确定糖原水平调节的性质：a）确定是否 最近合成的糖原的葡糖基单元是第一个 B）确定糖原合成和降解是否可以 同时发生。 第三个具体目标使我们能够测试这些 分隔的途径可能与代谢的其他组分相互作用， 特别是脂质利用的途径：a）我们将通过以下方式确定 外源性脂质影响糖原分解的机制是什么， 糖酵解; B）我们将测试是否改变葡萄糖和糖原（通过脂质 底物）导致代谢物区室化和途径改变 通量使用具体目标#1中的方法。 因此， 具体目标是密切相关的，最后，提供 获得的关于脂质代谢与 糖酵解和糖原分解，将用于开发进一步的具体 这些系统的操作，这将使我们能够严格定义 胞质区室化的性质和限制。

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.