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.