VASCULAR METABOLISM AND ITS RELATION TO FUNCTION

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Vascular smooth muscle (VSM) contractility, which underlies the regulation of circulation, is closely coupled to metabolism. We have shown that VSM metabolism is functionally compartmentalized, i.e., oxidative metabolism is strongly correlated with force whereas aerobic glycolysis is independently correlated with the Na-pump. Our central hypothesis is that this observed functional compartmentation is a reflection of the cytosolic compartmentation of metabolism in VSM. The testing of this hypothesis and its implications for VSM energetics, the coupling of metabolism with function and in particular, to our understanding of the mechanisms for control of VSM metabolism, forms the basis of this work. The first major goal is the elucidation of the compartmentation of glycolysis and glycogenolysis. Our studies will involve the determination of the metabolic fate of 14C from glycogen, the specific activities of glycolytic intermediates, and the mechanisms of its regulation. These studies will lead to the broader question of the nature of the substrate(s) for oxidative metabolism and its dependence on function. This will be addressed by measurement of oxygen consumption and CO2 production to establish the respiratory quotient, and, of the fate of 14C from various substrates. The second major specific aim involves elucidation of the functional compartmentation. The mechanism for the coupling of the Na-pump and aerobic glycolysis is poorly understood. Our hypothesis is that this coupling reflects the localization of a glycolytic enzyme cascade on the plasmalemma in close apposition to the Na+-K+ ATPase. This will be tested by quantitation of Na-pump function and aerobic glycolysis first in intact vessels, utilizing K+-electrodes to continuously monitor Na-pump function and high resolution fluorometric measurement of lactate. This will further be tested by cell fractionation studies including: localization and quantitation of the major oxidative and glycolytic enzymes, particularly, glycolytic enzymes in a purified plasma membrane preparation; and quantitation of Na-pump activity and glycolysis in this cell fraction. The last major specific aim involves the elucidation of the control mechanisms for coordination of metabolism with VSM function, testing the hypothesis that cyclic nucleotides and high energy phosphates, both likely metabolic regulators, are compartmentalized. Studies will include the correlation of the activities of cAMP-dependent protein kinases with the functional compartmentation, localization of these enzymes, determination of the role of cAMP in coordinating glycolysis and Na-pump function in purified plasma membranes, and isotachophoretic measurement of the tissue phosphagen and metabolites under various functional loading conditions. Coronary and carotid arteries will be the major vessels studied with the long term goal of relating metabolism to normal VSM function in order to detect and characterize the mechanisms underlying vascular disease.