An increasing body of evidence has linked abnormalities in the effective concentration of NO with accelerated atherosclerosis in diabetes and other diseases. Despite this, the nature of these abnormalities and the mechanisms by which they occur are incompletely understood. In recent studies, we have observed that hyperglycemia inhibits NO synthesis, as measured by arginine conversion to citrulline, in cultured porcine aortic endothelium, despite the fact it concurrently enhances the uptake of arginine. In this proposal, we will attempt to define the specific alterations in arginine transport and NO synthesis caused by hyperglycemia and the mechanisms responsible for them. In addition, we will examine how arginine transport and NO synthesis are altered, in vivo, in the aorta of control and alloxan-diabetic rabbits fed an atherogenic diet.
The specific aims are as follows: (1) To determine the effect of hyperglycemia on NO generation in cultured endothelial cells. Arginine transport through Na+ dependent and Na-independent transporters will be characterized and we 'will examine the relation of the two systems to NO synthesis. We will then assess the specific effects of hyperglycemia on these processes. To examine the implications of cellular NOS distribution on these events, we will also carry out analogous studies using human embryonic kidney cells stably transfected with myrostolation-deficient and wild type eNOS. (2) To define the changes in cell signalling and metabolism caused by hyperglycemia and to examine their relationship to its effects on the L-Arg/NO pathway. Changes in redox state, NADPH, DAG-PKC signalling, and Na pump activity will be related temporally to alterations in arginine transport and NO synthesis. In addition, if warranted, we will examine the effects of agents that inhibit or mimic these changes on the L-Argl/NO pathway. As part of these studies, we will compare the effects of chronic (days) versus acute hyperglycemia, and in collaboration with Project 1 of modified LDL, on these events. (3) To determine how the L-Arg/NO pathway in the intact aorta is affected by diabetes and atherosclerosis. We will determine whether hyperglycemia causes the same changes it does in cultured endothelium. In addition, in concert with project 3, we will map regional differences in the pathway in the aorta and assess whether they correlate with the propensity of a region to develop atherosclerosis in diabetic rabbits fed a Miller-Wilson diet. We will also determine whether changes in the L-Arg/NO pathway antedate the appearance of atherosclerotic lesions in these rabbits and whether they are prevented or reversed by interventions that diminish the severity of the atherosclerosis. These studies should provide basic information about the regulation of arginine transport and NO synthesis in the aortic endothelial cell and how they are altered by hyperglycemia. In concert with the efforts of project 3, they should also provide novel insights into the role of the L-ArgI/NO pathway in atherosclerosis, and its acceleration by diabetes.
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