The hypothesis of the proposed studies is that cholesterol enrichment alters the structure and function of the plasma membrane which, in turn, alters cation fluxes and cytoplasmic ion concentrations, thereby triggering altered vasomotion and cell proliferation. The studies will combine studies in isolated arteries from normal and cholesterol-fed rabbit aorta, isolated cells from the arteries, and cultured smooth muscle and endothelium.
Aim 1 will determine the effects of dietary atherosclerosis on basal and agonist activated transmembrane Ca++, K+, and Na++ movements and free cytosolic levels. Ion fluxes will be determined in isolated arteries, cytosolic calcium levels will be determined in dispersed cells with fura-2, cytosolic K+ and Na+ levels will be estimated from back extrapolation to zero time from the ion efflux data. Changes in the ions will be correlated to vasoactive responses to agonists and potassium channel activators. Comparisons will be made in cultured cells where purer membrane preps will be available. In addition, changes in microvascular responses to ischemia as well as to K+ activators in the Langendorff rabbit heart as well as blood pressure responses in the anesthetized rabbit will be studied.
Aim 2 will measure membrane lipids, fluidity will be assessed, and a collaborator will assess membrane structure by x-ray diffraction. These parameters will be correlated with the ion flux data in aim 1.
Aim 3 will examine the potential role of LDL obtained form normal and hypercholesterolemic subjects (as well as modified LDL) for its ability to cause similar changes in ion fluxes and membrane structure.
Aim 4 will determine the mechanisms responsible for the increased proliferation of vascular cells by determining the potency of calcium channel antagonists and performing parallel ion flux measurements, as well as determining the influence of conditioned media from cells enriched with cholesterol.
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