The 'selective' pathway for cellular uptake of lipoprotein-derived cholesteryl esters represents a high capacity, regulatable delivery system in which more cholesterol than apoprotein is internalized by cells. In this pathway, various cholesterol-rich lipoproteins bind to the surface of cells and cholesteryl esters are extracted without the lipoproteins entering the cells as intact particles. The 'selective' uptake of cholesterol has been shown to occur in intact steroidogenic organs, intact liver and a large variety of animal cells in culture. Yet, despite the extensive use of this pathway, we know very little about its mechanism of action, its time course, its intracellular targets, function, sites of regulation, dynamic interaction with other cholesterol-containing compartment of the cell, etc. It is the aim of this proposal to systematically study these issues using rat (and bovine?) steroidogenic and hepatic tissues. Our study is divided into 3 sections: In the first we will examine the itinerary of lipoprotein- derived cholesteryl ester transport and function in intact cell systems in which we ask whether cholesterol taken up by this pathway regulates cholesterol synthesizing enzymes, whether it is stored by the cells or used directly, how it is transported, where it is transported, to what extent it mixes with other intracellular cholesterol pools, and what regulates its amount and function. In the second section, we will use cell-free techniques to identify the various tissue factors and lipoprotein and membrane characteristics which influence 'selective' cholesterol uptake by cells. In the third section we will use cell transfection methods to modify certain intracellular proteins believed to influence the uptake and transfer of lipoprotein-cholesteryl esters during steroidogenesis. this study will combine the use of morphological, biochemical and molecular biology techniques which we believe bring a unique perspective to this field of research.
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