The research is aimed at the elucidation of the disposition and movement of cholesterol within mammalian cells. The major premise guiding the work is that unesterified cellular cholesterol exists in multiple discrete pools which do not mix. (1) The first goal is to verify a conclusion derived from previous studies that the human fibroblast plasma membrane contains nearly all the unesterified cholesterol in the cell. A two phase aqueous partition technique has been developed to purify plasma membrane for this purpose. It is proposed to demonstrate that cellular cholesterol is more enriched in these fractions than the universal cell surface marker 5'-nucleotidase. (2) The small pool of intracellular cholesterol will be distinguished from the massive background of plasma membrane cholesterol by oxidizing the latter selectively in intact cells with cholesterol oxidase. The fraction of intracellular cholesterol carried in lysosomes will be established through the analysis of purified fractions using two phase aqueous partition and equilibrium sucrose gradient centrifugation. (3) The kinetics of cholesterol movement will be used to examine whether intracellular cholesterol arising from cholesteryl ester droplet stores, ingested lipoproteins and de novo biosynthesis moves to the plasma membrane through a common pathway or through multiple non-mixing routes. Similarly, the divergence of intracellular cholesterol heading to the plasma membrane versus steroid hormone production will be examined in rat ovary granulosa cells. (4) In a parallel study, subcellular fractionation techniques will be used to examine whether cholesterol arising from the three sources identified in (3) above mix or remain separate. Digitonin will be used as a specific buoyant density shift reagent to detect sterol-rich membranes. (5) Using cholesterol oxidase, the hypothesis that the tight junctions of epithelial monolayers in culture pose a barrier to the diffusion of cholesterol between the apical and basolateral plasma membranes will be examined.
