Effects of sphingomyelin degradation on cell cholesterol oxidizability and steady-state distribution between the cell-surface and the cell interior

This study addresses questions related to (i) the distribution of cholesterol between the cell surface and intracellular membranes in cultured fibroblasts and (ii) the effects of plasma membrane sphingomyelin on this distribution. Cholesterol oxidase (Streptomyces sp.) converts cell cholesterol to cholestenone and was used in this study to probe the cellular distribution of cholesterol. The availability of cell cholesterol for oxidation by cholesterol oxidase was markedly influenced by the presence of sphingomyelin. In native, glutaraldehyde-fixed fibroblasts only about 20% of the cell cholesterol was oxidized under our experimental conditions. However, degradation of cell sphingomyelin with sphingomyelinase (Staphylococcus aureus) markedly enhanced the oxidation of cell surface cholesterol in glutaraldehyde-fixed fibroblasts. About 90% of the total unesterified cholesterol could be oxidized to cholestenone in confluent, sphingomyelin-depleted fibroblasts. These results suggest that about 90% of the unesterified cholesterol was at the cell surface in these cells. It was also observed that degradation of cell sphingomyelin exerted a dramatic effect on the distribution of cell cholesterol between the cell surface and intracellular membranes. Within 90 min after hydrolysis of cell sphingomyelin, about 30% of the total cell-associated unesterified cholesterol was transported from a cholesterol oxidase-susceptible pool to an oxidase-resistant pool. Together with the redistribution of cell cholesterol after sphingomyelin degradation, a marked enhancement of the endogenous cholesterol esterification reaction was observed. We conclude that the degradation of plasma membrane sphingomyelin resulted in a new apparent steady-state distribution of cellular cholesterol, with less cholesterol in the plasma membrane and more in intracellular membranes. It therefore appears that sphingomyelin is a major determinant of the distribution of cholesterol in intact cells.