EVIDENCE THAT A NEUTRAL CHOLESTERYL ESTER HYDROLASE IS RESPONSIBLE FOR THE EXTRALYSOSOMAL HYDROLYSIS OF HIGH-DENSITY-LIPOPROTEIN CHOLESTERYL ESTER IN RAT HEPATOMA-CELLS (FU5AH)

Diethylumbelliferyl phosphate (UBp) has been shown to inhibit the neut ral cholesteryl ester hydrolase activity responsible for hydrolysis of cellular lipid droplet cholesteryl ester (Harrison et al., 1990). The potential for (UBP) to inhibit uptake and hydrolysis of high density lipoprotein (HDL) cholestryl ester was studied in Fu5AH hepatoma cells , a model for HDL cholesterol delivery. Coincubation of H-3-cholestery l ester labeled HDL with UBP resulted in a 72% decrease in the cellula r free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free choleste rol. Total cellular H-3-CE uptake was modestly (27%) but significantly decreased by UBP. Pulse-chase experiments (15 min. pulse and 7 min. c hase) were used to study the hydrolysis of HDL H-3-CE in subcellular f ractions separated by percoll gradients. The conversion of H-3-CE to H -3-FC could be demonstrated in fractions that comigrated with the plas ma membrane/endosome fractions but were well separated from lysosomes. Neutral cholesteryl ester hydrolase activity was detected in those sa me fractions. These results suggest that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and its delivery to hep atoma cells. (C) 1993 Wiley-Liss, Inc.