Reversible high affinity uptake of apo E-free high density lipoproteins in cultured pig hepatocytes

We examined the high affinity binding, uptake, and degradation of apo E-free ¹²⁵I-high density lipoprotein (HDL) in cultured pig hepatocytes. At steady state, the cells degraded 9.4% of cell-associated ¹²⁵I-HDL/hour, compared with 41.7%/hour for ¹²⁵I-LDL. Pulse-chase experiments at 4° C revealed that high affinity ¹²⁵I-HDL binding was reversible. Similar experiments at 37° C revealed that about 70% of the cell-associated ¹²⁵I-HDL was released as a macromolecule; the remainder was degraded to acid-soluble products. In contrast, over 75% of the ¹²⁵I-LDL that was released had been degraded to acid soluble products. The amount of macromolecular ¹²⁵I-HDL released at 37° C was similar to the amount that was bound to the cell surface, as estimated from measurements of trypsin-releasable radioactivity. Density gradient ultracentrifugation and SDS-polyacrylamide gel electrophoretic analysis of macromolecular ¹²⁵I-HDL released to the medium revealed an increase in density, and the apparent partial proteolysis of apo A-I [M(r) 25,000] to products of apparent M(r) 12,000-14,000. The findings suggest that high affinity ¹²⁵I-HDL uptake had a reversible component in which HDL was concentrated temporarily at the cell surface, modified, and then released as a somewhat denser lipoprotein particle. Measurement of ¹²⁵I-HDL and ¹²⁵I-LDL degradation in cell homogenates revealed no difference in the inherent susceptibility of the two lipoproteins to proteolysis by lysosomal enzymes. The overall slower rate of degradation of ¹²⁵I-HDL compared to ¹²⁵I-LDL was therefore due in part to the smaller fraction of HDL that was committed to irreversible catabolism. The rate of catabolism of this fraction, however, was considerable. Cells pulsed at 4° C and subsequently warmed to 37° C released one-half the acid-soluble products from ¹²⁵I-HDL within about 4 hours, compared with 2 hours for cells pulsed with ¹²⁵I-LDL. These findings indicate that HDL was internalized, transported to lysosomes, and degraded at about one-half the rate of LDL.