High-affinity uptake and degradation of ApoE-free high-density lipoprotein and low-density lipoprotein in cultured porcine hepatocytes

Abstract

Isolated pig liver membranes contain a specific lipoprotein binding site that recognizes low-density lipoprotein (LDL) and apolipoprotein E (apoE) free high-density lipoprotein (HDL). A similar site exists in cultured porcine hepatocytes that mediates the uptake and degradation of apoE-free HDL. The binding of 125I-labeled HDL and 125I-labeled LDL (125I-HDL and 125I-LDL, respectively) at 4.degree. C and the uptake and degradation of the lipoproteins at 37.degree. C were time dependent and saturable and were not inhibited by unrelated proteins. Chloroquine (6 .times. 10-5 M) inhibited the degradation of 125I-HDL by 76% and of 125I-LDL by > 99%; leupeptin inhibited the degradation of both lipoproteins by .apprx. 25%. 125I-HDL binding (4.degree. C), uptake and degradation (37.degree. C) were inhibited by LDL, methyl-LDL and methyl-HDL about as well as by unlabeled HDL but were unaltered in Pronase-treated cells or in cells that were cultured for 24 h in either lipoprotein-free medium or medium containing HDL or LDL (200 .mu.g/ml). In contrast, these conditions affected the uptake and degradation of 125I-LDL disproportionately. HDL and methyl-LDL inhibited 125I-LDL uptake by 50% or more but had little effect on degradation. 125I-LDL binding was reduced by 12% and degradation by 57% in Pronase-treated cells. Preincubation of the cells with LDL (200 .mu.g/ml) reduced uptake by 35% and degradation by 68%. Similar preincubation with HDL (200 .mu.g/ml) increased 125I-LDL degradation by 60% but did not affect 125I-LDL uptake. Porcine hepatocytes probably were at least 2 distinct sites for lipoproteins. One site resembled the LDL receptor and mediated 125I-LDL degradation. A second, Pronase-insensitive site recognized both HDL and LDL. This site mediated almost all of the degradation of 125I-HDL but little if any degradation of 125I-LDL.