Behaviour of phospholipase-modified HDL towards cultured hepatocytes. I. Enhanced transfers of HDL sterols and apoproteins

Abstract

Human HDL subfractions (HDL 2, HDL 3, or HDL separated by heparin affinity chromatography) were labelled either on their apolipoprotein moiety with 125I or on their sterols: unesterified [ 14C]cholesterol and [ 3H]cholesteryl linoleyl ether, a non-hydrolysable analog of esterified cholesterol. HDL subfractions were then treated with or without phospholipase A 2 from Crotalus adamanteus in presence of albumin leading to a 72–82% phosphatidylcholine degradation. Control and treated HDL were reisolated and then addressed to cultured rat hepatocytes. (A) During incubations, unesterified [ 14C]cholesterol from HDL 3 readily appeared in hepatocytes. The specific uptake of HDL esterified cholesterol calculated from [ 3H]cholesteryl ether was 2–4-times less important. Uptake of HDL cholesterol tended to saturate at 150–200 μg/ml HDL protein. A prior phospholipase treatment of HDL 3 stimulated by 2–5-fold the uptake of [ 3H]cholesteryl ether, whereas the transfer of free [ 14C]cholesterol was minimally increased. The uptake of 3H/ 14C-labelled sterols from HDL 2 was 2–3-times higher than from HDL 3. (B) Parallel experiments were conducted with 125I-labelled HDL subfractions. At 37°C, the specific uptake and degradation of HDL 3 125I-apolipoprotein were about 2-fold enhanced following treatment of HDL 3 with phospholipase A 2. Uptakes of apolipoprotein and of esterified cholesterol were compared, indicating a preferential delivery of the sterol over apoprotein (×5). The dissociation was still more pronounced with phospholipase-treated HDL 3. Competition experiments showed that 12-times more unlabelled HDL 3 were required to half reduce the uptake of HDL 3 [ 3H]cholesteryl ether than to impede similarly the HDL 125I-apolipoprotein recovered in cells. Uptake of 125I-labelled apolipoprotein from HDL 2 was quantitatively comparable to that from HDL 3. (C) Binding of 125I-HDL subfractions was followed at 4°C. A specific binding was observed for HDL 2 and HDL 3, although kinetic parameters were quite different ( K D of 9 and 25 μg/ml, respectively). Following phospholipolysis, both the specific and non-specific contributions to total binding were increased. Hence, hepatocytes take up more 125I-labelled apolipoprotein and 3H/ 14C-labelled sterols from lipolysed HDL than from unmodified particles. This is associated to changes in the binding characteristics.