title: Behaviour of phospholipase-modified HDL towards cultured hepatocytes. I. Enhanced transfers of HDL sterols and apoproteins.
abstract: Human HDL subfractions (HDL2, HDL3, 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 A2 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 HDL3 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 micrograms/ml HDL protein. A prior phospholipase treatment of HDL3 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 HDL2 was 2-3-times higher than from HDL3. (B) Parallel experiments were conducted with 125I-labelled HDL subfractions. At 37 degrees C, the specific uptake and degradation of HDL3 125I-apolipoprotein were about 2-fold enhanced following treatment of HDL3 with phospholipase A2. Uptakes of apolipoprotein and of esterified cholesterol were compared, indicating a preferential delivery of the sterol over apoprotein (X5). The dissociation was still more pronounced with phospholipase-treated HDL3. Competition experiments showed that 12-times more unlabelled HDL3 were required to half reduce the uptake of HDL3 [3H]cholesteryl ether than to impede similarly the HDL 125I-apolipoprotein recovered in cells. Uptake of 125I-labelled apolipoprotein from HDL2 was quantitatively comparable to that from HDL3. (C) Binding of 125I-HDL subfractions was followed at 4 degrees C. A specific binding was observed for HDL2 and HDL3, although kinetic parameters were quite different (KD of 9 and 25 micrograms/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.
authors.affiliation: INSERM Unité 101, Hôpital Purpan, Toulouse, France.
authors.forename: X
authors.initials: X
authors.lastname: Collet
authors.forename: B P
authors.initials: BP
authors.lastname: Perret
authors.forename: G
authors.initials: G
authors.lastname: Simard
authors.forename: C
authors.initials: C
authors.lastname: Vieu
authors.forename: L
authors.initials: L
authors.lastname: Douste-Blazy
mesh_terms: D000818:Animals; D001053:Apolipoproteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D008076:Cholesterol, HDL / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D001053:Apolipoproteins; D008076:Cholesterol, HDL; D008075:Lipoproteins, HDL; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2