title: Efflux of cellular cholesterol and phospholipid to lipid-free apolipoproteins and class A amphipathic peptides.
abstract: The mechanism(s) by which lipid-free apolipoprotein (apo) AI is able to stimulate efflux of cholesterol and phospholipid from cells in cultures has (have) been examined. This process was found to be enhanced when macrophages were enriched with cholesterol. There were 12- and 4-fold increases in cholesterol and phospholipid efflux, respectively, from cholesterol-enriched mouse macrophages when compared to cells not loaded with cholesterol. This enhancement in cholesterol efflux to lipid-free apo AI from macrophages enriched with cholesterol was found to be controlled by the level of free cholesterol in the cells. When cholesterol-enriched mouse macrophages were exposed to lipid-free apo AI at 20 micrograms/mL (706 nM), there was significant efflux of [14C]cholesterol and [3H]phospholipid (20% +/- 0.5%/24 h and 6% +/- 0.3%/24 h, respectively). In comparison, HDL at equivalent protein concentrations only stimulated 11% and 4% efflux of cholesterol and phospholipid, respectively. Synthetic peptides containing amphipathic helical segments that mimic those present in apo AI were used to examine the structural features of the apoprotein which stimulate lipid efflux. Peptides containing only one (18A) or two (37pA) amphipathic helical segments stimulated as much cholesterol efflux from both mouse macrophages and L-cells as apo AI. The order of efficiency, as assessed by the mass concentration at which half-maximal efflux was reached (EC50), was apo AI > 37pA > 18A, indicating that acceptor efficiency was dependent on the number of amphipathic helical segments per molecule. When the helical content of 18A was increased by neutralizing the charges at the ends of the peptide (Ac-18A-NH2), there was a substantial increase in the efficiency for cholesterol efflux (EC50 18A = 17 micrograms/mL vs Ac-18A-NH2 = 6 micrograms/mL). In contrast, when the amphipathicity of the helix in 18A was decreased by scrambling the amino acid sequence, thereby reducing its lipid affinity, cholesterol and phospholipid efflux were not stimulated. The efficiency with which the peptides stimulated cholesterol efflux was in order of their lipid affinity (37pA > Ac-18A-NH2 > 18A), and this order was similar for phospholipid efflux. The time course of lipid release from mouse macrophages and L-cells indicated that phospholipid appeared in the extracellular medium before cholesterol. These results suggest that the apo AI or peptides first interacted with the cell to form protein/phospholipid complexes, that could then accept cholesterol.
authors.affiliation: Department of Biochemistry, Medical College of Pennsylvania, Philadelphia 19129, USA.
authors.forename: P G
authors.initials: PG
authors.lastname: Yancey
authors.forename: J K
authors.initials: JK
authors.lastname: Bielicki
authors.forename: W J
authors.initials: WJ
authors.lastname: Johnson
authors.forename: S
authors.initials: S
authors.lastname: Lund-Katz
authors.forename: M N
authors.initials: MN
authors.lastname: Palgunachari
authors.forename: G M
authors.initials: GM
authors.lastname: Anantharamaiah
authors.forename: J P
authors.initials: JP
authors.lastname: Segrest
authors.forename: M C
authors.initials: MC
authors.lastname: Phillips
authors.forename: G H
authors.initials: GH
authors.lastname: Rothblat
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport; D002784:Cholesterol / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D007739:L Cells; D008244:Lysophosphatidylcholines / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013109:Sphingomyelins / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D016632:Apolipoprotein A-I; D002788:Cholesterol Esters; D008244:Lysophosphatidylcholines; D009829:Oleic Acids; D010455:Peptides; D010713:Phosphatidylcholines; D010743:Phospholipids; D013109:Sphingomyelins; D019301:Oleic Acid; C014409:cholesteryl oleate; D002784:Cholesterol