Rinninger F, Greten H
University Hospital Hamburg Eppendorf, Department of Medicine, F.R.G.
Biochim Biophys Acta. 1990 Apr 17;1043(3):318-26. doi: 10.1016/0005-2760(90)90033-t.
High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.
高密度脂蛋白（HDL）胆固醇酯可通过HDL颗粒摄取和选择性摄取（即独立于HDL颗粒摄取的胆固醇酯摄取）被成纤维细胞摄取。在本研究中，我们调查了J774巨噬细胞对HDL的选择性摄取和HDL颗粒摄取。HDL3（密度=1.125 - 1.21 g/ml）用细胞内捕获的示踪剂进行标记：用125I标记的N - 甲基酪胺 - 纤维二糖 - 载脂蛋白A - I（125I - NMTC - apo A - I）追踪载脂蛋白A - I（apo A - I），并用[3H]胆固醇油醚追踪胆固醇酯。将J774巨噬细胞在含有双重标记的HDL3的培养基中于37℃孵育，其摄取了125I - NMTC - apo A - I，表明有HDL3颗粒摄取（在20μg/ml HDL3蛋白浓度下，每4小时每毫克细胞蛋白摄取102.7 ng HDL3蛋白）。根据[3H]胆固醇油醚的摄取情况计算出的表观HDL3摄取量（在20μg/ml HDL3蛋白浓度下，每4小时每毫克细胞蛋白摄取470.4 ng HDL3蛋白）显著超过125I - NMTC - apo A - I的摄取量，即J774巨噬细胞表现出对HDL3胆固醇酯的选择性摄取。为了研究HDL3摄取的调节，通过与低密度脂蛋白（LDL）或乙酰化LDL（乙酰 - LDL）预孵育来改变细胞胆固醇。之后，测定双重标记的HDL3摄取、LDL（apo B，E）受体活性或胆固醇含量。用LDL或乙酰 - LDL预孵育可使细胞胆固醇增加至比基础水平高约3.5倍。细胞胆固醇增加对HDL3颗粒摄取没有影响。相反，LDL和乙酰 - LDL负载降低了选择性摄取（在20μg HDL3蛋白/ml时，未负载细胞与乙酰 - LDL负载细胞每4小时每毫克细胞蛋白的表观摄取量分别为606 ng HDL3蛋白和366 ng HDL3蛋白）。与选择性摄取降低同时发生的是，特异性125I - LDL降解被下调。使用肝素以及过量未标记的LDL表明，HDL3摄取独立于LDL（apo B，E）受体。总之，J774巨噬细胞摄取HDL3颗粒。此外，J774细胞还选择性摄取与HDL3相关的胆固醇酯。HDL3的选择性摄取而非HDL3颗粒摄取可被调节。
