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American Journal of Pathology, Vol 121, 200-211, Copyright © 1985 by American Society for Investigative Pathology

REGULAR ARTICLES

A comparative microscopic and biochemical study of the uptake of fluorescent and 125I-labeled lipoproteins by skin fibroblasts, smooth muscle cells, and peritoneal macrophages in culture

GD Reynolds and RW St. Clair

Uptake of low density lipoprotein (LDL) and of acetyl LDL was compared in skin fibroblasts, smooth muscle cells, and peritoneal macrophages with the use of lipoproteins labeled with either 125I or the fluorescent probe 3,3'-dioctadecylindocarbocyanine (DiI). The uptake of DiI-labeled lipoproteins was assessed by quantitative spectrofluorometry and by fluorescence microscopy. The DiI was quantitatively retained by the cells, while the 125I-LDL was degraded and 125I-labeled degradation products were excreted from the cells. In smooth muscle cells and fibroblasts the uptake of LDL was virtually the same whether measured with the use of the DiI or 125I-label (sum of cell-associated and degraded 125I). The labeling of acetyl LDL with DiI enhanced its uptake in peritoneal macrophages by an average of 18%. With the DiI label, lipoprotein uptake (DiI-LDL for smooth muscle cells and skin fibroblasts and DiI-acetyl-LDL for mouse peritoneal macrophages) could be determined after as little as 10 minutes of incubation at 37 C. The pattern of uptake of the DiI-labeled lipoproteins was consistent with binding to specific receptors, because no DiI could be detected in mutant cells without LDL receptors, and uptake was competitively inhibited by addition of excess unlabeled lipoprotein. When the DiI-labeled lipoproteins were removed from the medium, there was a 5-15% loss of DiI from all cell types studied over the first 24 hours. Thereafter, DiI loss from cells was dependent on cell type and culture medium. No further loss of DiI occurred from skin fibroblasts for up to 96 hours of incubation in medium supplemented with either lipoprotein-deficient serum (LPDS) or 10% fetal bovine serum. During this same time period there was a 40-60% loss of DiI from smooth muscle cells and macrophages incubated in medium supplemented with LPDS. Most of the DiI lost from the cells (60-70%) could be recovered in the culture medium but was not the result of cell death, as was indicated by the relatively constant protein concentrations per dish. The loss of DiI was markedly reduced in smooth muscle cells and macrophages when 10% fetal bovine serum was substituted for the LPDS in the culture medium. This suggests that some cells incubated with LPDS undergo changes, perhaps in the plasma membrane, that alter their ability to retain the DiI. In the presence of 10% fetal bovine serum, however, the DiI label is quantitatively retained by all cells tested for up to 96 hours.(ABSTRACT TRUNCATED AT 400 WORDS)

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