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Journal of Lipid Research, Vol 29, 1117-1126, Copyright © 1988 by Lipid Research, Inc.

ARTICLES

Receptor-mediated internalization of high density lipoprotein by rat sinusoidal liver cells: identification of a nonlysosomal endocytic pathway by fluorescence-labeled ligand

K Takata, S Horiuchi, AT Rahim and Y Morino
Department of Biochemistry, Kumamoto University Medical School, Japan.

Rat sinusoidal liver cells possess the surface receptor for high density lipoprotein (HDL) (Murakami, M., S. Horiuchi, K. Takata, and Y. Morino. 1987. J. Biochem. (Tokyo) 101: 729-741). The present study was undertaken to determine whether cell surface-bound HDL underwent subsequent endocytic internalization by using 125I-labeled HDL and fluorescein isothiocyanate-labeled HDL (FITC-HDL). The cell-associated radioactivity obtained by a 40-min incubation with 125I-labeled HDL at 37 degrees C was released into the medium as acid-precipitable forms upon further incubation at 37 degrees C. When further incubated at 0 degree C instead of 37 degrees C, however, this release was significantly reduced. A similar phenomenon was observed after the cell- associated ligands had been treated with trypsin. The cell-associated ligands obtained after a 1-hr incubation with 125I-labeled HDL at 0 degree C were largely counted for by those bound to the outer surface of the cells, thus suggesting that HDL is internalized into cells at 37 degrees C but not at 0 degree C. Moreover, when cells were incubated with FITC-HDL at 0 degree C, the cell-associated ligands were found in a pH 7.2 +/- 0.1 compartment, whereas when incubated at 37 degrees C, its microenvironmental pH became much more acidic, exhibiting pH 6.2 +/- 0.1. Furthermore, this value returned to 7.1 +/- 0.1 upon treatment with carbonylcyanide m-chlorophenylhydrazone known to dissipate the total protonomotive force. These results suggest, therefore, that the internalization process does follow receptor-mediated binding of HDL in rat sinusoidal liver cells. This notion was also supported by fluorescence microscopic observations.
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