Receptor-mediated internalization of high density lipoprotein by rat sinusoidal liver cells: identification of a nonlysosomal endocytic pathway by fluorescence-labeled ligand.
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.
Takata
K
K
Department of Biochemistry, Kumamoto University Medical School, Japan.
Horiuchi
S
S
Rahim
A T
AT
Morino
Y
Y
D000818:Animals; D002352:Carrier Proteins; D004705:Endocytosis; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D008075:Lipoproteins, HDL; D008099:Liver / Q000166:cytology / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016601:RNA-Binding Proteins; D051381:Rats; D011956:Receptors, Cell Surface / Q000502:physiology*; D018110:Receptors, Lipoprotein; D013861:Thiocyanates
D002352:Carrier Proteins; D005452:Fluoresceins; D008075:Lipoproteins, HDL; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D018110:Receptors, Lipoprotein; D013861:Thiocyanates; C031616:high density lipoprotein receptors; C075197:high density lipoprotein binding protein; D016650:Fluorescein-5-isothiocyanate