Biochemical demonstration of endocytosis and subsequent resecretion of high-density lipoprotein by rat peritoneal macrophages.

To investigate the post-binding events of high-density lipoprotein (HDL), rat peritoneal macrophages were enriched by collagen gel-coated plates and incubated in a cell-suspension system with fluorescein isothiocyanate-labeled HDL (FITC-HDL), followed by fluorescence spectroscopic analyses. Upon incubation with FITC-HDL at 37 degrees C for 30 min, the microenvironmental pH of the cell-associated FITC-HDL was 6.50, whereas a 0 degree C-incubation gave a corresponding pH of 7.15. Carbonyl cyanide m-chlorophenylhydrazone, a protonophore known to dissipate the proton gradient, restored the former acidic pH (pH 6.40) to pH 7.20, but had no effect on the latter. This indicates that cell surface-bound HDL is internalized and exposed to an acidic compartment. When cells were incubated with FITC-HDL at 37 degrees C for 30 min and the cell-associated FITC-HDL was chased at 37 degrees C, the fluorescence intensity at 490 nm showed a time-dependent increase. This increase was explained by a release of endocytosed FITC-HDL into the extracellular medium but not by a simple outward dissociation of the cell-associated FITC-HDL. The microenvironmental acidic pH of the cell-associated FITC-HDL changed to a less acidic pH during the chase whereas that of FITC-HDL became constant after released into the medium, indicating that endocytosed HDL was resected back into the extracellular medium. This resection process was temperature-dependent and accelerated by HDL itself. These results provide the biochemical evidence for the presence of an endocytic-exocytic pathway for HDL in rat peritoneal macrophages.