Uptake, intracellular transport and release of 125I-poly(vinylpyrrolidone) and [ 14C]-sucrose-asialofetuin in rat liver parenchymal cells : Effects of ammonia on the intracellular transport

Abstract

We have studied the intracellular transport of 125I-labeled poly(vinylpyrrolidone) ( 125I-PVP) and [ 14C]sucrose-asialofetuin ( 14C-SAF) in isolated rat hepatocytes. 125I-PVP and 14C-SAF are taken up in the cells by fluid phase and receptor-mediated endocytosis, respectively. The labeled degradation products formed from 14C-SAF are trapped in the lysosomes. They can therefore serve as markers for lysosomes in subcellular fractionation studies. The accumulation of 125I-PVP in the cells was rapid initially and then decreased to a constant value. The diminished rate of accumulation was due to release (exocytosis †) of previously endocytosed 125I-PVP. The release of 125I-PVP was studied in cells that had accumulated 125I-PVP for various times and then after washing incubated in new medium at 37°. About 25% of the radioactivity associated with the cells after 1 hr was released to the medium subsequently. No such release was observed in cells that had taken up 14C-SAF. Subcellular distribution of 125I-PVP and 14C-SAF was studied by isopycnic centrifugation in sucrose gradients. Both compounds were sequentially associated with light (1.13 g/ml) and dense (1.19 g/ml) vesicles. Exocytosed 125I-PVP was derived from the light vesicles. The denser organelles were probably lysosomes as their distribution coincided with that of lysosomal enzymes. By measuring radioactivity soluble and precipitable in trichloroacetic acid it could be shown that only degraded 14C-SAF was associated with lysosomes. Undegraded 14C-SAF was associated with vesicles banding at 1.13 g/ml. Degraded 14C-SAF was, however, also seen first in this region of the gradient, suggesting that degradation started in a light lysosome. Both uptake and release of 125I-PVP were temperature dependent; both processes ceased at 10°. Ammonium ions had negligible effects on uptake and release of 125I-PVP. The amine inhibited, however, the transfer of both 125I-PVP and 14C-SAF to the lysosomes.