Uptake of pullulan in cultured rat liver parenchymal cells.

Uptake of pullulan, including a binding process followed by internalization, was examined in cultured rat liver parenchymal cells. A tyramine derivative of pullulan was labeled with [125I]iodine and used as a ligand. Pullulan bound to the cell surface was released by EDTA treatment, indicating that pullulan binding requires Ca2+ and a contribution from the asialoglycoprotein receptor. Binding of pullulan reached a steady state and internalization represented a biphasic mode, which included first- and zero-order processes in the initial stage and after 20 min incubation, respectively. The uptake of pullulan could be estimated by a similar model for intracellular disposition of asialofetuin. Kinetic parameters of pullulan constituting both binding and internalization were below those found for asialofetuin. These results suggest that pullulan is taken up by liver parenchymal cells via the asialoglycoprotein receptor; however, uptake availability is lower than that of asialofetuin.

Cellular disposition of arabinogalactan in primary cultured rat hepatocytes.

To characterize a targeting property of arabinogalactan (AG) as a carrier to the liver, we examined cellular disposition, such as binding and internalization in primary cultured rat hepatocytes, comparing them to those of asialofetuin (AF). A tyramine derivative of AG was synthesized to allow labeling with 125I. Binding of AG to the cells was concentration-dependent and saturable. The number of binding sites (n) of AG on the cell surface was 4.0 x 10(5) +/- 0.1 x 10(5) sites per cell which was about similar to that of AF. The value of Ka of AG was 2.2 x 10(8) +/- 0.1 x 10(8) M-1 being seven-fold higher than that of AF. The binding of AG was competitively inhibited by AF and was decreased by calcium depletion. These results indicate that AG can bind strongly to hepatocytes probably through the recognition by the asialoglycoprotein receptor (ASGP-R). Both 125I-labeled AG and fluorescein-labeled AG were internalized into the cells. The rate of internalization of AG was faster than that of AF, indicating that AG is effectively endocytosed. Microscopic observations showed that FITC labeled AG accumulated in granules within the primary cultured rat hepatocytes. Subcellular fractionation indicated that the internalized AG was mainly associated with the lysosomal fraction. However, the internalized AG seemed to remain intact in the hepatocytes. In conclusion, we found that AG is effectively internalized in primary cultured rat hepatocytes. Although AG seems a good candidate for targeting to the liver due to its high affinity binding and rapid internalization, it remains to be established whether the apparent lack of biodegradation will result in cytotoxic effects at chronic administration in vivo.