Diacytosis of asialoglycoprotein in isolated hepatocytes is dependent on the structure of ligand and cellular distribution of the receptors.

Diacytosis, degradation and retention of 125I-labeled asialoorosomucoid (ASOR), its reduced and carboxymethylated N-terminal cyanogen bromide-cleaved fragment (RC-ASCNBr-I) and asialofetuin preloaded into isolated rat or rabbit hepatocytes for various periods of time were compared. In rat hepatocytes preloaded with a saturating concentration (3.10(-8) M) of the ligands, the proportion of the preloaded ligands distributed to degradation and diacytosis was fairly constant during 4 h of preincubation. In addition, a small portion of the preloaded ligands was neither diacytosed nor degraded, but was retained intracellularly. Diacytosis of 125I-ASOR (29%) was greater than that of either 125I-RC-ASCNBr-I (23%) or 125I-asialofetuin (15%). Diacytosis of 125I-ASOR, when preloaded in the presence of 5 microM colchicine, was significantly enhanced by 79% (increasing from 29% to 52%), whereas those of 125I-RC-ASCNBr-I and 125I-asialofetuin were not significantly altered (with average increases of 14% and 19%, respectively). The fraction of the preloaded 125I-asialofetuin (69%) and 125I-RC-ASCNBr-I (68.6%) that was degraded was slightly higher than that of 125I-ASOR (64%) and all was decreased by colchicine. The fraction of all three ligands retained by the cells was increased 2- to 4-fold by colchicine. The extents of retention of 125I-asialofetuin and 125I-ASCNBr-I were greater than that of 125I-ASOR, particularly after preloaded for more than 2 h. Preloading of the cells with ligands at a non-saturating concentration (6.5.10(-10) M) did not change these patterns of ligand distribution. Conjugation of diphtheria toxin fragment A (DTA) to ASOR or RC-ASCNBr-I also did not significantly alter the pattern of ligand distribution. In rabbit hepatocytes containing more asialoglycoprotein receptors than rat cells, 125I-ASOR was diacytosed to a greater extent (50%, -colchicine; 61%, + cholchicine) but degraded to a lesser extent (33%, -colchicine; 13%, + colchicine) than was observed in rat cells. The extent of retention of 125I-ASOR in rabbit cells was also greater than that in rat cells. A similar pattern of differences between rabbit and rat hepatocytes was observed for 125I-DTA-ASOR. These results indicate that intracellular sorting of internalized asialglycoproteins between diacytosis and degradation is dependent on both the structure of the ligand and the distribution of the cellular receptors.