Electron microscope autoradiography was used to study the cellular localization of seven glycoproteins rapidly cleared from the circulating plasma of rats and taken up by the liver. I and 15 min after intravenous administration of the 125I-glycoproteins, livers were fixed in situ by perfusion and processed for autoradiography. Autoradiographic grains in the developed sections were found to represent the intact 125 I-ligand. A quantitative analysis of the distribution and concentration (density) of autoradiographic grains over the three major cell types of the liver was then performed. Three molecules, asialo-fetuin, asialo-orosomucoid, and lactosaminated RNase A dimer, the oligosaccharide chains of which terminate in galactose residues, were bound and internalized almost exclusively (>900 /0) by hepatocytes. Conversely, four molecules, the oligosaccharide chains of which terminate in either N-acetyl-glucosamine (agalacto-orosomucoid) or mannose (ahexosamino-orosomucoid, preputial,8-glucuronidase, and mannobiosaminated RNase A dimer), were specifically bound and internalized by cells lining the blood sinusoids-that is, by Kupffer cells and endothelial cells. Endothelial cells were two to six times more active (on a cell volume basis) than were Kupffer cells in the internalization of these four 125 I-ligands. Mannose and N-acetylglucosamine-terminated glycoproteins competed with each other for uptake into either endothelial cells or Kupffer cells, indicating that a single system recognized mannose or Nacetyl-glucosamine residues. Finally, agalacto-orosomucoid and ahexosaminoorosomucoid were also associated with hepatocytes, but competition experiments utilizing excess asialo-orosomucoid demonstrated that residual galactosyl residues were responsible for this association.J. CELL BIOLOGY

No abstract

Electron microscope autoradiographic and biochemical methods were used to study the intracellular fates of several '251-glycoproteins, known to be specifically bound and internalized by the different cell types in the liver. At the earliest times examined (1-2 min), l25í-asialo-glycoproteins (ASGP) were localized predominantly along the sinusoidal front of hepatocytes. Analysis of the distribution of autoradiographic grains indicated that: (a) -40-60% of the 1251-ligand could be ascribed to the plasmalemma; (b) a significant fraction had already been internalized; yet (c) very little 1251-ligand was present in the lysosome-Golgi region . Between 4 and 15 min after administration of 125 1-ASGPs, there was a dramatic redistribution of autoradiographic grains from regions of the plasmalemma and peripheral cytoplasm (30% decrease) to the lysosome-Golgi region (30% increase). At longer times (30 min), there was continued drainage of 1251-ASGP into this region . The grain density over secondary lysosomes was 60-90 times higher than that over recognizable Golgi elements, clearly indicating that lysosomes were the ultimate destination of the 1251-ASGP. However, no more than 60% of the total 125 1-ligand could be localized to lysosome-rich regions of the hepatocyte, with the remaining 40% primarily in the intermediate cytoplasm. Biochemical evidence for proteolysis of the internalized 1251-ASGP (presumably within lysosomes) was obtained when [1251]-mono-iodotyrosine was found in the liver (i.e., hepatocytes) at times later than 15 min.The temporal redistribution observed for mannose and N-acetylglucosamineterminated glycoproteins (ahexosamino-orosomucoid and agalacto-orosomucoid, respectively) in endothelial cells indicated that the 125 1-ligands resided in macropinocytic vesicles (1-15 min) before their ultimate residence in dense bodies (15 min) . The same 125 I-ligands were also localized to structures resembling secondary lysosomes in Kupffer cells. The lysosomal nature of these organelles was implied from the appearance of [1251]mono-iodotyrosine in the liver at later times. 1251-ß-glucuronidase followed the same intracellular pathway in both cell types but was not degraded .

A combination of biochemistry and morphology was used to demonstrate that >95% of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs) . Maximal specific binding of ' 25 1-asialoorosomucoid (' 25 1-ASOR) to dissociated hepatocytes at 5°C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell . Binding, uptake, and degradation of '25 1-ASOR at 37°C occurred at a rate of 1 x 10 6 molecules per cell over 2 h. Light and electron microscopic autoradiography (LM-and EM-ARG) of ' 25 1-ASOR were used to visualize the surface binding sites at 5°C and the intracellular pathway at 37°C. In the EM-ARG experiments, ARG grains corresponding to ' 25 1-ASOR were distributed randomly over the cell surface at 5°C but over time at 37°C were concentrated in the lysosome region . Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5°C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane . Such a result indicates that redistribution of ASGP surface receptors had occurred .Because the number of surface binding sites of '25 1-ASOR varied among cell preparations, the effect of collagenase on '25 1-ASOR binding was examined . When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37°C, 10-50% of control binding was observed . However, by measuring the extent of '25 1-ASOR binding at 5°C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested . Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca"-free pre-perfusion, were found to bind 110-240% more 125 1-ASOR after 1 h at 37°C that they did at 0 time . This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i .e ., basal medium or 1 mM cycloheximide) .Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo . >95% of these cells maintained the capacity to bind, internalize, and degrade ' 25 1-ASOR at levels comparable to those of the freshly isolated population . ASOR-HRP (at 5°C) was specifically THE JOURNAL OF CELL BIOLOGY " VOLUME 92 MARCH 1982 634-647

No abstract