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

The rapid removal of circulating desialylated glycoproteins from the blood plasma of mammals is well-documented, occurs exclusively in the liver and is mediated by a carbohydrate recognition system present only in hepatocytes. After binding to a plasma membrane receptor that recognizes galactose, glucose or N-acetylgalactosamine residues on their oligosaccharide chains, ligands are rapidly internalized, transported within membrane-bound structures to lysosomes and subsequently degraded. The fate of the receptor is less clear, although it is reutilized. Our recent experiments have addressed three questions: 1) What is the detailed intracellular pathway taken by asialo-glycoproteins (ASGP's)? 2) Where on the hepatocyte cell surface are ASGP's bound? and 3) What is the intracellular distribution of the ASGP receptor?Two electron microscopic tracers have been used to investigate the internalization of ASGP's by hepatocytes: asialoorosomucoid covalently coupled to horseradish peroxidase (ASOR-HRP); and lactosaminated ferritin (Lac-Fer), a ferritin molecule to which 90-110 lactose molecules have been attached via their glucose moieties.