Cell surface distribution and intracellular fate of asialoglycoproteins: A morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

P. L. Zeitlin, A. L. Hubbard

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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 125I-asialoorosomucoid (125I-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 125I-ASOR at 37°C occurred at a rate of 1x106 molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of 125I-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 125I-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 125I-ASOR varied among cell preparations, the effect of collagenase on 125I-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 125I-ASOR binding at 5°C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptor 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 125I-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 125I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5°C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor-mediated endocytosis of ASGPs.

Original languageEnglish (US)
Pages (from-to)634-647
Number of pages14
JournalJournal of Cell Biology
Issue number3
StatePublished - 1982
Externally publishedYes

ASJC Scopus subject areas

  • Cell Biology


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