Studies on the Galactose-Specific Recognition System of Mammalian Liver

Hubbard, Ann L.; Wall, Doris A.; Zeitlin, PL; Hong, S. W.

doi:10.1017/s0424820100099088

Cited by 1 publication

(3 citation statements)

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“…The experiments described above demonstrated that single hepatocytes and cells in monolayer cultures resembled hepatocytes in vivo, both quantitatively and qualitatively, in terms of binding activity, internalization, and fate of ASGPs. We next investigated the distribution of ASGP surface receptors on freshly isolated cells, because these cells have no recognizable blood front domain, the predominant location of the ASGP receptor in situ (6,7) . Qualitatively, the EM-ARG results indicated a random distribution of binding sites over the entire surface .…”

Section: Asor-hrp Cytochemistry In Dissociated Hepatocytesmentioning

confidence: 99%

“…The asialoglycoprotein (ASGP) receptor is localized exclusively to the parenchymal cells of mammalian liver and functions in the rapid removal of desialylated glycoproteins from the circulation by receptor-mediated endocytosis (1)(2)(3)(4)(5) . The distribution of the ASGP receptor along the plasma membrane of hepatocytes in situ is restricted to the blood sinusoidal surface and is preferentially concentrated in coated pits present in this domain (6,7) . During the preparation of single hepatocytes by collagenase perfusion of the liver, cells lose the three morphologically identifiable plasma membrane domains (8,9) (sinusoidal, bile canalicular, and lateral surfaces), yet retain the capacity to bind, internalize, and degrade 125 1-ASGPs (10)(11)(12)(13)(14)(15)(16) .…”

mentioning

confidence: 99%

“…FIGURE7 ' 25 1-ASOR binding at 5*C to dissociated cells and monolayer cultures . Dissociated hepatocytes and cells from the same preparation after 24 h in culture were preincubated at 5°C for 1 h, and total, nonspecific, and EGTA-inaccessible radioactivities were assayed as described in Materials and Methods.…”

mentioning

confidence: 99%

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

Zeitlin¹,

Hubbard²

1982

The Journal of Cell Biology

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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 ' 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

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Section: Asor-hrp Cytochemistry In Dissociated Hepatocytesmentioning

confidence: 99%