A morphological study of differentiated hepatocytes in vitro

Arterburn, Linda M.; Zurlo, Joanne; Yager, James D.; Overton, Regina M.; Heifetz, Aaron

doi:10.1002/hep.1840220128

Cited by 45 publications

(34 citation statements)

References 29 publications

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“…The present results indicate that G i2 may be part of the machinery that governs the maintenance of a polarized distribution of F-actin in hepatocytes. The observation that pertussis toxin pretreatment prevented the spreading of hepatocytes in primary culture provides further evidence that G i2 regulates F-actin organization, since it has been shown that hepatocyte spreading in culture requires F-actin organization (39).…”

Section: Fig 8 the Localization Of F-actin And G␣ I2 In Hepatocytesmentioning

confidence: 81%

Regulation of F-actin and Endoplasmic Reticulum Organization by the Trimeric G-protein Gi2 in Rat Hepatocytes

Wang¹,

Gregory

Barritt

2000

Journal of Biological Chemistry

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The roles of the heterotrimeric G-protein, G i2 , in regulating the actin cytoskeleton and the activation of store-operated Ca 2؉ channels in rat hepatocytes were investigated. G␣ i2 was principally associated with the plasma membrane and microsomes. Both F-actin and G␣ i2 were detected by Western blot analysis in a purified plasma membrane preparation, the supernatant and pellet obtained by treating the plasma membrane with Triton X-100, and after depolymerization and repolymerization of F-actin in the Triton X-100-insoluble pellet. Actin in the Triton X-100-soluble supernatant coprecipitated with G␣ i2 using either anti-G␣ i2 or antiactin antibodies. The principally cortical location of F-actin in hepatocytes cultured for 0.5 h changed to a pericanalicular distribution over a further 3.5 h. Some G␣ i2 co-localized with F-actin at the plasma membrane. Pretreatment with pertussis toxin ADP-ribosylated 70 -80% of G␣ i2 in the plasma membrane and microsomes, prevented the redistribution of F-actin, caused redistribution and fragmentation of the endoplasmic reticulum, and inhibited vasopressin-stimulated Ca 2؉ inflow. It is concluded that (i) a significant portion of hepatocyte G␣ i2 associates with, and regulates the arrangement of, cortical F-actin and the endoplasmic reticulum and (ii) either or both of these regulatory roles are likely to be required for normal vasopressin activation of Ca 2؉ inflow.

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Section: Fig 8 the Localization Of F-actin And G␣ I2 In Hepatocytesmentioning

confidence: 81%

Regulation of F-actin and Endoplasmic Reticulum Organization by the Trimeric G-protein Gi2 in Rat Hepatocytes

Wang¹,

Gregory

Barritt

2000

Journal of Biological Chemistry

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“…Freshly isolated adult hepatocytes in monolayer culture exhibit a flattened morphology, depolarize, and lose many of the surface characteristics of normal hepatocytes in vivo [407,408]. This physical change in cell shape upon hepatocyte adherence is accompanied by architectural changes in cytoskeletal components.…”

Section: Hepatocyte Spreading and Liver-specific Functioningmentioning

confidence: 98%

“…The larger areas of attachment between the cells in turn enhance the expression of intercellular gap junctions and E-cadherin-mediated cell adhesions, as well as the formation of bile canaliculus-like structures [16,418]. Actin fibers cluster at these canalicular surfaces [407]. Co-cultivation with another cell type or extracellular matrix overlay can partially reverse the effects seen in low density cultures [418][419][420], and stimulate a repolarization of cultivated hepatocytes [336,421].…”

Section: Hepatocyte Spreading and Liver-specific Functioningmentioning

confidence: 99%

“…Due to a difference in cytoarchitecture and the expression and distribution of cytoskeletal elements, hepatocytes plated at high densities are associated with less cell spreading, and acquire a more cuboidal morphology than those in low density cultures [355,407,418]. The larger areas of attachment between the cells in turn enhance the expression of intercellular gap junctions and E-cadherin-mediated cell adhesions, as well as the formation of bile canaliculus-like structures [16,418].…”

Section: Hepatocyte Spreading and Liver-specific Functioningmentioning

confidence: 99%

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Molecular Mechanisms Underlying the Dedifferentiation Process of Isolated Hepatocytes and Their Cultures

Elaut

Henkens²,

Papeleu³

et al. 2006

CDM

260

213

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Primary hepatocytes and their cultures are a simple but versatile, well-controlled, and relatively easy to handle in vitro system that is well-accepted for investigating xenobiotic biotransformation, enzyme induction and inhibition, and (biotransformation-mediated) hepatotoxicity. In addition, hepatocyte cultures have proven to be valuable tools in the study of liver physiology, viral hepatitis, and liver regeneration and are proposed as an alternative to orthotopic liver transplantation. It has been observed, however, that a number of liver-specific functions are progressively lost with time when hepatocytes are isolated and cultivated. These phenotypic changes are primarily the result of fundamental changes in gene expression concomitant with a diminished transcription of the relevant liver-specific genes, and can be interpreted as a 'dedifferentiation' of the isolated hepatocytes. Ischemia-reperfusion stress induced during the isolation process, disruption of the normal tissue architecture, as well as an adaptation to the in vitro environment are underlying factors and will be extensively discussed. A detailed description of the regulation of the hepatocyte phenotype in vivo in the first section of this review will help to understand the effect of these factors on hepatocyte gene expression. Although different approaches, mainly mimicking the in vivo hepatocyte environment, have been succesfully used to prevent or slow down the dedifferentiation of primary hepatocytes in monolayer culture, the ideal hepatocyte-based culture model, characterized by a long-term expression of hepatocyte-specific functions comparable to the in vivo level, does not exist at the moment. Consequently, alternative strategies should focus on the isolation procedure, during which dedifferentiation is already initiated. In addition, identification of the conditions needed for the full in vitro maturation of hepatic progenitor cells to quiescent, functional hepatocyte-like cells opens promising perspectives.

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“…1 However, in addition to altered gene expression, prominent remodeling of the cytoplasm occurs, characterized by changes of cell shape; cytoskeletal organization, polarity and cell-cell contacts. 2–4 Numerous enzyme activities decrease in the cytoplasm, including characteristic hepatic enzymes like cytochrome P 450 and tyrosine amino transferase. 4,5 Other liver-specific activities, such as albumin secretion also decline.…”

Section: Introductionmentioning

confidence: 99%

Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes

Rodrı́guez-Enrı́quez

Kai²,

Maldonado³

et al. 2009

Autophagy

106

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In primary culture, hepatocytes dedifferentiate, and their cytoplasm undergoes remodeling. Here, our aim was to characterize changes of mitochondria during remodeling. Hepatocytes were cultured one to five days in complete serum-containing Waymouth’s medium. In rat hepatocytes loaded with MitoTracker Green (MTG), tetramethylrhodamine methylester (TMRM), and/or LysoTracker Red (LTR), confocal microscopy revealed that mitochondria number and mass decreased by approximately 50% between Day 1 and Day 3 of culture. As mitochondria disappeared, lysosomes/autophagosomes proliferated five-fold. Decreased mitochondrial content correlated with (a) decreased cytochrome c oxidase activity and mitochondrial number observed by electron microscopy and (b) a profound decrease of PGC-1α mRNA expression. By contrast, mtDNA content per cell remained constant from the first to the third day of culture, although ethidium bromide (de novo mtDNA synthesis inhibitor) caused mtDNA to decrease by half from the first to the third culture day. As mitochondria disappeared, their MTG label moved into LTR-labeled lysosomes, which was indicative of autophagic degradation. A multiwell fluorescence assay revealed a 2.5-fold increase of autophagy on Day 3 of culture, which was decreased by 3-methyladenine, an inhibitor of autophagy, and also by cyclosporin A and NIM811, both selective inhibitors of the mitochondrial permeability transition (MPT). These findings indicate that mitochondrial autophagy (mitophagy) and the MPT underlie mitochondrial remodeling in cultured hepatocytes.

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A morphological study of differentiated hepatocytes in vitro

Cited by 45 publications

References 29 publications

Regulation of F-actin and Endoplasmic Reticulum Organization by the Trimeric G-protein Gi2 in Rat Hepatocytes

Regulation of F-actin and Endoplasmic Reticulum Organization by the Trimeric G-protein Gi2 in Rat Hepatocytes

Molecular Mechanisms Underlying the Dedifferentiation Process of Isolated Hepatocytes and Their Cultures

Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes

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