scholarly journals Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations.

1977 ◽  
Vol 74 (3) ◽  
pp. 858-877 ◽  
Author(s):  
J C Wanson ◽  
P Drochmans ◽  
R Mosselmans ◽  
M F Ronveaux
Keyword(s):  
Tight Junctions ◽  
De Novo ◽  
Calf Serum ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Ruthenium Red ◽  
Bile Canaliculi ◽  
Isolated Cells ◽  
Etching Technique ◽  
Primary Monolayer

Primary monolayer cultures were obtained in 60-mm petri dishes by incubating 3 X 10(6) isolated hepatocytes at 37 degrees C in Dulbecco's medium supplemented with 17% fetal calf serum. The ultrastructure of monolayer cells was examined after various incubation periods. Within 4 h of plating, the isolated spherical cells adhere to the plastic surface, establish their first contacts by numerous intertwined microvilli, and form new hemidesmosomes. After 12 h of culture, wide branched trabeculae of flattened polyhedral cells extend in all directions. Finally, after 24 h of culture, bile canaliculi are reconstituted, and a biliary polarity is recovered: the Golgi elements, which are scattered throughout the cytoplasm in the isolated cells, are reassembled in front of the newly formed bile canalculi, symmetrically in the adjacent cells; lysosomes are concentrated in that region, and microtubules reappear. Concomitantly, plasma membrane differentiations, namely desmosomes and tight junctions, develop. Tight junctions sealing the bile ducts constitute a barrier to the passage of ruthenium red and horseradish peroxidase. De novo formation of these junctions was studied by the freeze-etching technique: 10-nm particles compose a network of anastomosed linear arrays in the vicinity of the bile canaliculi; in the next step of differentiation, the particles fuse, form short ridge segments and finally continuous branched smooth strands, characteristic of the mature tight junction.

scholarly journals In vivo assembly of tight junctions in fetal rat liver.

1975 ◽  
Vol 67 (2) ◽  
pp. 310-319 ◽  
Author(s):  
R Montesano ◽  
D S Friend ◽  
A Perrelet ◽  
L Orci
Keyword(s):  
Tight Junctions ◽  
De Novo ◽  
Early Stage ◽  
Fetal Life ◽  
Bile Canaliculi ◽  
Linear Arrays ◽  
Fetal Rat ◽  
Particle Aggregates ◽  
Fetal Rat Liver

Examination of glutaraldehyde-fixed, freeze-fractured livers from 14-15-day rat fetuses provided the basis for the following observations. Membrane particles align in otherwise poorly particulated areas of the presumptive pericanalicular plasma membrane (A face), frequently forming a discontinuous "honey-comb" network joining small particle islands. Even at this early stage, contiguous B-fracture faces contain furrows, rather than rows of pits, distinguishing the linear particle aggregates on the A face as developing tight junctions rather than gap junctions. Short segments of these linear arrays merge with smooth ridges clearly identifiable as segments of discontinuous tight junctions. With the continuing confluence of particulate and smooth ridge segments, mature tight junctions become fully appreciable. We conclude that tight junctions form de novo by the alignment and fusion of separate particles into beaded ridges which, in turn, become confluent and are transformed into continuous smooth ones. At 21 days of fetal life, most of the images of assembly have disappeared, and the liver reveals well-formed bile canaliculi sealed by mature tight junctions.

Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures

In Vitro ◽  
1981 ◽  
Vol 17 (12) ◽  
pp. 1100-1110 ◽  
Author(s):  
Hugo O. Jauregui ◽  
Nancy T. Hayner ◽  
James L. Driscoll ◽  
Rhonda Williams-Holland ◽  
Milton H. Lipsky ◽  
...  
Keyword(s):  
Trypan Blue ◽  
Rat Hepatocytes ◽  
Blue Dye ◽  
Dye Uptake ◽  
Isolated Cells ◽  
Adult Rat ◽  
Monolayer Cultures ◽  
Adult Rat Hepatocytes ◽  
Primary Monolayer ◽  
Primary Monolayer Cultures

Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration

1994 ◽  
Vol 266 (6) ◽  
pp. C1764-C1774 ◽  
Author(s):  
E. L. LeCluyse ◽  
K. L. Audus ◽  
J. H. Hochman
Keyword(s):  
De Novo ◽  
Single Layer ◽  
Rat Hepatocytes ◽  
Hepatic Function ◽  
Cell Periphery ◽  
Bile Canaliculi ◽  
Rat Primary Hepatocytes ◽  
Representative Model ◽  
Sandwich Configuration

Rat primary hepatocytes were cultured under different extracellular matrix configurations and evaluated for the acquisition and maintenance of structural and functional cell polarity. De novo repolarization of the plasma membrane was variable in rate and extent in hepatocyte cultures maintained on a conventional single layer of either gelled or ungelled collagen. However, cultures maintained in a collagen-sandwich configuration initiated uniform formation of a contiguous anastomosing network of bile canaliculi throughout the entire culture. Localization of apical membrane markers demonstrated normal distribution at the canalicular membrane. A marked rearrangement of the intracellular microfilaments to the cell periphery was observed and coincided with the development of the bile canaliculi. Acquisition of normal bile canalicular function and integrity was observed within 3-4 days postoverlay as indicated by the concentration and retention of carboxyfluorescein within the canalicular network. These results demonstrate that cultures of hepatocytes maintained in a sandwich configuration may serve as a more reliable and representative model in which to study the physiology of hepatic function as well as the morphogenesis of polarized membrane domains in vitro.

Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration

1999 ◽  
Vol 277 (1) ◽  
pp. G12-G21 ◽  
Author(s):  
Xingrong Liu ◽  
Edward L. LeCluyse ◽  
Kenneth R. Brouwer ◽  
Liang-Sheng L. Gan ◽  
John J. Lemasters ◽  
...  
Keyword(s):  
Biliary Excretion ◽  
Organic Anion ◽  
Rat Hepatocytes ◽  
Immunoblot Analysis ◽  
Isolated Hepatocytes ◽  
Cultured Hepatocytes ◽  
Bile Canaliculi ◽  
Standard Buffer ◽  
Sandwich Configuration

The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca2+-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca2+-free buffer. The cumulative uptake of [3H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca2+-free buffer, due to accumulation of taurocholate in canalicular spaces. When [3H]taurocholate was preloaded in the day 5sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca2+-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from ∼8% at day 0 to ∼60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.

scholarly journals In vivo induction of tight junction proliferation in rat liver.

1976 ◽  
Vol 68 (3) ◽  
pp. 793-798 ◽  
Author(s):  
R Montesano ◽  
G Gabbiani ◽  
A Perrelet ◽  
L Orci
Keyword(s):  
Cell Membrane ◽  
Tight Junction ◽  
Tight Junctions ◽  
Rat Liver ◽  
Chronic Administration ◽  
Rat Hepatocytes ◽  
Bile Canaliculi ◽  
Parallel Orientation ◽  
In Vivo Induction

The chronic administration of phalloidin induces an extensive development of tight junctions between rat hepatocytes. The junctional strands lose their predominantly parallel orientation with respect to the canalicular lumen and extend abluminally in irregular patterns which cover large membrane areas at considerable distance from the bile canaliculi. These changes indicate both proliferation and provide further evidence that these junctions are not permanent differentiations of the cell membrane.

scholarly journals Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions.

1975 ◽  
Vol 66 (1) ◽  
pp. 1-22 ◽  
Author(s):  
P Drochmans ◽  
J C Wanson ◽  
R Mosselmans
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Parenchymal Cell ◽  
Liver Homogenate ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Isolated Cells ◽  
Adult Rat ◽  
Mean Diameter

The recirculating perfusion of adult rat liver with a Ca-++-free Hanks' solution produces a release of the adhesiveness of cells and a cleaving of the desmosomes. The addition of collagenase and hyaluronidase to the perfusion medium leads to complete dissociation of the liver tissue into a mixture of isolated cells and cell cords in which the hepatocytes remain connected with specific junctional differentiations, namely the gap and tight junctions. Individual cells are released by submitting the suspension of cell trabeculae to a gentle rolling. The gap junctions are ruptured at least in one of the two adjacent cells and remain generally attached to the other cell taking with them a small portion of cytoplasm. This technique of isolation of hepatocytes yields about 60-65% of the parenchymal cells contained in a liver; endothelial cells and other cells of the connective tissue are not recovered. The ultrastructural preservation of the isolated hepatocytes is excellent and the glucose-6-phosphatase activity, confined to the endoplasmic reticulum, appears unaltered in most cells. Protein, DNA and RNA recovery in the preparations of isolated hepatocytes is satisfactory, amounting to 70% of that found in liver homogenate; glycogen, the most labile component examined, is partly lost or degraded during the manipulations. Cell diameters measured by different methods confirm the preservation of the original volume of the in situ hepatocytes and the presence of more than one type of parenchymal cell. By submitting this heterogeneous cell population to an isopycnic density gradient centrifugation, two types of hepatocytes can be distinguished: the light hepatocytes, with a mean diameter of 20.5 mum and a mean density of 1.10, are characterized by an extended smooth-walled endoplasmic reticulum entrapping dispersed alpha-glycogen particles; the heavy hepatocytes, with a mean diameter of 19.0 mum and a mean density of 1.14, present a relatively reduced compartment of smooth endoplasmic reticulum, but large accumulations of glycogen. It is suggested that the cell fraction of low density is enriched in centrolobular cells and the high density fraction in perilobular hepatocytes.

scholarly journals Hormonal regulation of two urea-cycle enzymes in cultured foetal hepatocytes

1983 ◽  
Vol 216 (2) ◽  
pp. 281-285 ◽  
Author(s):  
A Husson ◽  
M Bouazza ◽  
C Buquet ◽  
R Vaillant
Keyword(s):  
Enzyme Activities ◽  
Hormonal Regulation ◽  
Cultured Cells ◽  
Urea Cycle ◽  
Rat Hepatocytes ◽  
Isolated Cells ◽  
Primary Monolayer Culture ◽  
Primary Monolayer ◽  
Urea Cycle Enzymes ◽  
Stimulation Of

Foetal-rat hepatocytes were cultured in primary monolayer culture, and activity changes of argininosuccinate synthetase (ASS, EC 6.3.4.5) and argininosuccinase (ASL, EC 4.3.2.1) were followed under defined hormone conditions. In hormone-free medium, cultured cells maintained the enzyme activities at values equal to those of freshly isolated cells for at least 3 days. Continuous addition of dexamethasone produced the development of the two enzyme activities, but only after the first 20h of culture. Under these conditions, urea production by the foetal hepatocytes was concomitantly increased in the culture medium. Pretreatment with dexamethasone for 20h was sufficient to produce the development of ASL activity within the 2 following days. Introduced alone, glucagon induced an increase of ASL activity, but did not affect the ASS activity. The most powerful stimulation of ASS and ASL could be observed in cultured hepatocytes if glucagon and dexamethasone were added simultaneously or sequentially. These results indicated that the development of the receptor complex for the induction of urea-cycle enzymes appears early before birth and established that glucocorticoids amplify the glucagon stimulation of these enzyme activities during foetal life.

Purine synthesis de novo and its regulation in rat hepatocytes

1980 ◽  
Vol 58 (8) ◽  
pp. 599-606 ◽  
Author(s):  
Christine Des Rosiers ◽  
Marcel Lalanne ◽  
Joan Willemot
Keyword(s):  
Pentose Phosphate Pathway ◽  
De Novo ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Pentose Phosphate ◽  
Male Rats ◽  
P Availability ◽  
Prolonged Incubation ◽  
Purine Synthesis ◽  
Stimulation Of

Purine synthesis de novo and its regulation were studied in freshly isolated hepatocytes from fed adult male rats. These cells incorporated [14C]formate mainly into purine ribonucleotides. The immediate effect of increasing the concentration of inorganic phosphate in the incubation medium was an increase in 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) availability and a stimulation of purine synthesis de novo. However, prolonged incubation of cells in 25 mM phosphate resulted in a decreased PP-ribose-P availability and purine synthesis de novo. Methylene blue and phenazine methosulfate decreased PP-ribose-P availability and purine synthesis de novo although they stimulated considerably the pentose phosphate pathway. In contrast, epinephrine and glucagon increased significantly PP-ribose-P availability and purine synthesis de novo, but they did not change the activity of the pentose phosphate pathway. These results show a relationship between PP-ribose-P availability and purine synthesis de novo in rat hepatocytes. They emphasize the complexity of the regulation of PP-ribose-P availability.

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