Normal Rat Liver Cells Research Articles

The number and distribution of hepatic natural killer cells (pit cells) in normal rat liver: An immunohistochemical study

Pit cells are a unique population of cells in sinusoids and peripheral blood, which can be considered natural killer (NK) cells with large granular lymphocyte (LGL) morphology. The aim of this study was to investigate the use of the monoclonal antibody (MAb) 3.2.3 as a specific marker of rat pit cells to detect their number and distribution in the liver. The number of 3.2.3-positive cells was comparable to the number of LGL in liver low-density (LD) and high-density (HD) pit cell fractions and in blood lymphocytes (P > 0.05). Immunoelectron microscopy showed that nearly all LGL in hepatic LD and HD and in blood fractions were 3.2.3 positive. Using MAb 3.2.3 immunoperoxidase staining, the mean number of pit cells in liver frozen sections was determined to be 13.7 ± 1.1/mm2. The number of pit cells was similar in the different liver lobes (P > 0.05). Reduced nicotinamide-adenine dinucleotide (NADH) oxidase histochemical staining, to visualize a portal to central vein gradient, combined with immunostaining was used to analyze the lobular distribution of pit cells. We found that 61.3% (17.1/mm2) of pit cells were in the periportal area and 38.7% (10.8/mm2) in the central area. We conclude that MAb 3.2.3 can be used as a specific marker of rat pit cells and therefore can be used to quantify rat pit cell number in various experimental models.

Acute effects of rat growth hormone (GH), human GH and prolactin on proliferating rat liver cells in vitro: A study of mitotic behaviour and ultrastructural alterations

Examination of livers from transgenic mice over expressing human growth hormone (hGH) revealed numerous alterations including a striking incidence of mitotic figures. The reason for increased proliferation is unclear, but could be related to effects of hGH, which is also acting as a lactogen in rodents. In order to identify some of the actions of GH, we have examined the effects of rat and human GH and rat prolactin on proliferation, as well as on morphological differentiation of normal rat liver cells in vitro. These cells, isolated from a 20-day-old rat, proliferate in culture, incorporate BrdU and are thus strikingly different from primary cultures of isolated hepatocytes, which typically are non-proliferating ceils. Monolayers of these cells were treated with rat prolactin (rPRL), rat growth hormone (rGH). rPRL and rGH in combination, or hGH, for 24 hr. Subsequently, mitotic figures were counted and the cultures were processed for transmission electron microscopy. The incidence of mitotic figures was significantly increased by rPRL (27.4%) versus control (19%), while rGH (13%) and hGH (9.6%) significantly decreased proliferation. In controls, 2% of the proliferating cells were in prophase, approximately 12% in metaphase and approximately 15% in telophase. In contrast, rPRL caused a significant increase in the number of cells in prophase (14%) and reduced the number of cells in the other mitotic stages. hGH and rGH reduced the overall number of mitotic figures. Unexpectedly, the effects of rGH plus rPRL were different from the effect of hGH. In addition, each treatment caused distinct morphological changes of liver cell organelles. Thus, after rPRL treatment mitochondria were generally large and bent, while rGH treatment was associated with irregularly shaped mitochondria and conspicuous microtubule/ microfilament arrangements. In contrast, after rPRL plus rGH treatment the chromatin pattern was altered. In summary, these results suggest specific hormone effects on both cell proliferation and metabolism as reflected by distinct changes in morphology. Unexpectedly, the combined effect of rGH and rPRL were not identical to the effects of hGH, suggesting a possibility of a unique effect of hGH in this system.

Immunohistochemical analysis of S-phase cells in normal human and rat liver by PC10 monoclonal antibody.

The expression of proliferating cell nuclear antigen (PCNA) was examined in normal human and rat liver fixed in either formaldehyde or methanol, and was compared with the incorporation of bromodeoxyuridine (BrdU) in S-phase cells. Codistribution of PCNA and BrdU was assessed in rat liver by double immunohistochemical staining using PC10 and anti-BrdU monoclonal antibodies to identify labelled nuclei of parenchymal and sinusoidal cells. In formaldehyde-fixed human biopsies (n = 13) PCNA-labelling index (PCNA LI) was 0.43 +/- 0.24% (mean +/- SEM) for hepatocytes and 0.09 +/- 0.03% for sinusoidal cells. A great interspecimen variability was observed and a preferential lobular distribution was not evident. In methanol-fixed human liver (n = 8) the immunostaining was strong. PCNA LI was 0.05 +/- 0.01% for hepatocytes and 0.14 +/- 0.01% for sinusoidal cells. 75% of labelled hepatocytes and 60% of labelled sinusoidal cells were found in acinar zone 1. In formaldehyde-fixed rat liver (n = 10) a weak nuclear staining and a great interspecimen variability were evident. LI was 0.13 +/- 0.07% for hepatocytes and 0.40 +/- 0.21% for sinusoidal cells without preferential acinar distribution. In methanol-fixed rat liver (n = 10), PCNA LI was 0.14 +/- 0.02% for hepatocytes and 0.40 +/- 0.04% for sinusoidal cells. 64% of labelled hepatocytes and 50% of labelled sinusoidal cells were found in zone 1. Only on methanol-fixed material did double immunohistochemistry show an almost complete overlap of BrdU and PCNA labelling. The PCNA LIs and the zonal distribution of labelled nuclei as obtained in methanol-fixed material are in keeping with previous reports using 3H-thymidine (3H-Thy) incorporation, suggesting that PCNA immunostaining represents a valid alternative to 3H-Thy.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis of smooth muscle α-actin positive perisinusoidal cells in normal human and rat liver

Quantitative analysis of smooth muscle α-actin positive perisinusoidal cells in normal human and rat liver

Ito cell heterogeneity: Desmin-negative Ito cells in normal rat liver

The presence of desmin is used to identify Ito cells in rat liver and to evaluate the purity of separated and cultured Ito cells. Heterogeneity of the normal Ito cell population has been suggested; this could include variations in the content of cytoskeletal components. For these reasons we decided to reevaluate the use of desmin staining as a phenotypical marker of Ito cells in normal rat liver. Our approach was to combine desmin staining with identification of vitamin A (autofluorescence), lipid droplets (Sudan III), vimentin, laminin and tenascin, using cryostat sections: Immunofluorescence, double-immunofluorescence or immunoperoxidase techniques were used. All the techniques described corroborate the existence of desmin-negative Ito cells, mainly located in pericentral areas. In fact, lobular desmin-positive cells showed uneven distribution because they were more frequent in periportal than in pericentral areas. On the contrary, Ito cells identified on the basis of morphological criteria or positivity for laminin were evenly distributed. Double immunofluorescence confirmed this observation, showing nearly complete codistribution of laminin and desmin in periportal areas. Outside this area, positivity for desmin was observed only in about 50 of lamininpositive cells. Our observations suggest that desmin cannot be viewed as a phenotypical marker but rather is a differentiation marker of Ito cells, possibly indicating a specific functional state. (Hepatology 1994;19:440–446).

Ito cell heterogeneity: Desmin-negative ito cells in normal rat liver

The presence of desmin is used to identify Ito cells in rat liver and to evaluate the purity of separated and cultured Ito cells. Heterogeneity of the normal Ito cell population has been suggested; this could include variations in the content of cytoskeletal components. For these reasons we decided to reevaluate the use of desmin staining as a phenotypical marker of Ito cells in normal rat liver. Our approach was to combine desmin staining with identification of vitamin A (autofluorescence), lipid droplets (Sudan III), vimentin, laminin and tenascin, using cryostat sections: Immunofluorescence, double-immunofluorescence or immunoperoxidase techniques were used. All the techniques described corroborate the existence of desmin-negative Ito cells, mainly located in pericentral areas. In fact, lobular desmin-positive cells showed uneven distribution because they were more frequent in periportal than in pericentral areas. On the contrary, Ito cells identified on the basis of morphological criteria or positivity for laminin were evenly distributed. Double immunofluorescence confirmed this observation, showing nearly complete codistribution of laminin and desmin in periportal areas. Outside this area, positivity for desmin was observed only in about 50% of laminin-positive cells. Our observations suggest that desmin cannot be viewed as a phenotypical marker but rather is a differentiation marker of Ito cells, possibly indicating a specific functional state.

Expression of P-glycoprotein in normal and malignant rat liver cells.

Expression of P-glycoprotein in normal and malignant rat liver cells.

Characterization of a mature bile duct antigen expressed on a subpopulation of biliary ductular cells but absent from oval cells

Hybridomas were produced with immune spleen cells generated by immunization of Balb/c mice with oval cell antigen (OC.2)-positive 17-day fetal liver cells isolated on antibody-coated magnetic beads. A primary screen by indirect immunofluorescence on frozen sections of fetal and adult liver identified several hybridomas secreting antibodies reactive with bile ducts and oval cells. One of these recognized an epitope, designated BD1, which was expressed on intrahepatic bile ducts in 16-day fetal and adult rat liver and in liver from rats fed a choline-deficient diet containing ethionine and rats treated with 2-acetylaminofluorine, but was absent from morphologically defined oval cells induced by a choline-deficient diet containing ethionine or by 2-acetylaminofluorine. Double-labeling immunofluorescence analysis with monoclonal antibody BD1 and OV6, a monoclonal antibody that reacts uniformly with all bile ducts and oval cells, revealed that BD1 expression on the intrahepatic bile ductular cells of normal adult rat liver was heterogeneous with a major ductal cell population (60% to 70%) expressing high levels of BD1 and a minor ductal cell population (30% to 40%) displaying undetectable or low levels of BD1 expression. Analysis of fetal liver demonstrated the presence of BD1-positive cells at day 16 of gestation on duct-like structures in contact with portal mesenchyme, an observation suggesting that expression of BD1 was associated with commitment of hepatoblasts to a ductular lineage. Taken together, our findings suggest that oval cells may be derived from an antigenically distinct subpopulation of bile ductal cells.

Long-term culture and characteristics of normal rat liver bile duct epithelial cells

Background: A method was established for isolation and long-term culture of bile duct epithelial cells (BDEC) of normal adult rat liver that does not require the preparation of highly purified BDEC. Methods: After dissociation of the liver parenchyma by collagenase perfusion, the liver remnant containing the intact biliary tree was minced into small fragments, embedded in a rat tail collagen gel, and cultured for 6 days in hormonally defined serum-free Dulbecco's Modified Eagle Medium/F12 medium (SFDM). BDEC cultures were subsequently subcultured and maintained on rat tail collagen gels in SFDM medium containing 5 μmol/L forskolin and 5%–10% Nu Serum IV (Collaborative Research, Bedford, MA). Results: Established BDEC lines continued to express ductal specific markers including γ-glutamyl transpeptidase, cytokeratins 7 and 19, and a number of monoclonal antibody-defined bile duct antigens, such as OC.2, OC.3, and OV6. Conclusions: The availability of a method to establish normal BDEC lines will allow further investigation of the function of bile duct cells and their role in normal liver differentiation and carcinogenesis.

Cross-linked Cytokeratin Polypeptides in Liver and Hepatoma Cells: Possible Association with the Process of Cell Degeneration and Death

We investigated transglutaminase-induced cross-linking of cytokeratin polypeptides in liver and hepatoma cells. To overcome the difficulties in the biochemical analysis of highly cross-linked polymers and aggregates of cytokeratins, cross-linked cytokeratin dimers were analyzed by immunoblotting to evaluate the degree of cross-linking of cytokeratins. Covalently cross-linked cytokeratin dimers were not detectable in normal rat liver cells. However, cytokeratin dimers and high-molecular-weight cytokeratin polymers were detected in liver tissue with histological evidence of coagulative necrosis induced by ischemia or carbon tetrachloride. Treatment of cultured hepatoma cells with the Ca2+ ionophore A23187 showed a dose-dependent, time-dependent decrease of cell viability. The appearance of cytokeratin dimers was shown to be correlated with cell death. These results suggest that the transglutaminase-induced cross-linking of cytokeratin polypeptides in liver and hepatoma cells is closely associated with the process of cell degeneration and death.

Cross-linked cytokeratin polypeptides in liver and hepatoma cells: Possible association with the process of cell degeneration and death

We investigated transglutaminase-induced crosslinking of cytokeratin polypeptides in liver and hepatoma cells. To overcome the difficulties in the biochemical analysis of highly cross-linked polymers and aggregates of cytokeratins, cross-linked cytokeratin dimers were analyzed by immunoblotting to evaluate the degree of cross-linking of cytokeratins. Covalently cross-linked cytokeratin dimers were not detectable in normal rat liver cells. However, cytokeratin dimers and high-molecular-weight cytokeratin polymers were detected in liver tissue with histological evidence of coagulative necrosis induced by ischemia or carbon tetrachloride. Treatment of cultured hepatoma cells with the Ca2+ ionophore A23187 showed a dosedependent, time-dependent decrease of cell viability. The appearance of cytokeratin dimers was shown to be correlated with cell death. These results suggest that the transglutaminase-induced cross-linking of cytokeratin polypeptides in liver and hepatoma cells is closely associated with the process of cell degeneration and death. (HEPATOLOGY 1993;17:118–124.)

Regulation of the 12‐o‐tetradecanoyl‐phorbol‐13‐acetate‐induced inhibition of intercellular communication

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and protein kinase C (PKC) inhibitors on cell-cell communication were studied in a normal rat liver cell line, clone 9. Communication was observed and quantitated with microspectofluorometric and image analysis techniques following scrape-loading of the cells with lucifer yellow. Lucifer yellow migrated as far as ten cells away from the scraped edge in control populations. Two minute TPA (25-50 micrograms/ml) treatment inhibited dye movement such that the dye remained mainly in the cells at the cut edge. The TPA-induced inhibition of cell-cell communication could be partially blocked by 15 min treatment of the cell populations with the PKC inhibitors trifluoperazine (30 micrograms/ml), staurosporine (2 x 10(-8) or 2 x 10(-6) M), sangivamycin (15 or 200 microM), or a PKC inhibitor peptide (20 micrograms/ml) scraped in at the same time as lucifer yellow. Normal communication was observed in cultures treated only with PKC inhibitors. Lower concentrations of TPA (50 ng/ml-1 micrograms/ml) used for 2 min did not inhibit dye communication. Our results demonstrate the phorbol ester-induced interruption of cell-cell communication. The inhibition of PKC by inhibitors eliminates the effect of TPA on communication. Our data are consistent with a role of PKC in the control of junctional communication.

Prostacyclin administration suppresses the increase in hepatic levels of COL1A(I) and glyceraldehyde-3-phosphate dehydrogenase mRNAs in the rat treated with carbon tetrachloride

Northern analysis using total RNAs from the component cells of normal rat liver indicated that COL1A(I) mRNA is present in fat-storing cells (Ito cells) and sinusoidal endothelial cells. A fraction for Kupffer cells also contained this mRNA. When CCl 4 was given, COL1A(I) mRNA was increased in a factor of 1.5 in the fractions of these component cells. After 48 h of the drug administration, hepatocytes appeared to possess over 60% of liver COL1A(I) mRNA, although in normal hepatocytes its level was below the range detectable by our procedures. Under this injured condition of liver, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level was elevated, while activity of this enzyme was lowered by 50% of the control value. All the changes were obviously suppressed by the simultaneous administration of prostacyclin.

Induction of zinc metallothionein by calcium ionophore in vivo and in vitro

The calcium ionophore, A23187, can induce rat hepatic metallothionein (MT) when administered in vivo (5.8-fold, 5.0 μM, 11 h) and rat hepatocyte MT when administered in vitro (10.70-fold, 1.0 μM, 24 h). Several rat hepatoma cell lines (2M, 4.55-fold; JM2, 12.29-fold; EC3, 14.12-fold; HTC, 7.99-fold) and a normal rat liver cell line (Clone 9, 39.67-fold) were tested for their inducibility of MT mRNA by Cd 2+ (10 μM, 8 h). Quantitatively, JM2 and 2M made the most MT mRNA, while HTC made the least. A23187 (0.1–7.0 μM) was studied as an inducer of MT mRNA in these cell lines (except for HTC) and in HeLa. A variety of responses and tolerances were seen with inductions ranging up to 32.11-fold. Quantitatively, the best responding cell lines were EC3 and 2M. A combination induction experiment, using TPA, a protein kinase C activator, and A23187 in EC3 cells revealed an additive effect of the two inducers on MT mRNA levels: TPA (10 nM), 11.71-fold; A23187 (3.0μM), 6.71-fold; and TPA + A23187, 20.00-fold. These studies have implicated perturbations in cytosolic calcium ion concentrations, caused by the ionophore A23187, as being involved in the complicated signaling systems which can lead to induction of MT mRNA and protein.

Enumeration of S-phase cells in normal rat liver by immunohistochemistry using bromodeoxyuridine-antibromodeoxyuridine system

The visualization of incorporation sites of the thymidine analog bromodeoxyuridine (BrdU) into DNA, detected by immunocytochemistry, has been proposed as an index of the percentage of S-phase cells in a variety of tissues and as an easy, less expensive alternative to autoradiography. This technique has not yet been applied to the study of physiological cell renewal in the normal liver. In the present study, results obtained with this method in the liver of normal young adult rats is reported. BrdU was administered in vivo and subsequent incorporation was detected by the PAP technique using monoclonal anti-BrdU antibodies. The nuclei exhibiting a positive reaction within the liver were few and accounted for about 0.45% of all hepatocytes. Positive cells were located preferentially in zone 1, which contained 82.7% of the labeled cells. Zone 2 contained 15.4%, while only 1.9% of the labeled cells were found in zone 3. Positive-staining Kupffer cell nuclei were rare (about 0.5% of all Kupffer cells) and were distributed randomly in the hepatic lobule. These findings provide quantitative data about hepatocyte renewal in the normal liver in the absence of a growth stimulus. The simplicity and the reproducibility of this technique suggests that further application of this method in situations assessing hepatic regeneration are indicated.

Demonstration of cross-linked cytokeratin polypeptides in transplantable rat hepatoma cells

Covalently cross-linked multimers of cytokeratins were shown to be present in transplantable Morris hepatoma 7777 cells. These high molecular weight antigens were not detectable in normal rat liver cells. However, identical high molecular weight antigens were also demonstrated in rat liver cells when the cells were homogenized in solutions containing Ca 2+. The cross-linking reaction was suggested to be mediated by the action of tissue transglutaminases.

Elevated cholesterol and decreased sterol carrier protein-2 in peroxisomes from AS-30D hepatoma compared to normal rat liver

Peroxisomes were isolated from AS-30D hepatoma and compared to normal rat liver cells for the purpose of investigating the cholesterol accumulation in the hepatoma cells. Cholesterol was found to be approximately 10-fold higher relative to protein in AS-30D peroxisomes as compared to peroxisomes from normal liver. The peroxisomes from the hepatoma cells were found to be more stable; catalase was not released from these peroxisomes during isolation or osmotic shock of the peroxisomal fraction. The elevated cholesterol level may stabilize the peroxisomal membrane. Sterol carrier protein-2 (SCP-2) levels were measured using a radioimmunoassay (RIA), which indicated the highest concentration of SCP-2 to be in peroxisomes. Hepatoma peroxisomes had a lower concentration of SCP-2 (2.5 μg/mg) than normal liver peroxisomes (8 μg/mg). Approximately half of all SCP-2 detected was found to be soluble in both hepatoma and normal rat liver cells. Immunoblots from both rat liver and AS-30D fractions demonstrated the presence of the 14-kDa form of SCP-2. The liver fractions also had a 57-kDa immunoreactive protein, which was barely detectable in the AS-30D fractions. The low abundance of the high molecular weight form of SCP-2 from hepatoma peroxisomes and the lower amounts of SCP-2 detected in the AS-30D peroxisomes may be related to the accumulation of cholesterol in the cells.

The origin and role of confronting cisternae in selected fetal mouse and rat tissues

Confronting cisternae (CC) are described for the first time in normal fetal rat and mouse liver and intestinal epithelial cells. In these cells, CC characteristically consist of 2 parallel cisternae which are devoid of ribosomes on their juxtaposed surfaces. The intracisternal spacing is consistently 20nm. While CC occur in rapidly proliferating tissues such as fetal liver and intestinal epithelium, they do not occur in hepatocytes following partial hepatectomy. Although it has been postulated that the intact CC profiles represent a mechanism of assuring the presence of performed nuclear envelope (NE) or rough endoplasmic reticulum (RER) in cells, it is more likely that the subunits which result from CC degradation serve as a pool of membrane precursors for new NE or RER.

Isolation and structure of a cDNA expressing a mammalian 3-methyladenine-DNA glycosylase.

A cDNA plasmid expression library was constructed from the poly(A)+ mRNA of H4 cells, a rat hepatoma cell line. The library was introduced into Escherichia coli strain BH290 deficient in the repair of 3-methyladenine (3-meAde) residues in DNA. This DNA repair deficiency renders the stain phenotypically sensitive to treatment with alkylating agents. The cDNA library was screened for survivors to methylmethane sulfonate. BH290 cells hosting one of the plasmids, pAPDG10 (Alkylated N-Purine-DNA Glycosylase), from surviving cells had a sensitivity to MMS equivalent to that of the wild type strain. Crude extracts of BH290 cells harboring the pAPDG10 plasmid released 3-meAde and 7-methylguanine residues from DNA methylated with [methyl-3H]dimethylsulfate. The cDNA sequence of 993 bp inserted in pAPDG10 has a single open reading frame greater than 85 amino acids in length. The derived APDG protein sequence of 253 amino acids and the 3-meAde-DNA glycosylase II of E. coli coded for by the alkA gene have regions of conserved sequences. Analysis of the genomic DNA using Southern hybridization suggests that the APDG gene has a minimal size of 6.5-12 kb. Northern blot analysis shows that the transcript produced in H4 cells is also present in normal rat liver cells.

Immunocytochemical localization of S-phase cells in normal rat liver

Immunocytochemical localization of S-phase cells in normal rat liver