Cultured Rat Liver Cells Research Articles

Peroxisome proliferator-activated receptor (PPAR) alpha-regulated growth responses and their importance to hepatocarcinogenesis

Peroxisome proliferators (PPs) are a class of non-genotoxic rodent hepatocarcinogens that act by perturbing liver growth regulation. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-β1 (TGFβ1). More recently, we have demonstrated that PPs can suppress apoptosis induced by more diverse stimuli such as DNA damage or ligation of Fas, a receptor related to the tumour necrosis factor α (TNFα) family of cell surface receptors. PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha, PPARα, a member of the nuclear hormone receptor superfamily. We investigated whether activation of PPARα mediates the suppression of rat hepatocyte apoptosis induced by PPs. We isolated a naturally occurring variant form of PPARα (hPPARα-6/29) from human liver by PCR cloning. hPPARα-6/29 shared the ability of mPPARα to bind to DNA but, unlike mPPARα, could not be activated by PPs. Furthermore, hPPARα-6/29 could act as a dominant negative regulator of PPAR-mediated gene transcription. When introduced into primary rat liver cell cultures by transient transfection, hPPARα-6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin, but not that seen in response to phenobarbitone (PB), a non-genotoxic carcinogen whose action does not involve PPARα. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. Recent data have suggested that TNFα, perhaps released by liver Kupffer cells in response to PPs, may play a key role in mediating the effects of PPs on hepatocyte growth regulation.

Ketoconazole-Induced Apoptosis through P53-Dependent Pathway in Human Colorectal and Hepatocellular Carcinoma Cell Lines

In this study, we first demonstrated that the widely used oral antifungal drug, ketoconazole (KT), can induce apoptosis in various type of human cancer cells and in a primary culture of rat liver cells. We further investigated the molecular mechanisms of KT-induced apoptosis. It was found that KT induced nuclear accumulation ofp53protein in a dose- and time-dependent manner. The level ofp53protein was elevated approximately three times as much in treated cells 24 h after KT (5 μM) exposure as in cells receiving mock treatment. We found that cells containing wild-typep53(COLO 205 and Hep G2) were more sensitive to KT exposure. Thebaxprotein was induced and thebcl-2protein was inhibited by KT in cells containing wild-typep53(Hep G2, COLO 205) but not in cells withoutp53(Hep 3B). The caspase-3 was activated 24 h after KT treatment. The Poly-(ADP ribose) polymerase (PARP) and thelaminA degradation was induced by KT, which promoted nuclear membrane disassembly and eventually caused apoptosis. Our results also indicated that none of the PKC gene family was involved in KT-induced apoptosis.

Insulin-like growth factor I reduces thyroid hormone receptors in the rat liver. Evidence for a feed-back loop regulating the peripheral thyroid hormone action.

Tri-iodothyronine (T3) is known to be involved in the regulation of the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis. In previous studies we demonstrated that IGF-I and GH reduced the metabolic response to T3 measured as the activity of two T3-dependent enzymes, mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytosolic malic enzyme (ME) in cultured rat liver cells. In this study we analysed in vivo the effect of IGF-I administered to rats on the activity of alpha-GPD and ME. IGF-I (240 micrograms/100 g body weight (BW) every 12 h for 48 h) significantly diminished alpha-GPD (P < 0.01) and ME (P < 0.05) activities. Serum basal glucose concentration was not significantly modified 12 h after the administration of recombinant human IGF-I (240 and 480 micrograms/100 g BW every 12 h for 48 h). Under similar conditions, no significant change in serum total thyroxine (TT4) concentration was observed, although free thyroxine (FT4) was diminished (P < 0.02) and total T3 (TT3) was increased (P < 0.03). To explore the participation of the nuclear thyroid hormone receptor (THR) in the mechanism of IGF-I action we measured the maximal binding capacity and the affinity constant (Ka) of THR by Scatchard analysis, and concentrations of messenger RNAs (mRNAs) that code for the isoforms of THR present in the liver (beta 1, alpha 1 and alpha 2) by Northern blot. IGF-I (240 micrograms/100 g BW every 12 h for 48 h) significantly reduced maximal binding capacity to 37% of the control value (P < 0.01) without changes in the Ka. beta 1, alpha 1 and alpha 2 THR mRNAs were significantly reduced (P < 0.01) by 120-480 micrograms/100 g BW IGF-I administration every 12 h for 48 h. Time-course studies indicated that this effect was obtained 12 h after the administration of 240 micrograms/100 g BW IGF-I (P < 0.05). These results indicate that IGF-I administration to rats diminishes the metabolic thyroid hormone action in the liver by a mechanism that involves, at least in part, a reduction in the number of THRs and in their level of expression.

Regulation of alkaline phosphatase gene expression in human hepatoma cells by bile acids.

Bile acid-dependent secretion and the translationally regulated synthesis of alkaline phosphatase (ALP) in rat liver cell culture and by bile duct ligation has already been demonstrated. With the advent of ALP cDNA cloned sequences, the mechanism of the effect of bile acids on ALP activity and the expression of the ALP gene in different hepatoma cells was investigated. The HuH7 and HepG2 cells were treated with taurine-conjugated cholic acid (CA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) at 0-1 mmol/L and the numbers of viable cells were counted at 24, 48 and 72 h after treatment. A [3H]-thymidine incorporation study was performed with different bile acids at a concentration of 1 mmol/L for 48 h. The cellular activity of ALP in HuH7 and HepG2 cells by bile acids was measured by a substrate-specific enzymatic assay. To elucidate the effects of bile acids on ALP gene expression, a northern blotting experiment using hybridization with mouse placental ALP cDNA was performed. Cellular ALP activity was time- and dose-dependently increased in both HuH7 and HepG2 cells treated by CA and CDCA; however, no change in ALP activity was observed following treatment with UDCA compared with controls. Induction of ALP activity was dominant in HepG2 cells and independent of cell growth and proliferation. The addition of UDCA synergistically reduced the increased activity of ALP produced by CA and CDCA in both HuH7 and HepG2 cells. By northern blot analysis, the level of ALP mRNA was elevated by CA and CDCA; however, levels of ALP mRNA were suppressed by UDCA. In conclusion, CA and CDCA cause up-regulation of ALP mRNA and UDCA leads to down-regulation of ALP mRNA by its interaction with either CA or CDCA. We assume that increased ALP synthesis in hepatoma cells after bile acid treatment results from an enhanced rate of transcription rather than translation of mRNA.

Comparative Toxicity of Eugenol and Its Quinone Methide Metabolite in Cultured Liver Cells Using Kinetic Fluorescence Bioassays

Comparative kinetic analyses of the mechanisms of toxicity of the alkylphenol eugenol and its putative toxic metabolite (quinone methide, EQM) were carried out in cultured rat liver cells (Clone 9, ATCC) using a variety of vital fluorescence bioassays with a Meridian Ultima laser cytometer. Parameters monitored included intracellular GSH and calcium levels ([Ca2+]i), mitochondrial and plasma membrane potentials (MMP and PMP), intracellular pH, reactive oxygen species (ROS) generation, and gap junction-mediated intercellular communication (GJIC). Cells were exposed to various concentrations of test compounds (1 to 1000 μM) and all parameters monitored directly after addition at 15 s intervals for at least 10 min. Eugenol depleted intracellular GSH, inhibited GJIC and generation of ROS, and had a modest effect on MMP at concentrations of 10 to 100 μM. At high concentrations (1000 μM), eugenol also affected [Ca2+]i, PMP, and pH. Effects of EQM were seen at lower concentrations (1 to 10 μM). The earliest and most potent effects of either eugenol or EQM were seen on GSH levels and GJIC. Coadministration of glutathione ethyl ester enhanced intracellular GSH levels by almost 100% and completely protected cells from cell death caused by eugenol and EQM. These results suggest that eugenol mediates its hepatotoxic effects primarily through depletion of cytoprotective thiols and interference in thiol-dependent processes such as GJIC. Furthermore, our results support the hypothesis that the toxic effects of eugenol are mediated through its quinone methide metabolite.

Evidence for the suppression of apoptosis by the peroxisome proliferator activated receptor alpha (PPAR alpha).

Peroxisome proliferators (PPs) are a class of nongenotoxic rodent hepatocarcinogens. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-beta1 (TGFbeta1). PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha (PPAR alpha), a member of the nuclear hormone receptor superfamily. Here, we investigate whether activation of PPAR alpha mediates the suppression of rat hepatocyte apoptosis induced by PPs. We isolated a naturally occurring variant form of PPAR alpha (hPPAR alpha-6/29) from human liver by PCR cloning. Electrophoretic mobility shift assays (EMSA) demonstrated that hPPAR alpha-6/29 shared the ability of mPPAR alpha to heterodimerise with the retinoid X receptor (RXR) and bind to DNA. When hPPAR alpha-6/29 was transfected into Hepa1c1c7 cells together with a reporter plasmid containing a PPAR response element (PPRE), hPPAR alpha-6/29, unlike mPPAR alpha, could not be activated by PPs. Furthermore, hPPAR alpha-6/29 could act as a dominant negative regulator of PPAR-mediated gene transcription since increasing concentrations of hPPAR alpha-6/29 abrogated the activation of co-transfected mPPAR alpha. When introduced into primary rat liver cell cultures by transient transfection, hPPAR alpha-6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin, but not that seen in response to phenobarbitone (PB), a nongenotoxic carcinogen whose action does not involve PPAR alpha. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. These data indicate that activation of rat liver PPAR alpha provides a survival signal for hepatocytes, preventing their death in response to apoptotic stimuli.

Up-regulation of P-glycoprotein expression in rat liver cells by acute doxorubicin treatment.

Expression of P-glycoprotein, a plasma-membrane glycoprotein involved in multidrug resistance and encoded by mdr genes, was investigated in cultured rat liver cells acutely exposed to doxorubicin. This anticancer drug was shown to increase mdr mRNA levels in a dose-dependent manner in both rat liver epithelial (RLE) cells and primary rat hepatocytes. This induction of mdr transcripts was detected as early as a 4-h exposure to doxorubicin used at 0.5 microg/ml. It occurred through increased expression of the mdr1 gene as assessed by northern blot analysis using rat mdr-gene-specific probes. In addition, RLE cells exposed to doxorubicin displayed an overexpression of a 140-kDa P-glycoprotein as demonstrated by western blotting. Moreover, doxorubicin-treated RLE cells displayed enhanced cellular efflux of the P-glycoprotein substrate rhodamine 123 that was inhibited by the P-glycoprotein blocker verapamil, thus providing evidence that doxorubicin-induced P-glycoprotein was functional in liver cells. Doxorubicin-mediated mdr mRNA induction was found to be fully inhibited by actinomycin D, thus indicating its dependence on RNA synthesis; it was demonstrated to be not associated with alteration of protein synthesis, suggesting it differed from the known mdr mRNA overexpression occurring in response to cycloheximide. In contrast to P-glycoprotein, other liver detoxification pathways such as cytochromes P-450 1A were not induced by doxorubicin treatment. These data indicate that doxorubicin can act as a potent acute inducer of functional P-glycoprotein in rat liver cells and therefore may modulate both chemosensitivity of hepatic cells and P-glycoprotein-mediated biliary secretion of xenobiotics.

Vasopressin synergistically stimulates DNA synthesis in normal and regenerating rat liver cell cultures in the presence of hepatocyte growth factor.

Liver regeneration is significantly impaired in rats with both alpha-adrenergic hepatic denervation and hereditary vasopressin deficiency. This may implicate a direct role for these agonists in the process of compensatory hyperplasia. The mitogenic capacities of norepinephrine, vasopressin and hepatocyte growth factor (HGF), either alone or in combination were investigated by [3H]thymidine incorporation into hepatocyte cultures prepared from normal and regenerating rat livers. The results show that normal hepatocytes incorporate less [3H]thymidine in response to HGF than do regenerating hepatocytes. In addition, physiological concentrations of vasopressin cause a synergistic stimulation of [3H]thymidine uptake in rat liver cells in the presence of HGF.

Proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha inhibit growth hormone stimulation of insulin-like growth factor I synthesis and growth hormone receptor mRNA levels in cultured rat liver cells.

Low levels of insulin-like growth factor I (IGF-I) in critical illness are observed despite increased or normal levels of growth hormone (GH). The mechanisms for this apparent GH resistance have not been elucidated. As many of the acute inflammatory responses in critical illness are mediated by the proinflammatory cytokines interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha), the present studies evaluated IL-1 beta and TNF-alpha effects on steady-state and GH-stimulated IGF-I synthesis and GH receptor mRNA levels. In rat hepatocytes in primary culture, IGF-I released into culture medium was determined by radioimmunoassay, and quantitative competitive polymerase chain reaction was used to measure IGF-I mRNA and GH receptor mRNA concentrations. Growth hormone increased GH receptor mRNA, IGF-I mRNA and IGF-I protein secreted into the culture medium. In cells not stimulated with GH, modest inhibitory effects of IL-1 beta on GH receptor mRNA, IGF-I mRNA and IGF-I protein levels were seen. However, the stimulatory effects of GH were inhibited in a dose-dependent manner both by IL-1 beta and TNF-alpha, and at higher cytokine concentrations no stimulatory effects of GH were observed. Both IL-1 beta and TNF-alpha in submaximal dose had additive inhibitory effects on IGF-I protein concentrations but these effects did not result in irreversible damage to cells, as indicated by restoration of IGF-I and GH receptor mRNA levels to normal after withdrawal of cytokines. In conclusion, we demonstrated that in rat hepatocytes in primary culture IL-1 beta and TNF-alpha inhibited GH-stimulated IGF-I synthesis. Diminished GH receptor mRNA concentrations in response to IL-1 beta and TNF-alpha indicate that low IGF-I levels during severe illness, despite high circulating GH levels, may at least partially be a consequence of suppression of hepatic GH receptor synthesis by IL-1 beta and TNF-alpha.

Lack of Effects of Ciprofloxacin and the Topoisomerase II Inhibitors,m-AMSA and Nalidixic Acid, on DNA Repair in Cultured Rat Liver Cells

Several quinolone antibiotics, including ciprofloxacin, have been reported to elicit autoradiographic unscheduled DNA synthesis (UDS) in cultured rat hepatocytes. In the present investigation, ciprofloxacin (CF), at 250–1500 μM, produced autoradiographic UDS in cultured rat hepatocytes, whereas neither the quinolone nalidixic acid norm-AMSA, both topoisomerase II inhibitors, produced autoradiographic UDS. CF also reduced cytoplasmic [3H]thymidine levels ([3H]TdR) relative to control at 250–1500 μMand concomitantly increased nuclear grain counts accounting for most of the net increase yielding positive UDS values. To obtain definitive information on whether the positive UDS observed with CF was due to DNA repair, DNA repair synthesis was measured in parental DNA separated from newly replicated DNA using a bromodeoxyuridine incorporation density gradient method. This method was used to measure DNA repair synthesis in parental DNA of both replicating rat liver epithelial cells (ARL-18) and nonproliferating rat hepatocytes in primary culture. Primary hepatocytes exposed to CF from 250 to 1500 μMdid not express DNA repair synthesis in parental DNA isolated by density gradient centrifugation but rather exhibited a concentration-related decrease in the level of [3H]TdR associated with DNA. In rat liver epithelial (ARL-18) cells, CF from 250 to 500 μMlikewise did not elicit DNA repair synthesis and also caused a concentration-related decrease in the level of [3H]TdR associated with parental DNA. In contrast, in both cell types a substantial level of repair synthesis occurred in parental DNA as a result of exposure to 2-acetylaminofluorene, a DNA-reactive carcinogen, and in hepatocytes a similar finding was made for the drug hydralazine. Also, after induction of DNA repair in hepatocytes by ultraviolet light, the DNA polymerase α inhibitor aphidicolin almost completely abolished repair synthesis, whereas CF had a negligible effect on the inhibition of repair relative to control. These results indicate that CF did not elicit authentic DNA repair and also did not inhibit DNA repair synthesis. The fact that CF elicited autoradiographic UDS and that the topoisomerase II inhibitorsm-AMSA and nalidixic acid did not indicates that effects on topoisomerase II are not the basis for the positive UDS result with CF as has been hypothesized in the past.

The influence of cyclosporine on lipid peroxidation and superoxide dismutase in adriamycine nephropathy in rats

The changes in the activity of nicotinamide: S-adenosylmethionine methyltransferase (nicotinamide methylase) were studied in rat liver which was subjected to different rates of cellular proliferation. The cytosolic enzyme activity increased 3–4-fold in the first 24–48 h after partial hepatectomy and decreased again to the basal levels until 4 days post-operatively, whereas it remained unchanged in the livers of sham-operated animals. A single administration of thioacetamide at a dose of 50–250 mg/kg body weight, a treatment which induces hepatocellular proliferation as well, also enhanced the enzyme activity 2–3-fold 24 h after drug administration. This activity increase was associated with a marked lowering of intracellular NAD content of as much as 50% of the control levels. d-Galactosamine, a known hepatotoxic agent causing acute hepatitis in experimental animals and preventing DNA synthesis in regenerating liver, blocked the activity increase in regenerating rat liver. The rate of 1-methylnicotinamide synthesis, as measured by incubating liver slices in the culture medium supplemented with [14C]nicotinamide as a precursor, was found to be 2–4 times higher in the slices from regenerating liver and thioacetamide-treated rat liver than those from non-proliferating control liver. These results, together with our previous finding on the enhancement by 1-methylnicotinamide of the growth of cultured rat liver cells (Hoshino, J., Kühne, U. and Kröger, H. (1982) Biochem. Biophys. Res. Commun. 105, 1446–1452), support the view that nicotinamide methylase and its product, 1-methylnicotinamide, are involved in the control of hepatocellular DNA synthesis and proliferation.

Regulation of type I 5'-deiodinase by thyroid hormone and dexamethasone in rat liver and kidney cells.

Type I 5'-deiodinase (5'D-I) is crucial for the generation of circulating 3,5,3'-triiodothyronine (T3) and so is a major determinant of thyroid hormone action. Liver and kidney 5'D-I activity is reduced in nonthyroidal illness (NTI), but the exact cause of reduced 5'D-I activity remains unknown. Elevated circulating glucocorticoid hormone concentrations are one factor postulated to play a role. We have studied regulation of 5'D-I expression in cultured rat liver (phi 1) and kidney (NRK-52E) cells in response to treatment with T3 and the glucocorticoid dexamethasone. 5'D-I mRNA was measured by Northern hybridization and 5'D-I activity was measured in a sub-strate conversion assay. Expression of the sodium pump alpha 1-subunit (Na+/K(+)-ATPase alpha 1-subunit) mRNA was measured as an index of T3 and dexamethasone effects. In ø1 liver cells, T3 increased 5'D-I mRNA by 76 +/- 17% and 5'D-I activity by 101 +/- 30% (all results mean +/- SEM; n = 9). Dexamethasone increased 5'D-I mRNA by 55 +/- 16% and 5'D-I activity by 128 +/- 6%. In NRK-52E rat kidney cells, 5'D-I mRNA increased by 87 +/- 15% with T3 treatment and by 76 +/- 14% with dexamethasone. 5'D-I activity in NRK-52E cells was measurable only after stimulation by combined T3 and dexamethasone treatment. Na+/K(+)-ATPase alpha 1-subunit mRNA expression was stimulated following T3 and dexamethasone treatment in both cell lines. These results provide evidence for direct pretranslational regulation of 5'D-I by T3 and dexamethasone in both rat liver and kidney cells. Our findings do not support the hypothesis that glucocorticoids are directly responsible for inhibition of 5'D-I enzyme activity in NTI.

Insulin-like growth factor II regulation of gene expression in rat and human hepatomas.

Insulin-like growth factor II (IGF II) regulated tissue-specific gene expression in hepatoma cell lines, but had no effect on expression of tissue-specific genes in primary cultures of E14 and newborn rat liver cells depleted of erythroid cells. No change was observed in these primary cultures with respect to alpha-fetoprotein (alpha-FP), albumin, cytokeratin 19 (CK19), gamma-glutamyltranspeptidase (GGT), and IGF II receptors. Two well-differentiated hepatoma, HepG2 and FTO-2B, and a poorly differentiated hepatoma, H4AzC2, did not show increased proliferation in the presence of IGF II, yet showed gene expression changes in response to IGF II. In HepG2 cells, IGF II increased albumin mRNA levels and resulted in a shift from clusters of cells positive to 100% of the cells expressing immunohistochemically detectable albumin. The transcription factor HNF-3 beta mRNA and protein levels of the bile duct markers, CK19 and GGT, were also increased in the presence of IGF II. Other genes tested were not affected, including alpha-1-antitrypsin, and two liver-specific transcription factors, HNF-4 and HNF-3 alpha. In FTO-2B cells, IGF II increased the expression of albumin, CK19, and GGT, without accompanying changes in albumin and GGT mRNAs. In H4A7C2 cells, IGF II reduced CK19 and OC.3 protein levels and GGT, transferrin, and HNF-3 beta mRNAs. The effects of IGF II on H4AZC2 cells were not blocked in the presence of an anti-rat IGF II receptor antibody. We conclude that IGF II affects tissue-specific gene expression of hepatomas and qualitative and quantitative aspects of its influence on the hepatomas is dependent on their degree of differentiation.

Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells.

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that appear to play an important role in the cellular defense system against cadmium toxicity. Although substantial evidence exists demonstrating a reduction in cadmium toxicity concomitant with MT induction, little is known about the possible effects of stimulation of MT synthesis on cadmium-induced genotoxicity. Thus, the alkaline elution technique was used to assess single-strand DNA damage (SSD) in TRL-1215 cells, a liver-derived cell line shown to have inducible MT gene expression. The SSD accumulated over a 2-h time period in a time-dependent manner following exposure to 500 microM CdCl2. Low-concentration cadmium pretreatment (10 microM CdCl2, 24 h) provided protection against the genotoxicity of high-concentration cadmium (500 microM CdCl2, 2 h). A 2-h exposure to 500 microM CdCl2 had no effect on viability, as assessed using a tetrazolium-dye based assay, in cells from either the pretreated or nonpretreated group. Metallothionein was induced in a time-dependent manner by low-concentration cadmium pretreatment: Exposure for 24 and 48 h resulted in 3.3- and 6.4-fold increases, respectively. In addition, a 24-h exposure to low-concentration cadmium resulted in an increase in MT-I gene expression. Cadmium accumulation was 2.6-fold greater in low-concentration cadmium-pretreated cells as compared to nonpretreated cells. These data demonstrate that low-concentration cadmium pretreatment provides protection against cadmium-induced single-strand DNA damage and support the hypothesis that this protection is due to stimulation of MT gene expression.

The mechanism of zinc uptake by cultured rat liver cells.

1. The initial rate of 65Zn uptake into cultured rat hepatocytes has been measured over a range of Zn2+ concentrations from 3 x 10(-10) M to 5 x 10(-6) M. Histidine and albumin were used to buffer Zn2+ ions at concentrations below 1 x 10(-6) M. 2. The results suggest there are two mechanisms for Zn2+ uptake; a high-affinity, saturable pathway, with a maximum velocity (Vmax) of 20-30 pmol (mg protein)-1 min-1 and a Michaelis-Menten constant (Km) of about 2 x 10(-9) M Zn2+ (with histidine), and a low-affinity, linear pathway, that only makes a significant contribution to Zn2+ uptake at Zn2+ concentrations above 1 x 10(-6) M. 3. Transport via the high-affinity pathway is dependent on the concentration of Zn2+ ions and not on the concentrations of Zn(2+)-ligand complexes, suggesting that Zn2+ is the transported species. 4. The affinity of the saturable pathway for Zn2+ is slightly lower in the presence of albumin, with a Km of about 1.3 x 10(-8) M. The reason for this is uncertain.

Vitisin A and cis-vitisin A, strongly hepatotoxic plant oligostilbenes from Vitis coignetiae (Vitaceae)

The methanolic extract of one of the common Vitaceaeous plants, Vitis coignetiae, showed marked prevention against injuries of primary cultured rat liver cells induced by carbon tetrachloride and D-galactosamine. Activity-guided fractionation of the extract resulted in the isolation of not only an antihepatotoxic stilbene derivative, e-viniferin, but also novel oligostilbenes, vitisin A and its stereoisomer cis-vitisin A as a mixture. The structures of the oligostilbenes have been determined on the basis of spectroscopic evidence, especially by 2D NMR methods such as HMBC spectra of degradative products

Release of polyamines in cultures of rat parotid and liver cells.

Rat parotid gland and liver cells were cultured for 6 and 24 h. The cells as well as their growth medium were analyzed on their content of the polyamines putrescine, spermidine and spermine. In control medium the content of polyamines was very low but already after 6 h substantial amounts of all three polyamines under study had been released into the medium from parotid as well as from liver cells. The release was much more pronounced from parotid compared to liver cells. Putrescine was accumulated in parotid cells as well as in the medium indicating that a new synthesis of this amine occurred in these cells.

Cytokeratin 14 expression in rat liver cells in culture and localization in vivo

Abstract Rat liver epithelial cells (LECs) are non-parenchymal proliferating cells that readily emerge in primary culture and can be established as cell lines, but their in vivo cell(s) of origin is unclear. We reported recently some evidence indicating that the LEC line, T51B, contains two cytokeratins (CKs) equivalent to human CK8 and CK14 respectively. T51B cells also contain vimentin assembled as a network of intermediate filaments distinct from that of the CKs. In the present study, we examined the expression of CK14 gene in various LEC preparations and a Triton-resistant rat skin cytoskeletal fraction, and then assessed its usefulness as an LEC specific marker in the liver. Northern and Western blot analyses with cDNAs and antibodies for CK8, CK14, CK18 and vimentin confirmed that rat hepatocytes express CK8 and CK18 genes only, whereas T51B cells express CK8, CK14 and vimentin genes in the absence of CK18. CK14 was also present in LECs derived as primary from embryonic-day 12 rat liver and secondary cultures from 4-day-old rat liver. Primary cultures of oval cells isolated from 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) treated rat liver (an enriched source of biliary epithelial cells) contained CK14 mRNAs which were slightly shorter than those in LECs. The analyses of CK5 (the usual partner of CK14) gene expression using specific cDNA and antibody clearly demonstrated its absence in LECs. In situ double immunolocalization analyses by laser scanning confocal microscopy showed that CK14 was not present in hepatocytes (HES + 6 cells) and was expressed in some biliary epithelial (BDS 7 + cells). CK14-positive cells were also found in the Glisson's capsule. However, CK14-positive cells of the portal region were vimentin negative, whereas those of the Glisson's capsule were vimentin positive. Our results suggest that CK14 gene expression is part of the differentiation program of two types of LECs and that this differential CK14 gene expression can be used as a new means to type LECs in culture and in vivo.

Regulation of insulin-like-growth-factor-II gene expression in rat liver cells.

The rat insulin-like-growth-factor-(IGF)-II gene is expressed at high levels during embryonic and fetal life and at low levels in adult animals. To study the regulation of IGF-II gene expression, we analyzed the synthesis and localization of the IGF-II transcripts in cultured rat liver cells either expressing (BRL3A cells) or not expressing (BRL30E and FAO cells) the IGF-II mRNA. The IGF-II gene is transcribed at a similar rate in expressing and non-expressing cells, whereas its nuclear and cytoplasmic RNA levels are diversely distributed in the cells. IGF-II RNA is more abundant in the cytoplasmic than in the nuclear RNA fraction of BRL3A cells and is present in the nucleus but not in the cytoplasm of the FAO cells. However, both precursor and mature IGF-II nuclear RNA levels are reduced in FAO cells. Our data indicate that the IGF-II gene expression is regulated by mechanisms affecting the subcellular distribution and the abundance of the transcripts.

Effects of vitamin A on collagen metabolism by cultured rat liver cells

Conflicting results have been reported concerning the phenotypes of collagen produced by cultured Ito cells. These variations may be attributed to differences in pretreatment, i.e., with or without vitamin A to facilitate the separation of Ito cells. In the present study, the effects of vitamin A on collagen metabolism by Ito cells and hepatocytes of rats were analyzed. In cultured Ito cells, staining reactions to type I collagen increased, and those to type IV collagen and laminin decreased after pretreatment with vitamin A. The rate of collagen synthesis by Ito cells decreases significantly by treatment with vitamin A. The decrease was clearer in degraded collagen than in intact collagen. The synthesis of type I collagen increased and that of type IV collagen significantly decreased in Ito cells by treatment with vitamin A. In the hepatocytes, the staining reaction to type I collagen increased with vitamin A pretreatment. The net collagen and type III collagen synthesis in hepatocytes decreased by treatment with vitamin A. These results indicate that vitamin A modifies collagen metabolism in different cell types in different ways.