Cultures Of Rat Liver Parenchymal Cells Research Articles

Uptake of Pullulan in Cultured Rat Liver Parenchymal Cells

Uptake of pullulan, including a binding process followed by internalization, was examined in cultured rat liver parenchymal cells. A tyramine derivative of pullulan was labeled with [125I]iodine and used as a ligand. Pullulan bound to the cell surface was released by EDTA treatment, indicating that pullulan binding requires Ca2+ and a contribution from the asialoglycoprotein receptor. Binding of pullulan reached a steady state and internalization represented a biphasic mode, which included first- and zero-order processes in the initial stage and after 20 min incubation, respectively. The uptake of pullulan could be estimated by a similar model for intracellular disposition of asialofetuin. Kinetic parameters of pullulan constituting both binding and internalization were below those found for asialofetuin. These results suggest that pullulan is taken up by liver parenchymal cells via the asialoglycoprotein receptor; however, uptake availability is lower than that of asialofetuin.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor α (TGFα), and 3,3ʹ,4,4ʹ,5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGFα stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGFα induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.

Identification of an enhancer element of class Pi glutathione S-transferase gene required for expression by a co-planar polychlorinated biphenyl

3,3´,4,4´,5-Pentachlorobiphenyl (PenCB), one of the most toxic co-planar polychlorinated biphenyl congeners, specifically induces class Pi glutathione S-transferase (GSTP1) as well as cytochrome P-450 1A1 in primary cultured rat liver parenchymal cells [Aoki, Matsumoto and Suzuki (1993) FEBS Lett. 333, 114–118]. However, the 5´-flanking sequence of the GSTP1 gene does not contain a xenobiotic responsive element, to which arylhydrocarbon receptor binds. Using a chloramphenicol acetyltransferase assay we demonstrate here that the enhancer termed GSTP1 enhancer I (GPEI) is necessary for the stimulation by PenCB of GSTP1 gene expression in primary cultured rat liver parenchymal cells. GPEI is already known to contain a dyad of PMA responsive element-like elements oriented palindromically. It is suggested that a novel signal transduction pathway activated by PenCB contributes to the stimulation of GSTP1 expression.

Identification of an enhancer element of class Pi glutathione S-transferase gene required for expression by a co-planar polychlorinated biphenyl

3,3',4,4',5-Pentachlorobiphenyl (PenCB), one of the most toxic co-planar polychlorinated biphenyl congeners, specifically induces class Pi glutathione S-transferase (GSTP1) as well as cytochrome P-450 1A1 in primary cultured rat liver parenchymal cells [Aoki, Matsumoto and Suzuki (1993) FEBS Lett. 333, 114-118]. However, the 5'-flanking sequence of the GSTP1 gene does not contain a xenobiotic responsive element, to which arylhydrocarbon receptor binds. Using a chloramphenicol acetyltransferase assay we demonstrate here that the enhancer termed GSTP1 enhancer I (GPEI) is necessary for the stimulation by PenCB of GSTP1 gene expression in primary cultured rat liver parenchymal cells. GPEI is already known to contain a dyad of PMA responsive element-like elements oriented palindromically. It is suggested that a novel signal transduction pathway activated by PenCB contributes to the stimulation of GSTP1 expression.

Differential expression and regulation of nucleoside transport systems in rat liver parenchymal and hepatoma cells.

Primary cultures of rat-liver parenchymal cells show carrier-mediated nucleoside uptake by a mechanism that mainly involves concentrative, Na+-dependent transport activity. In contrast, the hepatoma cell line FAO shows high nucleoside transport activity, although it is mostly accounted for by Na+-independent transport processes. This is associated with a low amount of sodium purine nucleoside transporter (SPNT) mRNA. SPNT encodes a purine-preferring transporter expressed in liver parenchymal cells. To analyze whether SPNT expression is modulated during cell proliferation, SPNT mRNA levels were determined in the early phase of liver growth after partial hepatectomy and in synchronized FAO cells that had been induced to proliferate. SPNT mRNA amounts increased as early as 2 hours after partial hepatectomy. FAO cells induced to proliferate after serum refeeding show an increase in SPNT mRNA levels, which is followed by an increase in Na+-dependent nucleoside uptake and occurs before the peak of 3H-thymidine incorporation into DNA. FAO cells also express significant equilibrative nucleoside transport activity, which may be accounted for by the expression of the nitrobenzylthioinosine (NBTI)-sensitive and -insensitive isoforms, rat equilibrative nucleoside transporter 1 (rENT1) and rENT2, respectively. Interestingly, rENT2 mRNA levels follow a similar pattern to that described for SPNT when FAO cells are induced to proliferate, whereas rENT1 appears to be constitutively expressed. Liver parenchymal cells show low and negligible mRNA levels for rENT1 and rENT2 transporters, respectively, although most of the equilibrative transport activity found in hepatocytes is NBTI-resistant. It is concluded that: 1) SPNT expression is regulated both in vivo and in vitro in a way that appears to be dependent on cell cycle progression; 2) SPNT expression may be a feature of differentiated hepatocytes; and 3) equilibrative transporters are differentially regulated, rENT2 expression being cell cycle-dependent. This is consistent with its putative role as a growth factor-induced delayed early response gene.

Phosphorylation of c-Jun stimulated in primary cultured rat liver parenchymal cells by a coplanar polychlorinated biphenyl.

Phosphorylation of c-Jun was stimulated in primary cultured rat liver parenchymal cells by treatment with a coplanar polychlorinated biphenyl congener, 3,3'4,4'5-pentachlorobiphenyl (PenCB), as well as by epidermal growth factor, but was not stimulated by the non-coplanar form. However, the amount of c-Jun mRNA did not increase with PenCB treatment. PenCB may activate a signal-transducing pathway consisting of protein kinases.

Transport of tyramine in short-term cultured rat liver parenchymal cells, endothelial cells and Kupffer cells

In a previous report, we have shown that the uptake of tyramine in isolated rat liver parenchymal cells (PC) was mainly mediated by an active transport process in addition to a simple diffusion. Our present work demonstrates that both free diffusion and transporter-mediated processes for tyramine uptake also co-exist in liver endothelial cells (LEC) and Kupffer cells (KC). Kinetic analysis for the transport-mediated process shows that the K t (μM) and V max (pmol/mg of proteins/min or pmol/10 6 cells/min) for PC are 23.5 and 748.4 or 1197.4, those for LEC are 29.2 and 141.2 or 6.6, and for KC are 76.3 and 72.0 or 8.5. Benztropine and N-ethylmaleimide (NEM), a thiol-group modifier, which potentially inhibit the activity of tyramine transporter in PC, equally block tyramine transport in LEC and KC. Thus, cysteine(s) having free thiol group(s) could be associated with the normal function of these transporters. The increase (or decrease) of the interior negative membrane potential caused by NO 3 − and SCN − (or SO 3 2− ) stimulates (or inhibits) the transport activity, indicating that membrane potential is the immediate driving force for the entry of the amine into the three types of cells. In addition to these, an interior-to-exterior proton gradient stimulates the tyramine transport in these cells, consistent with H + exchange or OH- cotransport, suggesting that the transport process could be neutrogenic. Taking these results together indicates that the tyramine transporters existing in PC, LEC and KC bear quite similar characteristics. The diversity of the efficient uptake of tyramine among the three types of cells could result from the difference of the amounts of the transporters expressed in these cells. Therefore, PC is the main type of liver cells for the clearance of tyramine from the portal vein blood.

Gap junctional intercellular communication of cultured rat liver parenchymal cells is stabilized by epithelial cells and their isolated plasma membranes.

The gap junctional intercellular communication (GJIC) determined by measuring dye coupling with Lucifer yellow, decreased within 3 d from 66% to 28% in monocultures of rat liver parenchymal cells. Coculturing of the parenchymal cells with a nonparenchymal epithelial cell line from rat liver resulted in increased and stabilized intercellular communication (83% after 3 d). The presence of isolated plasma membrane vesicles of the nonparenchymal epithelial cells also stabilized the intercellular communication between the liver parenchymal cells (70% after 3 d). When liver parenchymal cells were cocultured with a rat liver fibroblast cell line the gap junctional communication between the parenchymal cells was not stabilized (43% after 3 d), and isolated plasma membrane vesicles of the fibroblast were also unable to support the GJIC in parenchymal cells (35% after 3 d). It is concluded that plasma membrane constituents of the nonparenchymal epithelial cells were responsible for the stabilization of the GJIC between parenchymal cells. A heterotypic gap junctional communication between parenchymal and nonparenchymal cells was not observed.

Binding and intracellular fate of beta-very low density lipoprotein in isolated rat liver parenchymal cells.

Binding of rabbit 125I-tyramine-cellobiose-beta-very low density lipoprotein (125ITC-beta-VLDL) was saturable both in suspended and cultured rat liver parenchymal cells, and in isolated rat liver membrane fractions. The specific binding had KD values ranging from 8 to 10 micrograms/ml. At 37 degrees C beta-VLDL was internalized and degraded in parenchymal cells in culture, but only negligible degradation was measured in suspended cells. In cultured cells the degradation was inhibited by lysosomal and microtubular inhibitors, these agents had no effects in suspended cells. Studies of release suggested internalization in suspended cells and subcellular fractionation experiments confirmed that internalized 125ITC-beta-VLDL reached the lysosomal compartment in cultured cells, but not in suspended cells. Since lactosylated (lac-) beta-VLDL was taken up and degraded efficiently by suspended cells, these cells are not in general unable to transport large lipoprotein particles to the lysosomes. We believe that beta-VLDL does not dissociate from the receptor in the endosomes and therefore is retroendocytosed to the plasma membrane. In isolated parenchymal cells the beta-VLDL binding site is supposedly different from the methylamine-activated-alpha 2-macroglobulin (ma-alpha 2M) binding site, since; 1) Excess beta-VLDL did not reduce ma-alpha 2M binding; 2) In suspended parenchymal cells ma-alpha 2M was readily degraded, whereas beta-VLDL was not degraded and 3) The binding of beta-VLDL was not Ca+(+)-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of glutathione S-transferase P-form in primary cultured rat liver parenchymal cells by coplanar polychlorinated biphenyl congeners is suppressed by protein kinase inhibitors and dexamethasone

Glutathione S-transferase P-form (GST-P, EC 2.5.1.18) mRNA was expressed by epidermal growth factor as well as by 3,4,5,3',4'-penta-chlorinated biphenyl (PenCB) in primary cultured rat liver parenchymal cells. The expression of GST-P was suppressed by inhibitors of protein kinase C and dexamethasone, an antagonist of AP-1 transcription factor activity, whereas expression of cytochrome P4501A2 by PenCB was not affected by these reagents. The AP-1 related transcription factor may be essential for the expression of GST-P by PenCB as also may be a protein kinase C type enzyme.

Induction of glutathione S-transferase P-form in primary cultured rat liver parenchymal cells by co-planar polychlorinated biphenyl congeners

Alterations in protein synthesis in primary cultured rat liver parenchymal cells were examined after their exposure to the potent carcinogens, polychlorinated biphenyl (PCB) congeners. Co-planar PCB congeners (3,4,5,3',4'-PCB and 3,4,5,3',4',5'-PCB) (10 nM) induced a protein, the Mr of which was 25,000 (25 k protein) under denaturing conditions. However, non-co-planar PCB congeners and several xenobiotics, which induce microsomal proteins, did not induce the 25 k protein. By using immunoblotting, the 25 k protein was identified as glutathione S-transferase P-form (GST-P, 7-7, EC 2.5.1.18).

Alterations in protein synthesis in rat liver cells by in vitro and in vivo exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin

Alterations in protein synthesis in rat liver cells were examined following in vitro and in vivo exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Primary cultured rat liver parenchymal cells were exposed to 1 nM TCDD for 23 and 47 hr. Synthesis of two proteins with molecular weights ( M r) of 26,000 and 39,000 (designated 26k-P and 39k-P, respectively), other than cytochrome P450, was increased markedly in the cells. These proteins did not have the same antigens as cytochromes P450IA1 and P450IA2. Synthesis of three proteins with M rs of 24,000, 25,000 and 29,000, respectively, was decreased by TCDD. TCDD was administered to rats at a dose of 100 μg/kg body weight. The amount of five proteins (two proteins with M r of 26,000, one of 36,000 and two of 39,000) was increased in TCDD-treated rat liver. However, the proteins increased in vivo by TCDD were distinguishable from 26k-P and 39k-P by two dimensional gel electrophoresis.

A simple method for in situ freezing of anchorage-dependent cells including rat liver parenchymal cells.

We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish. The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at -80 degrees C. After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.

Induction of Protein Synthesis by Exposure to 2, 3, 7, 8-Tetrachlorodibenzo-p-Dioxin in Primary Cultured Rat Liver Parenchymal Cells and Rat Livers (Regular Presentations) (Proceedings of the 15 th Symposium on Environmental Pollutants and Toxicology)

Induction of Protein Synthesis by Exposure to 2, 3, 7, 8-Tetrachlorodibenzo-p-Dioxin in Primary Cultured Rat Liver Parenchymal Cells and Rat Livers (Regular Presentations) (Proceedings of the 15 th Symposium on Environmental Pollutants and Toxicology)

Metabolic activity of insulin-like growth factor I on cultured rat liver parenchymal cells

Journal Article Metabolic activity of insulin-like growth factor I on cultured rat liver parenchymal cells Get access H Hartmann, H Hartmann Medizinische Universitätsklinik Göttingen Search for other works by this author on: Oxford Academic Google Scholar S Zachmann, S Zachmann Medizinische Universitätsklinik Göttingen Search for other works by this author on: Oxford Academic Google Scholar W Creutzfeldt W Creutzfeldt Medizinische Universitätsklinik Göttingen Search for other works by this author on: Oxford Academic Google Scholar Acta Endocrinologica (Norway), Volume 120, Issue 3_Supplement, Jun 1989, Page S3, https://doi.org/10.1530/acta.0.120S0003 Published: 01 June 1989

Accumulation of newly synthesized serum proteins by cadmium in cultured rat liver parenchymal cells

Effects of cadmium on secretion of the two major serum proteins, albumin and transferrin were studied in primary cultured rat liver parenchymal cells. Exposure to 7 μM cadmium for 8 hr did not inhibit synthesis of both albumin and transferrin whereas secretions of the two proteins were effectively depressed. In the cells exposed to cadmium serum type albumin and proalbumin were accumulated. Cadmium may affect intracellular processing of secretory proteins and also retard the discharge of these proteins resulting in inhibition of protein secretion from the liver parenchymal cells.

Cadmium inhibits protein secretion from cultured rat liver parenchymal cells

Depressive action of cadmium (Cd) to the protein secretion from liver was studied in vitro using primary cultures of rat liver parenchymal cells. The cells were cultured in the presence of 0.1–5 μM cadmium chloride. Over this concentration range Cd had no effect on protein synthesis, but caused a dose-dependent inhibition of protein secretion. Although the metallothionein level was increased more than 4-fold by incubation with 10 −6 M dexamethasone, the depressive action of Cd was not prevented by the induction of metallothionein.

The effect of ethanol and acetaldehyde on [ 14C]pantothenate incorporation into CoA in cultured rat liver parenchymal cells

The effect of ethanol on [ 14C]pantothenate incorporation into CoA and on total CoA levels was measured in 3-day-old primary cultures of adult rat liver parenchymal cells. Ethanol decreased the incorporation of radioactivity into CoA a maximum of 67%, 5 m m ethanol was saturating for the inhibitory effect and 0.2 m m ethanol was sufficient for half-saturation. This inhibitory effect did not result from a loss of CoA precursors or from cell death. Ethanol concentrations up to 10 m m did not decrease the ATP content of cells or the total protein content of cells which adhered to the incubation flask. Ethanol (5 m m) had no effect on the cyteine + cystine content of the cells. Intracellular pantothenate concentrations were not affected by 5 m m ethanol, and increasing the pantothenate concentration did not affect ethanol inhibition. Ethanol inhibition of [ 14C]pantothenate conversion to CoA could be fully reversed by rinsing the cells free of ethanol. The ethanol inhibition could also be fully reversed by addition of 4-methylpyrazole, indicating that ethanol must be oxidized via alcohol dehydrogenase to exert its inhibitory effect. Acetaldehyde, the immediate product of alcohol dehydrogenase, was also an inhibitor of the incorporation of [ 14C]pantothenate into CoA; the maximum inhibition was 63%. Acetaldehyde concentrations maintained between 18 and 103 μ m inhibited incorporation by 57%. The inhibition by acetaldehyde did not correlate well with changes in the NADH and NAD + ratio of the cells (as determined by measuring changes in the lactate-to-pyruvate ratio). The ability of glucagon, dibutyryl cAMP + theophylline, or dexamethasone to stimulate [ 14C]pantothenate conversion to CoA was not decreased by the addition of ethanol or acetaldehyde, indicating that ethanol inhibition does not occur by reversal of the cAMP-mediated regulatory mechanism for CoA biosynthesis.

The role of actin in the surface integrity of cultured rat liver parenchymal cells

Adult rat hepatocytes grown in a primary culture showed a homogenous actin distribution during the first 24 hr in culture as seen with anti-actin antibodies in indirect immunofluorescence microscopy. When exposed to phalloidin the cells formed rapidly numerous blebs. In these cells bright fluorescent spots were seen at the base of the blebs with anti-actin antibodies. Aggregates of fibrillar material were analogously seen in electron microscopy both at the base of the blebs and in the cytoplasm of the phalloidin-treated cells. The results indicate that phalloidin-induced aggregation of actin in hepatocytes leads to a weakening and subsequent blebbing of hepatocyte surface, suggesting that the cortical meshwork of actin-containing filaments plays an important role in the surface integrity of hepatocytes.