Effect of Collagen Gel Configuration on the Cytoskeleton in Cultured Rat Hepatocytes

The cytoskeleton is important in the maintenance of cellular morphology and differentiated function in a number of cell types, including hepatocytes. In this study, adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single collagen gel for differences in the organization and expression of the cytoskeletal proteins actin and tubulin. Hepatocytes cultured between two layers of hydrated rat tail tendon collagen (sandwich gel) morphologically resembled cells in intact liver for several weeks. Actin filaments (F-actin) in these hepatocytes were concentrated under the plasma membrane in regions of cell-cell contact. In contrast, hepatocytes cultured on a single collagen gel were flattened and motile and had F-actin containing stress fibers. This was accompanied by a severalfold increase in actin mRNA. Microtubules formed an interwoven network in hepatocytes cultured in a sandwich gel, but in single gel cultures they formed long parallel arrays extending out to the cell periphery. Tubulin mRNA was severalfold greater in hepatocytes cultured on a single gel. Fibronectin and laminin staining were greater in single gel cultures, and these proteins were concentrated in fibrils radiating from the cell periphery. Overlaying a second collagen gel onto hepatocytes that had been cultured on a single gel (double gel rescue) reversed cell spreading and reduced stress fibers. Double gel rescue also resulted in a decrease in actin and tubulin mRNA to levels present in sandwich gel cultures and freshly isolated hepatocytes. These results show that the configuration of the external matrix has a dynamic effect on cytoskeletal proteins in cultured rat hepatocytes.     

Inhibition by Retinoic Acid of Albumin and DNA Synthesis in Adult Rat Hepatocytes

RA down-regulated albumin and DNA synthesis of adult rat hepatocytes in a dose-dependent manner at dose ranges of 10^?9–10^?5 m. 10^?5 m of RA decreased albumin synthesis as well as growth factors EGF, aFGF, bFGF, and HGF-induced DNA synthesis to half of the control. These inhibitory effects of RA were not a consequence of the toxicity of RA to hepatocytes.     

Ploidy-Dependent Growth and Binucleation in Cultured Rat Hepatocytes

The proliferative activity of rat hepatocytes, cultured in the presence of epidermal growth factor (EGF) and insulin, was examined by immunostaining of S-phase cells labeled with bromodeoxyuridine (BrdU) in culture. Proliferation rates of the different hepatocellular ploidy and nuclearity classes were measured by fluorescence image cytometry or by microscope counting of immunostained cells. Effects of EGF and insulin were largely additive, the binuclear cells being more growth factor-dependent (showing less growth in the absence of factors) than the mononuclear cells. A serial warm-washing procedure was used to remove excess BrdU from the culture medium, allowing the study of hepatocellular binucleation by a BrdU pulse-chase approach. A high rate of binucleation was detected (50%, possibly suggesting a quantal mechanism), indicating that the hormones induce a binucleating (polyploidizing) type of growth similar to that normally observed in the liver of growing rats. The highest proliferative activity (labeling index) in the hepatocyte cultures was found among the diploid cells, independent of the degree of mitogenic stimulation. The labeling index was inversely correlated with ploidy, suggesting that the ability of hepatocytes to proliferate decreases with increasing polyploidization.     

Propionate Enhances Synthesis and Secretion of Bile Acids in Primary Cultured Rat Hepatocytes via Succinyl CoA

To discover the role of propionate produced by colonic bacteria, this study examined the secretion of bile acids and cholesterol 7α-hydroxylase activity in the primary cultured hepatocytes. Addition of propionate (2 mM) to the medium for 48 h caused an increase in the bile acid secretion and enzyme activity, while acetate and butyrate had no significant influence. Bile acid secretion was increased by the addition of succinyl CoA and its precursor substances (α -ketoglutarate, valine, isoleucine, and methionine), but not malate and oxaloacetate, which are the metabolites of succinyl CoA. α -Ketoglutarate and valine also increased the activity of cholesterol 7α -hydroxylase. Since cholesterol 7α -hydroxylase is a microsomal cytochrome P-450 enzyme and the formation of δ-aminolevulinate from succinyl CoA in the mitochondria is the rate-controlling step for the subsequent synthesis of heme proteins, propionate may affect bile acid synthesis via elevation of mitochondrial succinyl CoA.     

Evidence for Fibrinogen Synthesis and Secretion by Rat Foetal Hepatocytes

Fibrinogen concentration in rat foetal plasma is very low at 18 days of gestation but increases rapidly thereafter. The present study provides evidence that this increase is due to synthesis by the foetus itself. (1) ¹²⁵I-labelled human fibrinogen, injected intravenously into the pregnant adult, did not reach the foetal circulation; (2) turpentine administration to the adult induced an increased maternal plasma fibrinogen concentration without affecting the foetal one; (3) conversely, in utero administration of turpentine to foetuses increased their plasma fibrinogen concentration without affecting the maternal one; (4) using sheep anti-rat fibrinogen antibodies labelled with peroxidase, in electron microscopy, fibrinogen was located in foetal hepatocytes within the organelles known to be responsible for the synthesis and the ultimate secretion of the protein.     

Synthesis of Sulfatide by Cultured Rat Schwann Cells

Abstract: The ³⁵S sulfolipids synthesized by purified cultures of rat Schwann cells, fibroblasts, and a rat cell line (RN2) were studied. Schwann cell ³⁵S sulfolipids were almost entirely [³⁵S]sulfatide, as shown by TLC in two different solvent systems with unlabeled authentic sulfatide run in the same track. RN2 and fibroblasts did not synthesize significant amounts of sulfatide, by the same criteria. Previous studies failed to detect any characteristic myelin components, including sulfatide, on Schwann cells after several days in culture (Brockes et al., 1980a; Mirsky et at., 1980). My results show that Schwann cells continue to synthesize some sulfatide in the absence of neurons.     

High Responsiveness to Thyroid Hormone of Adult Rat Primary Hepatocytes Cultured on EHS-Gel

The induction of malic enzyme gene expression by triiodothyronine and insulin was severely blunted in rat monolayer hepatocytes cultured on type I collagen compared with that in spherical hepatocytes cultured on a reconstituted basement membrane gel (EHS-gel). Although the mRNA level of thyroid hormone receptor β (TRβ) gradually decreased in the monolayer hepatocytes during culture, the mRNA level in the hepatocytes on EHS-gel was maintained at around the in vivo level. Our results suggest that the maintenance of TRβ mRNA on EHS-gel is responsible for the high responsiveness to thyroid hormone in a hepatocyte culture.     

Liposome Internalization by Isolated Rat Hepatocytes

Abstract

We studied the in vitro interaction and the endocytic process of peroxidase-loaded liposomes with isolated rat hepatocytes maintained in suspension culture. We report morphological (both at light and electron microscopy) and biochemical evidence that cationic egg PC (egg phosphatidylcholine)-liposomes are taken up by isolated rat hepatocytes via an endocytic pathway. The incubation of 2.5 mM liposomal lipids for at least 4 hours was not cytotoxic to the cells. The uptake of peroxidase-fluoresceine isothiocyanate conjugate-liposomes was not distributed homogeneously among the hepatocyte population. However the hepatocytes which have apparently internalized greater amount of probe were not damaged since cell shape and integrity of the membrane are retained as evaluated by conventional light microscopy. Therefore the fusion between liposomes and hepatocytes seems to be dependent on the viability of the isolated hepatocytes.     

Glandular Structure Formation of LS174T Human Colon Cancer Cells Cultured with Collagen Gels

LS174T human colon cancer cells formed glandular structures with microvilli, tight junctions, desmosomes and basal laminae arranged in order as in normal intestinal epithelial cells when combined with fetal rat mesenchymes in organ culture. When cultured with type I collagen gel, they also formed glandular structures, but less efficiently than with the mesenchyme. In collagen gel-induced glandular structures, microvilli, tight junctions and desmosomes were arranged in order as in mesenchyme-induced ones, but basal laminae were never observed in contrast with mesenchyme-induced ones. These results indicate that basal lamina formation is not necessary for the glandular structure formation and that mesenchymes promote glandular structure formation of LS174T cells by supplying components necessary for basal lamina formation.     

Cytotoxicity of 7-Ketocholesterol toward Cultured Rat Hepatocytes and the Effect of Vitamin E

The effects of 7-ketocholesterol on rat hepatocytes prepared by collagenase perfusion were examined. The viability of cells incubated with 100 μm 7-ketocholesterol was significantly lower than those with cholesterol, although the LDH activity in the cultured medium remained unchanged during the incubation. Hepatocytes treated with 7-ketocholesterol produced large amounts of ·NO and in the early stage of incubation. Treatment of the hepatocytes with Carboxy-PTIO, which selectively scavenged ·NO, or with l-NMMA, an inhibitor of ·NO synthase, increased the cell viability. The addition of 7-ketocholesterol to the culture medium tended to increase the ratio of total sterol to phospholipid of the hepatocytes in a time-dependent manner without changing the content of phospholipid. No lipid peroxidation or oxidation of the cellular SH groups, protein SH and glutathione, was apparent. Vitamin E added 1 h before the addition of 7-ketocholesterol prevented the hepatocytes from cell death by suppressing the incorporation of 7-ketocholesterol into the hepatocytes and by scavenging .     

cAMP and CaInvolvement in the Mitochondrial Response of Cultured Fetal Rat Hepatocytes to Adrenaline

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca²⁺concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca²⁺concentrations, suggesting that the effects of cAMP may be mediated by free Ca²⁺concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca²⁺act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both α- and β-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.     

Activation of Calcium Signaling in Isolated Rat Hepatocytes Is Accompanied by Shape Changes of Microvilli

Preceding studies using the hamster insulinoma cell line, HIT, and isolated rat hepatocytes have shown that two essential components of the Ca²⁺signaling pathway, the ATP-dependent Ca²⁺store and the store-coupled Ca²⁺influx pathway, are both located in microvilli covering the surface of these cells. Microvilli-derived vesicles from both cell types exhibited anion and cation pathways which could be inhibited by anion and cation channel-specific inhibitors. These findings suggested that the microvillar tip compartment forms a space which is freely accessible for external Ca²⁺, ATP, and IP₃. The entry of Ca²⁺into the cytoplasm, however, is largely restricted by the microvillar core structure, the dense bundle of actin microfilaments acting as a diffusion barrier between the microvillar tip compartment and the cell body. Moreover, evidence has been presented that F-actin may function as ATP-dependent and IP₃-sensitive Ca²⁺store that can be emptied by profilin-induced depolymerization or reorganization [K. Lange and U. Brandt (1996)FEBS Lett.395, 137–142]. Here we demonstrate the tight connection between microvillar shape changes and the activation of the Ca²⁺signaling system in isolated rat hepatocytes. Using a combination of scanning electron microscopy (SEM) and fura-2 fluorescence technique, we confirmed a consequence of the “diffusion barrier” concept of Ca²⁺signaling: Irrespective of the type of the applied stimulus, activation of the Ca²⁺influx pathway is accompanied by changes in the structural organization of microvilli indicative of the loss of their diffusion barrier function. We further show that the cell surfaces of unstimulated hepatocytes isolated by either the collagenase or the EDTA perfusion technique are densely covered with microvilli predominantly of a short and slender type. Beside this rather uniformly shaped type of microvilli, a number of dilated surface protrusions were observed. Under these conditions the cells displayed the well known rather high basal [Ca²⁺]_iof 200–250 nMas repeatedly demonstrated for freshly isolated hepatocytes. However, addition of the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), to the cell suspension immediately after its preparation reduced the basal cytoplasmic Ca²⁺level to about 100 nM.Concomitantly, dilated surface protrusions disappeared, and cell surfaces exclusively displayed short, slender microvilli. Activation of the Ca²⁺signaling pathway by vasopressin, as well as by the IP₃-independent acting Ca²⁺store inhibitor, thapsigargin, was accompanied by a conspicuous shortening and dilation of microvilli following the same time courses as the respective increases of [Ca²⁺]_iinduced by the effectors. Furthermore, the abundance of the large form of surface protrusions on isolated hepatocytes positively correlated with the size of a cellular Ca²⁺/Fura-2 compartment which is rapidly depleted from Ca²⁺by extracellular EGTA. These findings support the postulated localization of the store-coupled Ca²⁺influx pathway in microvilli of HIT cells also for hepatocytes and are in accord with the notion of a cytoskeletal diffusion barrier regulating the flux of external Ca²⁺via the microvillar tip region in the cytoplasm.     

Effects of High Zinc Levels on the Lipid Synthesis in Rat Hepatocytes

Zinc deficiency impairs the hepatic lipid metabolism. Previous studies were focused on the negative effects of zinc deficiency on the hepatic lipid metabolism. A few studies investigated the effects of high zinc levels on the lipid metabolism in hepatocytes. In this study, rat hepatocytes were cultured and treated with different and high concentrations of zinc to investigate the effects of high zinc levels on the lipid synthesis in hepatocytes in vitro. The levels of hepatocytes functional markers, including alkaline phosphatase, lactate dehydrogenase, and albumin, were significantly higher in the zinc treatment groups than in the control group (p?<?0.05, p?<?0.01). The mRNA and protein levels of sterol regulatory element-binding protein 1c (SREBP-1c) were significantly higher in the zinc treatment groups than in the control group (p?<?0.05, p?<?0.01). Furthermore, the mRNA expression levels of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) were significantly higher in the medium- and high-dose zinc treatment groups than in the control group (p?<?0.01). The mRNA levels of stearoyl-CoA desaturase-1 (SCD-1) were significantly higher in the high-dose group (p?<?0.01). These results indicate that high levels of zinc increase hepatocytes activity and SREBP-1c expression, which upregulate the expression of ACC1, FAS, and SCD-1, thereby improving the lipid metabolism in the hepatocytes.     

Antioxidative Activity of 5,6,7,8-Tetrahydrobiopterin and its Inhibitory Effect on Paraquat-Induced Cell Toxicity in Cultured Rat Hepatocytes

The in vitro antioxidative activity of 5,6,7,8-tetrahydrobiopterin (BPH4) was measured and the ability of BPH4 to prevent paraquat-induced cell damage was examined in cultured hepatocytes. The scavenging activity of BPH4 against superoxide anion radicals was assayed in two systems, i.e., xanthine/xanthine oxidase (X/XOD) and rat macrophage/phorbol myristate acetate (MξPMA) radical-generating systems. BPH4 showed an extremely strong superoxide anion radical-scavenging activity in both assay systems. Biopterin (BP) itself did not show any activity in the X/XOD system, but was effective in the MξPMA system. The antioxidative activities of BPH4 against both superoxide anion and hydroxyl radicals were confirmed by spin trapping-ESR spectrometry. BPH4 also protected rat brain homogenate against auto-oxidation. We further examined the effect of BPH4 on paraquat-induced cell toxicity in cultured rat hepatocytes. The paraquat-induced elevation of the release of lactate dehydrogenase (LDH), a marker enzyme for cytotoxicity from cultured hepatocytes was suppressed by BPH4 in a dose-dependent manner. The elevation of lipid peroxides simultaneously induced by paraquat was also inhibited by BPH4 in the same manner. These results suggest that BPH4 might be useful in the treatment of various diseases whose pathogenesis is active oxygen-related.     

Synthesis of IL-6 by Hepatocytes Is a Normal Response to Common Hepatic Stimuli

Exogenous interleukin 6 (IL-6), synthesized at the initiation of the acute phase response, is considered responsible for signaling hepatocytes to produce acute phase proteins. It is widely posited that IL-6 is either delivered to the liver in an endocrine fashion from immune cells at the site of injury, or alternatively, in a paracrine manner by hepatic immune cells within the liver. A recent publication showed there was a muted IL-6 response in lipopolysaccharide (LPS)-injured mice when nuclear NFκB was specifically inactivated in the hepatocytes. This indicates hepatocellular signaling is also involved in regulating the acute phase production of IL-6. Herein, we present extensive in vitro and in vivo evidence that normal hepatocytes are directly induced to synthesize IL-6 mRNAs and protein by challenge with LPS, a bacterial hepatotoxin, and by HGF, an important regulator of hepatic homeostasis. As the IL-6 receptor is found on the hepatocyte, these results reveal that induction of the acute phase response can be regulated in an autocrine as well as endocrine/paracrine fashion. Further, herein we provide data indicating that following partial hepatectomy (PHx), HGF differentially regulates IL-6 production in hepatocytes (induces) versus immune cells (suppresses), signifying disparate regulation of the cell sources involved in IL-6 production is a biologically relevant mechanism that has previously been overlooked. These findings have wide ranging ramifications regarding how we currently interpret a variety of in vivo and in vitro biological models involving elements of IL-6 signaling and the hepatic acute phase response.     

N-Methyl-d-Aspartate- or Glutamate-Mediated Toxicity in Cultured Rat Cortical Neurons Is Antagonized by FPL 15896AR

Abstract: The neuroprotective action of ( S )-α-phenyl-2-pyridineethanamine dihydrochloride (FPL 15896AR), a novel noncompetitive N -methyl- d -aspartate (NMDA) receptor antagonist, was examined in primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (50 µ M ) or glutamate (50 µ M ) for 15 min resulted in the death of 85–95% of the neurons during the next 24 h. This neurotoxicity was completely eliminated by adding FPL 15896AR (50 µ M ) to the cultures during the time of NMDA or glutamate exposure. Neuroprotective concentrations of FPL 15896AR also inhibited other acute effects of NMDA. FPL 15896AR (50 µ M ) prevented the loss of membrane-associated protein kinase C activity that developed by 4 h after transient exposure to 50 µ M NMDA or 50 µ M glutamate. FPL 15896AR also reduced by ∼35% the magnitude of NMDA-triggered increases in intracellular free Ca²⁺ concentration in the cortical cultures. These data indicate that NMDA-mediated toxicity in cultured cortical neurons can be blocked by the NMDA antagonist FPL 15896AR.     

Effects of Strontium on Collagen Content and Expression of Related Genes in Rat Chondrocytes Cultured In Vitro

Strontium stimulates cartilage matrix formation in vitro. However, the mechanisms governing these effects have not yet been extensively reported. In this study, chondrocytes were isolated from rat articular cartilage by enzymatic digestion and cultured for 24–72 h with 1–5 mM strontium. We investigated the effects of different concentrations of strontium on collagen content, type II collagen, insulin-like growth factor (IGF-1) and matrix metalloproteinase (MMP)-13 expression in rat cultured articular chondrocytes in vitro. The collagen content of the chondrocytes, determined as hydroxyproline, was measured by a colorimetry method. Type II collagen, IGF-1, and MMP-13 mRNA abundance and protein expression levels were determined by real-time polymerase chain reaction (real-time PCR) and western blot, respectively. The results showed that collagen content from the chondrocytes extracellular matrix increased with increasing strontium concentration. Moreover, 3 and 5 mM strontium strongly stimulated protein expression and mRNA levels of type II collagen and IGF-1. Conversely, MMP-13 expression in chondrocytes decreased dose-dependently with increasing strontium concentration. These results should provide insight into the ability of strontium to promote chondrocyte extracellular matrix synthesis. Strontium could promote collagen synthesis and suppress collagen degradation via the repression of MMP-13 expression.     

Regulation of Collagen Synthesis in Human Dermal Fibroblasts in Contracted Collagen Gels by Ascorbic Acid, Growth Factors, and Inhibitors of Lipid Peroxidation

Ascorbic acid has been shown to stimulate collagen synthesis in monolayer cultures of human dermal fibroblasts. In the present studies, we examined whether the presence of a collagen matrix influences this response of dermal fibroblasts to ascorbic acid. Fibroblasts and collagen were mixed and allowed to gel and contract for 6 days to form a matrix prior to determining the concentration and time dependence for ascorbic acid to affect collagen synthesis by fibroblasts within the matrix. Collagen synthesis was stimulated at levels at or above 10 μM ascorbic acid and was maximal after 2 days of treatment. This concentration and time dependence is similar to that of cells grown in monolayer cultures. The effects of transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF) were also examined in this model. TGF-β increased and FGF inhibited collagen synthesis in the gels, as has been shown for cells in monolayer cultures. The effects of potential inhibitors of lipid peroxidation induced by ascorbic acid were also examined in these matrices and compared to previous results obtained in monolayer cultures. Propyl gallate, cobalt chloride, α,α-dipyridyl, and α-tocopherol inhibited the ascorbic acid-mediated stimulation of collagen synthesis while mannitol had no effect. Natural retinoids inhibited total protein synthesis without the specific effect on collagen synthesis that was seen in monolayer cultures. These results indicate that ascorbic acid stimulates collagen synthesis in fibroblasts grown in a collagen matrix in a manner similar to that found in monolayer cultures. In contracting collagen gels, however, the magnitude of the effect is less and retinoids do not specifically inhibit collagen synthesis.     

Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies

In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm². The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm²) than on surfaces with a higher concentration of FGF-2 (120 ng/cm²).