Glycosidases of rat Kupffer cells, hepatocytes and peritoneal macrophages

The acid glycosidase content of rat liver Kupffer cells was compared with that of hepatocytes and resident peritoneal macrophages. Homogenates of all these cells were able to hydrolyze the p-nitrophenyl glycosides of N-acetylglucosamine, N-acetylgalactosamine, glucose, galactose, fucose and mannose, but not xylose. Activity was greatest against the N-acetylglucosaminoside. With Kupffer cell homogenates, most of the glycosidases behaved as if they were lysosomal enzymes.When expressed as rates of hydrolysis per 10⁶ cells, activities against a given substrate by homogenates from the three cell types generally agreed within a factor of 2–4. Significant differences between cell types were found, however, when ratios of glycosidase activities were compared. Furthermore, even though the quantity of glycosidase per cell was similar in Kupffer cells and hepatocytes, the glycosidase concentrations were much higher in the former cells, since Kupffer cells are much smaller than hepatocytes.     

Glycosidases of rat Kupffer cells, hepatocytes and peritoneal macrophages.

The acid glycosidase content of rat liver Kupffer cells was compared with that of hepatocytes and resident peritoneal macrophages. Homogenates of all these cells were able to hydrolyze the p-nitrophenyl glycosides of N-acetylglucosamine, N-acetylgalactosamine, glucose, galactose, fucose and mannose, but not xylose. Activity was greatest against the N-acetylglucosaminoside. With Kupffer cell homogenates, most of the glycosidases behaved as if they were lysosomal enzymes. When expressed as rates of hydrolysis per 10(6) cells, activities against a given substrate by homogenates from the three cell types generally agreed within a factor of 2-4. Significant differences between cell types were found, however, when ratios of glycosidase activities were compared. Furthermore, even though the quantity of glycosidase per cell was similar in Kupffer cells and hepatocytes, the glycosidase concentrations were much higher in the former cells, since Kupffer cells are much smaller than hepatocytes.     

Effect of praseodymium nitrate on hepatocytes and Kupffer cells in the rat.

Intravenous administration of the rare earth metal salt, praseodymium nitrate, induced hepatic damage in the rat, as assessed by morphologic examination (light and electron microscopy) and biochemical parameters (serum glutamic-pyruvic transaminase (EC 2.6.1.2) and glutamic-oxalacetic transaminase (EC 2.6.1.1) activity as well as hepatic triglyceride content). Praseodymium hepatotoxicity was only attained with lower doses (10, 20, or 40 mg/kg), whereas a larger dose (80 mg/kg) was inactive in this respect. As detected by electron microscopy, lower doses of the metal salt caused hepatocytic alterations consisting of degranulation and dilatation of rough endoplasmic reticulum, accumulation of smooth endoplasmic reticulum as well as numerous lipid droplets. No abnormalities were detected in the cell organelles following administration of a large dose of the metal salt; however, vacuoles containing markedly electron-dense material were seen in the cytoplasm of the hepatocytes and the sinusoidal Kupffer cells.     

N-alkylaminoacids and their derivatives interact with melanocortin receptors

Thirty four N-alkylaminoacids (Arg, Trp, Nal, Pro, Hyp, L and D) and derivatives were prepared by a process that included reductive alkylation of the amino function. Both solid phase and solution synthesis was used. Title substances displayed binding activity on melanocortin receptors MC(1,3-5) reaching the low micromolar range.     

Oxidative products from alcohol metabolism differentially modulate pro-inflammatory cytokine expression in Kupffer cells and hepatocytes

Pro-inflammatory cytokines play a vital role in the pathogenesis of alcoholic steatohepatitis. The present study was to determine the role of alcohol-induced oxidative stress in modulating cytokine production. A rat model of alcohol consumption was used to determine alcohol-induced hepatic cytokine expression. Chronic alcohol exposure caused lipid accumulation, oxidative stress, and inflammation in the livers of Wistar rats. The role of oxidative stress in regulating cell type-specific cytokine production was further dissected in vitro. Lipopolysaccharide (LPS) dose-dependently upregulated TNF-α, MIP-1α, MCP-1, and CINC-1 in Kupffer cells-SV40, whereas TNF-α dose-dependently induced CINC-1, IP-10, and MIP-2 expression in H4IIEC3 hepatoma cells. An additive effect on cytokine production was observed in both Kupffer cells-SV40 and hepatocytes when combined hydrogen peroxide with LPS or TNF-α, respectively, which was associated with NF-κB activation and histone H3 hyper-acetylation. Unexpectedly, an inhibitory effect of 4-hydroxynonenal on cytokine production was revealed in LPS-treated Kupffer cells-SV40. Mechanistic study showed that 4-hydroxynonenal significantly enhanced mRNA degradation of TNF-α, MCP-1, and MIP-1α, and decreased the protein levels of MCP-1 in LPS-stimulated Kupffer cells-SV40 through reducing the phosphorylation of mRNA binding proteins. This study suggests that Kupffer cells and hepatocytes express distinct pro-inflammatory cytokines/chemokines in response to alcohol intoxication, and oxidative products (4-hydroxynonenal) differentially modulate pro-inflammatory cytokine/chemokine production via NF-κB signaling, histone acetylation, and mRNA stability.     

Fc receptors mediate prostaglandin and superoxide synthesis in cultured rat Kupffer cells

Latex beads with covalently bound bovine serum albumin were prepared and coated with anti-BSA immunoglobulin G. These particles were shown to possess on their surfaces a defined quantity of the antibody with the Fc portions exposed to the medium. One homologous and two heterologous antibodies of the G class were used and compared in terms of their binding to the rat Kupffer cells and their ability to elicit the typical phagocytotic responses. These particles were phagocytosed by rat Kupffer cells and elicited synthesis of prostaglandins and superoxide anion radicals. A significant release of superoxide into the medium was observed in the presence of cytochalasin B only. The data presented here suggest that a) Fc-carrying particles can be bound to Kupffer cells and elicit responses via specific receptors; b) coating with the homologous antibody yields the most effective particles; c) superoxide release into the surrounding medium is most abundant when the particle-binding membrane areas are prevented from forming phagocytotic vesicles.     

The effect of a conditioned medium from neonatal rat hepatocytes on a mixed culture of Kupffer cells and fibroblast-like liver cells]

The study was carried out on primary coculture Kupffer cells and liver fibroblasts of newborn and mature rats. At first Kupffer cells and liver fibroblasts were taken on the equal quantity. There were observed significant decrease of Kupffer cells quantity and fibroblasts death after 4-5 days in coculture. Mitotic and functional activity liver fibroblasts, adhesion of Kupffer cells were increased by the using of conditioned medium of newborn rat hepatocytes. The rise of mitotic activity of liver fibroblasts and quantity of nucleolus in their nuclei forestalls of the increase Kupffer cells size, appearing of Kupffer cells with some nuclei.     

Enhanced DNA synthesis in rat hepatoma cells by conditioned media from Kupffer cells incubated with supernatants of tumor necrosis factor-alpha-pretreated hepatocytes.

The effects of tumor necrosis factor-alpha (TNF-alpha) on DNA synthesis in AH66 rat hepatoma cells and rat hepatocytes were analysed by means of [3H]thymidine incorporation. DNA synthesis in AH66 cells was suppressed when AH66 cells were directly incubated with TNF-alpha. When primary culture of rat Kupffer cells was incubated with hepatocyte conditioned media pretreated with TNF-alpha (0-200 U/ml), and AH66 cells were then treated with these hepatocyte/Kupffer cell-conditioned media, TNF-alpha used in the pretreatment caused a dose-dependent increase in DNA synthesis in AH66 cells with a maximum effect amounting to a more than 10-fold increase. In contrast, DNA synthesis in primary culture of rat hepatocytes was not stimulated by the TNF-alpha-pretreated hepatocyte/Kupffer cell conditioned media. These results suggest that TNF-alpha-mediated hepatocyte-Kupffer cell interaction selectively promotes proliferation of rat hepatoma cells.     

Differential regulation of insulin-like growth factor-II receptors in rat hepatocytes and hepatoma cells

This study compares the regulation of IGF-II receptors in three rat hepatoma lines, HTC, H-35 and 5123tc, and primary rat hepatocytes. In all cell types [125I]IGF-II bound solely to a species of approximately 250 kDa. Cell surface IGF-II receptors in hepatoma cells had slightly lower affinities (1-2 liters/nmol) than in hepatocytes (4 liters/nmol), but slightly higher IGF-I cross-reactivity (2-4% compared to 1% in hepatocytes). In confluent cultures, the three hepatoma lines expressed 5- to 15-fold more cell-surface receptors per cell than hepatocytes. However, while hepatocyte receptors showed marked inverse density-dependence, increasing over 6-fold between dense (3 x 10(5) cells/3.8 cm2) and sparse (0.16 x 10(5) cells/3.8 cm2) cultures, receptors in all hepatoma lines remained at a constant high level regardless of culture density. These distinct regulatory patterns resemble those described for growth-related functions in hepatocytes and hepatoma cells, and are thus consistent with a role for IGF-II receptors in liver cell proliferation.     

利用NASH大鼠原代肝细胞与原代Kupffer细胞共培养建立NASH原代细胞模型*

目的: 通过分离并提纯非酒精性脂肪性肝炎(NASH)大鼠原代肝细胞以及原代Kupffer细胞建立体外NASH原代细胞模型,为研究NASH提供可靠的细胞实验技术支持。方法: 选择SD大鼠40只,随机分为2组(n=20):对照组和NASH组,对照组大鼠利用普通饲料喂养,NASH组大鼠利用高脂饲料(88%基础饲料+10%猪油+ 2%胆固醇)喂养,6～8周后,利用NASH评分表,病理观察下肝组织切片脂肪变+小叶内炎症+气球样变评分≥4 分,表明大鼠NASH模型的成功建立,利用胶原酶原位灌注法分离并提纯NASH模型大鼠原代肝细胞以及原代Kupffer细胞,利用CK-18及CD68免疫荧光以及墨汁吞墨实验进行细胞鉴定,利用油红O染色、试剂盒测定谷丙转氨酶(ALT)、谷草转氨酶(AST)含量观察NASH大鼠原代肝细胞脂质累积和肝功情况,Western blot检测原代Kupffer细胞炎症因子表达情况,最后采用原代肝细胞:原代Kupffer细胞=6∶1比例共培养,显微镜下观察细胞状态。结果: 实验成功分离并提纯NASH原代肝细胞以及原代Kupffer细胞,通过油红O染色,NASH组大鼠原代肝细胞存在明显的脂肪沉积,且NASH组大鼠原代肝细胞中AST、ALT明显高于对照组,存在明显肝损伤(P＜0.05),Western blot测定原代Kupffer细胞TNF-α、IL-1β以及MCP-1,NASH组大鼠明显高于对照组(P＜0.05)。结论: 通过胶原酶原位灌注法可以成功分离NASH大鼠原代肝细胞以及原代Kupffer细胞,同时成功建立比例共培养大鼠体外原代细胞NASH模型。     

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM3.     

Protection against nitrofurantoin-induced oxidative stress by coelenterazine analogues and their oxidation products in rat hepatocytes

Coelenterazine (3,7-dihydro-2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzylimidazolo[1,2-a]pyrazin-3-one) is a substrate for the bioluminescence reaction in many marine animals. Recent work showed that CLZn, its synthetic analogue CLZm, and their common oxidation product coelenteramine (CLM) have strong antioxidative properties in acellular lipid peroxidation systems as well as in rat hepatocytes subjected to tert-butyl hydroperoxide (t-BHP). Here, we analyzed the ability of CLZm and several imidazolopyrazinone (IMPZs) analogues to protect primary cultures of rat hepatocytes against a nitrofurantoin (NF)-induced oxidative stress. Comparison of protection capabilities with reference antioxidants yielded the following ranking: CLZm >>> BHT > Trolox C® > probucol > α-tocopherol. The comparison of CLZm with analogues lacking the phenol group in R₁ revealed no differences although the presence of this phenol conferred superior protection against t-BHP. CLM, as well as its methoxylated analogue mCLM which lacks chain-breaking properties, were equally potent in preventing cellular damage caused by NF. mCLM and α-naphthoflavone, an inhibitor of cytochrome P450 (CYP450) IAI, similarly protected cells against NF-induced mortality and also equally inhibited EROD activity in methylcholanthrene-induced hepatocytes. The inhibition of EROD by CLZm and CLM was less pronounced. We suggest that the extent of protection conferred by IMPZs against NF-toxicity reflects both the occurrence of antioxidative properties detoxifying ROS produced within cells and inhibitory actions on CYP450 isoforms involved in the bioreduction of NF.     

Protection against nitrofurantoin-induced oxidative stress by coelenterazine analogues and their oxidation products in rat hepatocytes

Coelenterazine (3,7-dihydro-2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzylimidazolo[1,2-a]pyrazin-3- one) is a substrate for the bioluminescence reaction in many marine animals. Recent work showed that CLZn, its synthetic analogue CLZm, and their common oxidation product coelenteramine (CLM) have strong antioxidative properties in acellular lipid peroxidation systems as well as in rat hepatocytes subjected to tert-butyl hydroperoxide (t-BHP). Here, we analyzed the ability of CLZm and several imidazolopyrazinone (IMPZs) analogues to protect primary cultures of rat hepatocytes against a nitrofurantoin (NF)-induced oxidative stress. Comparison of protection capabilities with reference antioxidants yielded the following ranking: CLZm > BHT >Trolox C((R)) > probucol > alpha-tocopherol. The comparison of CLZm with analogues lacking the phenol group in R(1) revealed no differences although the presence of this phenol conferred superior protection against t-BHP. CLM, as well as its methoxylated analogue mCLM which lacks chain-breaking properties, were equally potent in preventing cellular damage caused by NF. mCLM and alpha-naphthoflavone, an inhibitor of cytochrome P450 (CYP450) IAI, similarly protected cells against NF-induced mortality and also equally inhibited EROD activity in methylcholanthrene-induced hepatocytes. The inhibition of EROD by CLZm and CLM was less pronounced. We suggest that the extent of protection conferred by IMPZs against NF-toxicity reflects both the occurrence of antioxidative properties detoxifying ROS produced within cells and inhibitory actions on CYP450 isoforms involved in the bioreduction of NF.     

CCK antagonists interact with CCK-B receptors on human small cell lung cancer cells

The ability of cholecystokinin (CCK) receptor antagonists to interact with CCK receptors in small cell lung cancer (SCLC) cells was investigated. L-365,260, CCK-8, L-364,718, CBZ-CCK(27-32)-NH2 and proglumide analogue 10 inhibited specific 125I-CCK-8 binding to SCLC cells with IC50 values of 0.2, 2, 500, 100,000 and 500,000 nM, respectively. Gastrin-I and CCK-8 elevated the cytosolic Ca2+ when SCLC cells were loaded with Fura 2-AM. L-365,260 inhibited the cytosolic Ca2+ increase caused by 10 nM CCK-8 in a dose-dependent manner. The effects of 10 nM L-365,260 were reversed by high concentrations of CCK-8. These data indicate that L-365,260 functions as a reversible CCK-8 antagonist using SCLC cells.     

Rabbit T cells with and without Fc receptors

Earlier, indirect evidence for rabbit subpopulations differing in Fc receptors and in response to mitogen has been directly tested. T cells were purified from spleen suspension by removal of adherent cells, followed by removal of Ig-bearing cells on petri dishes coated with antibody, directed against the light chain allotype of Ig receptors. The purified cells were further fractionated by formation of EA rosettes and separation on Ficoll-Hypaque. T cells which lacked Fc receptors had a larger response when stimulated with Con A or PHA than did T cells which possessed Fc receptors. Both subpopulations responded more when irradiated nonadherent B cells were added to the mixture, but the extent of help was the same for both cell populations. T cells which contained both Fc receptor-bearing cells and cells which lacked the receptor had a response which was intermediate between that of the two separated subpopulations.     

Induction of CYP3A and associated terfenadineN-dealkylation in rat hepatocytes cocultured with 3T3 cells

Long-term culture of hepatocytes has been challenged by the loss of differentiated functions. In particular, there is a rapid decline in cytochrome P450 (CYP). In this study, we cocultured rat hepatocytes with 3T3 fibroblasts for 10 days, and examined hepatocyte viability, morphology, and expression of CYP3A. Terfenadine was incubated with the cultures, and its biotransformation was quantitatively analyzed by HPLC. Terfenadine is metabolized by two major pathways:C-hydroxylation to an alcohol metabolite which is further oxidized to a carboxylic acid, andN-dealkylation to azacyclonol. In rat liver, only theN-dealkylation pathway appears to be mediated by CYP3A since anti-rat CYP3A antibody inhibited azacyclonol but not alcohol metabolite formation in incubations of terfenadine with liver microsomes. Freshly isolated rat hepatocytes were seeded on top of confluent 3T3 cells. Cultures were maintained in Williams' E medium supplemented with 10% fetal bovine serum and either 0.1 mol/L or 5 mol/L dexamethasone. In pure hepatocyte cultures, viability, as determined by lactate dehydrogenase (LDH) activity, decreased steadily to less than 30% of initial levels by day 10. In cocultures, LDH activity remained high and was 70% of initial levels on day 10. The half-life of terfenadine disappearance was optimally maintained in cocultures treated with 5 mol/L dexamethasone, and was associated with the increased formation of azacyclonol. On day 5, nearly 50% of added 5 mol/L terfenadine was converted to azacyclonol within 6 h, whereas the conversion was only 4% on day 1. Western and RNA-slot blot analyses confirmed that treatment with 5 mol/L dexamethasone induced CYP3A mRNA expression and CYP3A protein expression. This coculture system could offer a useful approach in the study of drugs and xenobiotics metabolized by CYP3A.Abbreviations BSA bovine serum albumin - CYP cytochrome P450 - DMSO dimethyl sulfoxide - LDH lactate dehydrogenase - PCN pregnenolone-16-carbonitrile - SDS sodium dodecyl sulfate - SSC saline sodium citrate     

Rat transformation-associated proteins (TAP) induced by Moloney murine sarcoma virus interact with specific receptors on normal rat kidney cells

In this report, data are presented to show that transformation-associated proteins (TAP) secreted from the transformed 6M2 cells have mitogenic activities in the stimulation of DNA synthesis and proliferation of normal rat kidney (NRK-2) cells and of nonpermissively grown 6M2 cells. TAP also bound specifically to NRK-2 cells with a binding dissociation constant (Kd) of 1.4 pM. Approximately 2 X 10(5) binding sites per cell were found. Therefore, TAP may represent a set of virally-induced growth stimulatory factors.     

Different beta 1-integrin collagen receptors on rat hepatocytes and cardiac fibroblasts

Detergent extracts of primary rat hepatocytes and neonatal cardiac fibroblasts were applied to collagen type I-Sepharose in the presence of 1 mM MnCl2. Elution of bound proteins by 10 mM EDTA yielded one beta 1-integrin heterodimer from hepatocytes with an Mr of 180,000/115,000 under nonreducing conditions. Two beta 1-integrins with Mr's (nonreduced) of 180,000/115,000 and 145,000/115,000 could be isolated from surface-iodinated fibroblasts. A monoclonal antibody, 3A3, directed against the rat homolog of the human integrin VLA-1, precipitated the affinity-purified Mr 180,000/115,000 heterodimer, establishing the relatedness of the Mr 180,000 subunit to the alpha 1-chain of the beta 1-integrin subfamily. Both the alpha 1 beta 1-integrin and the 145,000/beta 1-integrin heterodimers bound specifically to Sepharose beads derivatized with the collagen fragment alpha 1(I) CB3, which lacks RGD sequences. Immunofluorescence staining using the 3A3 monoclonal antibody revealed that the rat alpha 1 beta 1-integrin was present at focal adhesion sites of fibroblasts grown on native collagen type I- but not on fibronectin-coated substrates, although both types of substrates supported the formation of beta 1-integrin containing focal adhesions. Similarly, hepatocytes cultured on substrata coated with collagen type I (but not fibronectin) were stained in a patchy pattern localized to the cell periphery by 3A3 IgG. Furthermore, 3A3 IgG completely inhibited the attachment of hepatocytes to collagen type I, whereas under identical conditions the attachment of fibroblasts to these substrates was inhibited only by approximately 40%. The attachment of both hepatocytes and cardiac fibroblasts to fibronectin was unaffected by the presence of the 3A3 antibody. Collectively these data show that a rat homolog of the human VLA-1 heterodimer both biochemically and functionally fulfills the criteria of a single collagen receptor on rat hepatocytes. In contrast, rat cardiac fibroblasts utilize two different collagen-binding integrins to adhere to collagen, one of which is the rat homolog of the human VLA-1 heterodimer. Furthermore alpha 1(I) CB3 contains cell binding sites for beta 1-integrins.