OBJECTIVETo observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles.

METHODSThe localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods.

RESULTSH(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats.

CONCLUSIONThis finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.

Animals ; Cell Membrane ; enzymology ; Hepatocytes ; cytology ; enzymology ; ultrastructure ; Histocytochemistry ; methods ; Kidney ; cytology ; enzymology ; ultrastructure ; Lysosomes ; enzymology ; Male ; Organelles ; enzymology ; Rats ; Rats, Wistar ; Vacuolar Proton-Translocating ATPases ; metabolism

OBJECTIVETo investigate the characteristics of phase II metabolic enzymes in mouse embryonic stem (ES) cell-derived liver tissue.

METHODSMature hepatocytes were differentiated from embryonic stem cells in cultured mouse embryoid bodies (EB) at d18. Western blot was used to detect the expression of uridine 5'-diphosphate glucronosyl transferase (UGT1a1,UGT1a6) and microsomal glutathione S-transferases 1(mGST1) during the differentiation course.The derived liver tissue was incubated with UDPGA and 7-HFC,the formation of 7-HFC glucuronide was detected by HPLC to examine the total activities of UGT1a1 and UGT1a6. Furthermore, the microsomes were incubated with CDNB and GSH,and the mGST1 activity was measured by spectrometry.

RESULTSAn increase tendency of UGT1a1 expression was noticed during the differentiation course. UGT1a6 and mGST1 were not detected in the earlier stage until d18 of differentiation. The metabolic activity of mGST1 in the derived hepatocytes was 7.65 nmol/min/mg on d18.

CONCLUSIONThe ES cell-derived liver tissue possesses partial metabolic function of phase II enzymes on d18 of differentiation,which might be used as a model for in vitro research on hepatic pathophysiology and phase II drug metabolism.

Animals ; Cell Differentiation ; Embryoid Bodies ; cytology ; Embryonic Stem Cells ; cytology ; Glucuronosyltransferase ; physiology ; Glutathione Transferase ; physiology ; Hepatocytes ; cytology ; enzymology ; Mice

Thioredoxin reductase (TrxR), a component of the thioredoxin system, including thioredoxin (Trx) and NADPH, catalyzes the transfer of electrons from NADPH to Trx, acts as a reductant of disulfide-containing proteins and participates in the defense system against oxidative stresses. In this study, the regulation pattern of TrxR in the presence of various stressful reagents was compared between Chang (human normal hepatic cell) and HepG2 (human hepatoma cell) cell lines. Aluminum chloride (0.5 mM) and zinc chloride (0.5 mM) enhanced the TrxR activity in the Chang cell line to a higher degree than in the HepG2 cell line, but cupric chloride (0.2 mM) and cadmium chloride (0.1 mM) enhanced the TrxR activity in the HepG2 cell line to a greater degree. The TrxR activities in both Chang and HepG2 cell lines were similarly induced by treatment with sodium selenite (0.02 mM) and menadione (0.5 and 1.0 mM). Lipopolysaccharide (2microgram/m1) increased the TrxR activity upto 4.02- and 2.2-fold in the Chang and HepG2 cell lines, respectively, in time-dependent manners. Hydrogen peroxide (5 mM) markedly enhanced the TrxR activity in the HepG2 cell line, but not in the Chang cell line. NO-generating sodium nitroprusside (3.0 and 6.0 mM) induced TrxR activities in both human liver cell lines. The TrxR activity was also induced in human liver cells under limited growth conditions by serum deprivation. These results imply that the TrxR activities in normal hepatic and hepatoma cell lines are subject to different regulatory responses to various stresses.
Cell Line, Tumor ; Comparative Study ; Hepatocytes/cytology/*enzymology ; Human ; Oxidative Stress/*physiology ; Support, Non-U.S. Gov't ; Thioredoxin Reductase (NADPH) /*metabolism

OBJECTIVETo explore how arylamine N-acetyltransferases (NATs) is related to cell apoptosis.

METHODSNAT activity in apoptotic HepG2 cells was measured using high performance liquid chromatography (HPLC); the apoptosis rate of HepG2 cells acted upon by an NAT inhibitor was measured using flow cytometry.

RESULTSNAT activity was lowered in apoptotic HepG2 cells; apoptosis rate induced by camptothecin (CAM) increased after inhibition of NAT activity in HepG2 cells.

CONCLUSIONNAT can inhibit apoptosis in HepG2 cells.

Apoptosis ; drug effects ; Arylamine N-Acetyltransferase ; antagonists & inhibitors ; metabolism ; Camptothecin ; administration & dosage ; Cells, Cultured ; Dose-Response Relationship, Drug ; Hepatocytes ; cytology ; drug effects ; enzymology ; Humans

Primary rat hepatocytes were cultured using different in vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH was decreased over time in culture. However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC). The levels of AST and ALT did not change significantly over the investigated time. The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC. The decline of CYP 1A was faster in MC than in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC. In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC. Taken together, it was indicated each investigated model had its advantages and disadvantages. It was also underlined that various in vitro models may address different questions.
Albumins/*secretion ; Bioreactors ; Cell Culture Techniques/*methods ; Cell Separation ; Cytochrome P-450 CYP1A1/metabolism ; Hepatocytes/*cytology ; Hepatocytes/metabolism ; L-Lactate Dehydrogenase/metabolism ; Mitochondria, Liver/enzymology ; Mitochondrial Proteins/metabolism ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVETo study the effect of p38 MAPK activity on isolated rabbit liver during the period of cold preservation and reperfusion.

METHODSBased on the cold preservation-reperfusion model of isolated rabbit livers, according to the concentration of SB202190 in the preservation solution which was a specific p38 MAPK inhibitor, the isolated livers were divided into 4 groups, six in each A, B, C and D. Liver tissue samples and blood samples were harvested at different time points: before and end of cold preservation, reperfusion for 5, 10, 15, 30, 60, and 120 minutes. The activity levels of p38 MAPK were detected by both western blot and immunoprecipitation. The function markers of isolated livers were detected with automatic biochemistry analyzer, and the levels of total bile after reperfusion were measured.

RESULTSIn normal rabbit liver tissues, p38 MAPK had low activity. During the cold preservation period, the activity of p38 MAPK elevated slightly. But during the reperfusion period, the activity of p38 MAPK changed markedly which elevated rapidly at the early stage and reached its peak value at 10 minutes, then decreased gradually to the normal level (7.6 +/-0.9) at 120 minutes. SB202190 could inhibit the activity in a dose-dependent manner. The peak values of p38 MAPK activity in group B,C and D were 42.5 +/-2.4, 10.1+/-1.4, and 7.6 +/-0.6 respectively, while 78.6 +/-6.1 in group A. During the reperfusion period, the levels of serum ALT, AST and ALP were higher in group A than those in any other group, alike the p38 MAPK activity, especially at 15 and 30 minutes. On the other hand, the total bile secretion volume was (10.2 +/-2.9) ml/100 g, (13.9 +/-1.3) ml/100 g, (15.6 +/-1.2) ml/100 g, and (16.0 +/-1.3) ml/100 g in group A, B, C and D respectively.

CONCLUSIONSDuring the cold preservation- reperfusion period, p38 MAPK specific inhibitor SB202190 can lower the p38 MAPK activity, and ameliorate the isolated liver injury. Activation of p38 pathway is one of the important mechanisms to cause ischemia-reperfusion injury of isolated liver.

Animals ; Enzyme Inhibitors ; pharmacology ; Female ; Hepatocytes ; cytology ; physiology ; Imidazoles ; pharmacology ; In Vitro Techniques ; Liver ; blood supply ; cytology ; Male ; Protein Kinase C ; metabolism ; Pyridines ; pharmacology ; Rabbits ; Reperfusion Injury ; enzymology ; physiopathology ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

OBJECTIVETo elucidate gene expressions of phase II enzymes in the mouse embryonic stem (ES) cell-derived hepatocytes.

METHODSEmbryoid body (EB) formation cultures were applied in directed differentiation of ES to hepatic-like cells. The expressions of hepatic-specific genes, including AFP, ALB, Cyp7a1, were detected by RT-PCR during the differentiation course. Albumin was detected by immunocyto- chemistry. The gene expressions of mGST1 and UGTs family, including UGT1a1, UGT1a6, UGT1a9 and UGT2b5, were investigated using RT-PCR.

RESULTSA notable gene expression of AFP and ALB was observed on d 8. On d 18, AFP gene failed to express, while ALB and Cyp7a1 genes were detected.Albumin-positive staining was detected in hepatic-like cells. Phase II enzyme genes expressed in variance during the differentiation course, UGT1a1 and UGT1a9 were expressed stably, UGT1a6 expression increased gradually, and UGT2b5 failed to express. Little mGST1 gene expression could been detected in the early course until d 18. In addition, all the enzymes gene expressions in the derived hepatocytes on d 18 were similar to those from mature mouse hepatocytes.

CONCLUSIONSMouse ES cell-derived mature hepatocytes express phase II enzyme UGTs and mGST1 genes similar to those in mature hepatocytes. The system may offer an alternative animal testing model related to phase enzymes in further research.

Animals ; Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; enzymology ; Fluorescent Antibody Technique ; Glucuronosyltransferase ; genetics ; metabolism ; Glutathione S-Transferase pi ; genetics ; metabolism ; Hepatocytes ; cytology ; enzymology ; Isoenzymes ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Microsomes ; enzymology ; Reverse Transcriptase Polymerase Chain Reaction

Primary rat hepatocytes were cultured using different in vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH was decreased over time in culture. However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC). The levels of AST and ALT did not change significantly over the investigated time. The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC. The decline of CYP 1A was faster in MC than in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC. In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC. Taken together, it was indicated each investigated model had its advantages and disadvantages. It was also underlined that various in vitro models may address different questions.
Albumins ; secretion ; Animals ; Bioreactors ; Cell Culture Techniques ; methods ; Cell Separation ; Cytochrome P-450 CYP1A1 ; metabolism ; Hepatocytes ; cytology ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Male ; Mitochondria, Liver ; enzymology ; Mitochondrial Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Time Factors

Hepatic stellate cell (HSC) plays a pivotal role in liver fibrosis and isconsidered as one of the therapeutic targets for the treatment of hepatic fibrosis. Focal adhesion kinase (FAK) has been shown to play an important role in the HSC activation. The aim of the study is to explore the role of FAK in the proliferation and activation in culture-activated rat HSCs by using a specific antisense oligonucleotides targeting FAK (FAK-ASON). Rat HSCs were prepared from SD rats by in situ perfusion of pronase and collagenase and single-step density Nycodenze gradient. Culture-activated HSCs were transfected with the FAK-ASON (1 microM) by lipofectamine 2000 for 24, 48 or 72 hours. The proliferation of HSC was detected by MTT assay. The expression of the marker of HSC activation, alpha-smooth muscle actin (alpha-SMA), was assessed by reverse transcription- polymerase chain reaction (RT-PCR) and Western blot. The inhibition rates for HSC proliferation 24, 48 and 72 hours after transfection were 65.5% +/- 5.8%, 46.8% +/- 4.3% and 35.7% +/- 5.2% respectively. Transfection of FAK-ASON could significantly inhibit the proliferation of HSC. Meanwhile, treatment with the FAK-ASON could markedly decrease the mRNA and protein expression of alpha-SMA in rat HSC. The specific FAK-ASON may have an inhibitory effect on the proliferation and activation in rat HSC.
Actins ; biosynthesis ; genetics ; Animals ; Cell Proliferation ; Cells, Cultured ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Hepatocytes ; cytology ; enzymology ; Male ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection

OBJECTIVETo determine the roles of mitochondrial apoptosis and energy metabolism in hepatocytes during the pathogenic process of acute renal failure (ALF) by assessing disease-related differential activities of several key mitochondrial enzymes, including citrate synthase (CS), carnitine palmitoyltransferase-1 (CPT-1) and cytochrome c oxidase (COX).

METHODSThirty-two male Sprague Dawley rats were given D-galactosamine followed by and lipopolysaccharide (LPS) to induce acute liver failure and sacrificed after 4 (4 h group), 8 (8 h group) 12 (12 h group) and 24 hours (24 h group) of treatment. Eight unmodeled rats served as controls. Effects related to apoptosis were evaluated by pathological analysis of hepatic tissues and TUNEL staining. Ultrastructural changes in mitochondria were assessed by electron microscopy. The activity and expression of CS, CPT-1 and COX were measured.

RESULTSHepatocyte apoptosis was present in the 4 h treatment group and was increased obviously in the 8 h treatment group. Hepatocyte necrosis was first observed in the 12 h treatment group and was significantly higher in the 24 h treatment group, with inflammatory cell invasion. Ultrastructural changes in mitochondria were present in the 4 h treatment group, and the 24 h treatment group showed mitochondria with completely destroyed outer membranes, which resulted in mitochondrial collapse. Activity and protein expression of CS, CPT-1 and COX were increased in the 4 h group (vs. controls), were at their peak in the 8 h group (CS:t =1.481, P less than 0.01; CPT-1:t =2.619, P less than 0.05; COX:t =1.014, P less than 0.01) and showed a decreasing trend in the 12 h group. In addition, the activities of CS, CPT-1 and COX were enhanced at the stage of hepatocyte apoptosis, suggesting that these enzymes were involved in the initiation and development of ALF.

CONCLUSIONEnergy metabolism plays an important role in hepatocyte apoptosis during ALF.

Animals ; Apoptosis ; Carnitine O-Palmitoyltransferase ; metabolism ; Citrate (si)-Synthase ; metabolism ; Disease Models, Animal ; Electron Transport Complex IV ; metabolism ; Hepatocytes ; cytology ; enzymology ; Liver Failure, Acute ; metabolism ; pathology ; Male ; Mitochondria ; ultrastructure ; Rats ; Rats, Sprague-Dawley