Role of carbon monoxide and nitric oxide in adult rat hepatocytes proliferating in vitro: Effects of CAS 1609.

During liver regeneration in vivo carbon monoxide (CO) and nitric oxide (NO) are supposed to play a significant role. We raise the question whether CO and NO are involved in the growth process of cultured hepatocytes. Rat hepatocytes were stimulated into proliferation, growth being estimated by DNA content, mRNA by quantitative RT-PCR, and inducible NO synthase (iNOS) activity by GC-MS. Dexamethasone proved obligatory for fast proliferation. It suppressed the spontaneous rise of iNOS-mRNA in cultures devoid of glucocorticoids, but did not counteract the rise in mRNA in actively dividing cultures. Expression of iNOS-mRNA and cell growth were further enhanced by LiCl (10 mM). NOS activity was completely suppressed by the iNOS-specific inhibitors N-(3-(aminomethyl)benzyl) acetamidine (1400 W,100 microM) and L-N(6)-(1-iminoethyl)lysine (L-NIL, 500 microM), however, without a decrease in hepatocyte growth. Proliferation was attenuated only by very high concentrations (>0.5 mM) of N-nitro-L-arginine methyl ester (L-NAME) and asymmetric dimethylarginine (ADMA). Various NO donors (at 100 microM) did not stimulate cell growth. The furoxan CAS 1609 stimulated growth, decreased iNOS-mRNA expression and transiently increased haem oxygenase-1 (HO-1)-mRNA without releasing considerable amounts of NO. 1H-[1,2,4]Oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ) attenuated the action of CAS 1609. Proliferation was stimulated by Co-protoporphyrin and tricarbonyldichlororuthenium(II) dimer (CORM-2). We conclude that CAS 1609 triggers hepatocyte mitosis most likely via direct, NO-independent induction of HO-1 expression, pointing to CO as a growth-promoting signal in the proliferation cascade in cultured hepatocytes.

Cultured hepatocytes adopt progenitor characteristics and display bipotent capacity to repopulate the liver.

Clinical studies have proved the therapeutic potential of hepatocyte transplantation as a promising alternative to whole organ liver transplantation in the treatment of hereditary or end-stage liver disease. However, donor shortage seriously restricts cell availability, and the lack of appropriate cell culture protocols for the storage and maintenance of donor cells constitutes a significant obstacle. The aim of this study was to stimulate mature hepatocytes in culture to multiply in vitro and track their fate on transplantation. Rat hepatocytes isolated nonenzymatically were cultured serum free for up to 10 days. They were stimulated into proliferation in the presence of growth factors and conditioned media from nonparenchymal and hepatocyte culture supernatants, as well as 10 mM lithium chloride (LiCl). Cell proliferation was assessed by determining DNA content. Additionally, the extent of cell differentiation was estimated using immunofluorescence staining of hepatic, biliary, progenitor, and mesenchymal markers and gene expression analyses. Transplantation studies were performed on the Fischer CD26-mutant rat following pretreatment with retrorsine and partial hepatectomy. Proliferating hepatocytes increasingly adopted precursor characteristics, expressing progenitor (OV6, CD133), hepatic lineage (CK18), biliary (CD49f, CK7, CK19), and mesenchymal (vimentin) markers. The supplement of LiCl further enhanced the proliferative capacity by 30%. Transplantation studies revealed extensive repopulation by large donor hepatocyte clusters. Furthermore, bile duct-like structures deriving from donor cells proved to be immunoreactive to ductular markers and formed in close proximity to endogenous bile ducts. Mature hepatocytes reveal their potential to "switch" between phenotypes, adopting progenitor characteristics during proliferation in vitro. Following transplantation, these "retrodifferentiated" cells further expanded in vivo, thereby generating bipotentially differentiated progenies (hepatocytes and bile duct-like structures). This apparent plasticity of mature hepatocytes may open new approaches for cell-based strategies to treat liver disease.

Effects of paracetamol on NOS, COX, and CYP activity and on oxidative stress in healthy male subjects, rat hepatocytes, and recombinant NOS.

Paracetamol (acetaminophen) is a widely used analgesic drug. It interacts with various enzyme families including cytochrome P450 (CYP), cyclooxygenase (COX), and nitric oxide synthase (NOS), and this interplay may produce reactive oxygen species (ROS). We investigated the effects of paracetamol on prostacyclin, thromboxane, nitric oxide (NO), and oxidative stress in four male subjects who received a single 3 g oral dose of paracetamol. Thromboxane and prostacyclin synthesis was assessed by measuring their major urinary metabolites 2,3-dinor-thromboxane B2 and 2,3-dinor-6-ketoprostaglandin F(1α), respectively. Endothelial NO synthesis was assessed by measuring nitrite in plasma. Urinary 15(S)-8-iso-prostaglanding F(2α) was measured to assess oxidative stress. Plasma oleic acid oxide (cis-EpOA) was measured as a marker of cytochrome P450 activity. Upon paracetamol administration, prostacyclin synthesis was strongly inhibited, while NO synthesis increased and thromboxane synthesis remained almost unchanged. Paracetamol may shift the COX-dependent vasodilatation/vasoconstriction balance at the cost of vasodilatation. This effect may be antagonized by increasing endothelial NO synthesis. High-dosed paracetamol did not increase oxidative stress. At pharmacologically relevant concentrations, paracetamol did not affect NO synthesis/bioavailability by recombinant human endothelial NOS or inducible NOS in rat hepatocytes. We conclude that paracetamol does not increase oxidative stress in humans.

Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells.

Primary hepatocytes lose their differentiated functions rapidly when in culture. Our aim was to maintain the differentiated status of hepatocytes in vitro by means of vital hepatic stellate cells (HSCs), their soluble and particulate factors and lipid extracts. Hepatocytes were placed into collagen-coated culture dishes in the presence of HSCs at different ages of pre-culture, with or without direct cell to cell contacts, at different cell ratios and in monoculture with cellular HSC components in place of vital cells. Changes in morphology and enhancement of phosphoenolpyruvate carboxykinase (PCK) activity by glucagon were used to determine the differentiated status of hepatocytes in 2d-short-term culture. HSCs proved able to maintain the differentiated function of hepatocytes in co-culture either by direct cell contacts or via factors derived from HSC-conditioned medium. In comparison, however, without cellular contact to hepatocytes five to ten times as many HSCs were necessary to increase the PCK activity to the same degree as in the presence of intercellular contacts. Whereas stimulation in the presence of HSC/hepatocyte contacts was independent of HSC culture age only quiescent, resting HSCs (precultured for 1-2 d) were able to stimulate hepatocytes significantly via soluble factors. Culturing of hepatocytes with a lipid extract or a particulate fraction from HSCs clearly displayed a very strong beneficial effect on enzyme activity and morphology. HSCs maintain hepatocyte function and structure through preferentially cell-bound signalling and transfer of lipids.

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (alpha-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.

Mitochondrial inhibition prior to oxygen-withdrawal facilitates the occurrence of hypoxia-induced spreading depression in rat hippocampal slices.

Oxygen withdrawal blocks mitochondrial respiration. In rat hippocampal slices, this triggers a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). To unveil the contribution of mitochondria to the sensing of hypoxia and the ignition of HSD, we modified mitochondrial function. Mitochondrial uncoupling by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 microM) prior to hypoxia hastened the onset and shortened the duration of HSD. Blocking mitochondrial ATP synthesis by oligomycin (10 microg/ml) was without effect. Inhibition of mitochondrial respiration by rotenone (20 microM), diphenyleneiodonium (25 microM), or antimycin A (20 microM) also hastened HSD onset and shortened HSD duration. 3-nitropropionic acid (1 mM) increased HSD duration. Cyanide (100 microM) hastened HSD onset and increased HSD duration. At higher concentrations, cyanide (1 mM), azide (2 mM), and FCCP (10 microM) triggered SD episodes on their own. Compared with control HSD, the spatial extent of the intrinsic optical signals of cyanide- and azide-induced SDs was more pronounced. Monitoring NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) autofluorescence and mitochondrial membrane potential verified the mitochondrial targeting by the drugs used. Except 1 mM cyanide, no treatment reduced cellular ATP levels severely and no correlation was found between ATP, NADH, or FAD levels and the time to HSD onset. Therefore ATP depletion or a cytosolic reducing shift due to NADH/FADH2 accumulation cannot serve as a general explanation for the hastening of HSD onset on mitochondrial inhibition. Additional redox couples (glutathione) or events downstream of the mitochondrial depolarization need to be considered.

The expression of mesenchymal, neural and haematopoietic stem cell markers in adult hepatocytes proliferating in vitro.

BACKGROUND/AIMS: Cultured adult hepatocytes may be stimulated into clonal expansion. We raise the question whether adult hepatocytes proliferating in vitro recapitulate the early process of hepatic development. METHODS: A non-enzymatic method was used to isolate hepatocytes free of contamination with non-parenchymal cells. Hepatocytes were stimulated into proliferation in the presence of mitogens and conditioned media from non-parenchymal cell and hepatocyte culture supernatants. Immunofluorescence methods and PCR analysis were used to demonstrate immunophenotypical characteristics and gene expression profiles similar to those of progenitor cells. RESULTS: Rapid growth occurred during the first 7 days of culture. Cells continued to express hepatic markers (phosphoenolpyruvate carboxykinase, cytokeratin 18, transferrin and dipeptidylpeptidase IV), but the gap junction protein connexin 32 was down-regulated. In the early stage of proliferation, cells started to express biliary and extrahepatic progenitor markers (cytokeratin 19, CD49b, CD49f, nestin, vimentin, Thy1 and c-kit), followed by cytokeratin 7, connexin 43, and neural cell adhesion molecule. Co-expression of the epithelial liver progenitor marker alpha-foetoprotein with either nestin (neural marker) or Thy1 (mesenchymal marker) was also demonstrated. CONCLUSIONS: Mature hepatocytes reveal their potential to regain a spectrum of progenitor markers from different germ layers, suggesting enormous plasticity and differentiation potential of adult liver cells.

The diacylglycerol and protein kinase C pathways are not involved in insulin signalling in primary rat hepatocytes.

Diacylglycerol (DAG) and protein kinase C (PKC) isoforms have been implicated in insulin signalling in muscle and fat cells. We evaluated the involvement of DAG and PKC in the action of insulin in adult rat hepatocytes cultured with dexamethasone, but in the absence of serum, for 48 h. Our results show that although insulin stimulated glycolysis and glycogen synthesis, it had no effect on DAG mass or molecular species composition. Epidermal growth factor showed the expected insulin-mimetic effect on glycolysis, whereas ATP and exogenous phospholipase C acted as antagonists and abolished the insulin signal. Similarly to insulin, epidermal growth factor had no effect on DAG mass or molecular species composition. In contrast, both ATP and phospholipase C induced a prominent increase in several DAG molecular species, including 18:0/20:4, 18:0/20:5, 18:0/22:5 and a decrease in 18:1/18:1. These changes were paralleled by an increase in phospholipase D activity, which was absent in insulin-treated cells. By immunoblotting or by measuring PKC activity, we found that neither insulin nor ATP translocated the PKCalpha, -delta, -epsilon or -zeta isoforms from the cytosol to the membrane in cells cultured for six or 48 h. Similarly, insulin had no effect on immunoprecipitable PKCzeta. Suppression of the glycogenic insulin signal by phorbol 12-myristate 13-acetate, but not by ATP, could be completely alleviated by bisindolylmaleimide. Finally, insulin showed no effect on DAG mass or translocation of PKC isoforms in the perfused liver, although it reduced the glucagon-stimulated glucose output by 75%. Together these results indicate that phospholipases C and D or multiple PKC isoforms are not involved in the hepatic insulin signal chain.

Differential modulation of insulin actions by dexamethasone: studies in primary cultures of adult rat hepatocytes.

BACKGROUND/AIMS: Steroid diabetes is associated with hepatic insulin resistance; in hepatic cell models, however, mainly insulin-permissive effects have been described. Here we investigate modulation by dexamethasone of a larger number of insulin actions. METHODS: Adult rat hepatocytes were cultured+/-dexamethasone for 48 h; insulin actions were studied subsequently. RESULTS: Stimulation of glycolysis by insulin but not by glucose required culture with dexamethasone. Activation of glycogen synthesis by insulin or glucose was strongly enhanced by dexamethasone, the insulin effects on glycogenolysis and amino acid uptake were not modulated. When dexamethasone was omitted from the culture, insulin was incapable to activate glycogen synthase, inactivate glycogen phosphorylase or elevate the level of fructose 2,6-bisphosphate. Dexamethasone did not alter insulin binding, insulin receptor number or kinase activity, insulin receptor substrate-1 and Akt protein expression/phosphorylation. Insulin-stimulated association of phosphatidylinositol 3-kinase with insulin receptor substrates-1 and -2 was increased with dexamethasone, the increased association with IRS-2 may, at least partially, be explained by higher IRS-2 protein expression. CONCLUSIONS: The steroid does not cause hepatic resistance in vitro. The differential attenuation under steroid deprivation points to defects in branches of the insulin signal chain and/or loss of hormonal regulation at the level of target enzymes.