Cultures Of Adult Rat Hepatocytes Research Articles

317 Safety of panax ginseng aqua-acupunctures solution on P450-dependent drug metabolism in culture of adult rat hepatocytes

Both Ginkgo biloba and Panax ginseng exert a number of physiological effects and have been shown to modulate aspects of cognitive performance. Whilst a number of studies have examined ginkgo's effects on electroencephalograph (EEG) recordings, to date, none have investigated the EEG effects of ginseng. In this double-blind, placebo-controlled, balanced crossover experiment, the effects of single doses of G. biloba (360 mg GK501), P. ginseng (200 mg G115), and an identical placebo, on auditory-evoked potentials, contingent negative variation (CNV), and resting power within the delta, theta, alpha, and beta wavebands, were assessed in 15 healthy volunteers. Each participant was assessed on three separate occasions 4 h after consuming that day's treatment. The order of presentation of the treatments was dictated by a Latin square with 7 days between testing sessions. The results showed that ginseng led to a significant shortening of the latency of the P300 component of the evoked potential. Both ginseng and ginkgo also led to significant reductions in frontal ‘eyes closed’ theta and beta activity, with additional reduction for ginseng in the alpha waveband. These findings demonstrate for the first time that P. ginseng can directly modulate cerebroelectrical activity, and that these effects are more pronounced than those following G. biloba.

A Cdc25A Antagonizing K Vitamin Inhibits Hepatocyte DNA Synthesis in Vitro and in Vivo

Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.

Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein.

A 57-kDa protein in royal jelly (RJ) was previously shown to stimulate hepatocyte DNA synthesis and prolongs the proliferation of hepatocytes as well as increasing albumin production [Kamakura, M., Suenobu, N., and Fukushima, M. (2001) Biochem. Biophys. Res. Commun. 282, 865-874]. In this study, I investigated the signal transduction mechanisms involved in the induction of hepatocyte DNA synthesis and the promotion of cell survival by this 57-kDa protein in primary cultures of adult rat hepatocytes. Hepatocyte DNA synthesis induced by the 57-kDa protein was not influenced by several alpha- and beta-adrenoceptor antagonists, but was dose-dependently abolished by an inhibitor of a tyrosine-specific protein kinase, genistein. A phospholipase C inhibitor (U-73122) and a protein kinase C (PKC) inhibitor (sphingosine) inhibited 57-kDa protein-stimulated he-patocyte DNA synthesis, whereas a protein kinase A inhibitor (H-89) did not. The 57-kDa protein also activated PKC in rat hepatocytes. Various inhibitors of intracellular signal transduction elements (PD98059, p21 ras farnesyltransferase inhibitor, wortmannin and rapamycin) also blocked hepatocyte DNA synthesis induced by the 57-kDa protein. Furthermore, the 57-kDa protein activated mitogen-activated protein (MAP) kinase in rat hepatocytes. The activation of MAP kinase by the 57-kDa protein was inhibited by PD98059 and sphingosine. The 57-kDa protein also activated protein kinase B, which is a key regulator of cell survival. These results suggest that, like growth factors, the 57-kDa protein activates several important intracellular signaling factors involved in the stimulation of hepatocyte DNA synthesis and the protection of cells from apoptosis.

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.

Glycyrrhizin and some analogues induce growth of primary cultured adult rat hepatocytes via epidermal growth factor receptors

We investigated the effects of glycyrrhizin (GL-1) and some analogues on DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes. The hepatocytes underwent DNA synthesis and proliferation in response to GL-1 and some analogues. The effects of these agents occurred in a time- and dose-dependent manner. The proliferative potency as judged by half-maximal effective concentrations was in the following order: 18-β- H-glycyrrhetinic acid (GL-3; 4.5×10 −9 M)<18-β- H-glycyrrhizin (GL-1; 4.4×10 −8 M)<18-α- H-glycyrrhetinic acid (GL-6; 6.0×10 −8 M). The analogue 18-α- H-glycyrrhetinic acid 3- O-β- d-monoglucuronide (GL-5; 1.0×10 −7 M) weakly stimulated hepatocyte DNA synthesis and proliferation, whereas 18-α- H-glycyrrhizin (GL-4) and 18-β- H-glycyrrhetinic acid 3- O-β- d-monoglucuronide (GL-2) did not. The growth-promoting effects of GL-1, GL-3 and GL-6 were significantly inhibited at higher initial plating densities (7.0×10 4 and 10×10 4 cells/cm 2). A monoclonal antibody against epidermal growth factor (EGF) receptor (1–100 ng/ml), but not that against EGF (1–100 ng/ml), dose-dependently inhibited glycyrrhizin- and analogue-induced hepatocyte DNA synthesis and proliferation. Specific inhibitors of growth-related signal transducers, such as genistein, PD98059 (2′-amino-3′-methoxyflavone) and rapamycin, completely blocked glycyrrhizin- and analogue-induced hepatocyte DNA synthesis and proliferation. Treatment of hepatocytes with GL-1, GL-3 and GL-6 rapidly stimulated tyrosine phosphorylation of the EGF receptor and p42 MAP kinase, which were inhibited by genistein and PD98059, respectively. These results suggest that glycyrrhizin and some analogues are primary hepatocyte mitogens that bind to EGF receptors and subsequently stimulate the receptor tyrosine kinase/mitogen-activated protein kinase pathway to induce hepatocyte DNA synthesis and proliferation.

Prostaglandin E(2) (EP(1)) receptor agonist-induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes: the involvement of TGF-alpha.

We investigated the effects of prostaglandin (EP) receptor subtype agonists on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes to elucidate their mechanisms of action. Maintained in short-term cultures (i.e. 3.5 h) in a serum-free, defined medium, hepatocyte parenchymal cells underwent DNA synthesis and proliferation in the presence of sulprostone (10(-6) M), PGE(2) (10(-6) M), and 17-phenyl-trinor-PGE(2) (10(-9) M) in a time- and dose-dependent manner. PGE(2) was less potent than 17-phenyl-trinor-PGE(2) in stimulating hepatocyte mitogenesis. Sulprostone (10(-6) M) and 11-deoxy-PGE(1) (10(-6) M) showed weak and insignificant stimulation, respectively, for hepatocyte mitogenesis. These effects of PGE(2), 17-phenyl-trinor-PGE(2), and sulprostone were abolished by treatment with a specific EP(1) receptor antagonist, SC-51322, or the PLC inhibitor U-73122. The effects of these EP(1) receptor agonists were potentiated by ionomycin and blocked by verapamil. Hepatocyte mitogenesis was almost completely blocked by specific inhibitors of growth-related signal transducers, such as genistein, wortmannin, PD98059, and rapamycin. A monoclonal antibody against TGF-alpha dose-dependently inhibited PGE(2)- and 17-phenyl-trinor-PGE(2)-induced hepatocyte mitogenesis. Treatment with the EP(1) receptor agonists significantly increased the secretion of TGF-alpha, reaching a maximum within 5 min. The increase in TGF-alpha secretion was blocked by SC-51322, U-73122, somatostatin, and verapamil and potentiated by ionomycin. These results indicate that the proliferative mechanisms of action of EP(1) receptor agonists are mediated through an increase in the autocrine secretion of TGF-alpha, which is dependent on the EP(1) receptor/G-protein involved in PLC regulation/PLC/Ca(2+) system. The locally secreted TGF-alpha, in turn, acts as a complete mitogen that stimulates the tyrosine kinase/MAPK pathway in these cells.

Prostaglandin E2 (EP1) Receptor Agonist-Induced DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes: The Involvement of TGF-

Prostaglandin E2 (EP1) Receptor Agonist-Induced DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes: The Involvement of TGF-

Collagen type I gel cultures of adult rat hepatocytes as a screening induction model for cytochrome P450-dependent enzymes.

Albumin secretion, expression of cytochrome P450 dependent mono-oxygenases (CYPs) and their inducibility by well-known inducers were evaluated during 1 week in collagen type I gel sandwich and immobilisation cultures of adult primary rat hepatocytes. Albumin secretion increased during culture time and, following an initial decrease, CYP biotransformation activities remained stable for at least 7 days. Better preservation results were observed in the collagen gel sandwich culture than in the immobilisation model. The inducibility of CYPs by beta-naphthoflavone (beta-NF), 3- methylcholanthrene (3-MC), phenobarbital (PB) and dexamethasone (DEX) was studied in both collagen gel hepatocyte cultures. Exposure of the cells to either 5microM 3-MC or 25 microM beta-NF, added to the culture medium, resulted in strong increases of CYP1A1/2 activity in both culture models. Treatment with PB (3.2 mM) resulted in an increase in the CYP2B activity and a higher hydroxylation of testosterone in the 16alpha-position (CYP2B1/2 and CYP2C11), the 7alpha-position (CYP2A1/2), and the 6beta-position (CYP3A1). DEX (10 microM) markedly increased testosterone 6beta- and 7alpha-hydroxylation. Expression and induction experiments of CYP proteins exposed to these molecules confirmed the results of the CYP activity measurements. The patterns of CYP induction in collagen gel cultures of rat hepatocytes were similar to those observed in vivo. Consequently, collagen gel cultures and, more specifically, collagen gel sandwich cultures seem to be suitable as in vitro models for evaluating xenobiotics as potential inducers of CYP-enzymes.

Stimulation of DNA synthesis and proliferation by prostaglandins in primary cultures of adult rat hepatocytes

We studied the effects of several prostaglandins on DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes. Maintained in short-term cultures (i.e., 3.5 h), the hepatocyte parenchymal cells synthesized DNA and proliferated in the presence of various prostaglandins in a dose-dependent manner. The half-maximal effective concentrations (ED 50) of prostaglandin F 2α, prostaglandin E 1, prostaglandin E 2 and prostaglandin I 2 for proliferation were estimated to be 1.7×10 −9, 2.3×10 −8, 2.7×10 −8 and 3.3×10 −9 M, respectively. Prostaglandin E 2 and prostaglandin I 2 produced greater maximal responses than did either prostaglandin E 1 or prostaglandin F 2α. The cells responded only weakly to prostaglandin D 2. The stimulatory effects of 10 −6 M prostaglandin E 1 and 10 −6 M prostaglandin E 2 on hepatocyte DNA synthesis and proliferation were inhibited by a specific antagonist of the EP 1 receptor, 8-chlorodibenz[b, f][1, 4]oxazepine-10(11H)carboxylic acid, 2-[3-[(2-furanylmethyl)-thio]-1-oxopropyl]hydrazide (SC-51322; 10 −6 M). Specific inhibitors of signal transducing elements (e.g., 1-[6-[17β-3-methoxyestra-1, 3, 5(10)-trien-17-yl]amino] hexyl]-1H-pyrrol-2,5-dione (U-73122); 10 −6 M), 10 −6 M verapamil, 5×10 −6 M genistein) almost completely blocked the growth-promoting effects of the prostaglandins. These results suggest that prostaglandins stimulate hepatocyte DNA synthesis and proliferation by their own receptors and exert their effects through both phospholipase C/Ca 2+ and receptor tyrosine kinase pathways.

Transforming growth factor-p, inhibits the growth of primary adult rat hepatocyte cultures by increasing cAMP levels

Transforming growth factor-p, inhibits the growth of primary adult rat hepatocyte cultures by increasing cAMP levels

Transforming growth factor-β1 inhibits the growth of primary adult rat hepatocyte cultures by increasing cAMP levels

We investigated the mechanisms of transforming growth factor-β1 (TGF-β1) inhibition on transforming growth factor-α (TGF-α)-induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. TGF-α (1.0 ng/ml) produced a 4.2-fold elevation of DNA synthesis during 3 h of culture and a 1.2-fold increase in nucleus number (proliferation) during 4 h of culture. TGF-β1 dose dependently inhibited the TGF-α-induced hepatocyte DNA synthesis and proliferation: half-maximal inhibition occurred at a TGF-β1 concentration of 0.08 ng/ml. The inhibitory effects of 1.0 ng/ml TGF-β1 were almost completely reversed by adenylate cyclase inhibitors, 2,4-dideoxyadenosine (10 −6 M), and somatostatin (3×10 −7 M), or by a specific inhibitor of protein kinase A, H-89 ( N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; 10 −7 M). In addition, while TGF-α did not affect the basal cellular adenosine 3′,5′-monophosphate (cAMP) levels, TGF-β1 was found to produce dose-dependent increases in cellular cAMP levels. The cAMP-elevating effects of TGF-β1 were also reversed by 2,4-dideoxyadenosine (10 −6 M), and somatostatin (3×10 −7 M), but not by H-89 (10 −7 M). The present results suggest that the specific mechanisms involved in the growth inhibitory effect of TGF-β1 on TGF-α-induced hepatocyte DNA synthesis and proliferation are via stimulation of adenylate cyclase, which increases intracellular cAMP and subsequently activates protein kinase A.

Effect of Epidermal Growth Factor in Collagen Gel Cultures of Rat Hepatocytes

Collagen gel cultures of hepatocytes represent a promising in vitro model in pharmaco-toxicology. Epidermal growth factor (EGF) is usually added to the culture medium, although one could question its value in a culture model aiming at maintaining a maximum of differentiated functional capacities. In this study, the effects of EGF (20 ng/ml) on albumin secretion, morphology and pentoxyresorufin O-depentylase (PROD), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities have been examined in both collagen gel sandwich and immobilization gel cultures of adult rat hepatocytes. Transmission electron microscopy did not show an obvious influence of epidermal growth factor (EGF) on the intracellular organization of organelles of the rat hepatocytes. It was found that EGF addition had no effect on albumin secretion in both culture models. On the contrary, the presence of EGF in the culture medium provoked in collagen gel sandwich cultures, after 7 days, significant decreases of 66% and 25% in EROD and PROD activities, respectively. On GST activities, no effect of EGF could be observed in both collagen gel cultures. Removal of EGF from the culture medium seemed to have a positive effect on the maintenance of the phase 1 biotransformation capacity of rat hepatocytes. Its addition should therefore be avoided in collagen gel cultures used in pharmaco-toxicology.

Arsenite alters heme synthesis in long-term cultures of adult rat hepatocytes

Arsenite (As[III]) effects on the intermediate steps of heme biosynthesis were studied in adult rat hepatocytes seeded on a feeder layer of 3T3 cells (3T3-hepatocytes) and maintained for 2 weeks with culture medium non-supplemented or supplemented with 150 microM 5-aminolevulinic acid (ALA). The activities of the intracellular enzymes porphobilinogen deaminase (PBG-D), uroporphyrinogen III synthase (UROIII-S), and uroporphyrinogen III decarboxylase (URO-D), and the intermediary uroporphyrins (URO), coproporphyrins (COPRO) and protoporphyrin IX (PROTO) were determined in these cultures. The 3T3-hepatocytes maintained the activities of PBG-D, UROIII-S and URO-D during 2 weeks and ALA addition to the culture medium increased PBG-D (2-3-fold) and UROIII-S (50%) activities and porphyrin production, which accumulated as PROTO. Exposure to 3.9 microM As(III) inhibited UROIII-S activity (down to 34%), and PBG-D and URO-D activities to a lower extent; these effects were magnified by ALA supplementation. As(III) also produced an intracellular accumulation and a decreased excretion of PROTO, and a 31% reduction of the COPRO/URO ratio in the culture medium. Additionally, As(III) caused cytoplasmic vacuolization and lipid accumulation. Our results show that As(III) exposure selectively inhibits several intermediary enzymes of heme metabolism and affects the intra- and extracellular content of porphyrins and their ratio in the culture medium. They also confirm that 3T3-hepatocytes are a suitable in vitro model to study hepatic heme metabolism and its alterations by hepatotoxic chemicals.

Increased P-glycoprotein messenger RNA stability in rat liver tumors in vivo.

P-glycoproteins (Pgp) are comprised of a small family of plasma membrane proteins whose abundance in cultured cells is often associated with the multidrug resistance phenotype. Overexpression of Pgp has been observed in many types of human cancers, but the molecular basis for this overexpression has not been established. We have used primary monolayer cultures of adult rat hepatocytes and a stepwise model of rat liver carcinogenesis to study the regulation of Pgp gene expression. We observed a marked overexpression of Pgp, specifically the class II Pgp, in both systems. In addition, we observed that a number of unrelated genes including alpha-tubulin, beta-actin, gamma-actin, cytokeratin 8, cytokeratin 18, and c-myc are overexpressed in cultured hepatocytes, and they are also overexpressed during liver carcinogenesis and in transplantable tumors. Nuclear run-on assays showed no increase in the transcriptional activity of Pgp genes in transplantable liver tumors compared to normal liver. Studies of in vivo mRNA stability, however, revealed that all three Pgp mRNAs were relatively stable in transplantable liver tumors (t(1/2) > 12 h), in contrast to what was found in normal liver (t(1/2) < 2 h). In addition, mRNA for several other genes, including alpha-tubulin, c-myc, and cyclin D1, all appear to be stabilized in the tumors. These findings suggest that the overexpression of Pgp genes in rat liver tumors may be the result of a mechanism involving stabilization of a diverse group of mRNAs.

Signal transduction during liver regeneration.

Following partial hepatectomy (PH), there is a rapid and highly orchestrated series of biochemical events which occur prior to cellular proliferation. Some of these events are presumably intimately linked with the eventual regeneration of the liver, whereas others are likely to be stress related or required for the continued differentiated function of the liver while regeneration is occurring. The regulation of the AP-1 transcription factor c-Jun during hepatic regeneration has been studied here. There is a progressive increase in c-Jun-mRNA levels after sham operation, one-third PH, and two-thirds PH. A concomitant increase in activating protein 1 (AP-1) binding activity is also observed. The c-Jun protein is a major constituent of the AP-1 complex in quiescent and early regenerating liver. The activity of c-Jun amino-terminal kinase (JNK), which phosphorylates the activation domain of the c-Jun protein, is markedly stimulated after one-third and two-thirds PH. c-Jun amino-terminal kinase-1 is a constituent of this stimulated JNK activity after PH. When primary cultures of adult rat hepatocytes are incubated with epidermal growth factor or transforming growth factor-α, AP-1 transcriptional activity is increased and the activation domain of the c-Jun protein is further potentiated. Phosphopeptide mapping of the endogenous c-Jun protein in proliferating cultured hepatocytes demonstrates phosphorylation of the c-Jun activation domain. Pretreatment of animals prior to PH with a neutralizing antibody to tumour necrosis factor-α (TNFα), inhibits hepatocyte DNA synthesis and JNK activation. It is concluded that the stimulation of one-third or two-thirds PH activates JNK through a mechanism that requires TNFα, which phosphorylates the c-Jun activation domain in hepatocytes, resulting in enhanced transcription of AP-1-dependent genes. Although nuclear factor-kappa B (NFκB) binding activity is induced during liver regeneration following PH, the physiological consequence of this induction is unknown. The role of NFκB during liver regeneration has been assessed by delivering to the liver a super-repressor of NFκB activity using an adenoviral vector expressing a mutated form of IκB. This adenovirus (Ad5IκB) was almost exclusively expressed in the liver and inhibited NFκB DNA binding activity and transcriptional activity in cultured cells as well as in the liver in vivo. Following PH, Ad5IκB, but not a control adenovirus (Ad5βgal), resulted in the induction of apoptosis as demonstrated by histological staining and TUNEL analysis. In addition, infection with Ad5IκB but not Ad5βgal decreased the mitotic index following PH. These two phenomena, increased apoptosis and cell cycle arrest, were associated with liver failure in animals infected with the ad5IκB but not Ad5βgal as demonstrated by elevated serum bilirubin and ammonia levels. Thus, the induction of NFκB during liver regeneration following PH appears to be a required event to prevent apoptosis and to allow for normal cell cycle progression.

Effects of insulin-like growth factor I and II on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

We compared the effects of insulin-like growth factor I (IGF-I) and II (IGF-II) on DNA synthesis and proliferation and investigated various signal transduction mechanisms involved in insulin-like growth factor-induced mitogenesis in primary cultures of adult rat hepatocytes. IGF-I stimulated hepatocyte DNA synthesis and proliferation with an EC 50 of 75 ng/ml within 4 h of culture. These effects were sensitive to the IGF-I concentration and cell density. Hepatocyte proliferation induced by IGF-I was potentiated by metaproterenol (10 −6 M) as well as by 8-bromo-cAMP, phorbol 12-myristate 13-acetate (PMA; 10 −8 M) and was inhibited by U-73122 (1-(-[[17β-3-methoxyestra-1, 3, 5 (10)-triene-17-yl] amino] hexyl]-1 H pyrrol-2, 5-dione)), genistein, wortmannin, PD98059 (2′-amino-3′-methoxyflavone) and rapamycin. The IGF-I effect was independent of pertussis toxin (100 ng/ml). IGF-II also dose dependently stimulated hepatocyte DNA synthesis and proliferation with an EC 50 of 0.75 ng/ml within 4 h of culture. However, these effects were not dependent on the initial plating density. The stimulatory effects of IGF-II were potentiated by UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (10 −5 M) and inhibited by phenylephrine, PMA, metaproterenol, 8-bromo-cAMP, PD98059, rapamycin, and pertussis toxin. The IGF-II effects were not affected by genistein, U-73122, and wortmannin. These results suggest that IGF-I and IGF-II rapidly stimulate the DNA synthesis and proliferation of adult rat hepatocytes by separate mechanisms.

The inhibitory effect of interleukin 1beta on rat hepatocyte DNA synthesis is mediated by nitric oxide.

Interleukin 1beta (IL-1beta) and nitric oxide (NO) have potent growth-regulatory effects on different cell types. We found that epidermal growth factor-induced DNA synthesis in primary cultures of adult rat hepatocytes was inhibited by NO when it was provided by addition to the cultures of S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, as well as by addition of IL-1beta in a dose-dependent manner. IL-1beta also induced NO production and inducible NO synthase (iNOS) gene expression. The inhibition of DNA synthesis by IL-1beta was completely abrogated when NO production was inhibited by N-monomethyl-L-arginine (NMA), a competitive inhibitor of iNOS. IL-1beta-receptor antagonist (IL-1ra), which interferes with the interaction of IL-1beta with target cells, also abolished the inhibitory effects of IL-1beta on hepatocyte DNA synthesis as well as IL-1beta-induced iNOS gene expression. We also found that hepatocyte DNA synthesis inhibition by IL-1beta was completely antagonized by providing deoxynucleosides to bypass the block in ribonucleotide reductase, a rate-limiting step in DNA synthesis, thus implicating this enzyme in the mechanism of growth inhibition by IL-1beta. These experiments extended prior observations on the growth-inhibitory actions of IL-1beta on hepatocyte DNA synthesis, involving the IL-1beta receptor, NO production, and ribonucleotide reductase.

COMPARATIVE PHARMACODYNAMICS OF CYP2B INDUCTION BY DDT, DDE, AND DDD IN MALE RAT LIVER AND CULTURED RAT HEPATOCYTES

In this study the pharmacodynamics were characterized of rat hepatic cytochrome P450 2B (CYP2B) induction by the pesticide DDT \\[1,1,1-trichloro-2,2-bis(p -chlorophenyl) ethane] and its metabolites DDE \\[1,1-dichloro-2,2-bis(p -chlorophenyl)ethylene], which is bioretained, and DDD \\[1,1-dichloro-2,2-bis(p -chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction ( benzyloxyresorufin O -dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and 620 ppm in diet (14 d of exposure). The efficacies ( E values) for induction of benzyloxyresorufin O -dealkylation by DDT, max DDE, and DDD were 24-, 22-, and 21-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and 0.51 mu M, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and 5.9 mumol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and 2.7 mu M , respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.

The Ras/Rac1/Cdc42/SEK/JNK/c-Jun cascade is a key pathway by which agonists stimulate DNA synthesis in primary cultures of rat hepatocytes.

The ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor alpha (TNFalpha, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFalpha, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-L-lysine-coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 (N17), Cdc42 (N17), SEK1-, or JNK1- blunted the abilities of glucose, TNFalpha, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFalpha, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110alpha/p110gamma) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110alpha/p110gamma) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade, plays the pivotal role in the ability of agonists to stimulate DNA synthesis in primary cultures of rat hepatocytes.

Expression of alpha2- and beta2-adrenergic responses by hepatocyte growth factor and platelet-derived growth factor in primary cultures of adult rat hepatocytes.

We investigated the effects of hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) on the expression of alpha2- and beta2-adrenergic responses in primary cultures of adult rat hepatocytes. HGF (1 and 5 ng/ml) rapidly stimulated the expression of beta2-adrenergic responses and significantly increased alpha2-adrenergic responses with a maximal response at 21 h. The stimulatory effects of HGF were reduced dependent on the initial plating density and were completely blocked by cycloheximide (5 microM). On the other hand, PDGF (10 ng/ml) significantly increased the beta2-adrenergic response, but only slightly increased the alpha2-adrenergic responses. Expression of the alpha2- and beta2-adrenergic responses by PDGF was independent of the initial plating density. The expression of these responses was blocked by cycloheximide (5 microM). Northern blot analysis of the hepatocyte mRNA showed increased expression induced by the HGF and PDGF of the both alpha2- and beta2-adrenergic receptor mRNA, and this expression was inhibited by actinomycin D (5 ng/ml). These results indicate that the expression of alpha2- and beta2-adrenergic responses produced by these two growth factors is regulated differently by the cell density of the primary cultures. The results suggest that the expression of alpha2- and beta2-adrenergic responses is mediated through increased synthesis of these receptor proteins.