Hormonal regulation of aldolase B gene expression in rat primary cultured hepatocytes

A high performance liquid chromatography method for the determination of N3-methyl-5'-deoxy-5-fluorouridine, a possible metabolic product of the anticancer pro-drug 5'-deoxy-5-fluorouridine, in human serum and urine is described. Sample treatment involved addition of internal standard (5-bromouracil) and protein precipitation with ammonium sulphate (serum samples) followed by liquid-liquid extraction with ethyl acetate-isopropanol (90:10, v/v). The average recovery at 0.5 mg ml-1 level was (80 +/- 4%). A linear response extending over two decades of concentration was observed. Detection limits of 50 and 100 ng ml-1 were obtained in serum and urine, respectively.     

Sulfotransferase gene expression in primary cultures of rat hepatocytes

Hepatocyte cultures have been used in pharmacotoxicological studies, and sulfotransferases (ST) are important drug-metabolizing enzymes in liver. The expression of sulfotransferases in hepatocyte cultures has not been examined systematically. In the present study, the mRNA levels of different sulfotransferases in male and female rat hepatocytes were examined by northern-blot analyses. Various culture conditions such as different matrices (collagen, matrigel, collagen sandwich, or co-culture with epithelial cells), medium (Way-mouth's MB 752/1 and Modified Chee's Medium) and glucocorticoid supplementation (dexamethasone, 0.1 microM) were compared. Phenol ST (ST1A1) mRNA levels decreased to about 50% of initial mRNA levels within 10 hr of culture. At 96 hr, ST1A1 mRNA levels were approximately 20% of initial values when cultured on collagen, matrigel or co-culture. The two media did not differ in ability to maintain ST1A1 mRNA levels in the absence of dexamethasone (DEX); however, DEX addition to either medium resulted in ST1A1 mRNA levels greater than 100% of the initial mRNA levels at 96 hr, with the greatest increase observed using the matrigel substratum and Chee's medium. In the absence of DEX, the mRNA levels of N-hydroxy-2-acetylaminoflurene sulfortransferase (ST1C1), estrogen sulfotransferase (ST1E2) and hydroxysteroid sulfotransferase (ST-20/21, ST-40/41, ST-60) fell to approximately 20% of their initial levels within 24 hr, and to less than 5% at 96 hr. The loss of expression of these sulfotransferases was observed with all culture conditions. Addition of DEX to the media resulted in ST-40/41 and ST-60 mRNA expression at 20 and 35% of their initial values, respectively, in cultures maintained on matrigel and Chee's medium at 96 hr. These data suggest that sulfotransferases lose their constitutive expression in hepatocyte culture, but retain their inducibility.     

Stress protein expression in rat liver during tumour promotion: induction of heat-shock protein 27 in hepatocytes exposed to 2-acetylaminofluorene

Exposure of cells to a variety of stresses such as heat, radiation and xenobiotics leads to increased expression of heat-shock proteins (HSPs). HSPs protect cells against irreversible protein damage and are involved in adaptive responses to stress stimuli. Some HSPs are overexpressed in neoplasias, possibly contributing to the increased drug tolerance often observed in such lesions. We have studied HSP expression in two experimental rat hepatocarcinogenesis models. Our aim was to clarify whether they are involved in stress adaptation in hepatocytes during carcinogen exposure, and whether HSPs may contribute to xenobiotic resistance in preneoplastic lesions. The complete carcinogen 2-acetylaminofluorene (AAF) was used in a continuous feeding protocol, and in the resistant hepatocyte model where the growth of diethylnitrosamine initiated lesions is efficiently promoted. Of the HSPs tested, only heat-shock protein 27 (hsp27) was induced during continuous AAF exposure. After 4 weeks of feeding AAF, increased hsp27 expression was noted in hepatocytes in perivenous areas of the liver lobule, possibly mediating an adaptive response to stress caused by reactive AAF metabolites. Enzyme altered preneoplastic foci were not found to overexpress HSPs. Thus, HSP induction does not seem to be a general mechanism underlying the increased stress tolerance observed in such lesions.     

Xenobiotic-enhanced expression of cytochromes P450 2E1 and 2B in primary cultured rat hepatocytes

The purpose of this investigation was to determine whether long-term, heavy resistance training would cause adaptations in rat skeletal muscle structure and function. Ten male Wistar rats (3 weeks old) were trained to climb a 40-cm vertical ladder (4 days/week) while carrying progressively heavier loads secured to their tails. After 26 weeks of training the rats were capable of lifting up to 800 g or 140% of their individual body mass for four sets of 12-15 repetitions per session. No difference in body mass was observed between the trained rats and age-matched sedentary control rats. Absolute and relative heart mass were greater in trained rats than control rats. When expressed relative to body mass, the mass of the extensor digitorum longus (EDL) and soleus muscles was greater in trained rats than control rats. No difference in absolute muscle mass or maximum force-producing capacity was evident in either the EDL or soleus muscles after training, although both muscles exhibited an increased resistance to fatigue. Individual fibre hypertrophy was evident in all four skeletal muscles investigated, i.e. EDL, soleus, plantaris and rectus femoris muscles of trained rats, but muscle fibre type proportions within each of the muscles tested remained unchanged. Despite an increased ability of the rats to lift progressively heavier loads, this heavy resistance training model did not induce gross muscle hypertrophy nor did it increase the force-producing capacity of the EDL or soleus muscles.     

An okadaic acid-sensitive pathway involved in the phenobarbital-mediated induction of CYP2B gene expression in primary rat hepatocyte cultures

We have previously demonstrated that specific activation of a cAMP-dependent protein kinase A (PKA) pathway resulted in complete repression of phenobarbital (PB)-inducible CYP gene expression in primary rat hepatocyte cultures. In the current investigation, we examined the role of protein phosphatase pathways as potential co-regulators of this repressive response. Primary rat hepatocytes were treated with increasing concentrations (0.1-25 nM) of okadaic acid, a potent inhibitor of serine/threonine-specific protein phosphatases PP1 and PP2A. PB induction responses were assessed by use of specific hybridization probes to CYP2B1 and CYP2B2 mRNAs. Okadaic acid completely inhibited the PB induction process in a concentration-dependent manner (IC50, approximately 1.5-2 nM). Similar repression was obtained with low concentrations of other highly specific phosphatase inhibitors, tautomycin and calyculin A. In contrast, exposure of hepatocytes to 1-nor-okadaone or okadaol, negative analogs of okadaic acid largely devoid of phosphatase inhibitory activity, was without effect on the PB induction process. At similar concentrations, okadaic acid produced only comparatively weak modulation of the beta-naphthoflavone-inducible CYP1A1 gene expression pathway. In additional experiments, hepatocytes were treated with suboptimal concentrations of PKA activators together with phosphatase inhibitors. Okadaic acid markedly potentiated the repressive effects of dibutyryl-cAMP on the PB induction process. Together, these results indicate that both PKA and protein phosphatase (PP1 and/or PP2A) pathways exert potent and complementary control of the intracellular processes modulating the signaling of PB in cultured primary rat hepatocytes.     

Cell density-dependent mitogenic effect and -independent cellular handling of epidermal growth factor in primary cultured rat hepatocytes

Brain swelling is a serious complication associated with focal ischemia in stroke and severe head injury. Experimentally, reperfusion following focal cerebral ischemia exacerbates the level of brain swelling. In this study, the permeability of the blood-brain barrier has been investigated as a possible cause of reperfusion-related acute brain swelling. Blood-brain barrier disruption was investigated using Evans Blue dye and [14C]aminoisobutyric acid autoradiography in a rodent model of reversible middle cerebral artery (MCA) occlusion. Acute brain swelling and cerebral blood flow (CBF) during ischemia and reperfusion were analyzed from double-label CBF autoradiograms after application of the potent vasoconstrictor peptide endothelin-1 to the MCA. Ischemia was apparent within ipsilateral MCA territory, 5 min after endothelin-1 application to the exposed artery. Reperfusion, examined at 30 min and 1, 2, and 4 h, was gradual but incomplete within this time frame in the core of middle cerebral artery territory and associated with significant brain swelling. Ipsilateral hemispheric swelling increased over time to a maximum (>5%) at 1-2 h after endothelin-1 but was not associated with a significant increase in the ipsilateral transfer constant for [14C]aminoisobutyric acid over this time frame. These results indicate that endothelin-1 induced focal cerebral ischemia is associated with an acute but reversible hemispheric swelling during the early phase of reperfusion which is not associated with a disruption of the blood-brain barrier.     

Relationship between DNA synthesis and growth factor expression in primary cultures of adult rat hepatocytes

In this study we employed primary culture of adult rat hepatocytes to verify the effects of two different extracellular matrices (collagen, matrigel) on EGF-stimulated DNA synthesis and c-myc expression. Our results confirm that in adult rat hepatocytes EGF induces DNA synthesis, preceded by a transient increase of c-myc expression, when cells are cultured at low density on collagen. DNA synthesis appears to be in reciprocal relationship with hepatic expression of IGF-I, IGFBP-1, IGFBP-2 and IGFBP-4, suggesting that IGF-I/IGFBPs system is not involved in liver growth.     

Phorbol esters down-regulate alpha-fetoprotein gene expression without affecting growth in fetal hepatocytes in primary culture

The mammalian spermatozoon undergoes continuous modifications during spermatogenesis, maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are capable of binding the zona-intact egg and undergoing the acrosome reaction. The fertilization process is a net result of multiple molecular events which enable ejaculated spermatozoa to recognize and bind to the egg's extracellular coat, the zona pellucida (ZP). Sperm-egg interaction is a species-specific event which is initiated by the recognition and binding of complementary molecule(s) present on sperm plasma membrane (receptor) and the surface of the ZP (ligand). This is a carbohydrate-mediated event which initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be a prerequisite which enables the acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg. This review focuses on the formation and contents of the sperm acrosome as well as the mechanisms underlying the induction of the acrosome reaction. Special emphasis has been laid on the synthesis, processing, substrate specificity, and mechanism of action of the acid glycohydrolases present within the acrosome. The hydrolytic action of glycohydrolases and proteases released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of ZP. We have discussed the most recent studies which have attempted to explain signal transduction pathways leading to the acrosomal exocytosis.     

An immunologic approach to induction of epidermal growth factor deficiency: induction and characterization of autoantibodies to epidermal growth factor in rats

The role of glutamate as a possible mediator of neurodegeneration is well described, and the homeostasis of extracellular glutamate is considered of major importance when addressing the pathogenesis of excitatory neurodegeneration. Applying the 'indicator diffusion' method to the microdialysis technique, we present a method that is suitable for the in vivo investigation of the capacity of cellular uptake of glutamate. Using 14C-mannitol as reference, we measured the cellular extraction and the cell membrane permeability of the test substance 3H-D-aspartate in the corpus striatum of the rat brain. The cellular extraction fraction of 3H-D-aspartate was 0.29, and the cell membrane permeability 2.24 x 10(-4) cm/s. In the presence of the glutamate-uptake blocker DL-threo-beta-hydroxyaspartate (THA) the extraction of 3H-D-aspartate was completely abolished, indicating that extraction of 3H-D-aspartate was due to cellular uptake by glutamate transporters. The cell membrane permeability towards 3H-D-aspartate was reduced by approximately 98% due to THA, indicating that the cell membranes per se are highly resistant to diffusion of 3H-D-aspartate. It is concluded that the present method can be used in studying the capacity of the glutamate transporters in vivo.     

Transcription factor AP2 is required for expression of the rat transforming growth factor-alpha gene

DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.     

Differential induction of gamma-glutamyl transpeptidase in primary cultures of rat and mouse hepatocytes parallels induction during hepatocarcinogenesis

In carcinogen-treated rats, gamma-glutamyl transpeptidase (GGT) is induced in preneoplastic liver lesions and liver tumors. However, in mice, GGT is rarely detected during hepatocarcinogenesis. Data in this study reveal that GGT is not induced in mouse hepatocytes when they are maintained in vitro under the same conditions that induce GGT activity in primary cultures of rat hepatocytes. GGT activity in rat hepatocytes increased 20-fold during the first 7 days in culture, but there was no induction of GGT in primary cultures of mouse hepatocytes. Comparison of intracellular glutathione levels in rat and mouse liver cells showed that the glutathione level was higher in the mouse liver cells than the rat. Blocking glutathione synthesis with buthionine sulfoximine reduced the intracellular glutathione concentration in mouse liver cells but did not trigger an induction of GGT. Analysis of the GGT mRNA in primary cultures of rat hepatocytes showed that only GGT mRNA(III) is induced. This is the same GGT mRNA species present in preneoplastic hepatic lesions and liver tumors in the rat (1-3). Therefore activation of promoter III in the GGT gene is responsible for induction of GGT in both hepatocytes in vitro and liver tumors in vivo. These data show that primary cultures of rat and mouse hepatocytes provide a model system with which to study interspecies differences in the regulation of this enzyme and to better understand the role of GGT in normal and neoplastic processes.     

Adeno-associated virus type 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression

Adeno-associated virus type 2 (AAV), a single-stranded, DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays an important role in AAV-mediated transgene expression (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997) and that a strong correlation exists between the phosphorylation state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et al., J. Virol. 72:1593-1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is mediated by the EGF-R PTK. Treatment of cells with EGF results in phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes dephosphorylation of the ssD-BP and consequently leads to increased expression of the transgene. Furthermore, AAV transduction efficiency inversely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of the ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These data suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vectors in human gene therapy.     

Insulin effects on CYP2E1, 2B, 3A, and 4A expression in primary cultured rat hepatocytes

Although hyperketonemia and/or altered growth hormone secretion caused by diabetes have been implicated in enhanced CYP2E1, 2B, 3A and 4A expression, the effect of insulin on hepatic P450 expression, in the absence of associated metabolic/hormonal alterations, remains unknown. Primary cultured rat hepatocytes have been shown (Zangar et al., Drug Metab. Dispos., 23:681, 1995) to express stable and inducible CYP2E1 mRNA and protein levels, and provide an excellent system for mechanistic examination of the effect of insulin on CYP2E1, 2B, 3A and 4A expression. Maintaining primary rat hepatocytes in culture in the absence of insulin for 48, 72, or 96 h increased CYP2E1 mRNA levels 5-, 11-, and 4-fold, respectively, relative to cells maintained in the presence of the standard concentration of 1 microM insulin. In contrast, CYP2B mRNA levels increased only approximately 2-fold in the absence of insulin, when compared with the presence of 1 microM insulin. CYP2E1 and 2B protein levels were increased 6.7- and 3.8-fold, respectively, in cells cultured for 96 h in the absence of insulin as compared with those cultured in medium containing 1 microM insulin. Concentration-response studies revealed that decreasing the concentration of insulin below 10 nM (i.e. 1 nM, 0.1 nM, no insulin) increased CYP2E1 mRNA levels 4-, 7-, and 11-fold, respectively. In contrast, no such concentration-dependence was observed for CYP2B mRNA expression. As CYP3A and 4A expression is also elevated in diabetic rats, the effects of insulin on these P450s was also examined. CYP3A mRNA levels were unaltered and CYP4A mRNA levels were decreased marginally (approximately 50%) by the absence of insulin relative to levels in cells cultured in the presence of 1 microM insulin over 96 h in culture. The results of this study provide evidence that insulin itself, in the absence of other diabetes-induced metabolic or hormonal alterations, affects CYP2E1 and 2B, but not CYP3A or 4A, expression in primary cultured rat hepatocytes. Furthermore, CYP2E1 expression is differentially regulated by insulin relative to CYP2B, 3A or 4A. This study also demonstrates that decreasing the concentration of insulin in the culture medium provides a method by which CYP2E1 levels can be increased in primary cultured hepatocytes to facilitate mechanistic studies on the regulation of CYP2E1 expression.     

Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.     

Biphasic regulation of cytochrome P450 2B1/2 mRNA expression by dexamethasone in primary cultures of adult rat hepatocytes maintained on matrigel

We have demonstrated recently that although rat hepatocytes rapidly lose their cytochrome P450 mRNA content following their introduction into primary culture, hepatocytes cultured on Matrigel, a reconstituted basement membrane, subsequently spontaneously "reexpress" the mRNAs of some constitutive P450 forms (Kocarek et al., Mol Pharmacol 43: 328-334, 1993). In the present study, we used the Matrigel cell culture system to examine the dose-dependent effects of dexamethasone (DEX) treatments on the mRNAs for two of the P450 forms that are reexpressed spontaneously between days 3 and 5 in culture, 2B1/2 and 2C6. Treatment of cultured hepatocytes with low doses of DEX (10(-9) to 10(-8) M) that induced the mRNA for tyrosine aminotransferase, a model glucocorticoid-inducible gene, suppressed the spontaneous appearance of 2B1/2 mRNA while having little or no effect on the level of 2C6 mRNA or on beta-actin mRNA. However, treatment of the hepatocyte cultures with high doses of DEX (10(-6) to 10(-5) M) that induced P450 3A1 mRNA increased the amounts of the 2B1/2 and 2C6 mRNAs (4.1- and 2.4-fold, respectively, at 10(-5) M DEX). In contrast to the suppressive effects on the spontaneous increases in 2B1/2 mRNA, low doses of DEX (10(-8) to 10(-7) M) enhanced the induction of 2B1/2 mRNA by phenobarbital (2.5-fold at 10(-7) M DEX). Treatment of the hepatocyte cultures with triamcinolone acetonide, another potent glucocorticoid, suppressed spontaneous 2B1/2 mRNA expression at low doses, but did not induce 2B1/2 mRNA at high doses. Treatments with steroids of other classes, including dihydrotestosterone, 17 alpha-ethinylestradiol, fludrocortisone or R-5020, failed to suppress 2B1/2 mRNA levels at low doses. Additionally, treatment with RU-486, a glucocorticoid/progestin receptor antagonist, induced 2B1/2 mRNA at high doses (10(-6) to 10(-5) M). The suppressive effects of DEX on spontaneous 2B1/2 mRNA expression observed at low doses are consistent with a classical glucocorticoid-mediated mechanism, while the high-dose inductive effects of DEX appear to be exerted through a nonclassical mechanism, perhaps akin to that for induction of 3A1.     

Differential inhibition of epidermal growth factor signaling pathways in rat hepatocytes by long-term ethanol treatment

BACKGROUND & AIMS: Long-term ethanol intake suppresses liver regeneration in vivo and ethanol interferes with epidermal growth factor (EGF)-induced DNA synthesis in vitro. Therefore, the effects of long-term ethanol treatment on EGF-activated signaling reactions in rat hepatocytes were investigated. METHODS: Hepatocytes from long-term ethanol-fed rats and pair-fed controls were stimulated with EGF (0.5-20 nmol/L) for 15-120 seconds. Tyrosine phosphorylation of EGF receptor (EGFR), Shc, and phospholipase-C gamma1 (PLC gamma), and growth factor receptor binding protein 2 (Grb2) coprecipitation with EGFR and Shc were analyzed by Western blotting. RESULTS: EGFR autophosphorylation was suppressed at all EGF concentrations in ethanol-fed cells compared with pair-fed cells, without significant differences in total EGFR protein or EGFR tyrosine kinase activity detected in cell lysates, suggesting that intracellular factors suppressed EGFR function. EGF-induced PLC gamma tyrosine phosphorylation and inositol 1,4,5-trisphosphate (InsP3) formation were suppressed, but cytosolic [Ca2+]c elevation was little affected, indicating enhanced InsP3-mediated intracellular Ca2+ release in ethanol-fed cells. Grb2 binding to EGFR was suppressed, but EGF-induced Shc tyrosine phosphorylation and Grb2 association with Shc were not significantly decreased. CONCLUSIONS: Long-term ethanol feeding suppressed EGF-induced receptor autophosphorylation in rat hepatocytes with differential inhibition of downstream signaling processes mediated by PLC gamma, Shc, and Grb2. Altered patterns of downstream signals emanating from EGFR may contribute to deficient liver regeneration in chronic alcoholism.