Regulation of alpha2-macroglobulin expression in rat Sertoli cells and hepatocytes by germ cells in vitro

The combination of an immunohistochemical technique and a panel of monoclonal antibodies was used to investigate the presence of leukocyte populations in the distal jejunal lymph node of 3-4 week old calves and adult cattle. The application of computer-assisted morphometric analysis enabled information to be obtained on the distribution of leukocyte populations in lymphoid compartments of the lymph node cortex. Semi-quantitative estimates of the areas of staining in histological sections showed that calves possessed significantly fewer B-cells and CD4+ cells in the outer cortex and significantly fewer T-cells (CD4+, CD8+ and gamma delta T-cells) in the deep cortex. These findings were interpreted to be a possible consequence of immunosuppression resulting from the passive transfer of maternal immunity in colostrum. The presence of some B-cell follicles in the region defined as the deep cortex suggested the on-going differentiation of this predominantly T-cell compartment. The larger presence of interdigitating cells (IDC) in the deep cortex of calves than adults was suggested by significantly larger CD1+ populations and it was argued that this could be the result of the confrontation with exogenous antigen faced by calves in early postnatal life. Antigen presenting populations, pan MHC II+ and MHC II DQ+ populations, were increased in all compartments of calf lymph nodes but were not significantly different from the populations in adult lymph nodes. Variance component analysis of the data generated in the present study showed that the image analysis technique was an effective and statistically powerful approach to investigate leukocyte populations within the specific microenvironments of the lymph node.     

Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver

It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of rats with fluvastatin had no effect on hepatic levels of CYP3A1/2 mRNA or immunoreactive protein. However, treatment with 50 mg/kg/day fluvastatin induced CYP1A1 mRNA and protein. The effects of fluvastatin treatment on P450 expression seen in primary cultured rat hepatocytes thus largely recapitulated the effects seen in vivo. The differences in effects among the HMG-CoA reductase inhibitors suggest that simple inhibition of HMG-CoA reductase cannot explain all of the effects of these drugs on P450 expression.     

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.     

Regulation of rat hepatic 3 alpha-hydroxysteroid dehydrogenase in vivo and in primary cultures of rat hepatocytes

Lamoids in North America harbor a wide variety of parasites. Treatment and control methods based on previous experience with parasites of cattle and sheep have been successful, but problems do exist. First, the pharmacokinetics for most anthelmintics have not been evaluated in llamas. Second, even though llamas, sheep, and cattle share many parasites, the two most common nematodes found in llamas (C. mentulatus and T. tenuis) are not part of the parasitic fauna of livestock. This presents difficulties in basing treatment and control methods on those recommended for cattle and sheep. Variability in host response to the same parasite also hinders the use of cattle and sheep as models for the llama. This is best demonstrated by F. magna and F. hepatica; the reaction induced by the first more closely resembles those seen in cattle than sheep, but the reaction induced by the second more closely resembles those seen in sheep than cattle. Finally, parasites known to be pathogenic in livestock (e.g., N. battus) have unknown effects in llamas. These examples illustrate that we must use caution when extrapolating existing knowledge regarding the parasites of sheep and cattle to llamas. Further research on the epidemiology of parasites peculiar to the llama is needed to enhance control efforts. Improved methods of diagnosis and treatment of parasites also are areas in which further efforts are needed.     

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.     

Regulation of activity and apical targeting of the Cl-/HCO3- exchanger in rat hepatocytes

To test the hypothesis that rat hepatocyte canalicular Cl-/HCO3- exchange activity might be regulated by HCO3- or protein kinase-induced changes in the apical targeting of vesicles, isolated rat hepatocytes were cultured in the presence or absence of HCO3-/CO2.Cl-/HCO3- exchange activity increased in cells cultured in the presence of HCO3-/CO2 or when stimulated by dibutyryl cAMP. Both of these effects were blocked by either colchicine or the protein kinase C agonist phorbol 12,13-dibutyrate. Fluorescence and confocal microscopy, respectively, revealed increased pericanalicular-apical membrane localization of two canalicular markers, peanut agglutinin and a 110-kDa canalicular ecto-ATPase, when hepatocyte couplets were preincubated in HCO3-/CO2-containing medium, an effect that was again blocked by colchicine. Dibutyryl cAMP also stimulated canalicular localization of the 110-kDa protein. These findings suggest that hepatocyte Cl-/HCO3- exchange activity is regulated by HCO3-/CO2 and by protein kinase A and protein kinase C agonists through microtubule-dependent targeting of vesicles containing this exchanger to the canalicular domain.     

Pituitary activin receptor subtypes and follistatin gene expression in female rats: differential regulation by activin and follistatin

The activins, hormones produced in the gonads and extragonadal tissues (including the pituitary), rapidly increase FSH beta messenger RNA (mRNA) and FSH secretion. In the rat, activin acts via a family of activin receptor (ActR) subunits that includes at least one type I (ActRI or ALK-2) and two homologous type II (IIA and IIB) subunits. We have previously reported that ActRIIA mRNA rises after ovariectomy (OVX). Potentially, the OVX-induced increases in ActR mRNAs could result from altered activin or the activin-binding protein follistatin. It was the purpose of the current studies to determine whether activin and/or follistatin regulated activin receptor subunit mRNAs. Adult female rat pituitaries were dissociated and plated for 48 h, transferred to wells containing follistatin or activin for 2 or 24 h, then RNA extracted for measurement of ActRI, IIA, and IIB and follistatin mRNAs. All three ActR mRNAs were easily detectable in pituitary RNA, with the relative abundance of ActRI > IIA > IIB (18:9:1). Between 2-24 h, levels of all three ActR mRNAs increased 2- to 3-fold in wells containing medium alone, whereas levels of follistatin mRNA were unchanged. Follistatin significantly reduced FSH secretion and follistatin mRNA, but not the ActR mRNAs. Activin increased ActRI (4-fold, at 2 h), ActRIIB (2-fold, at 24 h), and follistatin (2-fold, at 24 h) mRNAs and FSH release (2-fold, at 24 h), but did not alter ActRIIA mRNA levels. We conclude that 1) pituitary ActR mRNA expression is under inhibitory tone in vivo, as suggested by the effect of pituitary removal and cell dispersion and an earlier report after OVX. 2) Pituitary-derived activin stimulates follistatin (but not ActR) mRNA production, and additional increases in follistatin mRNA can be induced by exogenous activin. 3) Higher concentrations of activin differentially regulate pituitary ActR mRNA expression, suggesting that activin exerts a positive feedback effect on its own receptor.     

Regulation of Mg2+ homeostasis by insulin in perfused rat livers and isolated hepatocytes

Several recent studies demonstrate that adrenergic receptor stimulation evokes marked changes in Mg2+ homeostasis. As insulin counter-regulates many of the metabolic consequences of adrenergic receptor stimulation, we evaluated the potential influence of insulin on Mg2+ movements in response to adrenergic stimulation. The data demonstrate that insulin is able to block the Mg2+ efflux from perfused rat livers stimulated by isoproterenol or 8-Br-cAMP, but has little or no effect on epinephrine or phenylephrine induced Mg2+ efflux. Thus, evidence is provided demonstrating that there are redundant adrenergic pathways regulating Mg2+ efflux from liver tissue. One of these pathways, the beta-adrenergic component, is selectively blocked by insulin. Furthermore, these findings may provide a cellular explanation for hypomagnesemia associated with diabetes.     

Regulation of expression of A-kinase anchoring proteins in rat granulosa cells

FSH action on granulosa cells involves the generation of cAMP and subsequent activation of the cAMP-dependent protein kinase (PKA). The PKA holoenzyme is targeted to specific subcellular sites through the interaction of the regulatory subunits with A-kinase anchoring proteins (AKAPs). We previously reported that FSH regulates expression of AKAPs. In this report we examine the relationship between AKAP expression and cell shape. Granulosa cells cultured in the absence of FSH tend to spread and flatten. Cell spreading is accompanied by an increased expression of a 140-kDa AKAP. This spreading/flattening phenotype is independent of the specific extracellular matrix proteins (fibronectin, polylysine, and gelatin) on which cells are plated. Addition of FSH prevents both cell spreading and induction of AKAP 140. Culturing cells on poly (2-hydroxyethyl methacrylate), a surface-coating agent that inhibits cell spreading and adhesion, also inhibits expression of AKAP 140. Addition of phorbol myristate acetate, an agent known to antagonize FSH actions, blocks FSH regulation of both cell shape and AKAP 140 expression. Addition of dexamethasone plus FSH causes a synergistic increase in progesterone levels but has no effect on cell shape or induction of AKAP 140. Dexamethasone produces a dose-dependent increase in AKAP 80 expression, which is blocked by FSH, suggesting cross talk between the glucocorticoid and FSH receptor signaling pathways. These data suggest that expression of AKAP 140 is linked to regulation of cell shape, and that changes in the expression of AKAPs are regulated by several different signaling pathways.     

Glucose inhibits phenobarbital-induced delta-aminolevulinate synthase expression in normal but not in diabetic rat hepatocytes

In the present work, we demonstrate the presence of a glucose inhibitory effect on the phenobarbital-mediated induction of the delta-aminolevulinate synthase mRNA in normal rat hepatocytes, consistent with the results obtained with the delta-aminolevulinate synthase activity previously reported. This "glucose effect" can be prevented by adding cAMP, adenylate cyclase activators, or a phosphodiesterase inhibitor. Delta-Aminolevulinate synthase mRNA half-life is not modified in the presence of phenobarbital or glucose. When the same experiments are performed using diabetic cells, no glucose effect is observed, even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study suggest that glucose decreases delta-aminolevulinate synthase biosynthesis by acting at a pretranslational step. Assuming that the glucose effect operates by a repression mechanism exerted by metabolites derived from or related to glucose, the present results may reflect a derangement in the formation of these metabolites as a result of the abnormal metabolism operating in the diabetic state.     

Regulation of tissue-type plasminogen activator (tPA) and type-1 plasminogen activator inhibitor (PAI-1) gene expression in rat hepatocytes in primary culture

We have performed a comparative study on tPA and PAI-1 mRNA expression in primary cultures of rat hepatocytes and elucidated the possible regulation of these factors by certain hormonal stimulation. The tPA mRNA increased 2- to 4-fold in the presence of cholera toxin (CT), dibutyryl cyclic AMP (dbcAMP), or 3-isobutyl-1-methyl xanthine (IBMX), but slightly decreased in the presence of dexamethasone. The tPA activity was also changed by these agents in a similar fashion. On the contrary, PAI-1 mRNA decreased with CT, dbcAMP, or IBMX, but increased transiently with dexamethasone. From results obtained with cycloheximide, ongoing protein synthesis was judged to be required for both PAI-1 induction with dexamethasone and PAI-1 suppression with IBMX, but not for the tPA induction with IBMX. Dexamethasone exerted opposite regulatory effects on the tPA mRNA expression depending on its concentration: at 10(-8) to 10(-6) M, it suppressed the expression; whereas at 10(-10) M, it elevated the expression.     

Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells

We have examined the rotational diffusion of the luteinizing hormone (LH) receptors binding human chorionic gonadotropin (hCG) or ovine luteinizing hormone (oLH) in MA-10 Leydig tumor cells using time-resolved phosphorescence anisotropy techniques. LH receptors binding erythrosin isothiocyanate (ErITC)-derivatized oLH were rotationally mobile with rotational correlation times of 62 micros, 48 micros, 38 micros, and 29 micros at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C, respectively. ErITC-hCG bound to the LH receptor was rotationally immobile, showing no anisotropy decay at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C. To determine whether cytoskeletal components influenced the rotational diffusion of LH receptors, we measured rotational diffusion of LH receptors on MA-10 cells treated with 20 microg/ml cytochalasin D and on plasma membrane preparations. Following 1 h exposure to cytochalasin D, the rotational correlation times for hCG-occupied LH receptors were typically 11 micros at 37 degrees C compared to > 1000 micros on untreated cells. Treatment of MA-10 cells with cytochalasin B or colchicine had no affect on LH receptor rotational diffusion. Rotational correlation times for LH-occupied receptors decreased from 29 micros to 12 micros at 37 degrees C following cytochalasin D treatment. The rotational diffusion of LH receptors on plasma membrane preparations was similar to that observed for LH- and hCG-occupied receptors on intact cells treated with cytochalasin D. These various results indicate that there are differential effects of LH and hCG binding on the interactions of LH receptors with plasma membrane proteins and that microfilaments anchor the hCG- and LH-occupied receptors.     

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.     

Immunogenicity of rat hepatocytes in vivo: effect of cholestasis-induced changes in major histocompatibility complex expression

Hepatocytes normally express few major histocompatibility complex (MHC) class I and no MHC class II molecules, a phenomenon which could explain their low immunogenicity. However, in pathological situations, such as allograft rejection and cholestasis, hepatocytes strongly express MHC class I molecules and their immunogenicity could be different. The aim of this study was to assess the role of MHC expression on the immunogenicity of hepatocytes in vivo. Hepatocytes were obtained from normal and cholestatic DA rats by whole-liver perfusion with EDTA. Cholestasis was induced by ligation-section of the common bile duct. MHC expression on hepatocytes was assessed by cytofluorimetry after labelling with monoclonal antibodies against MHC class I and class II antigens. The percentage of hepatocytes expressing MHC class I was 9.8 +/- 2.2% in normal rats and 77.2 +/- 3.3% in cholestatic rats (P = 2 x 10(-4)); MHC class II expression was present on 1 +/- 0.5% of normal hepatocytes and 0.4% +/- 0.1% of cholestatic hepatocytes (P > 0.05). Lewis rats received a DA or Wistar-Furth heart allograft 7 days after intravenous injection of 2 x 10(7) hepatocytes from normal or cholestatic DA rats. The DA heart allograft was rejected in 6.3 +/- 0.4 days in Lewis controls, 8.8 +/- 1.1 days (N.S.) in Lewis recipients that received normal DA hepatocytes and 17.6 +/- 3.0 days (P = 2 x 10(-4)) in Lewis recipients that received hepatocytes from cholestatic DA rats.(ABSTRACT TRUNCATED AT 250 WORDS)