Modulation of VIPergic phenotype of enteric neurons by colonic biopsy supernatants from patients with inflammatory bowel diseases: Involvement of IL-6 in Crohn's disease.

BACKGROUND: Neuroplastic changes in the enteric nervous system (ENS) observed during IBD might participate in physiopathological processes. Vasoactive intestinal polypeptide has been shown to be involved in intestinal inflammation and barrier functions. We aimed to investigate the modulation of VIP expression in colonic biopsies of IBD patient, the ability of soluble factors from biopsies to reproduce in vitro these modulations and identify soluble factors responsible. METHODS: VIP and cytokines mRNA expressions were assessed in colonic biopsies of healthy subjects (HS) and IBD patients from inflamed (I) and non-inflamed areas (NI). Supernatants (SUP) of biopsies were applied to primary culture of ENS and VIP and cytokines mRNA expressions were assessed. The role of cytokines in SUP induced changes in VIP expression was evaluated. KEY RESULTS: VIP mRNA expression was lower in biopsies of patients with Crohn's disease (CD) than Ulcerative Colitis (UC) but unchanged as compared to HS. VIP mRNA and protein expression were lower in primary culture of ENS incubated with SUP-CD than with SUP-UC. Furthermore, in CD but not UC, SUP-I reduced VIP expression in the ENS as compared to SUP-NI. Next, IL-6 but not IL-5, IL-10, IL-17, IFN-γ or TNF-α reduced VIP expression in the ENS. Finally, in CD, SUP-I incubated with anti-IL-6 antibody increased VIP expression as compared to SUP-I alone. CONCLUSIONS & INFERENCES: Mucosal soluble factors from IBD induce VIP neuroplastic changes in the ENS. IL-6 was identified as a putative soluble factor responsible in part for changes in VIP expression in CD.

IL-22BP is produced by eosinophils in human gut and blocks IL-22 protective actions during colitis.

Crohn's disease and ulcerative colitis, the two major forms of inflammatory bowel diseases (IBDs), are characterized by high levels of IL-22 production. Rodent studies revealed that this cytokine is protective during colitis but whether this is true in IBDs is unclear. We show here that levels of the soluble inhibitor of IL-22, interleukin 22-binding protein (IL-22BP), are significantly enhanced during IBDs owing to increased numbers of IL-22BP-producing eosinophils, that we unexpectedly identify as the most abundant source of IL-22BP protein in human gut. In addition, using IL-22BP-deficient rats, we confirm that endogenous IL-22BP is effective at blocking protective actions of IL-22 during acute colitis. In conclusion, our study provides new important insights regarding the biology of IL-22 and IL-22BP in the gut and indicates that protective actions of IL-22 are likely to be suboptimal in IBDs thus making IL-22BP a new relevant therapeutic target.

Neurochemical plasticity in the enteric nervous system of a primate animal model of experimental Parkinsonism.

Emerging evidences suggest that the enteric nervous system (ENS) is affected by the degenerative process in Parkinson's disease (PD). In addition lesions in the ENS could be associated with gastrointestinal (GI) dysfunctions, in particular constipation, observed in PD. However, the precise alterations of the ENS and especially the changes in the neurochemical phenotype remain largely unknown both in PD and experimental Parkinsonism. The aim of our study was thus to characterize the neurochemical coding of the ENS in the colon of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, a well-characterized model of PD. In the myenteric plexus, there was a significant increase in the number of neurons per ganglia (identified with Hu), especially nitric oxide synthase immunoreactives (IR) neurons in MPTP-treated monkeys compared to controls. A concomitant 72% decrease in the number of tyrosine hydroxylase-IR neurons was observed in MPTP-treated monkeys compared to controls. In contrast no change in the cholinergic or vasoactive intestinal peptide-IR population was observed. In addition, the density of enteric glial cells was not modified in MPTP-treated monkeys. Our results demonstrate that MPTP induces major changes in the myenteric plexus and to a lesser extent in the submucosal plexus of monkeys. They further reinforce the observation that lesions of the ENS occur in the course of PD that might be related to the GI dysfunction observed in this pathology.

Beclin 1 mRNA strongly correlates with Bcl-XLmRNA expression in human hepatocellular carcinoma.

Beclin 1 physically associates with Bcl-x(L) and is considered as a haploinsufficient tumor suppressor. As the role of Beclin 1 in hepatocellular carcinoma (HCC) is unknown, we determined Beclin 1 mRNA expression in 27 pairs of tumoral/nontumoral (T/NT) liver samples. The Beclin 1 mRNA T/NT ratio was less than 0.5 in 2 tumors and more than 2 in 1 tumor, and was positively correlated with the Bcl-X(L) mRNA T/NT ratio (P < 0.001), but not with the proliferating cell nuclear antigen mRNA T/NT ratio. Coregulation of Beclin 1 and Bcl-X(L) expression in HCC may suggest cooperation in the regulation of apoptosis.

Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-beta1-dependent pathway.

Although recent studies have shown that enteric neurons control intestinal barrier function, the role of enteric glial cells (EGCs) in this control remains unknown. Therefore, our goal was to characterize the role of EGCs in the control of intestinal epithelial cell proliferation using an in vivo transgenic and an in vitro coculture model. Assessment of intestinal epithelial cell proliferation after ablation of EGCs in transgenic mice demonstrated a significant increase in crypt cell hyperplasia. Furthermore, mucosal glial network (assessed by immunohistochemical detection of S-100beta) is altered in colon adenocarcinoma compared with control tissue. In an in vitro coculture model of subconfluent Caco-2 cells seeded onto Transwell filters with EGCs, Caco-2 cell density and [3H]thymidine incorporation were significantly lower than in control (Caco-2 cultured alone). Flow cytometry analysis showed that EGCs had no effect on Caco-2 cell viability. EGCs induced a significant increase in Caco-2 cell surface area without any sign of cellular hypertrophy. These effects by EGCs were also seen in various transformed or nontransformed intestinal epithelial cell lines. Furthermore, TGF-beta1 mRNA was expressed, and TGF-beta1 was secreted by EGCs. Exogenously added TGF-beta1 reproduced partly the EGC-mediated effects on cell density and surface area. In addition, EGC effects on Caco-2 cell density were significantly reduced by a neutralizing TGF-beta antibody. In conclusion, EGCs have profound antiproliferative effects on intestinal epithelial cells. Functional alterations in EGCs may therefore modify intestinal barrier functions and be involved in pathologies such as cancer or inflammatory bowel diseases.

Study of the effects of interferon a on several human hepatoma cell lines: analysis of the signalling pathway of the cytokine and of its effects on apoptosis and cell proliferation.

BACKGROUND: Interferon alpha (IFNalpha), currently used for the treatment of chronic viral hepatitis, is also known to prevent the development of hepatocellular carcinoma (HCC), the mechanism of this action being still debatable. AIMS: To study thoroughly in human hepatoma cell lines (HHL)--Hep3B, HepG2, HuH7, SKHep1, and Chang-Liver--submitted to rhIFNalpha, the signalling pathway of IFNalpha, the binding activity of the cytokine on specific gamma-activated sequence (GAS) and interferon-stimulated regulatory element (ISRE) nuclear sequences, and its effects on apoptosis and cell proliferation. METHODS: The behaviour of signal transducer and activator of transcription (STAT)1, STAT2, p48(IRF9) and the binding of nuclear proteins were investigated by immunoblot and electro-mobility shift assay. Expression of some IFNalpha-dependent proteins--p21/(WAF1), inducible nitric oxide synthase, IRF1 and 2--were studied by immunoblot. Apoptosis and the cell cycle were studied by morphological and biochemical methods. RESULTS: Transduction of INFalpha was unaltered, although there were some variations in the different HHL. Nuclear protein binding to GAS or ISRE showed that ISRE was mainly involved. Apoptosis did not occur. The cell cycle was slightly modified in HuH7. Three GAS- and/or ISRE-dependent proteins increased, suggesting that IFNalpha may have some biological effects on HHL. CONCLUSIONS: The IFNalpha signalling pathway is functional in several HHL, but the cytokine has no apoptotic effect and a moderate anti-proliferative effect. This suggests that the preventive role of IFNalpha on HCC cannot be explained by an apoptotic and/or an anti-proliferative effect, but possibly by its action on several specific nuclear sequences that protect liver cells from transformation.

Antral mucosa expresses functional leptin receptors coupled to STAT-3 signaling, which is involved in the control of gastric secretions in the rat.

BACKGROUND & AIMS: Leptin is a circulating hormone that communicates the peripheral nutritional status to the hypothalamus, which controls food intake, energy expenditure, and body weight. This study characterizes leptin receptors and leptin-sensitive STAT proteins in the antrum and investigates the effects of leptin on gastric secretions. METHODS: The effects of leptin on gastrin messenger RNA (mRNA), plasma gastrin, gastric acid in vivo in the rat, and on somatostatin and gastrin secretions by isolated antral cells were determined in vitro. Leptin receptors were investigated in isolated rat antral cells by reverse transcription-polymerase chain reaction and binding of [(125)I]-leptin studies. The effects of in vivo and in vitro leptin on transduction signal STAT proteins were investigated by immunoblotting antral extracts. RESULTS: Peripheral injection of leptin inhibited in a dose-dependent manner, basal gastric secretion, gastrinemia, and mucosal gastrin mRNA in vivo. mRNAs encoding the long (Ob-Rb) and short (Ob-Ra) receptor forms were detected in rat antral mucosa, as were STAT-1, -3, and -5b immunoreactive proteins. Isolated antral cells specifically bound [(125)I]-leptin, and addition of leptin to these cells inhibited the release of somatostatin and increased the release of gastrin. These effects were associated with an increase in nuclear STAT-3 proteins in vitro and in vivo. CONCLUSIONS: This study provides the first molecular evidence for the coexpression of leptin receptors and STAT-3 in antral mucosa. It provides further evidence for the involvement of leptin in the control of gastric secretions.

Functional cooperation between JunD and NF-kappaB in rat hepatocytes.

AP-1 and NF-kappaB are rapidly activated during liver regeneration. Whether these parallel inductions have potential functional implications is not known. Isolated rat hepatocytes were stimulated with two mitogens, epidermal growth factor or hepatocyte growth factor and with tumor necrosis factor alpha, a cytokine involved in the liver regenerative response in vivo and a strong inducer of NF-kappaB. All three cytokines increased AP-1 and NF-kappaB binding to their cognate cis-element and induced a 2.5-fold activation of NF-kappaB-dependent transcription. Inactivation of AP-1 by TAM67, a dominant negative mutant of AP-1 drastically inhibited basal and cytokine-induced NF-kappaB transactivation. Overexpression of Jun D, but not of the other Jun or Fos proteins increased by threefold NF-kappaB transactivation. Functional cooperation between JunD and p65 was demonstrated in a simple Gal-hybrid system. Finally, a twofold decrease in NF-kappaB transactivation was found in hepatocytes isolated from JunD(-/-) mice compared with hepatocytes from JunD(+/+) mice. Altogether these data demonstrate a functional cooperation of p65 with JunD, a major constituent of AP-1 in normal hepatocytes.

Potentiation of Smad transactivation by Jun proteins during a combined treatment with epidermal growth factor and transforming growth factor-beta in rat hepatocytes. role of phosphatidylinositol 3-kinase-induced AP-1 activation.

Cross-talk between Smad and mitogen-activated protein kinase pathways has been described recently, and evidence for Smad cooperation with AP-1 is emerging. Here we report that epidermal growth factor (EGF) potentializes transforming growth factor beta (TGF-beta)-induced Smad3 transactivation in rat hepatocytes, an effect abrogated by TAM-67, a dominant negative mutant of AP-1. Antisense transfection experiments indicated that c-Jun and JunB were involved in the synergistic effect, and endogenous c-Jun physically associated with Smad3 during a combined EGF/TGF-beta treatment. We next investigated which signaling pathway transduced by EGF was responsible for the Jun-induced synergism. Whereas inhibition of JNK had no effect, inhibition of the phosphatidylinositol-3' kinase (PI3-kinase) pathway by LY294002 or by expression of a dominant negative mutant of PI3-kinase reduced EGF/TGF-beta-induced Smad3 transcriptional activity. Transfection of an activated Ras with a mutation enabling the activation of the PI3-kinase pathway alone mimicked the EGF/TGF-beta potentiation of Smad3 transactivation, and TAM-67 abolished this effect, suggesting that the PI3-kinase pathway stimulates Smad3 via AP-1 stimulation. The EGF/TGF-beta-induced activation of Smad3 correlated with PI3-kinase and p38-dependent but not JNK-dependent phosphorylation of c-Jun. Since potentiation of a Smad-binding element-driven gene was also induced by EGF/TGF-beta treatment, this novel mechanism of Jun/Smad cooperation might be crucial for diversifying TGF-beta responses.

Differential effects of halothane and thiopental on surfactant protein C messenger RNA in vivo and in vitro in rats.

BACKGROUND: Pulmonary surfactant is a complex mixture of proteins and phospholipids synthetized by alveolar type II cells. Volatile anesthetics have been shown to reduce surfactant phospholipid biosynthesis by rat alveolar type II cells. Surfactant-associated protein C (SP-C) is critical for the alveolar surfactant functions. Our goal was to evaluate the effects of halothane and thiopental on SP-C messenger RNA (mRNA) expression in vitro in rat alveolar type II cells and in vivo in mechanically ventilated rats. METHODS: In vitro, freshly isolated alveolar type II cells were exposed to halothane during 4 h (1, 2, 4%) and 8 h (1%), and to thiopental during 4 h (10, 100 micrometer) and 8 h (100 micrometer). In vivo, rats were anesthetized with intraperitoneal thiopental or inhaled 1% halothane and mechanically ventilated for 4 or 8 h. SP-C mRNA expression was evaluated by ribonuclease protection assay. RESULTS: In vitro, 4-h exposure of alveolar type II cells to thiopental 10 and 100 micrometer increased their SP-C mRNA content to 145 and 197%, respectively, of the control values. In alveolar type II cells exposed for 4 h to halothane 1, 2, and 4%, the SP-C mRNA content increased dose-dependently to 160, 235, and 275%, respectively, of the control values. In vivo, in mechanically ventilated rats, 4 h of halothane anesthesia decreased the lung SP-C mRNA content to 53% of the value obtained in control (nonanesthetized, nonventilated) animals; thiopental anesthesia increased to 150% the lung SP-C mRNA content. CONCLUSIONS: These findings indicate that halothane and thiopental used at clinically relevant concentrations modulate the pulmonary SP-C mRNA content in rats. In vivo, the additive role of mechanical ventilation is suggested.

Stimulation of liver RNA and protein breakdown in endotoxemic rats: role of glucocorticoids.

In systemic or localized acute inflammation, liver ribosomal RNA (rRNA) and protein contents increase. We first determined whether changes in RNA, more specifically rRNA, and protein breakdown rates were involved in the accumulation of both types of macromolecules 24 h after induction of endotoxemia. Liver RNA and protein contents were enhanced by 35 and 19%, respectively, in the endotoxemic rats. RNA and protein degradation rates measured during in situ cyclic perfusions of the livers were significantly higher in the endotoxemic rats than in the controls (42 and 46%, respectively). In order to check that the stimulation of RNA and protein degradation corresponded to an activation of the hepatocyte autophagic pathway, the fractional cytoplasmic volume (FCV) of autophagosomes, digestive autophagic vacuoles and dense bodies was measured by morphometry in electron microscopy. The FCV of the sum of these lysosomal structures was significantly increased in the endotoxemic rats. We next tried to identify the factor(s) responsible for the high breakdown rates. The increase in macromolecular degradation did not result from reduced portal amino acid supply. The effects of dexamethasone, interleukin-6, interleukin-1beta, and tumor necrosis factor alpha on RNA degradation were then investigated in primary cultures of hepatocytes isolated from control rats. Only dexamethasone stimulated RNA breakdown. Finally, pretreatment of endotoxemic rats with RU 38486, a glucocorticoid receptor antagonist, completely abolished the stimulation of RNA degradation observed in the sham-gavaged LPS-treated rats. Our data suggest an important role of glucocorticoids in the high levels of RNA and protein breakdown in endotoxemic rats.

Hepatocyte growth factor activates the AP-1 complex: a comparison between normal and transformed rat hepatocytes.

BACKGROUND/AIMS: Stimulation of activator protein-1 (AP-1), a Fos/Jun complex, is a key event in the cell response to growth factors. We have investigated whether hepatocyte growth factor (HGF) induces differential AP-1 responses in normal and transformed rat hepatocytes, the 7777 cells. METHODS: Primocultures of isolated hepatocytes or 7777 cells were stimulated with HGF. Gene expression was evaluated by ribonuclease protection assay and Western blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay. Identification of the proteins bound to the probes was made by supershift assays with specific antibodies. Cells were electroporated with plasmids containing an AP-1-dependent chloramphenicol acetyl transferase (CAT) gene, and CAT activity was measured 24 h after treatment with medium alone or HGF. RESULTS: In both cell types, HGF triggered the same program of jun family mRNA activation, but distinct Fos/Jun proteins accumulated in the nucleus. HGF increased DNA-binding activity to the phorbol 12-O-tetradecanoate-13-acetate responsive element (TRE) in both cell types, but distinct TRE-binding proteins were recruited in the AP-1 dimers. HGF also increased consistently binding to a cAMP responsive element (CRE) in hepatocytes only. Finally, HGF triggered TRE- and CRE-dependent gene activations in hepatocytes but TRE-dependent gene activation alone in 7777 cells. CONCLUSIONS: HGF-induced AP-1 activation leads to the formation of distinct dimers with different functional capacities in normal and transformed hepatocytes. These data suggest the importance of qualitative abnormalities of the AP-1 complex for the establishment or maintainance of a transformed phenotype.

Down-regulation of liver RNA breakdown by turpentine administration in the starved rat: autophagy and relevant factors.

OBJECTIVE AND DESIGN: To determine whether the inhibition of RNA breakdown observed in ad libitum fed rats 24 h after turpentine administration still occurs in inflamed rats fasted for 24 h and to examine the mechanism and factors involved. METHODS: RNA breakdown was measured during cyclic in situ perfusion of livers by the accumulation of [14C] cytidine after in vivo RNA labelling. Autophagic activity was determined by the morphometric analysis of lysosomal structures. RESULTS: The decrease in RNA breakdown (53%) observed in the inflamed rats was accompanied by a 38% drop in the fractional cytoplasmic volume of initial and digestive autophagic vacuoles. Among amino acids, only the portal levels of glutamate were significantly enhanced by 83%. In vivo suppression of glucocorticoid activity using RU 38486 in inflamed rats did not affect the inhibition of RNA breakdown. CONCLUSIONS: The results show that turpentine-induced inflammation in fasted rats inhibits RNA degradation as well as autophagy and that glucocorticoids do not seem to be involved.

Hepatocyte differentiation of WIF-B cells includes a high capacity of interleukin-6-mediated induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin.

Responsiveness to cytokine-mediated acute inflammatory stimuli of the highly differentiated and polarized WIF-B hybrid cell line was studied by measuring the induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin mRNAs after interleukin-1, interleukin-6 and tumor necrosis factor-alpha treatments in the presence of dexamethasone. Compared with their Fao parent, WIF-B cells were 10 times more responsive to 24-h interleukin-6 induction regarding alpha 2-macroglobulin induction. At variance from the response measured in Fao cells, the late effects of interleukin-6 treatment confirmed the higher sensitivity of WIF-B cells to this cytokine as a 72-h treatment as 10 times more effective than a 24-h treatment at inducting alpha 1-acid glycoprotein mRNA. These findings highlight the hepatocyte differentiation of WIF-B cells compared with other hepatoma cell lines, with respect to the regulation of acute-phase protein gene expression. They also make WIF-B cells a convenient model to study the molecular effects of interleukin-6 in terms of transduction and/or transcription, and the many cross-talks that occur during the regulation of acute-phase protein gene expression.

Growth hormone inhibits rat liver alpha-1-acid glycoprotein gene expression in vivo and in vitro.

The gene encoding alpha-1-acid glycoprotein (AGP), one of the major acute-phase proteins, is positively controlled at the transcriptional level by cytokines (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) and glucocorticoids. Here, we show that growth hormone (GH) treatment of isolated rat hepatocytes in vitro reduces AGP messenger RNA (mRNA) expression. AGP gene expression remained inducible by IL-1, IL-6, and phenobarbital (PB) in GH-treated hepatocytes. Interestingly, the repressive effect of GH on AGP gene expression was also observed in vivo: liver AGP mRNA content was strongly increased in hypophysectomized rats, and GH treatment of these animals led to a decrease in mRNA to levels lower than those in untreated control animals. Moreover, the inhibitory effect of GH mainly occurs at the transcriptional level and can be observed as little as 0.5 hours after GH adding in vitro to isolated hepatocytes. These results show negative regulation of AGP gene expression and strongly suggest that GH is a major endogenous regulator of constitutive AGP gene expression. Moreover, transfection assays showed that the region of the AGP promoter located at position -147 to -123 is involved in AGP gene regulation by GH. Furthermore, GH deeply modifies the pattern of nuclear protein binding to this region. GH treatment of hypophysectomized rats led to the release of proteins of 42 to 45 and 80 kd and to the binding of proteins of 48 to 50 and 90 kd.

In vivo stimulation of polymeric Ig receptor transcytosis by circulating polymeric IgA in rat liver.

Binding of human polymeric IgA ligand to its epithelial cell polymeric Ig receptor, pIgR, has been shown to stimulate pIgR apical transcytosis in an in vitro system, based on polarized confluent MDCK cells expressing rabbit pIgR. The present study aimed at testing whether such a stimulation also occurs in vivo. Transcytosis of pIgR was monitored by rat liver output of total secretory component (SC) into bile, measured by radial immunodiffusion as the sum of free SC and pIgA-bound SC. Whereas in the perfused rat liver system addition of pIgA to the perfusate showed no effect, i.v. injection of human and rat pIgA, but not of monomeric IgA nor PBS, in living rats significantly increased total bile SC output for more than 1 h. Furthermore, depletion of the normal pIgA level circulating in the liver before injecting more pIgA was not required to show the stimulation. Our data thus strongly suggest that stimulation of liver pIgR transcytosis by pIgA ligand binding is physiologically relevant, helping to quickly adjust pIgA transport into bile to increase circulating pIgA levels, without need for increased SC/pIgR synthesis.

Inducible expression of the alpha1-acid glycoprotein by rat and human type II alveolar epithelial cells.

Alpha1-acid glycoprotein (AGP) is a major acute phase protein in rat and human. AGP has important immunomodulatory functions that are potentially important for pulmonary inflammatory response. The liver is the main tissue for AGP synthesis in the organism, but the expression of AGP in the rat lung has not been investigated. We show that AGP mRNA was induced in the lung of dexamethasone-, turpentine-, or LPS-treated rats, whereas AGP mRNA was not detected in the lung of control rats. In the lung of animals treated intratracheally with LPS, in situ hybridization showed that AGP gene expression was restricted to cells located in the corners of the alveolus, consistent with an alveolar type II (ATII) cell localization. The inducible expression of the AGP gene was confirmed in vitro with SV40 T2 cells and rat ATII cells in primary culture: maximal expression required the presence of dexamethasone. IL-1 and the conditioned medium of alveolar macrophages acted synergistically with dexamethasone. Rat ATII cells secreted immunoreactive AGP in vitro when stimulated with dexamethasone or with a combination of dexamethasone and the conditioned medium of alveolar macrophages. In vivo, in the human lung, we detected immunoreactive AGP in hyperplastic ATII cells, whereas we did not detect AGP in the normal lung. We conclude that AGP is expressed in the lung in cases of inflammation and that ATII cells are the main source of AGP in the lung.

Presence of distinct AP-1 dimers in normal and transformed rat hepatocytes under basal conditions and after epidermal growth factor stimulation.

Activation of the transcriptional regulator AP-1, a dimeric complex formed of various combinations of Fos and Jun proteins, is a key step in the cellular response to mitogens. Because different dimers are believed to display different regulatory functions, we hypothesized that transformed cells that lack normal growth constraints might display AP-1 dimers that are different from those of normal cells. We therefore compared in primary and transformed rat hepatocytes (1) the composition of AP-1 dimers under basal conditions and (2) AP-1 induction by epidermal growth factor (EGF). Under basal conditions, AP-1 contained predominantly Jun homodimers in both cell types. However, whereas normal hepatocytes contained only JunD, both JunD and JunB were present in the AP-1 complex of 7777 cells. EGF treatment triggered almost identical programs of fos and jun gene activation at the messenger RNA (mRNA) level in both cell types, with an early accumulation of c-fos, c-jun, and junB mRNAs, but no change in junD mRNA levels. In both cell types, c-Fos and Fra-1 proteins increased after EGF treatment, but differences in the induction of Jun proteins were noted, with an increase of c-Jun in hepatocytes and an increase of JunB in 7777 cells. In both cell types, activation of AP-1 DNA binding activity by EGF was accompanied by the recruitment of Fra-1 into AP-1, detected earlier in 7777 cells than in hepatocytes, and with the transient appearance of c-Fos in 7777 cells only. Finally, EGF activated AP-1-dependent transcription in 7777 cells but not in hepatocytes. These data indicate important differences in the functional activity of AP-1 in transformed hepatocytes.

An improved digitonin-collagenase perfusion technique for the isolation of periportal and perivenous hepatocytes from a single rat liver: physiological implications for lobular heterogeneity.

Morphological and functional heterogeneity of hepatocytes according to their position in the liver lobule has been known for many years. The digitonin-collagenase perfusion technique is widely used to study hepatocyte heterogeneity and has yielded reliable data. However, with this procedure, periportal (PP) or perivenous (PV) hepatocytes are isolated from different livers, allowing only comparison between cell populations issued from two separate animals. To overcome this drawback, we have modified this technique by perfusing the two main rat liver lobes of a single animal in succession. The procedure involved alternate clamping of the median and the left lateral lobes, restricting digitonin infusion to one lobe via the portal vein, and to the other via the caudal vena cava. Lobe exclusion during digitonin perfusion, and zonal restriction of digitonin-induced damage, were monitored using macroscopic and histological controls. We compared our results with previous data on PP and PV hepatocytes issued from two different livers using the conventional digitonin-collagenase perfusion technique. First, we found that the cellular sensitivity to angiotensin II, a calcium-mobilizing agonist, was 60% to 80% higher in PV than in PP hepatocytes, whereas, previously, no difference had been recorded. Second, we found that albumin messenger RNAs (mRNAs) were 35% more abundant in PP than in PV hepatocytes, whereas, previously, larger differences had been reported. Our results show that PP and PV hepatocytes may be isolated from a single liver using an improved digitonin-collagenase perfusion technique. Furthermore, we suggest that zonal differences can be artificially masked or amplified when comparing PP and PV cell populations from two different livers, indicating that it is preferable to use a single liver for accurate zonal comparisons between hepatocytes.

Inhibition of liver RNA breakdown during acute inflammation in the rat.

Liver RNA- and protein-degradation rates were measured after the induction of acute inflammation in the rat. A preliminary study determined changes in hepatic RNA and protein content 12, 18 and 24 h after a turpentine oil injection. The RNA content in turpentine-treated rats compared with pair-fed animals increased significantly and sharply from 12 h (+ 11%) to 18 h (+ 32%) and slightly thereafter (+ 37% at 24 h). The liver protein content was significantly enhanced only at 24 h (+ 11%) in response to inflammation. RNA-degradation rates were determined in livers perfused cyclically in situ for 15 min by measuring the accumulation of radioactive cytidine in the medium 60 h after in vivo labelling of RNA by [5-3H]cytidine instead of [6-14C]orotic acid, the most commonly used radioactive marker. Several validation procedures showed that the method employed was a valid alternative to the use of radioactive orotic acid. RNA-degradation rates, which mainly reflect rRNA breakdown, were significantly lower in the turpentine-treated rats than in respective pair-fed animals at 18 and 24 h (57 and 45% decrease respectively). Proteolysis rates measured at 24 h together with RNA breakdown by valine accumulation in the perfusion medium were not modified after turpentine treatment. The main factors known to regulate RNA degradation (amino acids, insulin/glucagon ratio) were measured in the portal blood 24 h after induction of acute inflammation. Of the known regulatory amino acids, only glutamine and to a lesser extent methionine were increased in the turpentine-treated rats as compared with their pair-fed counterparts. The insulin/glucagon molar ratio was similar in both groups. In conclusion, the reduced breakdown of RNA, especially rRNA, is largely responsible for the accumulation of hepatic RNA during acute inflammation. This inhibition of RNA degradation could possibly be related to the increase in glutamine.