Three rhodamine 6G derivatives (REHA, RETA and REDA) were designed and synthesized by connecting rhodamine 6G and 3-methyl-2-thiophenal with hydrazine hydrate, ethylenediamine and diethylenetriamine, respectively. In CH3CN/H2O (50/50, v/v), the absorbance of REHA, RETA and REDA at 528 nm was suddenly enhanced by 3.2, 3.8 and 7.2 times within the pH range of 3.03-2.31, 3.05-2.32 and 3.06-2.34, respectively, and the solution changed from colorless to pink. Meanwhile, the maximal fluorescence intensity sharply increased by 53.9, 26.6 and 24.9 times in the pH range of 3.86-3.46, 3.88-3.47 and 3.89-3.48, respectively, and the solution changed from dark to bright yellow-green fluorescence. REHA, RETA and REDA can act as highly selective and sensitive colorimetric and fluorescent pH switches with good recyclability and anti-interference ability. The response mechanism of REHA, RETA and REDA to pH was studied by 1H NMR spectroscopy, and their application in indicating small pH changes in dyeing wastewater was investigated.

Cholestatic liver diseases, accompanied by the hepatic accumulation of bile salts, frequently lead to liver fibrosis, while underlying profibrogenic mechanisms remain incompletely understood. Here, we evaluated the role of extracellular pH (pHe) on bile salt entry and hepatic stellate cell (HSC) activation and proliferation. As modulators of intracellular pH (pHi), various proton pump inhibitors (PPI) were tested for their ability to prevent bile salt entry and HSC activation. Lastly, the PPI pantoprazole was employed in the 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine (DDC)-diet model of cholestatic liver fibrosis. We found in vitro, that slightly acidic pHe (7.2-7.3) enhanced bile salt accumulation in HSC and was a prerequisite to bile salt-induced HSC activation. Pantoprazole in the DDC model exhibited antifibrotic effects. We conclude that bile salt-induced activation of HSC may depend on the slightly acidic microenvironment present in the perisinusoidal space and modulation of pHi in HSC may offer a novel pharmacological target in cholestatic disease.

Eosinophilic esophagitis (EoE) is characterized by histopathologic modifications of esophageal tissue, including eosinophil-rich inflammation, basal zone hyperplasia, and dilated intercellular spaces (DIS). The underlying molecular processes that drive the histopathologic features of EoE remain largely unexplored.

We sought to investigate the involvement of solute carrier family 9, subfamily A, member 3 (SLC9A3) in esophageal epithelial intracellular pH (pHi) and DIS formation and the histopathologic features of EoE.

We examined expression of esophageal epithelial gene networks associated with regulation of pHi in the EoE transcriptome of primary esophageal epithelial cells and an in vitro esophageal epithelial 3-dimensional model system (EPC2-ALI). Molecular and cellular analyses and ion transport assays were used to evaluate the expression and function of SLC9A3.

We identified altered expression of gene networks associated with regulation of pHi and acid-protective mechanisms in esophageal biopsy specimens from pediatric patients with EoE (healthy subjects, n = 6; patients with EoE, n = 10). The most dysregulated gene central to regulating pHi was SLC9A3. SLC9A3 expression was increased within the basal layer of esophageal biopsy specimens from patients with EoE, and expression positively correlated with disease severity (eosinophils/high-power field) and DIS (healthy subjects, n = 10; patients with EoE, n = 10). Analyses of esophageal epithelial cells revealed IL-13-induced, signal transducer and activator of transcription 6-dependent SLC9A3 expression and Na+-dependent proton secretion and that SLC9A3 activity correlated positively with DIS formation. Finally, we showed that IL-13-mediated, Na+-dependent proton secretion was the primary intracellular acid-protective mechanism within the esophageal epithelium and that blockade of SLC9A3 transport abrogated IL-13-induced DIS formation.

SLC9A3 plays a functional role in DIS formation, and pharmacologic interventions targeting SLC9A3 function may suppress the histopathologic manifestations in patients with EoE.

Liver fibrosis and cirrhosis are characterized by activation of hepatic stellate cells (HSCs), which is associated with higher intracellular pH (pHi). The vacuolar H⁺ adenosine-triphosphatase (v-ATPase) multisubunit complex is a key regulator of pHi homeostasis. The present work investigated the functional role of v-ATPase in primary human HSC (hHSC) activation and its modulation by specific adenosine monophosphate-activated protein kinase (AMPK) subunits. We demonstrate that the expression of different v-ATPase subunits was increased in in vivo and in vitro activated hHSCs compared to nonactivated hHSCs. Specific inhibition of v-ATPase with bafilomycin and KM91104 induced a down-regulation of the HSC fibrogenic gene profile, which coincided with increased lysosomal pH, decreased pHi, activation of AMPK, reduced proliferation, and lower metabolic activity. Similarly, pharmacological activation of AMPK by treatment with diflunisal, A769662, and ZLN024 reduced the expression of v-ATPase subunits and profibrogenic markers. v-ATPase expression was differently regulated by the AMPK α1 subunit (AMPKα1) and AMPKα2, as demonstrated in mouse embryo fibroblasts specifically deficient for AMPK α subunits. In addition, activation of v-ATPase in hHSCs was shown to be AMPKα1-dependent. Accordingly, pharmacological activation of AMPK in AMPKα1-depleted hHSCs prevented v-ATPase down-regulation. Finally, we showed that v-ATPase expression was increased in fibrotic livers from bile duct-ligated mice and in human cirrhotic livers.

The down-regulation of v-ATPase might represent a promising target for the development of antifibrotic strategies. (Hepatology 2018).

The hepatic stellate cell (HSC) is the primary cell type in the liver responsible for excess collagen deposition during fibrosis. Following a fibrogenic stimulus the cell changes from a quiescent vitamin A-storing cell to an activated cell type associated with increased extracellular matrix synthesis and increased cell proliferation. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been shown to regulate several aspects of HSC activation in vitro, including collagen synthesis and cell proliferation. Using a targeted approach to inhibit PI3K signaling specifically in HSCs, we investigated the role of PI3K in HSCs using a rodent model of hepatic fibrosis. An adenovirus expressing a dominant negative form of PI3K under control of the smooth muscle alpha-actin (alphaSMA) promoter was generated (Ad-SMAdnPI3K). Transducing HSCs with Ad-SMAdnPI3K resulted in decreased proliferation, migration, collagen expression, and several additional profibrogenic genes, while also promoting cell death. Inhibition of PI3K signaling was also associated with reduced activation of Akt, p70 S6 kinase, and extracellular regulated kinase signaling as well as reduced cyclin D1 expression. Administering Ad-SMAdnPI3K to mice following bile duct ligation resulted in reduced HSC activation and decreased extracellular matrix deposition, including collagen expression. A reduction in profibrogenic mediators, including transforming growth factor beta, tissue inhibitor of metalloproteinase 1, and connective tissue growth factor was also noted. However, liver damage, assessed by alanine aminotransferase levels, was not reduced.

Inhibition of PI3K signaling in HSCs during active fibrogenesis inhibits extracellular matrix deposition, including synthesis of type I collagen, and reduces expression of profibrogenic factors. These data suggest that targeting PI3K signaling in HSCs may represent an effective therapeutic target for hepatic fibrosis.

The kinase Akt mediates signals from growth factor receptors for increased cell proliferation, survival, and migration, which contribute to the positive effects of Akt in cancer progression. Substrates are generally inhibited when phosphorylated by Akt; however, we show phosphorylation of the plasma membrane sodium-hydrogen exchanger NHE1 by Akt increases exchanger activity (H(+) efflux). Our data fulfill criteria for NHE1 being a bona fide Akt substrate, including direct phosphorylation in vitro, using mass spectrometry and Akt phospho-substrate antibodies to identify Ser(648) as the Akt phosphorylation site and loss of increased exchanger phosphorylation and activity by insulin and platelet-derived growth factor in fibroblasts expressing a mutant NHE1-S648A. How Akt induces actin cytoskeleton remodeling to promote cell migration and tumor cell metastasis is unclear, but disassembly of actin stress fibers by platelet-derived growth factor and insulin and increased proliferation in growth medium are inhibited in fibroblasts expressing NHE1-S648A. We predict that other functions shared by Akt and NHE1, including cell growth and survival, might be regulated by increased H(+) efflux.

Hepatic fibrogenesis, a consequence of chronic liver tissue damage, is characterized by activation of the hepatic stellate cells (HSC). Silybin has been shown to exert anti-fibrogenic effects in animal models. However, scant information is available on the fine cellular and molecular events responsible for this effect. The aim of this study was to assess the mechanisms regulating the anti-fibrogenic and anti-inflammatory activity of Silybin.

Experiments were performed on HSC isolated from human liver and activated by culture on plastic.

Silybin was able to inhibit dose-dependently (25-50 microM) growth factor-induced pro-fibrogenic actions of activated human HSC, including cell proliferation (P < 0.001), cell motility (P < 0.001), and de novo synthesis of extracellular matrix components (P < 0.05). Silybin (25-50 microM), inhibited the IL-1-induced synthesis of MCP-1 (P < 0.01) and IL-8 (P < 0.01) showing a potent anti-inflammatory activity. Silybin exerts its effects by directly inhibiting the ERK, MEK and Raf phosphorylation, reducing the activation of NHE1 (Na+/H+ exchanger, P < 0.05) and the IkBalpha phosphorylation. In addition, Silybin was confirmed to act as a potent anti-oxidant agent.

The results of the study provide molecular insights into the potential therapeutic action of Silybin in chronic liver disease. This action seems to be mostly related to a marked inhibition of the production of pro-inflammatory cytokines, a clear anti-oxidant effect and a reduction of the direct and indirect pro-fibrogenic potential of HSC.

A novel and integral approach to the understanding of human neurodegenerative diseases (HNDDs) and cancer based upon the disruption of the intracellular dynamics of the hydrogen ion (H(+)) and its physiopathology, is advanced. From an etiopathological perspective, the activity and/or deficiency of different growth factors (GFs) in these pathologies are studied, and their relationships to intracellular acid-base homeostasis reviewed. Growth and trophic factor withdrawal in HNDDs indicate the need to further investigate the potential utilization of certain GFs in the treatment of Alzheimer disease and other neurodegenerative diseases. Platelet abnormalities and the therapeutic potential of platelet-derived growth factors in these pathologies, either through platelet transfusions or other clinical methods, are considered. Finally, the etiopathogenic mechanisms of apoptosis and antiapoptosis in HNDDs and cancer are viewed as opposite biochemical and biological disorders of cellular acid-base balance and their secondary effects on intracellular signaling pathways and aberrant cell metabolism are considered in the light of the both the seminal and most recent data available. The "trophic factor withdrawal syndrome" is described for the first time in English-speaking medical literature, as well as a Darwinian-like interpretation of cellular behavior related to specific and nonspecific aspects of cell biology.

Liver fibrosis, a wound-healing response to a variety of chronic stimuli, is characterized by excessive deposition of extracellular matrix (ECM) proteins, of which type I collagen predominates. This alters the structure of the liver leading to organ dysfunction. The activated hepatic stellate cell (HSC) is primarily responsible for excess collagen deposition during liver fibrosis. Two important aspects are involved in mediating the fibrogenic response: first the HSC becomes directly fibrogenic by synthesizing ECM proteins; second, the activated HSC proliferates, effectively amplifying the fibrogenic response. Although the precise mechanisms responsible for HSC activation remain elusive, substantial insight is being gained into the molecular mechanisms responsible for ECM production and cell proliferation in the HSC. The activated HSC becomes responsive to both proliferative (platelet-derived growth factor) and fibrogenic (transforming growth factor-beta[TGF-beta]) cytokines. It is becoming clear that these cytokines activate both mitogen-activated protein kinase (MAPK) signaling, involving p38, and focal adhesion kinase-phosphatidylinositol 3-kinase-Akt-p70 S6 kinase (FAK-PI3K-Akt-p70(S6K)) signaling cascades. Together, these regulate the proliferative response, activating cell cycle progression as well as collagen gene expression. In addition, signaling by both TGF-beta, mediated by Smad proteins, and p38 MAPK influence collagen gene expression. Smad and p38 MAPK signaling have been found to independently and additively regulate alpha1(I) collagen gene expression by transcriptional activation while p38 MAPK, but not Smad signaling, increases alpha1(I) collagen mRNA stability, leading to increased synthesis and deposition of type I collagen. It is anticipated that by understanding the molecular mechanisms responsible for HSC proliferation and excess ECM production new therapeutic targets will be identified for the treatment of liver fibrosis.

Liver fibrosis may be considered as a dynamic and integrated cellular response to chronic liver injury. The activation of hepatic stellate cells and the consequent deposition of large amounts of extracellular matrix play a major role in the fibrogenic process, but it has been shown that other cellular components of the liver are also involved. Although the pathogenesis of liver damage usually depends on the underlying disease, oxidative damage of biologically relevant molecules might represent a common link between different forms of chronic liver injury and hepatic fibrosis. In fact, oxidative stress-related molecules may act as mediators able to modulate all the events involved in the progression of liver fibrosis. In addition, chronic liver diseases are often associated with decreased antioxidant defenses. Although vitamin E levels have been shown to be decreased in chronic liver diseases of different etiology, the role of vitamin E supplementation in these clinical conditions is still controversial. In fact, the increased serum levels of alpha-tocopherol following vitamin E supplementation not always result in a protective effect on liver damage. In addition, clinical trials have usually been performed in small cohorts of patients, thus making definitive conclusions impossible. At present, treatment with vitamin E or other antioxidant compounds could be proposed for nonalcoholic fatty liver disease (NAFLD), the most frequent hepatic lesion in western countries which can progress to nonalcoholic steatohepatitis and cirrhosis due to the production of large amounts of oxidative stress products. However, although some studies have shown encouraging results, multicentric and long-term clinical trials are needed.

Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH through the activity of the Na(+)/H(+) exchanger and the Na(+) dependent Cl(-)/HCO(3)(-) exchanger. The inhibition of these mechanisms could therefore inhibit cancer cell growth.

We evaluated the effect of two selective inhibitors of these transporters (cariporide and S3705) on proliferation and apoptosis of human cholangiocarcinoma cells (HUH-28 and Mz-ChA-1 cells) as a function of external pH (7.4 and 6.8).

HUH-28 cells incubated for 24h at external pH 7.4 or 6.8 without inhibitors maintained intracellular pH at physiological level, whereas incubation with cariporide and/or S3705 caused the intracellular pH of cells to drop. Incubation of HUH-28 cells with cariporide and/or S3705 was able to reduce proliferation, evaluated by a colorimetric ELISA method, and to induce apoptosis, evaluated by measuring caspase-3 activity and Annexin-V staining, and these effects were more evident at external pH 6.8. S3705 but not cariporide was able to inhibit serum-induced phosphorylation of ERK1/2, AKT and BAD, intracellular molecules involved in cancer cell proliferation and survival. Similar results were obtained in Mz-ChA-1 cells.

(1) Inhibition of intracellular pH regulatory mechanisms by cariporide and S3705 reduces proliferation and induces apoptosis in cholangiocarcinoma cells; and (2) these drugs might have potential therapeutic value against cholangiocarcinoma.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes involved in diverse aspects of cellular and integrative physiology, including energy metabolism, cell growth, survival, invasion, migration or angiogenesis. The activity of this transcription factor is known to be increased by hypoxia, but also by a growing number of apparently unrelated factors that can activate it even in nonhypoxic conditions. Here I propose a model in which an alteration in oxygen metabolism is the key cellular event involved in HIF-1 activation under hypoxic and nonhypoxic conditions. This new perspective unifies previously unrelated observations and predicts cellular processes and therapeutic strategies that may modify HIF-1 activity. This may have relevance, for instance, to cancer, as HIF-1 overexpression is observed in many human cancers and has been associated with increased patient mortality.

Looked at from the genetic point-of-view cancer represents a daunting and, frankly, confusing multiplicity of diseases (at least 100) that require an equally large variety of therapeutic strategies and substances designed to treat the particular tumor. However, when analyzed phenotypically cancer is a relatively uniform disease of very conserved 'hallmark' behaviors across the entire spectrum of tissue and genetic differences [D. Hanahan, R.A. Weinberg, Hallmarks of cancer, Cell 100 (2000) 57-70]. This suggests that cancers do, indeed, share common biochemical and physiological characteristics that are independent of the varied genetic backgrounds, and that there may be a common mechanism underlying both the neoplastic transformation/progression side and the antineoplastic/therapy side of oncology. The challenge of modern oncology is to integrate all the diverse experimental data to create a physiological/metabolic/energetic paradigm that can unite our thinking in order to understand how both neoplastic progression and therapies function. This reductionist view gives the hope that, as in chemistry and physics, it will possible to identify common underlying driving forces that define a tumor and will permit, for the first time, the actual calculated manipulation of their state. That is, a rational therapeutic design. In the present review, we present evidence, obtained from a great number of studies, for a fundamental, underlying mechanism involved in the initiation and evolution of the neoplastic process. There is an ever growing body of evidence that all the important neoplastic phenotypes are driven by an alkalization of the transformed cell, a process which seems specific for transformed cells since the same alkalinization has no effect in cells that have not been transformed. Seen in that light, different fields of cancer research, from etiopathogenesis, cancer cell metabolism and neovascularization, to multiple drug resistance (MDR), selective apoptosis, modern cancer chemotherapy and the spontaneous regression of cancer (SRC) all appear to have in common a pivotal characteristic, the aberrant regulation of hydrogen ion dynamics [S. Harguindey, J.L. Pedraz, R. García Cañero, J. Pérez de Diego, E.J. Cragoe Jr., Hydrogen ion-dependent oncogenesis and parallel new avenues to cancer prevention and treatment using a H+-mediated unifying approach: pH-related and pH-unrelated mechanisms, Crit. Rev. Oncog. 6 (1) (1995) 1-33]. Cancer cells have an acid-base disturbance that is completely different than observed in normal tissues and that increases in correspondence with increasing neoplastic state: an interstitial acid microenvironment linked to an intracellular alkalosis.

Pirfenidone (5 methyl-1-phenyl-2(1H)-pyridone) is a novel anti-fibrotic agent, which has been shown to decrease collagen deposition in a variety of animal models in vivo, and recently in hepatic fibrosis also. At cellular level, we have recently demonstrated that pirfenidone is able to inhibit proliferation of hepatic stellate cells induced by platelet-derived growth factor, as well as collagen type I accumulation and alpha1(I) procollagen mRNA expression.

To evaluate if pirfenidone maintains its anti-fibrotic properties also when administered after the induction of hepatic damage and to further investigate the molecular mechanisms leading to the anti-fibrotic effect of pirfenidone.

Rats treated with dimethylnitrosamine (10 mg/kg) for 5 weeks received a liquid diet containing 0.5% pirfenidone starting from the third week. Pirfenidone treatment reduced the degree of liver injury, as determined by alanine aminotransferase values and necro-inflammatory score, which was associated with reduced hepatic stellate cells proliferation and collagen deposition. Treatment with dimethylnitrosamine increased transcripts levels for transforming growth factorbeta1, procollagen alpha1(I), tissue inhibitors of metalloproteinase-1 and matrix metalloproteinase-2 by 7-, 7-, 4- and 15-fold, respectively. Pirfenidone administration downregulated elevated levels of those transcripts by 50-60%, and this was associated with a 70% reduction in collagen deposition.

(1) Pirfenidone is effective also if administered after the induction of the hepatic damage; (2) the anti-fibrotic effect of pirfenidone is mainly due to the reduced expression of profibrogenic procollagen alpha1(I) and TIMP-1, most likely through the downregulation of transforming growth factorbeta1 mRNA, and of matrix metalloproteinase-2, which is mainly implicated in the degradation of the normal extracellular matrix.

Progressive liver fibrosis is the main cause of organ failure in chronic liver diseases of any aetiology. Fibrosis develops with different spatial patterns and is a consequence of different prevalent mechanisms according to the diverse causes of parenchymal damage. Indeed, fibrosis, observed as a consequence of chronic viral infection is initially concentrated within and around the portal tract, while fibrosis secondary to toxic/metabolic damage is located mainly in the centrolobular areas. In addition, it is increasingly evident that different cell types are involved in the deposition of fibrillar extracellular matrix during active hepatic fibrogenesis: hepatic stellate cells are mainly involved when hepatocellular damage is limited or concentrated within the liver lobule, whereas portal myofibroblasts and fibroblasts provide a predominant contribution when the damage is located in the proximity of the portal tracts. In the later stages of evolution (septal fibrosis) it is likely that all extracellular matrix-producing cells contribute to fibrogenesis. Recruitment and activation of extracellular matrix-producing cells to the site of tissue damage can be due to different major mechanisms: (1) Chronic activation of the tissue repair process. In this case, as a consequence of the reiterated damage, accumulation of fibrillar extracellular matrix reflects the impossibility of an effective remodelling and regeneration. (2) Effect of oxidative stress products, including reactive oxygen intermediates and reactive aldehydes. These products, whose concentration become critical in toxic/metabolic liver injury, are able to induce the synthesis of fibrillar extracellular matrix even in the absence of significant hepatocyte damage and inflammation. (3) Derangement of normal the epithelial/mesenchymal interaction. This typically occurs in all conditions characterised by cholangiocyte damage/proliferation, where a consensual proliferation of extracellular matrix-producing cells and progressive fibrogenesis is commonly observed. A major advancement towards the understanding of the molecular mechanisms of fibrogenesis is derived from a consistent number of in vitro studies investigating the biological role of growth factors/cytokines and other soluble factors and their intracellular signalling pathways. The relevance of these factors has been confirmed by studies performed on animal models and by studies performed on pathological human liver. Along these lines, the elucidation of a consistent number of cellular and molecular mechanisms responsible for the progression of liver fibrosis has provided sound basis for the development of pharmacological strategies able to modulate this important pathophysiological process. Finally, there are several clinically relevant issues that need re-evaluation and/or further investigation, and in particular: (1) the need of an accurate and effective monitoring of the fibrotic progression of chronic liver diseases and of the effectiveness of the currently proposed treatments; (2) the identification of general or individual factors potentially relevant for a faster progression of the disease.

We tested the hypothesis that the dietary energy-dependent alterations of the rumen papillae size are accompanied by corresponding changes in systemic insulin-like growth factor (IGF)-1 concentration and in rumen papillary IGF type 1 receptors (IGF-1R). Young male goats (n=24) were randomly allocated to two groups (n=12) and fed a high level (HL) metabolizable energy [1200 kJ/(kg(0.75).d)] or a low level (LL) [500 kJ/(kg(0.75).d)] diet for 42 d. The concentration of ruminal total SCFA did not differ between the groups, but the molar proportion of butyric acid was enhanced by 70% in the HL group (P<0.05). Both the length and width of the papillae were greater (P<0.05) in the HL group, and the surface was 50-100% larger (P<0.05) in the tissue sampled from the artrium ruminis, the ventral ruminal sac and the ventral blind sac. Transport of Na+ across the rumen epithelium, which is amiloride sensitive, was higher (P<0.05) in the HL than in the LL group. Furthermore, the plasma IGF-1 concentration was about twofold higher in the HL group (P<0.05), and the maximal rumen epithelial IGF-1R binding was also higher in the HL (P<0.05) than in the LL group. IGF-1R mRNA and IGF-1 mRNA were detected in rumen papillae; however, they were unaffected by dietary treatments. DNA synthesis and cell proliferation of cultured rumen epithelial cells were higher (P<0.05) after IGF-1 treatment (25 or 50 microg/L) compared with those in the medium without IGF-1. Thus dietary energy-dependent alterations of rumen morphology and function are accompanied by corresponding changes in systemic IGF-1 and ruminal IGF-1R.

Mrp3(Abcc3) is markedly induced following bile duct ligation (BDL) in the rat and in some human cholestatic liver diseases and is believed to ameliorate liver injury in this setting. Recently, the orphan nuclear receptor fetoprotein transcription factor/cholesterol-7alpha-hydroxylase promoter factor (CPF/FTF/Lrh-1) has been shown to activate Mrp3 expression. However, whether inflammatory cytokines or elevated bile acid levels increased Lrh-1/Mrp3 expression in obstructive cholestasis was not known. We hypothesized that induction of Mrp3 would be associated with Lrh-1 up-regulation and would require intact cytokine signaling. Male tumor necrosis factor (Tnf) receptor I (Tnfr-/-) mice and C57BLJ wild type (WT) controls were subjected to sham surgery or bile duct ligation. HepG2 cells were treated with bile acids or cytokines. Immunoblot assay and real time reverse transcriptase-PCR were used to determine expression of MRP3/Mrp3, CPF/Lrh-1, Mrp2, and Bsep. CPF/Lrh-1 DNA binding to the MRP3/Mrp3 promoter was assessed using electrophoretic mobility shift assay, and promoter activity was determined by luciferase assay. Total bile acids and lactate dehydrogenase were measured using colorimetric assays, and cytokine abundance was determined by enzyme-linked immunosorbent assay. Lrh-1 and Mrp3 were significantly induced after BDL in WT but not Tnfr-/- mice. This was associated with more severe hepatocellular necrosis in Tnfr-/- mice. Lrh-1 binding to the Mrp3 promoter increased after BDL in WT but not in Tnfr-/- mice. Tnfalpha treatment of HepG2 cells also up-regulated CPF and MRP3, increased CPF binding to the MRP3 promoter, and up-regulated MRP3 promoter activity. These results indicate that induction of Mrp3 after BDL is due to Tnfalpha-dependent up-regulation of Lrh-1. They provide strong evidence that induction of Mrp3 plays a significant role in hepatocyte protection during obstructive cholestasis.

The mechanisms of tumor promotion in liver by various xenobiotics of diverse structure are not well understood. However, these tumor promoters share the ability to exert growth-stimulatory effects on hepatocytes. Our laboratory has been utilizing normal rat hepatocytes under defined conditions of primary cultures, to investigate growth-stimulatory actions of liver tumor promoters. We have shown that most, if not all, of the liver tumor promoters tested stimulate hepatocyte DNA synthesis when added in combination with epidermal growth factor (EGF), insulin, and glucocorticoids. In the present study, we sought evidence for the role of the Na(+)/H(+) antiporter and cytoplasmic alkalinization in the direct growth-stimulatory actions of tumor promoters on hepatocytes. Hepatocytes cultured under conditions (bicarbonate-buffered medium) where intracellular pH (pH(i)) was independent of extracellular pH (pH(e)), EGF- and insulin-stimulated rates of DNA synthesis were unaffected by modest changes in pH(e). However, under conditions (HEPES-buffered medium) where pH(i) varied in a linear fashion with pH(e), rates of EGF- and insulin-stimulated DNA synthesis were highly dependent on pH(e). Similarly, 12-O-tetradecanoylphorbol-13-acetate (TPA) and alpha-hexachlorocyclohexane (HCH)-stimulated DNA synthesis were pH(e)-dependent but were stimulatory over different pH(e) ranges, suggesting that these promoters may act by distinct mechanisms. Chemicals that are capable of inducing rapid cytoplasmic alkalinization, ammonium chloride (1 and 15 mM) and monensin (0.5 microM), were found to stimulate hepatocyte DNA synthesis. The role of the Na(+)/H(+) antiport in controlling pH(i) of hepatocytes was demonstrated by artificially acidifying 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl (BCECF)-loaded isolated hepatocytes with 20 mM sodium acetate and the use of specific inhibitors. Amiloride and its analogues inhibited pH(i) recovery from the acid load in a dose dependent manner and the relative potency of these inhibitors paralleled their K(i) values for the Na(+)/H(+) antiport. At concentrations that stimulate hepatocyte DNA synthesis, some liver tumor promoters phenobarbital (PB) and HCH, were found to cause a rapid rise pH(i) in isolated hepatocytes which was sensitive to amiloride and its analogues. Taken together, our data suggest that activation of Na(+)/H(+) antiport activity may be one mechanism whereby some liver tumor promoters stimulate hepatocytes DNA synthesis. This study has implications for the mechanisms of tumor promotion in liver carcinogenesis.

The aim of this study was to evaluate the effect of cariporide, a selective Na(+)/H(+) exchange inhibitor, on isolated and cultured hepatic stellate cells (HSCs) and in 2 in vivo models of rat liver fibrosis. Platelet-derived growth factor (PDGF)-induced HSC proliferation, evaluated by measuring the percentage of bromodeoxyuridine-positive cells, was significantly inhibited by cariporide, with a maximal effect at 10 micromol/L. Incubation with cariporide did not inhibit PDGF-induced extracellular-regulated kinase 1/2 (ERK1/2), Akt (a downstream component of the phosphatidylinositol [PI]-3 kinase pathway), and protein kinase C (PKC) activation but reduced PDGF-induced activation of the Na(+)/H(+) exchanger, with a maximal effect at 10 micromol/L. Rats treated with dimethylnitrosamine (DMN; 10 mg/kg) for 1 and 5 weeks received a diet with or without 6 ppm cariporide. Treatment with cariporide reduced the degree of liver injury, as determined by alanine aminotransferase (ALT) values, also when administered after the induction of hepatic damage. This was associated with reduced HSC activation and proliferation and reduced collagen deposition, as determined by morphometric evaluation of alpha-smooth muscle actin (SMA)/proliferating cell nuclear antigen-positive cells and percentage of Sirius red-positive parenchyma, respectively. Moreover, cariporide was also able to reduce alpha(1)I procollagen messenger RNA (mRNA) expression. Similar effects were observed in bile duct-ligated (BDL) rats. In conclusion, selective inhibition of the Na(+)/H(+) exchanger by cariporide may represent an effective therapeutic strategy in the treatment of hepatic fibrosis.

Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC).

HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70(S6K) activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and alpha1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively.

Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 microM, with a maximal effect at 1000 microM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70(S6K) activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na(+)/H(+) exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 microM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 microM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor(beta1) by 54% and 92%, respectively, as well as TGF(beta1)-induced alpha1(I) procollagen mRNA expression.

Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases.

Cytoskeletal reorganization plays an important role in the regulation of different cell functions, such as proliferation and migration. Since platelet-derived growth factor (PDGF) stimulates both proliferation and chemotaxis of hepatic stellate cells (HSC), we investigated the effects of this cytokine on cytoskeletal components of cultured rat HSC.

Exposure of HSC to PDGF induced the formation of stress fibres and of a ruffled configuration of the plasma membrane, evaluated by both fluorescence and electron microscopy. These modifications were also induced by exposure to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) and abolished by pretreatment with the PKC inhibitor calphostin C, with the Rho inhibitor C3 exoenzyme and with the intracellular calcium chelator MAPTAM, but not with the PI-3 kinase inhibitor wortmannin or with the mitogen-activated protein kinase kinase inhibitor PD 98059. PDGF induced a translocation of Rho from the cytosol to the membrane which was inhibited by C3 exoenzyme and by calpostin C, and which was also induced by PMA. Moreover, PDGF induced a rearrangement of vinculin which was prevented by C3 exoenzyme and calphostin C.

PDGF-induced cytoskeletal reorganization in HSC is dependent on PKC and Rho, thus suggesting that these two pathways may play an important role in the response of liver to injury.