Tauroursodesoxycholate-induced choleresis involves p38(MAPK) activation and translocation of the bile salt export pump in rats

BACKGROUND & AIMS: Canalicular secretion of bile acids is stimulated by tauroursodesoxycholate (TUDC). This study investigates the underlying mechanisms. METHODS: TUDC effects on mitogen-activated protein (MAP) kinases, taurocholate (TC) excretion, proteolysis, and the localization of the bile salt export pump (Bsep) were studied in rat hepatocytes and perfused liver. RESULTS: TUDC induced a transient and concentration-dependent activation of p38(MAPK) and of extracellular signal-regulated kinase 2 (Erk-2), but not of c-Jun-N-terminal kinase (JNK). In perfused liver, TUDC concentrations of 20 micromol/L was sufficient to elicit the MAP kinase responses and TC choleresis. SB 202190, a specific inhibitor of p38(MAPK), had no effect on TUDC- induced Erk activation but abolished the stimulatory effect of TUDC on TC excretion in perfused liver, indicating the requirement of p38(MAPK) in addition to the reported Erk dependence for the choleretic response. TUDC-induced stimulation of TC excretion was accompanied by a p38(MAPK)-dependent insertion of subcanalicular immunoreactive Bsep into the canalicular membrane. In addition TUDC induced a p38(MAPK)-sensitive inhibition of proteolysis. CONCLUSIONS: TUDC-induced stimulation of canalicular TC excretion involves a MAP kinase-dependent translocation of subcanalicular Bsep to the canalicular membrane. Dual activation of Erks and p38(MAPK) is required for the choleretic effect of both TUDC and hypo-osmotic cell swelling.     

Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis

BACKGROUND & AIMS: Stimulation of canalicular secretion by tauroursodeoxycholate (TUDC) involves dual activation of p38 mitogen-activated protein kinase (p38(MAPK)) and extracellular signal-regulated kinase (ERK). This study investigates the sensing and upstream signaling events of TUDC-induced choleresis. METHODS: TUDC and hypo-osmolarity effects on protein kinase activities and taurocholate excretion were studied in perfused rat liver. RESULTS: TUDC induced a rapid activation of focal adhesion kinase (FAK) and Src, as shown by an increase in Y418 phosphorylation and a decrease in Y529 phosphorylation of Src. Inhibition of Src by PP-2 abolished the TUDC-induced activation of p38(MAPK) but not of FAK and ERKs. An integrin-inhibitory peptide with an RGD motif blocked TUDC-induced FAK, Src, ERK, and p38(MAPK) activation, suggesting that integrin signaling toward FAK/Src is required for TUDC-induced MAPK activation. The RGD peptide and PP-2 also abolished the stimulation of taurocholate excretion in perfused rat liver in response to TUDC. Integrin-dependent Src activation was also identified as an upstream event in hypo-osmotic signaling toward MAPKs and choleresis. CONCLUSIONS: TUDC-induced stimulation of canalicular taurocholate excretion involves integrin sensing, FAK, and Src activation as upstream events for dual MAPK activation. Integrins may also represent one long-searched sensor for cell hydration changes in response to hypo-osmolarity.     

Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N-terminal kinase-dependent manner

BACKGROUND & AIMS: Prevention of bile acid-induced apoptosis is of therapeutic interest and requires the understanding of underlying mechanisms. METHODS: The effect of tauroursodeoxycholate (TUDC) on taurolithocholic acid-3 sulfate (TLCS)-induced apoptosis was studied in cultured rat hepatocytes. RESULTS: TLCS induced activation of caspases 8, 9, and 3 and hepatocyte apoptosis. These effects were abolished by TUDC in a PI 3-kinase-/protein kinase B (PKB)-, p38(MAPK)-, and extracellular signal-regulated kinase-2 (Erk-2)-independent manner. These protein kinases were activated by both TLCS and TUDC, however, with different kinetics. TLCS, but not TUDC, led to a sustained activation of c-Jun N-terminal kinase (JNK) and CD95 trafficking to the plasma membrane; both TLCS effects were prevented by TUDC. Inhibition of JNK1 or protein kinase C prevented TLCS-induced CD95 membrane trafficking and blunted the apoptotic response. The apoptotic potency of other bile acids paralleled their ability to induce sustained JNK activation. CONCLUSIONS: Protection by TUDC against TLCS-induced apoptosis starts upstream of caspase 8 activation and is independent of a PI 3-kinase-dependent survival pathway. JNK activation may be important for bile acid-induced apoptosis by triggering ligand-independent CD95 surface trafficking and activation of apoptosis.     

Effects of colchicine on the maximum biliary excretion of cholephilic compounds in rats

BACKGROUND AND AIM: Colchicine, an inhibitor of intracellular vesicular transport, has been reported to inhibit the biliary excretion of bile acids and organic anions, but the previous findings are controversial. In order to systematically evaluate the effect of colchicine on the biliary excretion of cholephilic compounds, we studied the effect of colchicine on the biliary excretion of substrates of various canalicular transporters, which were administered at or above the excretory maximum in rats. METHODS: Substrates of various canalicular adenosine triphosphate-binding-cassette transporters were infused at or above the rate of maximum excretion into rats, and the effect of colchicine (0.2 mg/100 g), which was intraperitoneally injected 3 h before, on the biliary excretion was studied. Furthermore, the effect of tauroursodeoxycholate (TUDC) co-infusion on the biliary excretion of taurocholate (TC) after colchicine treatment was also studied. RESULTS: The biliary excretion of TC and cholate administered at the rate of 1 micro mol/min/100 g was markedly inhibited by colchicine, whereas that of TUDC was not inhibited even with the infusion rate of 2 micro mol/min/100 g. TUDC co-infusion at the rate of 1 micro mol/min/100 g increased the biliary excretion of TC (1 micro mol/min/100 g), which was decreased by the colchicine pretreatment. The biliary excretory maximum of taurolithocholate-sulfate and sulfobromophthalein, substrates of the multidrug resistance protein 2, of erythromycin, a substrate of the P-glycoprotein, and of indocyanine green were not affected by colchicine. CONCLUSIONS: The different excretory maximums of TC and TUDC and the different effect of colchicine on the excretion of these bile acids are considered to be a result of different regulatory mechanisms of vesicular targeting of the bile salt export pump to the canalicular membrane by these bile acid conjugates. The vesicular targeting of the multidrug resistance protein 2 and the P-glycoprotein to the canalicular membrane is considered to be colchicine insensitive in the absence of bile acid coadministration.     

Hepatobiliary transport of bile acids and organic anions

Recent studies have elucidated the mechanism and regulation of hepatic transport of bile acids and organic anions. Bile acids are taken up into hepatocytes by basolateral transporters, Na⁺‐dependently by Na⁺/taurocholate cotransporting polypeptide (NTCP) and Na⁺‐independently by organic anion transporting polypeptide (OATP) families. Organic anions are taken up into hepatocytes by OATP families. These compounds are then transported in hepatocytes bound to cytosolic binders, and subjected to transport by ATP binding cassette (ABC) transporters at the canalicular membrane. Amidated bile acids are excreted into bile by bile salt export pump (BSEP), and organic anions and bile acid sulfates and glucuronides are excreted by multidrug resistance protein 2 (MRP2). Hepatic transporters are downregulated under cholestasis in rats and humans, except for MRP3, a basolateral ABC transporter, which is upregulated and may have a role in removing bile acids and organic anions from hepatocytes to the blood under cholestatic conditions. Nuclear receptors, which bind bile acids, have been shown to regulate the expression of hepatic transporters. Farnesoid X receptor (FXR), which downregulates CYP7A1, the rate‐limiting enzyme of bile acid biosynthesis, upregulates BSEP and downregulates NTCP. MRP2 is upregulated by both FXR and pregnane X receptor (PXR), which upregulates CYP3A.     

p38丝裂原活化蛋白激酶与慢性气道疾病

支气管哮喘和慢性阻塞性肺疾病是两大主要的慢性气道疾病.作为一种细胞内信号转导通路,p38丝裂原活化蛋白激酶(MAPK)介导基因表达、炎性因子产生,在炎性细胞的增殖、分化、凋亡等方面扮有关键性作用,参与慢性气道疾病的气道炎症机制.体外实验表明p38 MAPK抑制剂有抗炎作用,动物实验表明p38 MAPK抑制剂可有效抑制慢性气道疾病模型的气道炎症、气道高反应性和重塑.此外,p38 MAPK的活性异常还与激素不敏感密切相关,p38 MAPK抑制剂可提高激素的抗炎效应.     

p38 mitogen‐activated protein kinase impairment of innate immune cells is a characteristic feature of HBeAg‐negative chronic hepatitis B

The mitogen‐activated protein kinase p38 (MAPK) is implicated in the induction of immune responses by regulating the differentiation of T lymphocytes and production of cytokines. Our aim was to investigate p38MAPK phosphorylation in different stages of the natural history of hepatitis B virus (HBV) infection. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll‐Hypaque density‐based centrifugation from 10 patients with HBeAg‐negative chronic hepatitis B [HBeAg(?) CHB;HBV‐DNA>2000IU/mL], eight patients with HBeAg‐negative chronic HBV infection [HBeAg(?) CI;undetectable HBV‐DNA] and 8 healthy controls (HCs). p38MAPK phosphorylation was assessed by phospho‐specific flow cytometry in PBMCs and cell subsets (CD3+,CD3?,CD56+,CD56?) after stimulation with cytokines (IL‐12+IL‐2 and IL‐12+IL‐18) or nonspecific stimuli [arsenite, phorbol 12‐myristate 13‐acetate (PMA) and ionomycin] at 0,30,60,120 and 240 minutes using p38 phospho‐specific conjugated antibodies. ΙFN‐γ was determined by ELISA in PBMCs culture supernatants after stimulation with rhIL‐2, rhIL‐12 and rhIL‐18, with and without pre‐treatment with the p38 MAPK inhibitor, SB203580. HBeAg(?) CI patients showed the highest expression of phosphor‐p38 MAPK in total PBMCs and subpopulations compared to HBeAg(?) CHB and HCs. A striking impairment in p38 phosphorylation was noted in CD56+ cells and in especially in NK cells (CD3‐CD56+). SB203580‐induced inhibition of p38MAPK phosphorylation was associated with suppression of IFN‐γ production in all groups. The universal lack of p38 MAPK activation in CD56+ and in particular in NK cells from HBeAg(?) CHB patients during viremia suggests a potential cell‐dependent implication of this pathway in the natural history of HBV infection.     

OSU-DY7, a novel D-tyrosinol derivative, mediates cytotoxicity in chronic lymphocytic leukaemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway

Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative targeting p38 mitogen-activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC-1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose- and time-dependent manner. The OSU-DY7-induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z-VAD-FMK. Interestingly, OSU-DY7-induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B-CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU-DY7-mediated cytotoxicity. Furthermore, OSU-DY7-induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU-DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies.     

p38MAPK信号转导通路在EGF诱导食管腺癌SEG-1细胞表达u-PA中的作用

目的:探讨表皮生长因子(epidermal growth factor,EGF)对人食管腺癌SEG-1细胞尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator,u-PA)mRNA和蛋白表达的影响及p38MAPK信号转导通路在其中的作用.方法:以相同浓度的EGF(100g/L)按时间梯度刺激SEG-1细胞,应用Western blot法测定各时间点总p38MAPK蛋白、磷酸化p38MAPK蛋白、u-PA蛋白表达,并应用RT-PCR方法检测各时间点u-PAmRNA表达.用p38MAPK特异抑制剂SB203580预处理细胞后,观察上述指标变化.结果:EGF可明显增强SEG-1细胞(u-PA)mRNA和蛋白的表达,并可激活p38MAPK蛋白的磷酸化,具有时间依赖性.SB203580能明显抑制EGF诱导的p38MAPK蛋白的磷酸化,用其阻断p38MAPK信号转导通路后,EGF对u-PAmRNA和蛋白表达的诱导作用受到显著抑制,并且具有剂量依赖性.结论:EGF可通过p38MAPK信号转导通路诱导SEG-1细胞表达u-PA.     

Effects of lipopolysaccharide on the biliary excretion of bile acids and organic anions in rats

BACKGROUND: Lipopolysaccharide is known to be a cause of cholestasis associated with sepsis. It has also recently been reported to down-regulate the basolateral and canalicular transporters. The aim of the present study was to examine simultaneously the effect of lipopolysaccharide on the biliary excretion of typical substrates of bile salt export pump and multidrug resistance protein 2 in vivo, and the effect of lipopolysaccharide on the amount of these transporters. METHODS: After intravenous administration of O127:B8-derived lipopolysaccharide (2.5 mg/kg), the biliary excretion of taurocholate and various organic anions was studied, and the protein levels of bile salt export pump and multidrug resistance protein 2 in the crude liver membrane was determined by western blot analysis. RESULTS: Lipopolysaccharide decreased the biliary excretion of tracer amounts of taurocholate, leukotriene C4, taurolithocholate-3-sulfate and temocapril without affecting bile flow. The biliary excretion of high doses of taurocholate and sulfobromophthalein was markedly inhibited by lipopolysaccharide. Lipopolysaccharide decreased bile salt export pump levels in the liver plasma membrane fraction to 48% of control rats, and markedly decreased multidrug resistance protein 2 levels to 17% of control rats. CONCLUSIONS: These findings support the hypothesis that down-regulation of the canalicular transporters by lipopolysaccharide causes the impairment of the biliary excretion of bile acids and organic anions in cholestasis of sepsis prior to the decrease of bile flow.     

A progressive familial intrahepatic cholestasis type 2 mutation causes an unstable, temperature-sensitive bile salt export pump

BACKGROUND/AIMS: Progressive familial intrahepatic cholestasis type 2 (PFIC-2) patients have a defect in the hepatocanalicular bile salt secretion. The disease is caused by mutations in the bile salt export pump (BSEP). Ten different missense mutations have been described. In this study, we analysed the effect of the D482G PFIC-2 mutation on BSEP function. METHODS: Adenosine triphosphatase (ATPase) and taurocholate transport assays were performed with full-length mouse Bsep (mBsep) with and without the D482G mutation. The effect on expression and subcellular sorting was studied in HepG2 cells, stably expressing enhanced green fluorescent protein (EGFP)-tagged mBsep proteins. RESULTS: The D482G mutation did not significantly affect the taurocholate transport activity of mBsep, even though the bile salt-inducible ATPase activity of the mutant protein was slightly reduced. Protein expression and canalicular sorting were strongly affected by the D482G mutation. Mutant EGFP-mBsep protein was only partly glycosylated and detected in both the canalicular membrane and the cytoplasm. At 30 degrees C, the mutant mRNA and protein levels were strongly increased, and the protein was predominantly glycosylated and efficiently targeted to the canalicular membrane. CONCLUSIONS: These data suggest that PFIC-2 patients with the D482G mutation express a functional, but highly unstable, temperature-sensitive bile salt export pump.     

Protein kinase C-dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells

The subcellular localization of hepatobiliary transport proteins directly affects the rate of bile formation, e.g., the conjugate export pump multidrug resistance protein 2 (MRP2) is regulated on a short-term scale by retrieval from and insertion into the canalicular membrane in the liver. This study reports on the effects of protein kinase C on MRP2 localization and activity in human hepatoblastoma HepG2 cells. MRP2 was detected in HepG2 cells by immunocytochemistry and Western blot analysis. Functional activity was assessed by confocal laser scanning microscopy using fluorescent MRP2 substrates. In untreated HepG2 cells MRP2 was almost exclusively localized at the apical membrane. Treatment of HepG2 cells with phorbol-12-myristate-13-acetate (PMA) resulted in a rapid decrease of apically localized MRP2 and a loss of more than 90% of pseudocanaliculi within 4 hours. This was accompanied by a reduced pseudocanalicular secretion of the MRP2 substrate glutathione-methylfluorescein. Interestingly, PMA treatment (1-100 nmol/L) led to the appearance of immunoreactive MRP2 at the basolateral membrane within 30 minutes. This was shown by its colocalization with MRP1, human dipeptidylpeptidase IV (DPPIV), and transfected rat Ntcp. The effects of PMA on MRP2 localization were sensitive to the protein kinase C (PKC) inhibitor G?6850 but insensitive to inhibition of MEK by PD098059. Basolateral MRP2-appearance was not inhibited by cycloheximide or by disruption of microtubules or microfilaments. In rat livers cholestasis was induced by PMA (100 nmol) and MRP2 was detected at the basolateral membrane in some areas, colocalizing with Ntcp. The data suggest that retargeting of canalicular MRP2 to the basolateral membrane due to PKC activation may represent a novel mechanism that may contribute to cholestasis.     

在Fas诱导Bel-7402细胞凋亡中p38MAPK调节Bcl-2的表达

目的:探讨p38MPAK是否参与Fas和AD诱导Bel-7402细胞的凋亡过程,以及p38MPAK和bcl-2的关系,进一步揭示p38MAPK的凋亡途径.方法:在Fas和AD作用24h后,用MTT法检测Bel-7402细胞的活力,用Western-blot和RT- PCR法检测p38MAPK,p-p38MAPK和Bcl-2 expression,用免疫荧光法对p-p38MAPK进行细胞定位.结果:随着Fas浓度的增加,Bel-7402细胞的活力明显抑制,p38MAPK和p-p38MAPK表达明显增高(P<0.01),且p-p38MAPK由胞质易位到胞核.Bcl-2的表达明显降低(P<0.01),并且这种降低趋势被p38MAPK抑制剂SB203580所阻止.结论:p38MAPK参与Fas诱导的凋亡途径,以磷酸化形式激活后抑制Bcl一2的表达,进而促进细胞凋亡.     

Dynamic changes of mitogen‐activated protein kinase signal transduction in rats with severe acute pancreatitis

OBJECTIVE: To investigate the dynamic changes of mitogen‐activated protein kinase (MAPK) signal transduction in rats with severe acute pancreatitis (SAP). METHODS: The SAP model was induced by infusing the bilio‐pancreatic duct of 56 Sprague‐Dawley rats with 5% sterile sodium taurocholate solution. The rats were randomly divided into seven groups: control group, 0.5 h postoperative group, 1 h group, 3 h group, 6 h group, 12 h group and 24 h group. Western blot analysis was used to determine the activities of p38 MAPK and c‐Jun N‐terminal kinase (JNK) in the pancreas and lungs. RESULTS: In the rats of the control group, basal p38MAPK activity could be detected but not that of JNK. After SAP was induced, the p38MAPK activity in the pancreas increased markedly and peaked at 3 h, but in the lung it peaked at 6 h. The p38MAPK activity in the pancreas and lungs was significantly higher than the basal activity at the 24 h time point. The activity of JNK was only increased at the 12 h point and was not detectable at 24 h. CONCLUSION: The MAPK signal transduction pathway, in particular p38MAPK, plays an important role in the pathogenesis of SAP.     

Role of mitogen-activated protein kinases in tauroursodeoxycholic acid-induced bile formation in cholestatic rat liver.

Aim: Ursodeoxycholic acid exerts anticholestatic effects in various cholestatic disorders and experimental models of cholestasis. Its taurine conjugate (TUDCA) stimulates bile salt secretion in isolated perfused rat livers (IPRL) under physiological, non-cholestatic conditions, in part by mitogen-activated protein kinase (MAPK)-dependent mechanisms. The role of MAPK in the anticholestatic effect of TUDCA, however, is unclear. Therefore, we studied the role of MAPK in the anticholestatic effect of TUDCA in IPRL and isolated rat hepatocytes (IRH) in taurolithocholic acid (TLCA)-induced cholestasis. Methods: Bile flow, biliary levels of 2,4-dinitrophenyl-S-glutathione (GS-DNP) as a marker of hepatobiliary organic anion secretion and activity of lactate dehydrogenase (LDH) in hepatovenous effluate as a marker of hepatocellular damage in IPRL perfused with TUDCA and/or TLCA were determined in the presence or absence of MAPK inhibitors. In addition, phosphorylation of Erk 1/2 and p38(MAPK) induced by TUDCA and/or TLCA was studied by Western immunoblot in IPRL and IRH. Results: TUDCA-induced bile flow was impaired by the Erk 1/2 inhibitor PD98059 in normal livers (-28%), but not in livers made cholestatic by TLCA. GS-DNP secretion was unaffected by PD98059 under both conditions. TUDCA-induced bile formation and organic anion secretion both in the presence and absence of TLCA were unaffected by the p38(MAPK) inhibitor SB202190. Erk 1/2 phosphorylation in liver tissue was unchanged after bile salt exposure for 70 min, but was transiently enhanced by TUDCA in IRH. Conclusion: MAPK do not mediate the anticholestatic effects of TUDCA in TLCA-induced cholestasis.     

Protease-activated receptor-2 regulates cyclooxygenase-2 expression in human bile duct cancer via the pathways of mitogen-activated protein kinases and nuclear factor kappa B

Background/purpose

Recent studies have suggested that protease-activated receptor-2 (PAR-2) activity correlates with cell proliferation and tumor growth, and its activation induces expression of cyclooxygenase-2 (COX-2). However, no previous reports have investigated PAR-2 signaling pathways in bile duct cancer. The aim of this study was to determine whether PAR-2 activation can regulate COX-2 expression via mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-??B) in human bile duct cancer cells.

Methods

We immunohistochemically examined PAR-2 and COX-2 expression in 104 resected human specimens of extrahepatic bile duct cancer. We then determined how inhibitors of MAPKs and NF-??B signaling pathways influence COX-2 expression under PAR-2 activation in HuCCT1 and TKKK, human bile duct cancer cell lines.

Results

PAR-2 and COX-2 proteins were immunohistochemically recognized in 63 and 57% of specimens and were significantly correlated. PAR-2 agonist peptide activated mRNA and protein expression of COX-2 in HuCCT1 and TKKK. Pharmacologic blockade of p44/42 or p38 MAPK significantly inhibited PAR-2-activated mRNA and protein expression of COX-2 in both cells. COX-2 protein expression was also inhibited by the blocker of NF-??B pathway in both cells.

Conclusions

PAR-2 may regulate COX-2 expression in human bile duct cancer via the MAPKs and NF-??B pathways.     

Melatonin decreases cell proliferation and induces melanogenesis in human melanoma SK‐MEL‐1 cells

Abstract: Melatonin is an indoleamine synthesized in the pineal gland, and after its release into the blood, it has an extensive repertoire of biological activities, including antitumoral properties. In this study, we found that melatonin reduced the growth of the human melanoma cells SK‐MEL‐1. The antiproliferative effect was associated with an alteration in the progression of the phases of the cell cycle and also with an increase in tyrosinase activity, the key regulatory enzyme of melanogenesis. Antagonists for melatonin membrane receptors (luzindole and 4‐P‐PDOT) and the general G‐coupled receptor inhibitor, pertussis toxin, did not prevent the melatonin‐induced cell growth arrest; this suggests a mechanism independent of G‐coupled membrane receptors. In contrast, p38 mitogen‐activated protein kinase (p38 MAPK) signaling pathway seems to play a significant role in cell growth inhibition by melatonin. The indoleamine‐induced phosphorylation of p38 MAPK and the effect on cell proliferation were abrogated by the specific inhibitor SB203580 . Furthermore, comparative studies with known antioxidants such as N‐acetyl‐l ‐cysteine and trolox indicate that the growth of SK‐MEL‐1 cells is highly sensitive to antioxidants.     

Effect of genipin on the biliary excretion of cholephilic compounds in rats.

Aim: Genipin, a metabolite of geniposide, is reported to stimulate the insertion of multidrug resistance protein 2 (Mrp2) in the bile canalicular membrane, and to cause choleresis by increasing the biliary excretion of glutathione, which has been considered to be a substrate of Mrp2. In the present study, the effect of colchicine on the choleretic effect of genipin was investigated. The effect of genipin on the biliary excretion of the substrates of bile salt export pump and Mrp2 was also studied. Methods: After bile duct cannulation into rats, genipin was administered at the rate of 0.2 mumol/min/100 g, and the effect of colchicine pretreatment (0.2 mg/100 g) was examined. Metabolites of genipin in the bile were examined by a thin layer chromatography. Taurocholate (TC), sulfobromophthalein (BSP), and pravastatin were infused at the rate of 1.0, 0.2 and 0.3 mumol/min/100 g, respectively, and the effect of genipin co-administration was examined. Results: Genipin increased bile flow and the biliary glutathione excretion, and those increases were not inhibited by colchicine. The biliary excretion of genipin glucuronide was less than 10% of the genipin excreted into bile. The biliary excretion of TC, BSP, and pravastatin was unchanged by genipin co-administration. Conclusion: It was indicated that colchicine-sensitive vesicular transport has no role on the genipin-induced insertion of Mrp2 to the canalicular membrane. Choleresis of genipin is considered to be mainly due to the increased biliary glutathione excretion by genipin, not by the biliary excretion of glucuronide. TC had no effect on the biliary glutathione excretion.     

ATP7B mediates vesicular sequestration of copper: insight into biliary copper excretion

BACKGROUND & AIMS: The Wilson protein (ATP7B) regulates levels of systemic copper by excreting excess copper into bile. It is not clear whether ATP7B translocates excess intrahepatic copper directly across the canalicular membrane or sequesters this copper into exocytic vesicles, which subsequently fuse with canalicular membrane to expel their contents into bile. The aim of this study was to clarify the mechanism underlying ATP7B-mediated copper detoxification by investigating endogenous ATP7B localization in the HepG2 hepatoma cell line and its ability to mediate vesicular sequestration of excess intracellular copper. METHODS: Immunofluorescence microscopy was used to investigate the effect of copper concentration on the localization of endogenous ATP7B in HepG2 cells. Copper accumulation studies to determine whether ATP7B can mediate vesicular sequestration of excess intracellular copper were performed using Chinese hamster ovary cells that exogenously expressed wild-type and mutant ATP7B proteins. RESULTS: In HepG2 cells, elevated copper levels stimulated trafficking of ATP7B to pericanalicular vesicles and not to the canalicular membrane as previously reported. Mutation of an endocytic retrieval signal in ATP7B caused the protein to constitutively localize to vesicles and not to the plasma membrane, suggesting that a vesicular compartment(s) is the final trafficking destination for ATP7B. Expression of wild-type and mutant ATP7B caused Chinese hamster ovary cells to accumulate copper in vesicles, which subsequently undergo exocytosis, releasing copper across the plasma membrane. CONCLUSIONS: This report provides compelling evidence that the primary mechanism of biliary copper excretion involves ATP7B-mediated vesicular sequestration of copper rather than direct copper translocation across the canalicular membrane.