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Bruno Stieger

Publications and source records attributed to Bruno Stieger.

33 records · Page 2Linked to original sources
PubMed2004

Differential expression of bile salt and organic anion transporters in developing rat liver.

BACKGROUND/AIMS: Differentiated hepatocytes express distinct transport systems at their basolateral and canalicular membrane domains. Here, we investigated the ontogenesis of the polar expression of hepatocellular organic anion and bile salt transport systems in rat liver. METHODS: mRNA levels (real time PCR) and protein expression (immunofluorescence microscopy) were investigated for the Na(+)-taurocholate cotransport protein (Ntcp), the organic anion transporting polypeptides (Oatp1a1, Oatp1a4, Oatp1b2), the multidrug resistance associated proteins (Mrp2, Mrp6) and the bile salt export pump (Bsep). RESULTS: Expression of mRNA and protein was detected first for Oatp1b2, Mrp2 and Mrp6 at embryonic day 16 (E16), followed by Ntcp, Oatp1a1 and Bsep at E20 and by Oatp1a4 at postnatal day 5 (P5). Intracellular localization of Oatps (e.g. Oatp1b2) preceded expression at the plasma membrane. Approximate adult phenotypes of polarized expression were achieved for Ntcp by P5, for Bsep, Mrp2 and Mrp6 by P12 and for Oatp1a1, Oatp1a4 and Oatp1b2 by P29. CONCLUSIONS: The data demonstrate that full maturation of polarized transporter expression in rat liver requires several weeks. The findings provide a molecular explanation for the previously observed chronology of the functional maturation of bile salt-independent and dependent bile formation and of hepatic detoxification functions in developing rat liver.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Enterohepatic bile salt transporters in normal physiology and liver disease.

The vectorial transport of bile salts from blood into bile is essential for the generation of bile flow, solubilization of cholesterol in bile, and emulsification of lipids in the intestine. Major transport proteins involved in the enterohepatic circulation of bile salts include the hepatocellular bile salt export pump (BSEP, ABCB11), the apical sodium-dependent bile salt transporter (ASBT, SLC10A2) in cholangiocytes and enterocytes, the sodium-dependent hepatocyte bile salt uptake system NTCP (SLC10A1), the organic anion transporting polypeptides OATP-C (SLC21A6), OATP8 (SLC21A8) and OATP-A (SLC21A3), and the multidrug resistance protein MRP3 (ABCC3). Synthesis and transport of bile salts are intricately linked processes that undergo extensive feedback and feed-forward regulation by transcriptional and posttranscriptional mechanisms. A key regulator of hepatocellular bile salt homeostasis is the bile acid receptor/farnesoid X receptor FXR, which activates transcription of the BSEP and OATP8 genes and of the small heterodimer partner 1 (SHP). SHP is a transcriptional repressor that mediates bile acid-induced repression of the bile salt uptake systems rat Ntcp and human OATP-C. A nuclear receptor that activates rodent Oatp2 (Slc21a5) and human MRP2 (ABCC2) is the pregnane X receptor/steroid X receptor PXR/SXR. Intracellular trafficking and membrane insertion of bile salt transporters is regulated by lipid, protein, and extracellular signal-related kinases in response to physiologic stimuli such as cyclic adenosine monophosphate or taurocholate. Finally, dysfunction of individual bile salt transporters such as BSEP, on account of genetic mutations, steric inhibition, suppression of gene expression, or disturbed signaling, is an important cause of cholestatic liver disease.

Animals↗
PubMed2004

Metal-responsive transcription factor-1 (MTF-1) is essential for embryonic liver development and heavy metal detoxification in the adult liver.

Metal-responsive transcription factor-1 (MTF-1) activates the transcription of metallothionein genes and other target genes in response to heavy metal load and other stresses such as hypoxia and oxidative stress. It also has an essential function during embryogenesis: targeted disruption of Mtf1 in the mouse results in lethal liver degeneration on day 14 of gestation. Here we studied Mtf1 knockout mice at embryonic and adult stages, the latter by means of conditional knockout. Hepatocytes from Mtf1 null mutant and wild-type embryos were taken into culture on day 12.5 of gestation. Both initially appeared normal, but mutant cells were lost within a few days. Furthermore, Mtf1 null hepatocytes were poorly, if at all, rescued by cocultivation with wild-type rat embryo hepatocytes, indicating a cell-autonomous defect. When the Mtf1 gene was excised by Cre recombinase after birth in liver and bone marrow and to a lesser extent in other organs, mice were viable under non-stress conditions but highly susceptible to cadmium toxicity, in support of a role of MTF-1 in coping with heavy metal stress. An additional MTF-1 function was revealed upon analysis of the hematopoietic system in conditional knockout mice where leukocytes, especially lymphocytes, were found to be severely underrepresented. Together, these findings point to a critical role of MTF-1 in embryonic liver formation, heavy metal toxicity, and hematopoiesis.

Animals↗
PubMed2004

Gd-BOPTA transport into rat hepatocytes: pharmacokinetic analysis of dynamic magnetic resonance images using a hollow-fiber bioreactor.

RATIONALE AND OBJECTIVES: To investigate the transport of the hepatobiliary magnetic resonance (MR) imaging contrast agent Gd-BOPTA into rat hepatocytes. MATERIALS AND METHODS: In a MR-compatible hollow-fiber bioreactor containing hepatocytes, MR signal intensity was measured over time during the perfusion of Gd-BOPTA. For comparison, the perfusion of an extracellular contrast agent (Gd-DTPA) was also studied. A compartmental pharmacokinetic model was developed to describe dynamic signal intensity-time curves. RESULTS: The dynamic signal intensity-time curves of the hepatocyte hollow-fiber bioreactor during Gd-BOPTA perfusion were adequately fitted by 2 compartmental models. Modeling permitted to discriminate between the behaviors of the extracellular contrast agent (Gd-DTPA) and the hepatobiliary contrast agent (Gd-BOPTA). It allowed the successfully quantification of the parameters involved in such differences. Gd-BOPTA uptake was saturable at high substrate concentrations. CONCLUSIONS: The transport of Gd-BOPTA into rat hepatocytes was successfully described by compartmental analysis of the signal intensity recorded over time and supported the hypothesis of a transporter-mediated uptake.

Animals↗
PubMed2003

Regulation of basolateral organic anion transporters in ethinylestradiol-induced cholestasis in the rat.

BACKGROUND/AIMS: Estrogen-mediated cholestasis is an important clinical entity, but its molecular pathophysiology is still not fully understood. Impaired sodium-dependent uptake of bile acids has been associated with diminished expression of a basolateral Na(+)/bile acid cotransporter (Ntcp), whereas sodium-independent uptake is maintained despite a down-regulation of the organic anion transporter Oatp1. Thus, expression of the two other rat Oatps (Oatps2 and -4) was determined in estrogen-induced cholestasis. In addition, known transactivators of Oatp2 and Ntcp were studied to further characterize transcriptional regulation of these transporter genes. METHODS: Hepatic protein and mRNA expression of various Oatps (1, 2, 4) in comparison to Ntcp were analyzed after 0.5, 1, 3 and 5 days of ethinylestradiol (EE) treatment (5 mg/kg) in rats. Binding activities of Oatp2 and Ntcp transactivators were assessed by electrophoretic mobility shift assays. RESULTS: All basolateral Oatps (1, 2 and 4) were specifically down-regulated at the protein level by 30-40% of controls, but less pronounced than Ntcp (minus 70-80%). In contrast to unaltered Oatp4 mRNA levels, Oatp1 and Oatp2 mRNAs were reduced to various extents (minus 40-90% of controls). Binding activity of known transactivators of Ntcp and Oatp2 such as hepatocyte nuclear factor 1 (HNF1), CAAT enhancer binding protein alpha (C/EBPalpha) and pregnane X receptor (PXR) were also diminished during the time of cholestasis. CONCLUSIONS: Estrogen-induced cholestasis results in a down-regulation of all basolateral organic anion transporters. The moderate decline in expression of Oatp1, -2 and -4 may explain the unchanged sodium-independent transport of bile acids due to overlapping substrate specificity. Reduction in transporter gene expression seems to be mediated by a diminished nuclear binding activity of transactivators such as HNF1, C/EBP and PXR by estrogens.

Animals↗
PubMed2003

Regulation of rat organic anion transporters in bile salt-induced cholestatic hepatitis: effect of ursodeoxycholate.

Hepatic uptake of organic anions, including bile salts, is mediated by members of the organic anion-transporting polypeptide (Oatp) family. In rat liver, Oatp1 (Slc21a1), Oatp2 (Slc21a5), and Oatp4 (Slca10) are expressed at the basolateral membrane of hepatocytes and may be differentially regulated under pathophysiologic conditions such as cholestasis. The aim of this study was to determine the effects of cholic acid (CA) and ursodeoxycholic acid (UDCA) on the expression of Oatp4 compared with Ntcp, Oatp1, and Oatp2. Wistar rats were fed with CA (0.5%) or both CA (0.5%) and UDCA (0.25%) for 3 weeks. Oatp expression was studied by Northern and Western blot analysis as well as immunofluorescence analysis. Transport function was compared measuring biliary secretion of (14)C-CA and (14)C-taurocholic acid (TCA). In CA-fed animals, biliary secretion of (14)C-CA and (14)C-TCA was markedly delayed over 40 minutes compared with controls. Accordingly, Oatp4 protein was significantly down-regulated in CA-fed animals together with Oatp1 and Ntcp. Cofeeding of CA plus UDCA prevented the impairment of (14)C-CA and (14)C-TCA secretion and the down-regulation of Oatp4. Oatp4 messenger RNA (mRNA) levels did not differ significantly between bile salt-fed groups, suggesting a posttranscriptional effect of CA on Oatp4 expression. In contrast to Oatp1 and Oatp4, Oatp2 protein expression was increased by CA feeding, indicating a differential regulation of Oatp transporters. In conclusion, we show that CA feeding may cause cholestasis associated with a posttranscriptional down-regulation of Oatp4. UDCA may prevent impairment of hepatic function by restoring hepatic transporter expression.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2003

Phenobarbital alters hepatic Mrp2 function by direct and indirect interactions.

Phenobarbital (PB) treatment impairs the biliary excretion of some organic anions. One mechanism may involve direct competition for biliary excretion by PB and/or a PB metabolite. Alternatively, PB may alter the expression and/or function of hepatic organic anion transport proteins. The role of multidrug resistance-associated protein 2 (Mrp2) in the biliary excretion of PB and metabolites was studied using isolated perfused livers (IPLs) from Wistar and Mrp2-deficient TR- rats. In normal livers, 4.19 +/- 0.53% of the PB dose was recovered in bile as PB metabolites [2.21 +/- 0.69% as 5-ethyl-5-(4-OH phenyl) barbituric acid (PBOH)-glucuronide; 1.98 +/- 0.09% as PBOH-sulfate]. In TR- livers, only PBOH-sulfate was recovered in bile (0.35 +/- 0.16% of dose) during the 2-h perfusion. Mrp2 message was increased (2.3-fold) by PB pretreatment (80 mg/kg i.p. x 4 days) but decreased to control values after a 48-h washout. Mrp2 protein was increased slightly in PB-treated livers and remained slightly elevated after a 24-h washout, but it was decreased significantly to 62 +/-7% of control values after a 48-h washout. The 120-min cumulative biliary excretion of the Mrp2 substrate 5-(and-6)-carboxy-2', 7'-dichlorofluorescein in IPLs from PB-treated rats after a 48-h washout was significantly lower than in vehicle-treated livers (66.3 +/- 9.2% versus 83.4 +/- 2.4% of the dose, respectively). These data support two mechanisms for impaired biliary excretion of some organic anions by PB treatment: 1) PBOH-glucuronide is a substrate for Mrp2 and may compete with other organic anions for biliary excretion and 2) Mrp2 protein expression and functional capacity is decreased 48 h after PB treatment.

ATP-Binding Cassette Transporters↗
PubMed2003

Physiological characteristics of allo-cholic acid.

The physiological characterstics of allo-cholic acid (ACA), a typically fetal bile acid that reappears during liver regeneration and carcinogenesis were investigated. [(14)C] Tauro-ACA (TACA) uptake by Chinese hamster ovary cells expressing rat organic anion transporter polypeptide (Oatp)1 or sodium-taurocholate cotransporter polypeptide (Ntcp) was lower than that of [(14)C]taurocholic acid (TCA). Although TACA inhibited ATP-dependent TCA transport across plasma membrane vesicles from Sf9 cells expressing rat or mouse bile salt export pump (Bsep), no ATP-dependent TACA transport was found. In rats, TACA was secreted into bile with no major biotransformation and it had lower clearance and longer half-life than TCA. In mice, TACA bile output was lower (-50%) than that of TCA, whereas TACA induced 9-fold higher bile flow than TCA. Even though the intracellular levels were lower for TACA, translocation into the hepatocyte nucleus was higher for TACA than for TCA; however, rate of DNA synthesis, expression levels of alpha-fetoprotein, albumin, Ntcp, and Bsep, cell viability, and apoptosis in rat hepatocytes were similarly affected by both isomers. In conclusion, TACA partly shares hepatocellular uptake system(s) for TCA. Furthermore, in contrast to other "flat" bile acids, TACA is efficiently secreted into bile via transport system(s) other than Bsep and is highly choleretic, hence its appearance during certain situations may prevent accumulation of cholestatic precursors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Hepatobiliary organic anion transporters are differentially regulated in acute toxic liver injury induced by carbon tetrachloride.

BACKGROUND/AIMS: Hepatobiliary transporters are down-regulated in cholestasis, but their expression in acute, non-cholestatic, cytokine-mediated liver injury is unknown. Thus we studied the molecular mechanisms, by which sodium taurocholate cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, multidrug-resistance protein 2 (Mrp2) and bile salt export pump (Bsep) are regulated in liver injury induced by carbon tetrachloride (CCl(4)). METHODS: mRNA and protein levels were determined in rats 24 and 72h after CCl(4) injection. Transporter gene transcription and binding activities of Ntcp and Mrp2 transactivators were assessed by nuclear runoff and electrophoretic mobility shift assays. RESULTS: mRNA levels significantly declined to 41+/-44% for Ntcp, 65+/-41% for Oatp1 and 64+/-28% for Oatp2, but remained unchanged for Oatp4, canalicular Mrp2 and Bsep. Protein levels declined only for Oatp4 (-50+/-17%) and Ntcp (-23+/-13%) at 24h. Reduced mRNA levels (Ntcp, Oatp1, Oatp2) were associated with decreased transcriptional activities. Binding activity of Ntcp transactivators (hepatocyte nuclear factor 1 alpha (HNF1alpha) and CAAT enhancer binding protein alpha (C/EBPalpha) were reduced by 24h, whereas retinoid X receptor alpha (RXRalpha):retinoid acid receptor alpha (RARalpha) as transactivator of both Ntcp and Mrp2 remained unaltered. Recovery of acute hepatitis and changes in gene expression occurred after 72h. CONCLUSIONS: Acute liver injury results in down-regulation of basolateral organic anion transporters similar to liver regeneration after partial hepatectomy, but in contrast to endotoxin-induced cholestasis. Maintained binding activity of RXRalpha:RARalpha may explain differences in Mrp2 expression.

Acute Disease↗
PubMed2002

Effects of bile salt flux variations on the expression of hepatic bile salt transporters in vivo in mice.

BACKGROUND/AIMS: Expression of hepatic bile salt transporters is partly regulated by bile salts via activation of nuclear farnesoid X-activated receptor (Fxr). We investigated the physiological relevance of this regulation by evaluating transporter expression in mice experiencing different transhepatic bile salt fluxes. METHODS: Bile salt flux was manipulated by dietary supplementation with taurocholate (0.5% w/w) or cholestyramine (2% w/w) or by disruption of the cholesterol 7alpha-hydroxylase-gene (Cyp7A(-/-) mice) leading to reduced bile salt pool size. Expression of hepatic transporters was assessed (polymerase chain reaction (PCR), immunoblotting, and immunohistochemistry). RESULTS: Biliary bile salt secretion was increased (+350%) or decreased (-50%) after taurocholate or cholestyramine feeding, respectively, but plasma bile salt concentrations and hepatic Fxr expression were not affected. The bile salt uptake system Na(+)-taurocholate co-transporting polypeptide (Ntcp) and organic anion transporting polypeptide-1 (Oatp1) were down-regulated by taurocholate and not affected by cholestyramine feeding. Cyp7A(-/-) mice did not show altered Ntcp or Oatp1 expression. Canalicular bile salt export pump (Bsep) was up-regulated by 65% in taurocholate-fed mice, and slightly down-regulated in Cyp7A(-/-) mice. CONCLUSIONS: Large variations in hepatic bile salt flux have minor effects on expression of murine Ntcp and Bsep in vivo, suggesting that these transporters are abundantly expressed and able to accommodate a wide range of 'physiological' bile salt fluxes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Functional expression of the canalicular bile salt export pump of human liver.

BACKGROUND & AIMS: Hepatic bile salt secretion is an essential function of vertebrate liver. Rat and mouse bile salt export pump (Bsep) are adenosine triphosphate (ATP)-dependent bile salt transporters. Mutations in human BSEP were identified as the cause of progressive familial intrahepatic cholestasis type 2. BSEP protein is highly identical with its rat and mouse orthologs and has not yet been functionally characterized; the effect of BSEP mutations on its function has also not been studied. Therefore, the aim of this study was to functionally characterize human BSEP. METHODS: Complementary DNA for BSEP was isolated from human liver and expressed with the baculovirus system in Sf9 cells. ATP-dependent bile salt transport assays were performed with Sf9 cell vesicles expressing BSEP and a rapid filtration assay. RESULTS: Cloning of human BSEP required the inactivation of a bacterial cryptic promoter motif within its coding region. BSEP expressed in Sf9 cells transports different bile salts in an ATP-dependent manner with Michaelis constant values as follows: taurocholate, 7.9 +/- 2.1 micromol/L; glycocholate, 11.1 +/- 3.3 micromol/L; taurochenodeoxycholate, 4.8 +/- 1.7 micromol/L; tauroursodeoxycholate, 11.9 +/- 1.8 micromol/L. The rank order of the intrinsic clearance of bile salts was taurochenodeoxycholate > taurocholate > tauroursodeoxycholate > glycocholate. CONCLUSIONS: This study characterizes human BSEP as an ATP-dependent bile salt export pump with transport properties similar to its rat and mouse orthologs. Expression of BSEP in Sf9 cells will enable functional characterization of the consequences of mutations in the human BSEP gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Gender difference in the Oatp1-mediated tubular reabsorption of estradiol 17beta-D-glucuronide in rats.

The gender difference in the urinary excretion of estradiol-17beta-glucuronide (E(2)-17betaG) was examined in rats. The urinary clearance of E(2)-17betaG was >250 times lower in male than in female rats. No such major gender difference was observed in its biliary excretion or metabolism in kidney homogenate. Both plasma protein binding and inulin clearance were comparable in male and female rats, suggesting that this gender difference cannot be explained by glomerular filtration. The urinary clearance with respect to the plasma unbound E(2)-17betaG in male rats was <1% of the glomerular filtration rate, indicating its potential reabsorption by the kidney, and this increased to a level comparable with that found in female rats when dibromosulfophthalein was coinfused. A marked increase in E(2)-17betaG urinary excretion was also observed in male rats that had undergone orchidectomy. Testosterone injections given to female rats reduced the urinary excretion to a level comparable with that of control male rats. The concomitant change in the expression of the gene product for organic anion-transporting polypeptide Oatp1, of which E(2)-17betaG is a typical substrate, was found in the kidney membrane fractions after these treatments. These results suggest that urinary E(2)-17betaG excretion is subject to hormonal regulation and that the large gender difference can be explained by regulation in Oatp1-mediated reabsorption.

Absorption↗
PubMed2002

Expression of rat hepatic multidrug resistance-associated proteins and organic anion transporters in pregnancy.

The expression of hepatic multidrug resistance-associated protein (Mrp)1, 2, 3, and 6 and organic anion transporting polypeptides (Oatp)1 and 2 were examined in control and 20- to 21-day pregnant rats. Western analysis showed that expression of Oatp2 was decreased 50% in pregnancy, whereas expression of Oatp1 did not change. Expression of Mrp2 protein determined by Western analysis of total liver homogenate decreased to 50% of control levels in pregnant rats, consistent with studies using plasma membranes. Confocal immunohistochemistry showed that Mrp2 expression was confined to the canalicular membrane in both control and pregnant rats and was not detectable in intracellular compartments. In isolated perfused liver, the biliary excretion of 2,4-dintrophenyl-glutathione was significantly decreased in pregnancy, consistent with decreased expression of Mrp2. The expression of the basolateral transporter Mrp1 was not altered in pregnancy, whereas expression of Mrp6 mRNA was decreased by 60%. Expression of Mrp3 was also decreased by 50% in pregnant rat liver, indicating differential regulation of Mrp isoforms in pregnancy. These data also demonstrate that decreased Mrp2 expression is not necessarily accompanied by increased Mrp3 expression.

Animals↗
PubMed2000

Molecular Mechanisms in Bile Formation.

Active canalicular secretion of bile salts and non-bile salt organic anions represents the major driving force of hepatic bile formation. The most important carriers involved have now been cloned on both the basolateral and canalicular sides of hepatocytes. Elucidation of their structure, transport properties, and regulation is an important step forward in the ultimate understanding of the molecular physiology of bile formation.

Journal Article↗