Browsing by Author "Chianale, J"
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- ItemDifferences between nuclear run-off and mRNA levels for multidrug resistance gene expression in the cephalocaudal axis of the mouse Intestine(ELSEVIER SCIENCE BV, 1995) Chianale, J; Vollrath, V; Wielandt, AM; Miranda, S; Gonzalez, R; Fresno, AM; Quintana, C; Gonzalez, S; Andrade, L; Guzman, SP-glycoprotein is a multidrug transporter encoded by the mdr3 gene in the mouse intestinal epithelium. The aims of this study were to characterize the mdr3 gene expression in the cephalocaudal axis of the intestine in adult animals and during perinatal development, and to define the molecular mechanism responsible for the heterogeneous expression of the gene along the cephalocaudal axis. RNA extracted from stomach, duodenum, jejunum, ileum, cecum and colon was hybridized by slot blot and Northern blot using a mdr3 cDNA probe. The regulation of gene expression was investigated examining the rate of transcription by nuclear run-off analysis. Transport studies of rhodamine 123, a substrate of P-glycoprotein, were performed in everted jejunum and ileum. The level of mdr3 mRNA and P-glycoprotein found in ileum was 6-fold higher than the level found in duodenum. The regional pattern of mdr3 gene expression is established in the intestine of 10-day-old animals. Similar mdr3 hybridization signal in nuclear run-off assay was found in nuclei of enterocytes isolated from jejunum and ileum, suggesting that the heterogeneous expression of the mdr3 gene in the cephalocaudal axis of the small bowel may be predominantly regulated at the post-transcriptional level. Transport rate of rhodamine 123 from the serosal to mucosal side in everted ileum was higher than the rate of transport found in jejunum. These results indicate that enterocytes of the ileum may be more actively involved in the P-glycoprotein-mediated transport of xenobiotics into the intestinal lumen.
- ItemDifferential expression of canalicular membrane Ca2+/Mg2+-ecto-ATPase in estrogen-induced and obstructive cholestasis in the rat(MOSBY, INC, 2000) Accatino, L; Pizarro, M; Solis, N; Arrese, M; Vollrath, V; Ananthanarayanan, M; Chianale, J; Koenig, CSExtracellular adenosine triphosphate (ATP) may regulate hepatocyte and cholangiocyte functions, and under some conditions it may have deleterious effects on bile secretion and cause cholestasis. The canalicular membrane enzyme Ca2+/Mg2+-ecto-ATPase (ecto-ATPase) hydrolyzes ATP/adenosine diphosphate (ATP/ADP) and regulates hepatic extracellular ATP concentration. Changes in liver ecto-ATPase in estrogen-induced cholestasis were examined in male rats receiving 17 alpha-ethinylestradiol (E groups) for 1, 3, or 5 days (5 mg/kg/day, sc) and compared with changes in rats subjected to obstructive cholestasis (O groups) for 1, 3, or 8 days. Activity of ecto-ATPase, protein mass in canalicular membranes and bile (estimated by Western blotting), steady state mRNA levels (by Northern blotting), and cellular and acinar distributions of the enzyme (histochemistry and immunocytochemistry) were assessed in these groups. Activity of ecto-ATPase, protein mass in isolated canalicular membranes, and enzyme mRNA levels were significantly increased in E group rats as compared with controls. In contrast, these parameters were markedly decreased in O group rats, and the enzyme protein was undetectable in bile. The ecto-ATPase histochemical reaction was markedly increased in the canalicular membrane of E group rats, extending from acinar zone 2 to zone 1,whereas it decreased in the O group. The ecto-ATPase immunocytochemical reaction was present in the canalicular membrane and pericanalicular vesicles in control and E group hepatocytes, but it decreased in obstructive cholestasis and was localized only to the canalicular membrane. Thus, significant changes in liver ecto-ATPase were apparent in 17 alpha-ethinylestradiol-induced cholestasis that were opposite to those observed in obstructive cholestasis. Assuming that the alterations observed in obstructive cholestasis are the result of the cholestatic phenomenon, we conclude that changes in ecto-ATPase in 17 alpha-ethinylestradiol-treated rats might be either primary events or part of an adaptive response in 17 alpha-ethinylestradiol-induced cholestasis.
- ItemFibrates induce mdr2 gene expression and biliary phospholipid secretion in the mouse(PORTLAND PRESS LTD, 1996) Chianale, J; Vollrath, V; Wielandt, AM; Amigo, L; Rigotti, A; Nervi, F; Gonzalez, S; Andrade, L; Pizarro, M; Accatino, LDisruption of the murine mdr2 gene leads to the complete absence of biliary phospholipids. We tested the hypothesis that the increase in biliary phospholipid output induced by fibrates is mediated via induction of the hepatic mdr2 gene and its encoded product, the P-glycoprotein canalicular flippase. Increased levels of mdr2 mRNA were observed in the liver of mice treated with different fibrates: ciprofibrate, 660+/-155% (as compared with control group); clofibrate, 611+/-77 %; bezafibrate, 410+/-47 %; fenofibrate, 310+/-52 %; gemfibrozil, 190+/-25 % (P < 0.05 compared with control group). Induction of expression of the mdr gene family was specific to the mdr2 gene. Two- to three-fold increases in P-glycoprotein immunodetection were evident on the canalicular plasma-membrane domain of clofibrate- and ciprofibrate-treated mice. Biliary phospholipid output increased from 4.2+/-1.2 nmol/min per g of liver in the control group to 8.5+/-0.6, 7.1+/-2.9 and 5.8+/-2.5 in ciprofibrate-, clofibrate- and bezafibrate-treated mice respectively (P < 0.05 compared with control group). Moreover, a significant correlation between biliary phospholipid output and the relative levels of mdr2 mRNA was found (r = 0.86; P < 0.05). In treated animals, bile flow as well as cholesterol and bile acid outputs remained unchanged. Our findings constitute the first evidence that pharmacological modulation of biliary lipid secretion mediated by fibrates can be related to the overexpression of a specific liver gene product, the mdr2 P-glycoprotein, and are consistent with the hypothesis that the mdr2 P-glycoprotein isoform plays a crucial role in the secretion of biliary phospholipid.
- ItemInduction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver(PORTLAND PRESS, 1999) Wielandt, AM; Vollrath, V; Manzano, M; Miranda, S; Accatino, L; Chianale, JThe canalicular multispecific organic anion transporter. cMoat, is an ATP-binding-cassette protein expressed in the canalicular domain of hepatocytes. In addition to the transport of endo- and xenobiotics, cMoat has also been proposed to transport GSH into bile, the major driving force of bile-acid-independent bile flow. We have shown previously that the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a peroxisome-proliferator agent, significantly increases bile-acid-independent bile flow in mice. On this basis, the effect of the herbicide on cMoat gene expression was studied. A 3.6-fold increase in cMoat mRNA levels and a 2.5-fold increase in cMoat protein content were observed in the liver of mice fed on a diet supplemented with 0.125% 2,4,5-T. These effects were due to an increased rate of gene transcription (3.9-fold) and were not associated with peroxisome proliferation. Significant increases in bile flow (2.23 +/- 0.39 versus 1.13 +/- 0.15 mu l/min per g of liver: P < 0.05) and biliary GSH output (7.40 +/- 3.30 versus 2.65 +/- 0.34 nmol/min per g of liver; P < 0.05) were observed in treated animals. The hepatocellular concentration of total glutathione also increased in hepatocytes of treated mice (10.95 +/- 0.84 versus 5.12 +/- 0.47 mM; P < 0.05), because of the induction (2.4-fold) of the heavy subunit of the gamma-glutamylcysteine synthetase (GCS-HS) gene. This is the first model of co-induction of cMoat and GCS-HS genes in vivo in the mouse liver, associated with increased glutathione synthesis and biliary glutathione output. Our observations are consistent with the hypothesis that the cMoat transporter plays a crucial role in the secretion of biliary GSH.
- ItemModulation of hepatic content and biliary excretion of P-glycoproteins in hepatocellular and obstructive cholestasis in the rat(1996) Accatino, L; Pizarro, M; Solis, N; Koenig, CS; Vollrath, V; Chianale, JBackground/Aims: Release into bile of canalicular membrane enzymes, such as alkaline phosphatase and gamma-glutamyl transpeptidase, is significantly increased in rats subjected to experimental models of hepatocellular or obstructive cholestasis. This effect appears to be related to a greater susceptibility of these membrane intrinsic proteins to the solubilizing effects of secreted bile acids. It is not known whether canalicular membrane transport proteins, such as P-glycoprotein isoforms, involved in ATP-dependent xenobiotic biliary excretion and phospholipid secretion, are excreted into bile and whether this process is modified in cholestasis. The aims of this work have been to investigate in the rat: a) whether P-glycoproteins are normally excreted into bile, b) whether their excretion is modified in two experimental models of cholestasis, i.e., hepatocellular cholestasis induced by ethynylestradiol and obstructive cholestasis, and c) whether observed changes correlate with bile acid and phospholipid secretion and enzyme release into bile and with relative P-glycoprotein content in hepatic tissue and isolated and purified canalicular membranes.
- ItemRole of Nrf2 in the regulation of the Mrp2 (ABCC2) gene(2006) Vollrath, V; Wielandt, AM; Iruretagoyena, M; Chianale, JThe Nrf2 (nuclear factor-erythroid 2 p45-related factor 2) transcription factor regulates gene expression of the GCLC (glutamate-cysteine ligase catalytic subunit), which is a key enzyme in glutathione synthesis, and GSTs (glutathione S-transferases) via the ARE (antioxidant-response element). The Mrp2 (multidrug-resistance protein 2) pump mediates the excretion of GSH and GSSG excretion as well as endo- and xeno-biotics that are conjugated with GSH, glucuronate or sulphate. Considering that Mrp2 acts synergistically with these enzymes, we hypothesized that the regulation of Mrp2 gene expression is also dependent on Nrf`2. Using BHA (butylated hydroxyanisole), which is a classical activator of the ARE-Nrf2 pathway, we observed an increase in the transcriptional activity of Mrp2, GCLC and Gstal/Gsta2 genes in the mouse liver. A similar pattern of co-induction of Mrp2 and GCLC genes was also observed in mouse (Hepa 1-6) and human (HepG2) hepatoma cells treated with BHA, beta-NF (beta-naphthoflavone), 2,4,5-T (trichlorophenoxyacetic acid) or 2AAF (2-acetylaminofluorene), suggesting that these genes share common mechanism(s) of transcriptional activation in response to exposure to xenobiotics. To define the mechanism of Mrp2 gene induction, the 5'-flanking region of the mouse Mrp2 gene (2.0 kb) was isolated, and two ARE-like sequences were found: ARE-2 (-1391 to -1381) and ARE-1 (-95 to -85). Deletion analyses demonstrated that the proximal region (-185 to +99) contains the elements for the basal expression and xenobiotic-mediated induction of the Mrp2 gene. Gel-shift and supershift assays indicated that Nrf2-protein complexes bind ARE sequences of the Mrp2 promoter, preferentially to the ARE-1 sequence. Overexpression of Nrf2 increased ARE-1-mediated CAT (chloramphenicol acetyltransferase) gene activity, while overexpression of mutant Nrf2 protein repressed the activity. Thus Nrf2 appears to regulate Mrp2 gene expression via an ARE element located at the proximal region of its promoter in response to exposure to xenobiotics.