Increased levels of the multidrug resistance protein in lateral membranes of proliferating hepatocyte-derived cells

Roelofsen, Han; Vos, T. A.; Schippers, IJ; Kuipers, Folkert; Koning, Henk; Moshage, Han; Jansen, Petér L. M.; Müller, Marius

doi:10.1053/gast.1997.v112.pm9024305

Cited by 132 publications

(105 citation statements)

References 6 publications

(8 reference statements)

Supporting

Mentioning

101

Contrasting

Order By: Relevance

“…Specific polyclonal antibodies against mrp1 (k5) were raised by immunizing rabbits with the peptide, RGLFYSMAKDA-GLV, located at the carboxy terminal of human MRP1. This antibody does not cross-react with mrp2, as described by Roelofsen et al 12 In addition, no cross-reactivity with rat mrp3 was found (Roelofsen et al, February 1997, unpublished data). Polyclonal antibodies against mrp2 (k4) were raised by immunizing rabbits with the peptide, AKEAGIENVNHTEL, located at the carboxy terminal of rat mrp2.…”

Section: Methodssupporting

confidence: 53%

“…9,10 MRP1/mrp1 is present at very low levels at the lateral membrane of the hepatocyte. 11,12 The human MRP1 and its murine homologue, mrp1, have been shown to confer multidrug resistance to natural product drugs. 13,14 Moreover, MRP1 is able to transport glutathione disulfide 15 and glutathione (GSH) S-conjugates including the lipid peroxidation products, leukotriene C 4 16,17 and 4-hydroxynonenal S-GSH.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Up–Regulation of the Multidrug Resistance Genes, Mrp1 And Mdr1b , and Down–Regulation of the Organic Anion Transporter, Mrp2, and the Bile Salt Transporter, Spgp, in Endotoxemic Rat Liver

Vos¹,

Hooiveld²,

Koning³

et al. 1998

Hepatology

333

View full text Add to dashboard Cite

Endotoxin-induced cholestasis is mainly caused by an impaired canalicular secretion. Mrp2, the canalicular multispecific organic anion transporter, is strongly downregulated in this situation, and canalicular bile salt secretion is also reduced. We hypothesized that other adenosine triphosphate-binding cassette (ABC) transporters may compensate for the decreased transport activity to protect the cell from cytokine-induced oxidative damage. Therefore, we examined the expression of ABC-transport proteins in membrane fractions of whole liver and of isolated hepatocytes of endotoxin-treated rats and performed reversetranscriptase polymerase chain reaction (RT-PCR) on mRNA isolated from these livers. In addition, the localization of these transporters was examined using confocal scanning laser microscopy. By 6 hours after endotoxin administration, we found a clear increase of mrp1 mRNA and protein, whereas mrp2 mRNA and protein were decreased. This was confirmed in isolated hepatocytes. In addition, mdr1b mRNA was strongly increased, whereas mdr1a and mdr2 mRNA did not change significantly. Both the mRNA and protein levels of the sister of P-glycoprotein (spgp), the recently cloned bile salt transporter, decreased. After endotoxin treatment, the normally sharply delineated canalicular staining of mrp2 and spgp had changed to a fuzzy pattern, suggesting localization in a subapical compartment. We conclude that endotoxin-induced cholestasis is caused by decreased mrp2 and spgp levels, as well as an abnormal localization of these proteins. The simultaneous up-regulation of mrp1 and mdr1b may confer resistance to hepatocytes against cytokine-induced metabolic stress. (HEPATOLOGY 1998;28:1637-1644.)Excretion of a large variety of endogenous and exogenous compounds from hepatocytes into bile is an adenosine triphosphate (ATP)-dependent process, predominantly performed by members of the P-glycoprotein (Pgp) subfamily and the multidrug-resistance protein (MRP) subfamily of the ATP-binding cassette (ABC) protein superfamily. 1,2 At least four members of the Pgp subfamily are located at the canalicular membrane of rodent liver: mdr1a, mdr1b, mdr2, and spgp. In normal rodent liver, mdr1a and mdr1b are present at low levels. Overexpression of mouse mdr1a/mdr1b confers multidrug resistance against a broad variety of natural product drugs. 3,4 The main physiological function of these multidrug-resistance proteins is presumably the transport of bulky amphiphilic compounds, such as cationic drugs, hydrophobic peptides, steroids, and atypical glycolipids, across the canalicular membrane. 1,4,5 The expression of mdr2 in normal rodent liver is high. This transporter functions as a flippase that translocates phosphatidylcholine across the membrane. 6 From pig liver, Childs et al. cloned part of another member of the Pgp subfamily: the sister gene of Pgp (spgp). 7 Most recently, Gerloff et al. cloned the full-length cDNA of spgp from rat liver. 8 They provided evidence that spgp, which is exclusively present in the liver, most likely is ...

show abstract

Section: Methodssupporting

confidence: 53%

mentioning

confidence: 99%

Up–Regulation of the Multidrug Resistance Genes, Mrp1 And Mdr1b , and Down–Regulation of the Organic Anion Transporter, Mrp2, and the Bile Salt Transporter, Spgp, in Endotoxemic Rat Liver

Vos¹,

Hooiveld²,

Koning³

et al. 1998

Hepatology

333

View full text Add to dashboard Cite

show abstract

“…21,25 MRP1, a MRP family member with a similar substrate specificity as MRP2, 22 has been considered as an isoform in the basolateral membrane of hepatocyte-derived cells. 42 Immunoblotting and immunofluorescence microscopy have suggested a low expression of MRP1 in normal hepatocytes and an enhanced expression in proliferating hepatocytederived cells. 42 However, Northern blot analysis and RNase protection assays 18 did not provide evidence for expression of MRP1 mRNA in human liver.…”

Section: Discussionmentioning

confidence: 99%

“…42 Immunoblotting and immunofluorescence microscopy have suggested a low expression of MRP1 in normal hepatocytes and an enhanced expression in proliferating hepatocytederived cells. 42 However, Northern blot analysis and RNase protection assays 18 did not provide evidence for expression of MRP1 mRNA in human liver. Recent studies rather showed that, in contrast to MRP1, another isoform is expressed in liver.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Human Multidrug Resistance Protein Isoform Mrp3 Localized to the Basolateral Hepatocyte Membrane

et al. 1999

View full text Add to dashboard Cite

The membrane proteins mediating the adenosine triphosphate (ATP)-dependent transport of anionic conjugates and amphiphilic anions, such as the bilirubin glucuronosides, 1 have been identified as members of the multidrug resistance protein (MRP) family. 2-11 Export pumps for anionic conjugates in yeast, 12 plants, 13 and Caenorhabditis elegans 14 were subsequently recognized too as members of the MRP family of ATP-dependent membrane transporters (for reviews Keppler and Kö nig 15 and Ishikawa et al. 16 ). In humans, six members of the MRP family, designated MRP1 through 6, have been identified. 17,18 At present, two of these, human MRP1 19 and MRP2,7,20,21 have been functionally characterized as conjugate export pumps. 2,3,10,11,22 The occurrence of distinct MRP isoforms in hepatocytes has been initially shown by amplification and sequencing of novel complementary DNA (cDNA) fragments, by immunoblotting, and by immunofluorescence microscopy. 5 The canalicular MRP isoform deficient in Dubin-Johnson syndrome was identified as MRP2, 21,23,24 however, the molecular identity of the MRP isoform(s) localized to the lateral or basolateral hepatocyte membrane 5,23,25 remained uncertain. RNase protection assays indicated that the liver expresses MRP2, MRP3, MRP5, and MRP6, but hardly MRP1. 18,26 Identification of MRP isoforms in liver by immunoblotting or immunofluorescence microscopy has been difficult because of the cross-reactivity of some antibodies with different MRP isoforms. 5,7,23 MRP3 has been recognized as a prominent MRP family member expressed in rat liver. 26,27 Human MRP3 has been cloned recently by Kiuchi et al. 28 and by our group (GenBank/EMBL accession Y17151 and this article), however, its localization and function in the hepatocyte remained unknown. It has been our hypothesis that a MRP isoform localized to the lateral or basolateral membrane may serve to excrete conjugates, which are formed inside the hepatocyte, into the sinusoidal blood under conditions of extrahepatic cholestasis or hereditary deficiency of the apical MRP isoform, MRP2. 5,25 Such a compensatory excretion across the basolateral membrane may be fulfilled by basolateral organic anion transporters functioning bidirectionally, in addition to MRP isoforms localized to the basolateral membrane domain. Cloning of MRP3 in the present study has enabled us to raise antibodies specifically directed against this MRP isoform and to localize MRP3 to the basolateral membrane domain of human hepatocytes. MATERIALS AND METHODSMaterials. Aprotinin, leupeptin, pepstatin, fetal calf serum, agar, and the protein standard mixture (M r 26,600 to 180,000) for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were from Sigma (Deisenhofen, Germany). Agarose was from Roth (Karlsruhe, Germany) and lysozyme and ampicillin were from Boehringer Mannheim (Mannheim, Germany). Marathon-Ready cDNA and Advantage cDNA Polymerase Mix were from Clontech (Heidelberg, Germany). RNase inhibitor RNAguard, StrataScript Moloney murine leukemia virus reverse trans...

show abstract

“…These cells are well differentiated such that they retain specific hepatic functions such as albumin and bile acid synthesis (14,15). In monolayer cultures, bile caniculus-like structures, apical vacuoles, can be observed optically, and the uptake of fluorescent glutathione conjugates into these structures has been reported (16,17). Because these cells originate from a malignant source, they express high levels of three of the multidrug resistance protein (MRP) members including MRP1, whose expression level is usually extremely low in normal hepatocytes (16,18).…”

mentioning

confidence: 99%

Menadione metabolism to thiodione in hepatoblastoma by scanning electrochemical microscopy

Mauzeroll

Bard

Owhadian

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The cytotoxicity of menadione on hepatocytes was studied by using the substrate generation͞tip collection mode of scanning electrochemical microscopy by exposing the cells to menadione and detecting the menadione-S-glutathione conjugate (thiodione) that is formed during the cellular detoxication process and is exported from the cell by an ATP-dependent pump. This efflux was electrochemically detected and allowed scanning electrochemical microscopy monitoring and imaging of single cells and groups of highly confluent live cells. Based on a constant flux model, Ϸ6 ؋ 10 6 molecules of thiodione per cell per second are exported from monolayer cultures of Hep G2 cells.electrochemistry ͉ SECM ͉ oxidative stress ͉ ultramicroelectrode ͉ glutathione C ytotoxic effects of menadione on hepatocytes were investigated by scanning electrochemical microscopy (SECM), a technique that allows one to detect electroactive species at an ultramicroelectrode tip that can be positioned with high resolution. Here, the substrate generation͞tip collection mode of SECM was used to monitor the concentration of thiodione from both highly confluent and isolated Hep G2 cells. Thiodione is a biological metabolite that is actively exported out of the cells by a glutathione S-conjugate pump after the application of oxidative stress by menadione on the hepatocytes (Fig. 1). The efflux of thiodione from both isolated and highly confluent (75-100%) cells can be imaged by SECM, and time profiles of the export can also be obtained after exposure to a cytotoxic concentration of menadione. A simplified model was used to treat the concentration of thiodione from highly confluent cells and to estimate the flux per cell. A similar model has previously been applied to an electrochemical study of doxorubicin transport from Chinese hamster ovarian cells (1).SECM has previously been used with mammalian and other cells and has provided useful information about the permeability of cellular membranes to a wide variety of redox couples. The regeneration reaction of different mediators at human breast cells (2) and Rhodobacter sphaeroides (3) was monitored by the feedback mode of SECM, allowing one to distinguish between normal and malignant cells. A theoretical kinetic treatment of these processes is reported in ref. [4][5][6][7][8][9]) also carried out live-cell studies and monitored respiration rate changes from oxygen reduction profiles for different cell types. In these experiments, oxygen present in solution is consumed by the living organism, so that its concentration is lower near the cell surface. This consumption leads to a lower measured oxygen reduction current when the tip is in close proximity to the cells. These SECM studies monitoring efflux from cells used the feedback approach. Menadione (10) has been used in previous studies because it readily enters the cell through the cell membrane, and we have used it in a study closely related to this one involving the use of SECM to monitor the behavior of yeast cells by using a substrate generation͞tip coll...

show abstract

Increased levels of the multidrug resistance protein in lateral membranes of proliferating hepatocyte-derived cells

Cited by 132 publications

References 6 publications

Up–Regulation of the Multidrug Resistance Genes, Mrp1 And Mdr1b , and Down–Regulation of the Organic Anion Transporter, Mrp2, and the Bile Salt Transporter, Spgp, in Endotoxemic Rat Liver

Up–Regulation of the Multidrug Resistance Genes, Mrp1 And Mdr1b , and Down–Regulation of the Organic Anion Transporter, Mrp2, and the Bile Salt Transporter, Spgp, in Endotoxemic Rat Liver

Characterization of the Human Multidrug Resistance Protein Isoform Mrp3 Localized to the Basolateral Hepatocyte Membrane

Menadione metabolism to thiodione in hepatoblastoma by scanning electrochemical microscopy

Contact Info

Product

Resources

About