Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1)

Florinda Gennuso, Cristina Fernetti, Cataldo Tirolo, Nuccio Testa, Francesca L'Episcopo, Salvo Caniglia, Maria Concetta Morale, J. Donald Ostrow, Lorella Pascolo, Claudio Tiribelli, Bianca Marchetti

Research output: Contribution to journalArticle

120 Citations (Scopus)

Abstract

Unconjugated bilirubin (UCB) causes encephalopathy in severely jaundiced neonates by damaging astrocytes and neurons. Astrocytes, which help defend the brain against cytotoxic insults, express the ATP-dependent transporter, multidrug resistance-associated protein 1 (Mrp1), which mediates export of organic anions, probably including UCB. We therefore studied whether exposure to UCB affects the expression and intracellular localization of Mrp1 in cultured mouse astroglial cells (>95% astrocytes). Mrp1 was localized and quantitated by confocal laser scanning microscopy and double immunofluorescence labeling by using specific antibodies against Mrp1 and the astrocyte marker glial fibrillary acidic protein, plus the Golgi marker wheat germ agglutinin (WGA). In unexposed astrocytes, Mrp1 colocalized with WGA in the Golgi apparatus. Exposure to UCB at a low unbound concentration (Bf) of 40 nM caused rapid redistribution of Mrp1 from the Golgi throughout the cytoplasm to the plasma membrane, with a peak 5-fold increase in Mrp1 immunofluorescence intensity from 30 to 120 min. Bf above aqueous saturation produced a similar but aborted response. Exposure to this higher Bf for 16 h markedly decreased Trypan blue exclusion and methylthiazoletetrazoilum activity and increased apoptosis 5-fold by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. These toxic effects were modestly increased by inhibition of Mrp1 activity with 3-([3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl-(3-dimethylamino-3-oxopropyl) -thio-methyl]thio)propanoic acid (MK571). By contrast, Bf = 40 nM caused injury only if Mrp1 activity was inhibited by MK571, which also blocked translocation of Mrp1. Our conclusion is that in astrocytes, UCB up-regulates expression of Mrp1 and promotes its trafficking from the Golgi to the plasma membrane, thus moderating cytotoxicity from UCB, presumably by limiting its intracellular accumulation.

Original languageEnglish
Pages (from-to)2470-2475
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number8
DOIs
Publication statusPublished - Feb 22 2004

Fingerprint

Bilirubin
Astrocytes
Up-Regulation
Wheat Germ Agglutinins
Fluorescent Antibody Technique
multidrug resistance-associated protein 1
Cell Membrane
Kernicterus
Trypan Blue
DNA Nucleotidylexotransferase
Poisons
Glial Fibrillary Acidic Protein
Golgi Apparatus
Jaundice
Confocal Microscopy
Anions
Cytoplasm
Adenosine Triphosphate
Apoptosis
Neurons

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

@article{4aed5e43176f4d7d8d7ae880be2d1f1b,
title = "Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1)",
abstract = "Unconjugated bilirubin (UCB) causes encephalopathy in severely jaundiced neonates by damaging astrocytes and neurons. Astrocytes, which help defend the brain against cytotoxic insults, express the ATP-dependent transporter, multidrug resistance-associated protein 1 (Mrp1), which mediates export of organic anions, probably including UCB. We therefore studied whether exposure to UCB affects the expression and intracellular localization of Mrp1 in cultured mouse astroglial cells (>95{\%} astrocytes). Mrp1 was localized and quantitated by confocal laser scanning microscopy and double immunofluorescence labeling by using specific antibodies against Mrp1 and the astrocyte marker glial fibrillary acidic protein, plus the Golgi marker wheat germ agglutinin (WGA). In unexposed astrocytes, Mrp1 colocalized with WGA in the Golgi apparatus. Exposure to UCB at a low unbound concentration (Bf) of 40 nM caused rapid redistribution of Mrp1 from the Golgi throughout the cytoplasm to the plasma membrane, with a peak 5-fold increase in Mrp1 immunofluorescence intensity from 30 to 120 min. Bf above aqueous saturation produced a similar but aborted response. Exposure to this higher Bf for 16 h markedly decreased Trypan blue exclusion and methylthiazoletetrazoilum activity and increased apoptosis 5-fold by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. These toxic effects were modestly increased by inhibition of Mrp1 activity with 3-([3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl-(3-dimethylamino-3-oxopropyl) -thio-methyl]thio)propanoic acid (MK571). By contrast, Bf = 40 nM caused injury only if Mrp1 activity was inhibited by MK571, which also blocked translocation of Mrp1. Our conclusion is that in astrocytes, UCB up-regulates expression of Mrp1 and promotes its trafficking from the Golgi to the plasma membrane, thus moderating cytotoxicity from UCB, presumably by limiting its intracellular accumulation.",
author = "Florinda Gennuso and Cristina Fernetti and Cataldo Tirolo and Nuccio Testa and Francesca L'Episcopo and Salvo Caniglia and Morale, {Maria Concetta} and Ostrow, {J. Donald} and Lorella Pascolo and Claudio Tiribelli and Bianca Marchetti",
year = "2004",
month = "2",
day = "22",
doi = "10.1073/pnas.0308452100",
language = "English",
volume = "101",
pages = "2470--2475",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "8",

}

TY - JOUR

T1 - Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1)

AU - Gennuso, Florinda

AU - Fernetti, Cristina

AU - Tirolo, Cataldo

AU - Testa, Nuccio

AU - L'Episcopo, Francesca

AU - Caniglia, Salvo

AU - Morale, Maria Concetta

AU - Ostrow, J. Donald

AU - Pascolo, Lorella

AU - Tiribelli, Claudio

AU - Marchetti, Bianca

PY - 2004/2/22

Y1 - 2004/2/22

N2 - Unconjugated bilirubin (UCB) causes encephalopathy in severely jaundiced neonates by damaging astrocytes and neurons. Astrocytes, which help defend the brain against cytotoxic insults, express the ATP-dependent transporter, multidrug resistance-associated protein 1 (Mrp1), which mediates export of organic anions, probably including UCB. We therefore studied whether exposure to UCB affects the expression and intracellular localization of Mrp1 in cultured mouse astroglial cells (>95% astrocytes). Mrp1 was localized and quantitated by confocal laser scanning microscopy and double immunofluorescence labeling by using specific antibodies against Mrp1 and the astrocyte marker glial fibrillary acidic protein, plus the Golgi marker wheat germ agglutinin (WGA). In unexposed astrocytes, Mrp1 colocalized with WGA in the Golgi apparatus. Exposure to UCB at a low unbound concentration (Bf) of 40 nM caused rapid redistribution of Mrp1 from the Golgi throughout the cytoplasm to the plasma membrane, with a peak 5-fold increase in Mrp1 immunofluorescence intensity from 30 to 120 min. Bf above aqueous saturation produced a similar but aborted response. Exposure to this higher Bf for 16 h markedly decreased Trypan blue exclusion and methylthiazoletetrazoilum activity and increased apoptosis 5-fold by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. These toxic effects were modestly increased by inhibition of Mrp1 activity with 3-([3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl-(3-dimethylamino-3-oxopropyl) -thio-methyl]thio)propanoic acid (MK571). By contrast, Bf = 40 nM caused injury only if Mrp1 activity was inhibited by MK571, which also blocked translocation of Mrp1. Our conclusion is that in astrocytes, UCB up-regulates expression of Mrp1 and promotes its trafficking from the Golgi to the plasma membrane, thus moderating cytotoxicity from UCB, presumably by limiting its intracellular accumulation.

AB - Unconjugated bilirubin (UCB) causes encephalopathy in severely jaundiced neonates by damaging astrocytes and neurons. Astrocytes, which help defend the brain against cytotoxic insults, express the ATP-dependent transporter, multidrug resistance-associated protein 1 (Mrp1), which mediates export of organic anions, probably including UCB. We therefore studied whether exposure to UCB affects the expression and intracellular localization of Mrp1 in cultured mouse astroglial cells (>95% astrocytes). Mrp1 was localized and quantitated by confocal laser scanning microscopy and double immunofluorescence labeling by using specific antibodies against Mrp1 and the astrocyte marker glial fibrillary acidic protein, plus the Golgi marker wheat germ agglutinin (WGA). In unexposed astrocytes, Mrp1 colocalized with WGA in the Golgi apparatus. Exposure to UCB at a low unbound concentration (Bf) of 40 nM caused rapid redistribution of Mrp1 from the Golgi throughout the cytoplasm to the plasma membrane, with a peak 5-fold increase in Mrp1 immunofluorescence intensity from 30 to 120 min. Bf above aqueous saturation produced a similar but aborted response. Exposure to this higher Bf for 16 h markedly decreased Trypan blue exclusion and methylthiazoletetrazoilum activity and increased apoptosis 5-fold by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. These toxic effects were modestly increased by inhibition of Mrp1 activity with 3-([3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl-(3-dimethylamino-3-oxopropyl) -thio-methyl]thio)propanoic acid (MK571). By contrast, Bf = 40 nM caused injury only if Mrp1 activity was inhibited by MK571, which also blocked translocation of Mrp1. Our conclusion is that in astrocytes, UCB up-regulates expression of Mrp1 and promotes its trafficking from the Golgi to the plasma membrane, thus moderating cytotoxicity from UCB, presumably by limiting its intracellular accumulation.

UR - http://www.scopus.com/inward/record.url?scp=10744221309&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=10744221309&partnerID=8YFLogxK

U2 - 10.1073/pnas.0308452100

DO - 10.1073/pnas.0308452100

M3 - Article

VL - 101

SP - 2470

EP - 2475

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 8

ER -