Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1).
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.
Neuropharmacology Section, OASI Institute for Research and Care on Mental Retardation and Brain Aging, 94018 Troina, Italy.
Florinda
F
Gennuso
Cristina
C
Fernetti
Cataldo
C
Tirolo
Nuccio
N
Testa
Francesca
F
L'Episcopo
Salvo
S
Caniglia
Maria Concetta
MC
Morale
J Donald
JD
Ostrow
Lorella
L
Pascolo
Claudio
C
Tiribelli
Bianca
B
Marchetti
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects / Q000473:pathology; D001663:Bilirubin / Q000502:physiology* / Q000633:toxicity; D001921:Brain / Q000166:cytology; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006056:Golgi Apparatus / Q000502:physiology / Q000648:ultrastructure; D020024:Leukotriene Antagonists / Q000494:pharmacology; D051379:Mice; D011422:Propionates / Q000494:pharmacology; D021381:Protein Transport; D011804:Quinolines / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D020024:Leukotriene Antagonists; D011422:Propionates; D011804:Quinolines; C059141:verlukast; D001663:Bilirubin