Bilirubin induces apoptosis via activation of NMDA receptors in developing rat brain neurons.
Increased amounts of bilirubin, the end product of heme degradation, are known to be detrimental to the central nervous system, especially in preterm newborns. In an attempt to delineate the cellular mechanisms by which unconjugated bilirubin exerts its toxic effects on neuronal cells in the developing brain, bilirubin (0.25-5 microM) was added to the extracellular medium of 6-day-old primary cultured neurons from the embryonic rat forebrain, and cell alterations were studied over the ensuing 96 h. Bilirubin decreased cell viability dose dependently with an ED(50) around 1 microM. At the dose of 0.5 microM, it triggered delayed cell death that affected 24% of the neurons. Nuclear incorporation of the fluorescent dye DAPI (4,6-diamidino-2-phenylindole) depicted the presence of apoptosis (16%). Apoptosis features were confirmed by DNA fragmentation reflected by a progressive loss of [(3)H]thymidine and sequential changes in macromolecular synthesis, as shown by the time course of [(3)H]leucine incorporation, as well as by the beneficial effects of cycloheximide and caspase inhibitors. In parallel, treatments with glutamate receptor antagonists showed that MK-801, but not NBQX, protected neurons against bilirubin neurotoxicity, suggesting a role for NMDA receptors in bilirubin effects. Coupled with previous work about glutamate toxicity in the same culture model, these data support the hypothesis that low levels of free bilirubin may promote programmed neuronal death corresponding to an apoptotic process which involves caspase activation and requires the participation of NMDA receptors, along with bilirubin-induced inhibition of protein kinase C activity.
Grojean
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JE 2164, Université Henri Poincaré, 30 rue Lionnois, Nancy Cédex, 54013, France.
Koziel
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Vert
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Daval
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JL
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001663:Bilirubin / Q000633:toxicity*; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D007211:Indoles / Q000493:pharmacokinetics; D008297:Male; D009474:Neurons / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D009842:Oligopeptides / Q000494:pharmacology; D011247:Pregnancy; D016548:Prosencephalon / Q000166:cytology / Q000196:embryology; D011493:Protein Kinase C / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism / Q000494:pharmacology; D014316:Tritium
D015853:Cysteine Proteinase Inhibitors; D018691:Excitatory Amino Acid Antagonists; D005456:Fluorescent Dyes; D007211:Indoles; D009842:Oligopeptides; D011500:Protein Synthesis Inhibitors; D011810:Quinoxalines; D016194:Receptors, N-Methyl-D-Aspartate; C108637:aspartyl-glutamyl-valyl-aspartal; D014316:Tritium; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; C096429:L 709049; C007293:DAPI; D016291:Dizocilpine Maleate; D003513:Cycloheximide; D011493:Protein Kinase C; D020169:Caspases; D001663:Bilirubin; D013936:Thymidine