title: N-methyl-aspartate receptor and neuronal nitric oxide synthase activation mediate bilirubin-induced neurotoxicity.
abstract: Hyperbilirubinemia may lead to neurotoxicity and neuronal death. Although the mechanisms of nerve cell damage by unconjugated bilirubin (UCB) appear to involve a disruption of the redox status and excitotoxicity, the contribution of nitric oxide (NO·) and of N-methyl-D-aspartate (NMDA) glutamate receptors is unclear. We investigated the role of NO· and NMDA glutamate receptors in the pathways of nerve cell demise by UCB. Neurons were incubated with 100 micromol/L UCB, in the presence of 100 micromol/L human serum albumin for 4 h at 37ºC, alone or in combination with N-ω-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of neuronal nitric oxide synthase [nNOS]), hemoglobin (an NO· scavenger) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (an NMDA-receptor antagonist). Exposure to UCB led to increased expression of nNOS and production of both NO· and cyclic guanosine 3',5'-monophosphate (cGMP), along with protein oxidation and depletion of glutathione. These events concurred for cell dysfunction and death and were counteracted by L-NAME. Moreover, the UCB-induced loss of neuronal viability was abolished by hemoglobin, whereas the activation of nNOS and production of both NO· and cGMP were counteracted by MK-801, resulting in significant protection from cell dysfunction and death. These results reinforce the involvement of oxidative stress by showing that nerve cell damage by UCB is mediated by NO· and therefore is counteracted by NO· inhibitors or scavengers. Our findings strongly suggest that the activation of nNOS and neurotoxicity occur through the engagement of NMDA receptors. These data reveal a role for overstimulation of glutamate receptors in mediating oxidative damage by UCB.
authors.affiliation: Research Institute for Medicines and Pharmaceutical Sciences, Faculdade de Farmácia, University of Lisbon, Lisbon, Portugal. abrito@ff.ul.pt
authors.forename: Maria A
authors.initials: MA
authors.lastname: Brito
authors.forename: Ana R
authors.initials: AR
authors.lastname: Vaz
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Silva
authors.forename: Ana S
authors.initials: AS
authors.lastname: Falcão
authors.forename: Adelaide
authors.initials: A
authors.lastname: Fernandes
authors.forename: Rui F M
authors.initials: RF
authors.lastname: Silva
authors.forename: Dora
authors.initials: D
authors.lastname: Brites
mesh_terms: D000818:Animals; D001663:Bilirubin / Q000633:toxicity*; D016923:Cell Death / Q000187:drug effects; D006152:Cyclic GMP / Q000096:biosynthesis; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects; D008954:Models, Biological; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology* / Q000473:pathology; D009498:Neurotoxins / Q000633:toxicity*; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000096:biosynthesis / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*
chemical_list: D009498:Neurotoxins; D009573:Nitrites; D016194:Receptors, N-Methyl-D-Aspartate; D009569:Nitric Oxide; D016291:Dizocilpine Maleate; D052248:Nitric Oxide Synthase Type I; D005978:Glutathione; D006152:Cyclic GMP; D001663:Bilirubin; D019331:NG-Nitroarginine Methyl Ester