Molecular Medicine

, Volume 16, Issue 9–10, pp 372–380 | Cite as

N-Methyl-d-Aspartate Receptor and Neuronal Nitric Oxide Synthase Activation Mediate Bilirubin-Induced Neurotoxicity

  • Maria A. Brito
  • Ana R. Vaz
  • Sandra L. Silva
  • Ana S. Falcão
  • Adelaide Fernandes
  • Rui F. M. Silva
  • Dora Brites
Research Article


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 µmol/L UCB, in the presence of 100 µmol/L human serum albumin for 4 h at 37°C, 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.



This work was supported by grant PTDC/SAU-NEU/64385/2006, from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal, and European Regional Development Fund (FEDER) (to D Brites).


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Copyright information

© The Feinstein Institute for Medical Research 2010

Authors and Affiliations

  • Maria A. Brito
    • 1
  • Ana R. Vaz
    • 1
  • Sandra L. Silva
    • 1
  • Ana S. Falcão
    • 1
  • Adelaide Fernandes
    • 1
  • Rui F. M. Silva
    • 1
  • Dora Brites
    • 1
  1. 1.Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farmácia daUniversity of LisbonLisboaPortugal

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