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.
Highlights
Oxidative stress occurs when the production of reactive oxygen and/or nitrogen species overwhelms the antioxidant defense capacity [1]
When 100 μmol/L L-NAME was associated with unconjugated bilirubin (UCB) the values returned to levels similar to those observed in controls (1.1-fold, not significant), indicating that UCB-induced increased expression of neuronal NOS (nNOS) was blocked by approximately 90% (P < 0.01)
The results of the present study demonstrated that exposure of neurons to clinically relevant concentrations of UCB increases the expression of nNOS, as well as the production of nitrites and cyclic guanosine monophosphate (cGMP), which are accompanied by protein oxidation and cell demise, whereas the major cellular antioxidant defense system, provided by GSH, is impaired
Summary
Oxidative stress occurs when the production of reactive oxygen and/or nitrogen species overwhelms the antioxidant defense capacity [1]. Among the reactive nitrogen species is nitric oxide (NO·), a free radical formed by a group of constitutive and inducible nitric oxide synthase (NOS) enzymes [2]. NO· synthesis occurs in a complex sequence of events involving the stimulation of glutamate receptors in the postsynaptic neuron, especially the N-methyl-D-aspartate (NMDA) subtype, which leads to increased intracellular free Ca2+ that, upon binding to calmodulin, activates neuronal NOS (nNOS), originating NO· and L-citrulline from L-arginine and oxygen [3,4]. The excess NO· formed can permeate cell membranes and diffuse a distance of a few cell diameters from its site of synthesis to neighboring cells [3]; it can impair numerous biological events by reacting with various metals, thiols and oxygen species, modifying proteins, DNA and lipids, inducing neurotoxicity and playing a role in neurodegeneration [7]. NO· leads to mitochondrial dysfunction and consequent energy deficiency that may constitute a driving pathway to neurodegeneration [11]
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