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.

Figure 1

UCB induces neuronal nNOS expression in neurons, which is counteracted by l-NAME. Primary cultures of rat neurons were treated for 4 h at 37ºC with either 100 μmol/L UCB, 100 μmol/L l-NAME, UCB plus l-NAME or no addition (control), in the presence of 100 μmol/L HSA. The expression of nNOS was evaluated by Western blotting, as described in Materials and Methods. (A) Blot representative of at least four separate experiments. (B) The intensity of the bands was quantified by scanning densitometry, normalized with respect to endogenous β-actin and expressed as fold change compared with control. **P < 0.01 from control; ^$$P < 0.01 from UCB alone.

Figure 2

UCB induces nitrite formation in neurons, which is abrogated by l-NAME. Primary cultures of rat neurons were treated for 4 h at 37ºC with 100 μmol/L UCB, 100 μmol/L l-NAME, UCB plus l-NAME or no addition (control), in the presence of 100 μmol/L HSA. The formation of NO· was indirectly evaluated by measuring nitrites in the incubation medium using the Griess reagent, as described in Materials and Methods. **P < 0.01 from control; ^$$P < 0.01 from UCB alone.

Figure 3

UCB induces cGMP production in neurons, which is prevented by l-NAME. Primary cultures of rat neurons were treated for 4 h at 37ºC with 100 μmol/L UCB, 100 μmol/L l-NAME, UCB plus l-NAME or no addition (control), in the presence of 100 μmol/L HSA. Formation of cGMP was assessed by using a commercial kit, as described in Materials and Methods. **P < 0.01 from control; ^$P < 0.05 from UCB alone.

Figure 4

UCB induces protein oxidation in neurons, which is prevented by l-NAME. Primary cultures of rat neurons were treated for 4 h at 37ºC with either 100 μmol/L UCB, 100 μmol/L l-NAME, UCB plus l-NAME or no addition (control), in the presence of 100 μmol/L HSA. Protein oxidation was assessed by quantification of carbonyls by slot blot analysis, as described in Materials and Methods. **P < 0.01 from control; ^$P < 0.05 from UCB alone.

Figure 5

UCB impairs GSH homeostasis in neurons, which is restored by l-NAME. Primary cultures of rat neurons were treated for 4 h at 37ºC with 100 μmol/L UCB, 100 μmol/L l-NAME, UCB plus l-NAME or no addition (control), in the presence of 100 μmol/L HSA. Intracellular levels of reduced GSH were determined by an enzymatic recycling procedure, as described in Materials and Methods. *P < 0.05 from control; ^$P < 0.05 from UCB alone.

Figure 6

UCB impairs cell function in neurons, which is restored by l-NAME. Primary cultures of rat neurons were treated for 4 h at 37ºC with either 100 μmol/L UCB, 100 μmol/L l-NAME, UCB plus l-NAME or no addition (control), in the presence of 100 μmol/L HSA. Cell function was assessed by measurement of MTT conversion to a blue formazan product, as described in Materials and Methods. **P < 0.01 from control; ^$$P < 0.05 from UCB alone.

Figure 7

UCB induces neuronal death, which is prevented by l-NAME or by hemoglobin (Hb). Primary cultures of rat neurons were treated for 4 h at 37ºC with 100 μmol/L UCB, 100 μmol/L l-NAME, UCB plus l-NAME, 2 μmol/L Hb, UCB plus Hb or no addition (control), in the presence of 100 μmol/L HSA. Cell death was evaluated by measuring the activity of LDH released by nonviable cells to the incubation medium by using the LDH cytotoxicity detection kit, as described in Materials and Methods. **P < 0.01 from control; ^$P < 0.05 from UCB alone.

Figure 8

Schematic representation of key steps in neuronal oxidative damage by UCB. UCB interaction with neurons causes the release of the neurotransmitter glutamate, which stimulates the NMDA glutamate-subtype receptor. Upon stimulation of NMDA receptors, neuronal nNOS is activated and NO· is produced. NO· leads to increased formation of cGMP, through activation of soluble guanylate cyclase, and to disruption of the redox status. As a corollary of these events, mitochondrial function is impaired, ultimately leading to cell death. Inhibition of nNOS by l-NAME prevents downstream events from occurring, thus pointing to NO· as a key mediator in UCB-induced oxidative disruption. Preclusion of UCB-induced cell death by hemoglobin sequestration of NO· corroborates the involvement of this species in neuronal demise by UCB. Upstream blockade of NMDA receptors by MK-801 abrogates the neurotoxicity mediated by NO·, therefore indicating that excitotoxicity plays a key role in UCB-induced oxidative damage to neurons.