NMDA receptor activation increases free radical production through nitric oxide and NOX2

Abstract

Reactive oxygen species (ROS) and nitric oxide (NO) participate in NMDA receptor signaling. However, the source(s) of the ROS and their role in the increase in cerebral blood flow (CBF) induced by NMDA receptor activation have not been firmly established. NADPH oxidase generates ROS in neurons, but there is no direct evidence that this enzyme is present in neurons containing NMDA receptors, or that is involved in NMDA receptor-dependent ROS production and CBF increase. We addressed these questions using a combination of in vivo and in vitro approaches. We found that the CBF and ROS increases elicited by topical application of NMDA to the mouse neocortex were both dependent on neuronal NO synthase (nNOS), cGMP, and the cGMP effector kinase protein kinase G (PKG). In mice lacking the NADPH oxidase subunit NOX2, the ROS increase was not observed, but the CBF increase was still present. Electron microscopy of the neocortex revealed NOX2 immunolabeling in postsynaptic somata and dendrites that also expressed the NMDA receptor NR1 subunit and nNOS. In neuronal cultures, the NMDA-induced increase in ROS was mediated by NADPH oxidase through NO, cGMP and PKG. We conclude that NADPH oxidase in postsynaptic neurons generates ROS during NMDA receptor activation. However, NMDA receptor-derived ROS do not contribute to the CBF increase. The findings establish a NOX2-containing NADPH oxidase as a major source of ROS produced by NMDA receptor activation, and identify NO as the critical link between NMDA receptor activity and NOX2-dependent ROS production.

Figure 1.

Neocortical superfusion of NMDA increases local CBF and ROS production. A, Superfusion with NMDA (40 μm) increases CBF. The left y-axis lists CBF as percentage increase (relative to baseline before NMDA application) and the right y-axis lists raw CBF values (perfusion units). B, The increase in CBF induced by NMDA is stable over the 30 min experimental period. C, The increase in CBF induced by NMDA is blocked by topical application of the NMDA receptor antagonist MK-801 (200 μm) (control:16.0 ± 2.0; NMDA+MK-801: 16.3 ± 1.7 perfusion units; p > 0.05 from control) (*p < 0.05 from NMDA; n = 5/group). MK-801 reduced resting CBF (from 19 ± 1 to 16 ± 2 perfusion units; p < 0.05). Superfusion with kainate (10 μm) induces increases in CBF comparable to those elicited by NMDA. D, NMDA increases ROS at the superfusion site, an effect blocked by MK-801. Kainate does not increase ROS production (*p < 0.05; ANOVA and Tukey's test; n = 5/group).

Figure 2.

The increase in CBF and ROS induced by NMDA depends on nNOS-derived NO. A, The nNOS inhibitor 7-NI (50 mg/kg; i.p.) reduces resting CBF from 18.9 ± 1.4 to 16.7 ± 1.2 perfusion units (p < 0.05), and attenuates the increase in CBF induced by NMDA (control: 16.7 ± 1.2; NMDA + 7-NI: 17.6 ± 0.9 perfusion units; p > 0.05 from control) (*p < 0.05 from NMDA; t test; n = 5/group). B, 7-NI attenuates the increase in ROS induced by NMDA, but not that induced by topical application of AngII (50 nm) (*p < 0.05 from Ringer; ^#p < 0.05 from NMDA and AngII + 7-NI; ANOVA and Tukey's test; n = 5/group). C, NMDA does not increase CBF in nNOS^−/− mice (*p < 0.05 from nNOS^+/+; t test; n = 5/group). D, NMDA does not increase ROS in nNOS^−/− mice (*p < 0.05 from nNOS^+/+ and p > 0.05 from Ringer in F; ANOVA and Tukey's test; n = 5/group). E, Tat-NR2B9c, a peptide that disrupts the association between the NMDA receptor complex and nNOS, but not its scrambled control (sTat-NR2B9c), attenuates the increase in CBF induced by NMDA (*p < 0.05 from sTat-NR2B9c; n = 5/group). F, Tat-NR2B9c, but not sTat-NR2B9c, attenuated NMDA induced ROS production (*p < 0.05 from Ringer and Tat-NR2B9c; ANOVA and Tukey's test; n = 5/group).

Figure 3.

The sGC inhibitor ODQ and the PKG inhibitor KT-5823 attenuate the increase in CBF and ROS induced by NMDA. A, ODQ (100 μm) reduces resting CBF (vehicle 19 ± 2; ODQ: 16 ± 1 perfusion units; p < 0.05; n = 5) and attenuates the increase in CBF evoked by NMDA, an effect reversed by pCPT-cGMP (cGMP; 10 μm). KT-5823 (5 μm) also attenuates the CBF increase (^#p < 0.05 from NMDA and NMDA+ODQ+cGMP; ANOVA and Tukey's test; n = 5/group). B, ODQ reduces the increase in ROS evoked by NMDA, an effect reversed by cGMP. KT-5823 also attenuates the ROS increase (*p < 0.05 from Ringer; ^#p < 0.05 from Ringer; ANOVA and Tukey's test; n = 5/group).

Figure 4.

NMDA increases CBF, but not ROS production, in NOX2^−/− mice. A, The increases in CBF induced by NMDA in NOX2^−/− mice are attenuated by 7-NI (*p < 0.05 from NMDA; t test; n = 5/group). B, The increases in ROS induced by NMDA are not observed in NOX2^−/− mice. 7-NI attenuates the increase in ROS in NOX2^+/+ mice. (*p < 0.05 from NOX2^+/+ Ringer and NMDA + 7-NI; ANOVA and Tukey's test; n = 5/group). C, The ROS scavenger MnTBAP (100 μm) does not attenuate NMDA-induced CBF increase. D, MnTBAP blocks the NMDA-induced increase in ROS (*p < 0.05 from Ringer and NMDA+MnTBAP; ANOVA and Tukey's test; n = 5/group).

Figure 5.

Electron micrographs showing somatodendritic colocalization of NOX2 and nNOS (A) or NR1 (B, C) in somatosensory cortex. A shows immunogold labeling (small arrows) for NOX2 located near endomembranes (em) and mitochondria (m) in two somata that also exhibit diffuse cytoplasmic immunoperoxidase labeling for nNOS (Nox2/nNOS-so). Within the neuropil, NOX2-immunogold (arrows) is seen near endomembranes (em) in a small dendrite (Nox2-de) without nNOS labeling. B, C, immunogold labeling (small arrows for NR1 in the cytoplasmic compartment of dendrites showing dense aggregated endomembrane (em) labeling for NOX2 (NOX2/NR1-de). These dendrites also contain several mitochondria (m) and coated vesicles (cv). Scale bars, 500 nm.

Figure 6.

NMDA increases ROS in neuronal cultures via NO, cGMP, PKG, and NADPH oxidase. A, Bright-field images (a, d) and ROS-dependent fluorescence (DHE) (b, c, e, f) in neuronal cultures treated with vehicle (b), NMDA (40 μm) (c), MK-801 (5 μm) (e), or MK-801+NMDA (f). NMDA increases the ROS signal in neurons (arrows in a, b, c), but not after pretreatment with MK-801 (e, f). B, NMDA (40 μm) increases ROS production (n = 7). The increase is blocked by MK-801 (5 μm; n = 5), MnTBAP (100 μm; n = 9), the NOS inhibitor l-NNA (100 μm; n = 14), the peptide inhibitor of NADPH oxidase gp91ds-tat (gp91ds; 1 μm; n = 10), but not a scrambled control peptide (sgp91ds; 1 μm; n = 7) (*p < 0.05 from vehicle; ANOVA and Tukey's test). C, The increase in ROS induced by NMDA is attenuated by ODQ (100 μm; n = 10) or KT5823 (5 μm; n = 6) (*p < 0.05 from vehicle; ANOVA and Tukey's test). D, NMDA (40 μm) increases NO release. The increase is blocked by MK-801 or l-NNA (100 μm). gp91ds-tat (gp91ds; 1 μm), ODQ, and KT5823 do not affect NMDA-induced NO release (*p < 0.05 from NMDA; ANOVA and Tukey's test; n = 4/group). Scale bar, 10 μm.