Neuronal Nitric Oxide Synthase Activation and Peroxynitrite Formation in Ischemic Stroke Linked to Neural Damage

Abstract

Nitric oxide (NO) is a new intercellular messenger that occurs naturally in the brain without causing overt toxicity. Yet, NO has been implicated as a mediator of cell death in cell death. One explanation is that ischemia causes overproduction of NO, allowing it to react with superoxide to form the potent oxidant peroxynitrite. To address this question, we used immunohistochemistry for citrulline, a marker for NO synthase activity, and 3-nitrotyrosine, a marker for peroxynitrite formation, in mice subjected to reversible middle cerebral artery occlusion. We show that ischemia triggers a marked augmentation in citrulline immunoreactivity but more so in the peri-infarct than the infarcted tissue. This increase is attributable to the activation of a large population (approximately 80%) of the neuronal isoform of NO synthase (nNOS) that is catalytically inactive during basal conditions, indicating a tight regulation of physiological NO production in the brain. In contrast, 3-nitrotyrosine immunoreactivity is restricted to the infarcted tissue and is not present in the peri-infarct tissue. In nNOS(Delta/Delta) mice, known to be protected against ischemia, no 3-nitrotyrosine immunoreactivity is detected. Our findings provide a cellular localization for nNOS activation in association with ischemic stroke and establish that NO is not likely a direct neurotoxin, whereas its conversion to peroxynitrite is associated with cell death.

Influence of duration of focal cerebral ischemia and neuronal nitric oxide synthase on translocation of apoptosis-inducing factor to the nucleus

“…Nuclear staining for AIF has also been reported in the rat brain at 4 hours of reperfusion after 1 hour of MCAO (Ferrer et al, 2003). An increase in the production of reactive oxygen species (ROS) leads to DNA damage and activation of PARP after MCAO (Zhang et al, 1994;Eliasson et al, 1999). In neuronal culture, activation of PARP is linked to a signaling process that translocates AIF from the mitochondria to the nucleus (Yu et al, 2002;Wang et al, 2004).…”

“…In experimental stroke, both nNOS and PARP activity were found to be increased (Eliasson et al, 1997;Endres et al, 1997;Eliasson et al, 1999;Goyagi et al, 2003), and gene deletion or pharmacologic inhibition of nNOS or PARP-1 reduced infarct volume (Huang et al, 1994;Eliasson et al, 1997;Endres et al, 1997;Goto et al, 2002). In addition, inhibition of PARP decreased AIF translocation, and knockdown of AIF in Harlequin mice reduced infarct volume after 45 minutes of MCAO (Culmsee et al, 2005).…”

“…Activation of PARP, in turn, is triggered by damage to DNA from oxidizing agents, such as peroxynitrite formed from nitric oxide (NO) and superoxide (Zhang et al, 1994). Gene deletion of neuronal NO synthase (nNOS) results in decreased formation of peroxynitrite (Eliasson et al, 1999) and decreased formation of poly(ADP-ribose) polymers (PAR) by PARP activity after MCAO (Endres et al, 1998). The second objective of the present study was to determine the role of nNOS in nuclear AIF translocation.…”

Modulation of multiple pathways involved in the maintenance of neuronal function during aging by fisetin

“…Peroxynitrite levels increase during aging and may contribute to some of the nerve cell damage associated with normal aging [27]. In addition, peroxynitrite-mediated toxicity has been implicated in many age-related neurological disorders including ischemic stroke [26], AD, Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) (reviewed in [87]). Although peroxynitrite itself is not a free radical, it is a uniquely damaging molecule because it can initiate strong oxidation reactions through decomposition into a hydroxyl radical and nitrogen dioxide [5].…”

Inhibition of Skeletal Muscle S1-Myosin ATPase by Peroxynitrite

“…Furthermore, Andrade et al (4) have shown that exposure of muscle fibers to low peroxide concentrations induced an altered cross-bridge kinetics. Because peroxynitrite is a major oxidative agent in ischemia/reperfusion insults such as heart infarct or atrial fibrillation (1,5,6) and produces a reaction likely to be irreversible in vivo, the nitration of protein tyrosines to 3-nitrotyrosine (7)(8)(9), the level of protein nitration has gained acceptance as a fingerprint of in vivo peroxynitrite-mediated oxidative-stress damage (8). Recent studies have shown that myosin is one of the muscle proteins showing higher levels of 3-nitrotyrosine after atrial fibrillation (1,6) and aging (10).…”