Reactive nitrogen species

Nitric oxide (NO) influences many aspects of the normal physiology or pathophysiology of the CNS being either beneficial or detrimental to the nervous tissue. It is produced from oxidation of L-arginine to L-citrulline via an enzymatically catalyzed process (Bredt 1999).

The enzymes that produce NO have been divided into two classes depending on their special features: constitutive and inducible NO synthases (Wang and Marsden 1995). The brain expresses all three identified nitric oxide synthases (NOS) isoforms. Moreover NOS activity in the brain is higher than in any other tissue of the body (Duncan and Heales 2005).

The constitutively expressed isoforms of nitric oxide synthase (cNOS) include neuronal NOS (nNOS or type 1) and endothelial NOS (eNOS or type 3). Their function is dependent on the changes in intracellular Ca²⁺ levels and binding to calmoduline (Moncada et al. 1995).

These enzymes can produce relatively small amounts of NO, mainly under physiological

conditions. Nitric oxide has several physiological functions in the CNS. It can act as a neurotransmitter, as well as co-transmitter. A subset of neurons termed as “nitrergic nerves”

were shown to release NO (Moncada et al. 1995) and a large body of evidence suggests that these neurons play a role in the control of neuroendocrine secretion in the hypothalamus, behavioural changes, as well as in learning and memory (Holscher 1997). NO’s role as a neurotransmitter in the hippocampus appears to be important in the phenomenon of “long term potentiation”, which is a form of synaptic plasticity associated with learning and memory (Arancio et al. 1996). NO produced by eNOS also functions as a vasodilator in the brain, acting on smooth muscle cells of the arteries and arterioles. It regulates the local blood flow and is believed to help to preserve the cerebral blood flow in cases of brain ischemia (Alderton et al. 2001). The role in maintaining uninterrupted circulation is vital, since the brain (and especially neurons) have high metabolic demand and do not tolerate reduced blood flow (Sims and Anderson 2002).

In contrary to the constitutive nNOS and eNOS, the expression of the inducible form of the enzyme (iNOS or type 2) is evoked only by appropriate stimuli such as bacterial lipopolysaccharide (LPS), pro-inflammatory cytokines (IFN-γ, IL-1β, TNF-α, IL-6) (Forstermann and Kleinert 1995; MacMicking et al. 1997). iNOS, first identified in macrophages, is expressed in many brain cells, however, not under normal, physiological conditions. Astrocytes in vitro can be stimulated to express iNOS and generate up to 1 µM NO in the extracellular medium within few hours (Brown 1995). Human microglial cells have also been shown to express iNOS both in vivo and in vitro (Kitamura et al. 1998). Some neurons can express iNOS and release NO at least under in vitro conditions (Moro et al.

1998).

Unlike the constitutive forms of nitric oxide synthases, iNOS can produce high amounts of NO for relatively long periods. Induction of iNOS may have either toxic or protective effects, depending on the type of the insult, the tissue type, the level and duration of iNOS expression. In high local concentration NO can be cytostatic and cytotoxic for fungal, bacterial and protozoal organisms, as well as for tumour cells (Xu et al. 2002; Colasanti et al.

2002; Ascenzi et al. 2003). On the other hand, prolonged exposure to high concentration of NO may be also cytotoxic for the host cells (Abramson et al. 2001; Wink et al. 2001).

The iNOS is regulated at the expression level by transcriptional and post-transcriptional mechanisms (Kleinert et al. 2003). The main regulator of iNOS expression at transcriptional level seems to be nuclear factor-κB (NF-κB). Pharmacological inhibition of NF-κB significantly attenuates iNOS mRNA expression and NO production in cytokine stimulated

cells (de Vera et al. 1996; Salzman et al. 1996; Taylor et al. 1998). The important role for NF-κB binding sites in the induction of iNOS has been shown in murine (Blanchette et al. 2003), rat (Eberhardt et al. 1998) and human cells (Chu et al. 1998; Marks-Konczalik et al. 1998).

Several other transcription factors were demonstrated to have an important function in the regulation of iNOS at the promoter level, among which are the octamer factor (Oct), interferon regulatory factor-1 (IRF-1), signal transducer and activator of transcription-1α (STAT-1 α), cAMP-induced transcription factors CREB and C/EBP, activating protein-1 (AP-1), peroxisome proliferator-activated receptors (PPAR) (extensively reviewed by Kleinert et al. 2003). Along with the transcriptional control, post-transcriptional mechanisms play an important role in regulation of iNOS expression. Several RNA binding proteins seem to destabilize iNOS mRNA and inhibit translational efficiency (reviewed by Taylor and Geller 2000). Also transforming growth factor β1 (TGF-β1) was shown to destabilise iNOS mRNA, retard the synthesis of iNOS protein and accelerate its degradation (Vodovotz et al. 1996).

Once produced, NO has a short half-life, and it decomposes rapidly to nitrite (NO2-) and nitrate (NO3-) (Singh and Evans 1997). These two NO-metabolites can be detected using a colorimetric assay – Griess reaction (Green et al. 1982). However, NO can also combine rapidly with the superoxide anion (O2-) and form peroxynitrite (OONO^-), which can damage DNA and proteins, cause lipid peroxidation as well as inhibition of cellular respiration.

Peroxynitrite can diffuse much faster through the cell membrane and exerts more toxic effects than hydroxyl radicals. Many of the toxic effects previously attributed to superoxide and nitric oxide alone may be in fact due to peroxynitrite. Peroxynitrite was described to cause cell death and tissue damage in number of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS) and brain ischemia (Torreilles et al. 1999; Ebadi and Sharma 2003), as well as in toxic shock, arthritis, acute reperfusion injury (Bauerova and Bezek 1999; Boveris et al. 2002; Wang et al. 2003).

There are in vitro observations showing that NO and peroxynitrite can trigger DNA damage as a result of nitrosylation and deamination of nucleic acids leading to single strand and double strand breaks in DNA. In addition, damage to DNA can cause activation of the poly-(ADP-ribose) polymerase (PARP). PARP is an enzyme that is activated during DNA damage and is suggested to regulate gene expression and gene amplification, cellular differentiation, cellular division and malignant transformation as well as apoptotic cell death.

Upon binding to DNA PARP becomes activated and cleaves NAD⁺ in an ATP dependent manner. Therefore, excessive activation of PARP leads to the depletion of cellular NAD⁺ and ATP pools, and contributes to cellular energy depletion (Cosi and Marien 1999). It was shown

that ROS and RNS activate PARP not only via DNA damage but also directly (Hasko et al.

2002). Evidence shows that PARP activation resulting from oxidative/nitrosative stress is involved in the pathogenesis of neurodegenerative diseases (Virag and Szabo 2002).

Another mechanism leading to neuronal damage induced by NO and peroxynitrite is the inhibition of mitochondrial respiration compromising cellular energy metabolism. RNS are known to interact and inhibit irreversibly components of the electron transport chain in mitochondria and in particular complex I, complex II and complex IV (Duncan and Heales 2005).

There are differences in the susceptibility of brain cells to NO. Upon in vitro exposure to NO astrocytes appear more resistant to the effects of RNS, when compared to neurons. This may be explained by the astrocytes’ ability to up-regulate glycolysis, as following NO-mediated inhibition of respiration these cells rapidly increase the activity of key regulatory enzymes of glycolysis (Almeida et al. 2004). Another factor influencing the difference in susceptibility to RNS between astrocytes and neurons appears to be related to the cellular availability of anti-oxidative reduced glutathione (GSH). In astrocytic cultures the levels of GSH are approximately double than in neuronal cultures (Bolanos et al. 1995). Moreover astrocytes were shown to up-regulate quickly the biosynthesis of GSH upon exposure to NO (Heales et al. 2004).

Although astrocytes may have a neuroprotective role, increasing the GSH availability for neuronal cells, prolonged co-culture of neurons with NO generating astrocytes leads to mitochondrial dysfunction and to neuronal cell death (Stewart et al. 2000) probably due to the formation of more potent oxidizing species such as peroxynitrite.