Mechanism of Action of Nitric Oxide: S-nitrosylation.

A second method by which nitric oxide exerts it effects on cells is by the process of S-nitrosylation. In this signaling mechanism, nitric oxide modifies the sulfur atom of a protein cysteine residue, forming an S-nitrosothiol group (Fig. 1.8-4). This process requires no enzyme, and many S-nitrosylated proteins have altered function. Proteins that
Mechanism of Action of Nitric Oxide: S-nitrosylation.

FIGURE1.8-3. Neurotransmitter and signaling functions of nitric oxide (NO) via production of cyclic guanosine monophosphate (cGMP). Gaseous nitric oxide is enzymatically generated and freely diffuses into an adjacent neuron (upper right). In comparison to traditional neurotransmitters (upper left), NO does not act via a specific neurotransmitter receptor on the surface membrane of a neuron. In contrast, NO freely diffuses across the neuronal membrane and activates the enzyme, guanylyl cyclase, which converts guanosine 5’-triphosphate (GTP)into the second messenger, cGMP. Nitric oxide effects are mediated, in part, by cGMP activation of neuronal protein kinases, new gene expression, and effects on neuronal long-term potentiation (LTD) and long-term depression (LTD). ATP, adenosine triphosphate.

have been nitrosylated vary in their response to this modification; some are activated and others inactivated.

The number of protein targets of S-nitrosylation is rapidly expanding and includes molecules involved in signal transduction, programmed cell death, transcription factors, cytoskeletal proteins, ion pumps, and ion channels. In many cases modification of a single

cysteine residue in a target protein is sufficient for nitric oxide to regulate its activity. Specific targets that are activated by S-nitrosylation include L-type calcium channels, calcium activated potassium channels, and Y -aminobutyric acid type A (GABAa) receptors. Proteins inhibited by nitrosylation include several types of sodium channel, the N-methyl-d-aspartate (NMDA) subtype of the glutamate

Mechanism of Action of Nitric Oxide: S-nitrosylation.

FIGURE 1.8-4. Nitric oxide (NO) signaling via S-nitrosylation. In addition to NO activation of guanylyl cyclase, NO may also directly alter protein function via the process of S-nitrosylation. In this process, which does not require enzymatic catalysis, NO reacts with -SH groups of protein cysteine residues, resulting in an -SNO modification and altered protein function. Some proteins demonstrate robust activation following S-nitrosylation, whereas others are inhibited by the process.

neurotransmitter receptor, and several metabolic enzymes. S-nitro-sylation as a means of signal transduction is somewhat analogous to protein phosphorylation, as both are reversible covalent modifications that regulate protein function to change cell activity. S-nitrosylation may play roles in memory, learning, and behavior, as many brain proteins are nitrosylated through the activity of neuronal nitric oxide generation.

Nitric Oxide and Neuro transmission. Long-term potentiation (LTP) is the process by which repetitive stimulation of a presynaptic neuron leads to stronger firing of a postsynaptic neuron, a process that underlies changes in learning and behavior. Induction of LTP depends on activation of postsynaptic NMDA receptors, while LTP maintenance relies on presynaptic mechanisms. Neurotransmission through the NMDA receptor facilitates LTP, in part, through the activity of nitric oxide. Activation of the NMDA receptor leads to a cellular calcium increase, promoting nitric oxide synthesis and cGMP formation in the postsynaptic cell (Fig. 1.8-5).

Pharmacological inhibitors of NOS have revealed deficits in LTP in rodent, bird, and honeybee models, and nitric oxide has been implicated in both short- and long-term memory acquisition. Genetically modified mice lacking either nNOS or eNOS demonstrate no changes in LTP in the hippocampus; however, mice deficient in both nNOS and eNOS show decreased LTP in the CA1 region of the hippocampus. One form of NOS may compensate for the absence of the other, or the two may function cooperatively in LTP. Studies of the enteric nervous system have also revealed roles for nitric oxide in relaxation of the pyloric sphincter, and mice deficient in nNOS reveal a marked hypertrophy of the pylorus. Nitric oxide may also regulate monoaminergic neurotransmission. Inhibition of NOS in rats enhances the effects of cocaine and amphetamine, while the reverse is observed by increasing nitric oxide.

N itric O xide and Be havio r. Nitric oxide neurotransmission can play a role in behavior, as nNOS-deficient male mice display exaggerated aggressive tendencies and increased sexual activity. In
Mechanism of Action of Nitric Oxide: S-nitrosylation.

FIGURE 1.8-5. Nitric oxide (NO) generation following N-methyl-D-aspartate (NMDA) receptor activation. The presynaptic neuron (top) releases glutamate (not shown), activating the NMDA glutamate receptor and allowing for calcium entry into the postsynaptic neuron. Calcium binds to the protein calmodulin (CaM), which in turn activates neuronal nitric oxide synthase (nNOS) to synthesize NO. A freely diffusible gas, NO exerts effects upon the target neuron via formation of cyclic guanosine monophosphate (cGMP) and S-nitrosylation (Figs. 1.8-3 and 1.8-4).

female mice the contrary is true, as they have reduced aggression. As manic bipolar patients may show both hypersexuality and aggression, the nitric oxide pathway may participate in the psychopathology of affective states.

In the periphery, nNOS localizes to neurons that innervate blood vessels of the penis, including the corpus cavernosa. Stimulation of these nerves releases nitric oxide, leading to cGMP formation, blood vessel wall relaxation and vasodilatation, penile engorgement, and initial erection. The sustained phase of erection also depends on nitric oxide; turbulent blood flow leads to phosphorylation of eNOS and sustained nitric oxide production. Drugs used in treatment of erectile dysfunction, sildenafil (Viagra), tadalafil (Cialis), and varde-nafil (Levitra), act to inhibit type 5 phosphodiesterase, an enzyme that degrades cGMP in the penis (Fig. 1.8-3), thereby potentiating nitric oxide neurotransmission and penile erection.

Numerous lines of evidence have suggested a role for nitric oxide in the regulation of sleep-wake cycles. nNOS expressing neurons occur in several areas that initiate rapid eye movement (REM) sleep, including the pons, dorsal raphe nucleus, laterodorsal tegmentum, and pedunculopontine tegmentum. In animal models, microinjection of compounds that release nitric oxide result in decreased wakefulness and increased slow wave sleep. Consistent with this, NOS inhibitors show a trend toward decreasing slow wave and REM sleep. Studies of NOS-deficient mice suggest that nitric oxide may serve a more complex role than merely promoting sleep. nNOS-deficient animals also show reduced REM sleep; however, iNOS-deficient mice demonstrate the reverse, suggesting a complex interplay between NOS enzymatic isoforms.

Nitric Oxide and Mood Disorders. NOS-expressingneu-rons are well represented in areas implicated in depression, including the dorsal raphe nucleus and prefrontal cortex. A role for nitric oxide has been suggested in antidepressant response as selective serotonin reuptake inhibitor (SSRI) antidepressants can directly inhibit NOS activity. Moreover, in animal studies such as the forced swim test, NOS and soluble guanylyl cyclase inhibitors can achieve antidepressant-like effects. Plasma nitric oxide levels were elevated in patients with bipolar disorder compared to healthy control subjects. However, in depressed subjects, studies have found decreased nitric oxide levels and increased plasma nitrite, a byproduct of nitric oxide. Reduced NOS has also been described in the paraventricular nucleus of patients with schizophrenia and depression compared to controls.

Neurogenesis, the process by which new neurons are generated in the adult brain, is increasingly appreciated to participate in both mood disorder pathophysiology and antidepressant response. Increased hippocampal neurogenesis is associated with antidepressant response, and smaller hippocampal volume may be a risk factor for mood and anxiety disorders. Serotonin, itself, appears to promote neurogenesis in the hippocampus, while nitric oxide has been found to inhibit neurogenesis. Pharmacologic inhibitors of NOS result in increased serotonin and neurogenesis in the dentate gyrus of the hippocampus, a paramount site of this process. These NOS inhibitors also lead to an increase in serotonin in the dentate gyrus. Unsurprisingly, nNOS-deficient animals also manifest increased proliferation in the dentate gyrus. As steroids appear to induce NOS expression, nitric oxide may contribute to effects on mood and anxiety often observed in those treated with these agents.

Nitric oxide has been questioned as to its ability to regulate neurotransmission at serotonin, norepinephrine, and dopamine nerve termini. No clear consensus has been arrived at, and nitric oxide appears to possess the capability of increasing or decreasing activity at these neurons depending on the timing of its activation and the region of the brain studied.

Nitric Oxide and Schizophrenia. Nitric oxide has been investigated as a candidate molecule contributing to symptoms of schizophrenia. Two genetic studies have identified schizophrenia-associated single nucleotide polymorphisms (SNPs) in CAPON, a protein that associates with nNOS. SNPs in nNOS itself have been associated with schizophrenia, although others have not been able to reproduce such findings. Changes in NOS levels have been reported in postmortem brain samples of individuals with schizophrenia. Abnormalities have been noted in the cortex, cerebellum, hypothalamus, and brainstem, although no specific trend can be discerned. Elevated NOS activity has been noted in platelets from drug-naive and drug-treated individuals with schizophrenia. Some investigators find increased nitric oxide activity and others the reverse. In autopsy samples, schizophrenic patients were found to have abnormally localized NOS expressing neurons in the prefrontal cortex, hippocampus, and lateral temporal lobe, consistent with abnormal migration of these neuronal types during development. In a rat model, prenatal stress led to reduced NOS expressing neurons in the fascia dentate and hippocampus.

Neuropathologic Roles of Nitric Oxide. Abundant evidence exists that nitric oxide is a direct participant in a variety of neuropathic events. Superoxide, a byproduct of cellular metabolism, can react with nitric oxide to form peroxynitrite (chemical formula ONOO-). This labile and toxic compound forms chemical adducts with protein tyrosine residues, a process termed protein nitration, and deoxyribonucleic acid (DNA), leading to cellular dysfunction.

Cell loss resulting from ischemic stroke is mediated in part by overstimulation of the glutamate NMDA receptor, a process termed excitotoxicity. Nitric oxide produced by NMDA activation appears to mediate a significant portion of this excitotoxic neuronal death, and stroke damage is reduced in mice with a genetic deletion of nNOS.

S-nitrosylation has also been implicated in pathologic processes in the brain. Mutations in the Parkin protein are associated with early onset Parkinson’s disease. Parkin is an E3 ubiquitin ligase, adding ubiquitin molecules to proteins and targeting them for destruction in the cell proteasome. In sporadic Parkinson’s disease (i.e., without the early onset mutation), nitric oxide can nitrosylate the Parkin protein and inhibit its protective E3 ubiquitin ligase function. An overabundance of nitric oxide signaling may thus predispose to the dysfunction and cell death of dopaminergic neurons in Parkinson’s disease by interfering with proteins essential for cell functioning. In Alzheimer’s disease excess oxidation of brain protein, lipids, and carbohydrates has long been appreciated, but nitrosative stress from excess nitric oxide also appears to participate in the disease. Protein disulfide iso-merase (PDI) is a cellular protective protein that may help combat the accumulation of misfolded proteins such as the amyloid fibrils occurring in the disease. In both Alzheimer’s and Parkinson’s disease brains, PDI appears to be S-nitrosylated in a harmful way that impedes its cellular protective function.

The discovery that nitric oxide participates in neurodegenerative processes raises the possibility for improved diagnostic processes, such as detecting damage to cellular components produced by nitric oxide prior to the onset of full-blown symptoms. In addition, drugs may be designed to attenuate the damage to crucial neuronal proteins that protect against disease onset. However, completely and nonspecifically inhibiting or stimulating nitric oxide synthesis is likely to produce significant side effects because of its wide-ranging activities throughout the body.

Mechanism of Action of Nitric Oxide: S-nitrosylation.

FIGURE 1.8-6. Synthesis of carbon monoxide (CO), an unexpected neurotransmitter. Gaseous carbon monoxide is enzymatically synthesized in neurons via the enzyme heme oxygenase, also converting heme into the molecule biliverdin and liberating free iron (Fe). Similar to nitric oxide, CO is not stored in neuronal vesicles and can freely diffuse across neuronal membranes. CO also similarly activates soluble guanylyl cyclase, and leads to activation of multiple intracellular signaling molecules such as p38 MAP kinase. CO exerts its neurotransmitter and signaling functions at concentrations far below that at which classical CO toxicity occurs. The significance of this pathway in neurons is underlined by the existence of two distinct heme oxygenase enzymes, one of which is predominantly expressed in the brain. Biliverdin is converted to bilirubin via the enzyme biliverdin reductase. Similar to CO, bilirubin is no longer relegated to the status of toxic byproduct and may be an important antioxidant.

Nitric oxide is one of the most intensively studied compounds in the body, and it possesses pleiotropic activities in different organs. Although nitric oxide has physiologic roles in the brain, vasculature, and immune system, it is a complex and incompletely understood molecule that also participates in disease. However, oxygen, which is a highly reactive molecule like nitric oxide, also possesses the capacity to contribute to disease pathogenesis, such as in oxidative damage, while still being essential for life.
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