Multidisciplinary approach to nitric oxide signaling : Focus on the gastrointestinal and the central nervous system

Nitric oxide (NO), with close historical ties to cardiovascular physiology, is an important endogenous mediator, the most potent natural vasorelaxant known, involved in many biological functions . NO acts as an intra/intercellular signalling molecule and is a mediator in almost all organ systems . NO is known to occur in many cells types, including vascular endothelial cells, neurons, and epithelial cells . The transmitter of nonadrenergic, noncholinergic (NANC) inhibitory neurons has been the subject of hundreds of investigations over the past 3 decades. Recent evidence suggests that NO may serve as a NANC inhibitory signaling molecule in the gastrointestinal (GI) tract. NO serves as the primary enteric inhibitory neurotransmitter in GI muscles, and nitrergic neurons regulate gut tone, phasic contractile amplitude and frequency, and inhibitory reflexes . In the central nervous system (CNS) NO is involved in some major processes such as memory through longterm potentiation (LTP) and learning . This gasotransmitter also contributes to a pathogenesis of epilepsy. The role of NO in the generation of epilepsy is contradictory since there is evidence of its proconvulsive and anticonvulsive effects . In this review, we will discuss about the possible role of NO as neurotransmitter in the GI and CNS, with focus on the contribution of NO-mediated signaling pathways in the GI motility and CNS excitability. Physiological functions of nitric oxide


Introduction
Nitric oxide (NO), with close historical ties to cardiovascular physiology, is an important endogenous mediator, the most potent natural vasorelaxant known, involved in many biological functions 1 .NO acts as an intra/intercellular signalling molecule and is a mediator in almost all organ systems 2 .NO is known to occur in many cells types, including vascular endothelial cells, neurons, and epithelial cells 3 .The transmitter of nonadrenergic, noncholinergic (NANC) inhibitory neurons has been the subject of hundreds of investigations over the past 3 decades.Recent evidence suggests that NO may serve as a NANC inhibitory signaling molecule in the gastrointestinal (GI) tract.NO serves as the primary enteric inhibitory neurotransmitter in GI muscles, and nitrergic neurons regulate gut tone, phasic contractile amplitude and frequency, and inhibitory reflexes 4 .
In the central nervous system (CNS) NO is involved in some major processes such as memory through longterm potentiation (LTP) and learning 5 .This gasotransmitter also contributes to a pathogenesis of epilepsy.The role of NO in the generation of epilepsy is contradictory since there is evidence of its proconvulsive and anticonvulsive effects 6 .In this review, we will discuss about the possible role of NO as neurotransmitter in the GI and CNS, with focus on the contribution of NO-mediated signaling pathways in the GI motility and CNS excitability.

Physiological functions of nitric oxide
In 1978, Furchgott 7 , discovered a substance in endothelial cells that relaxes blood vessels, calling it endothelium-derived relaxing factor (EDRF), then he had worked out EDRF's nature and mechanism of action.It has been identified to be NO, an important compound in many aspects in both physiological and pathological conditions.NO is biosynthesied from L-arginine by the NO synthase.NO synthesis and release is mediated through the activation of NO synthase (3 isoforms of NO synthase, NOS enzymes) by an elevation of cytoplasmic Ca 2+ , conversion of L-arginine to NO and L-citrulline, NO diffuses passively into the extracellular fluid.Although NO is made by a cytosolic enzyme, it is a highly permeable molecule that can easily diffuse out of the cell that makes it 8 .NO is a tasteless, colourless gas.It is rapidly absorbed via the pulmonary vasculature directly into the bloodstream.The mechanism of NO action is not fully understood, but many of its actions are mediated by the activation of guanylate cyclase, which results in an increase in the concentration of cyclic guanosine 3',5'-monophosphatate (GMP) in smooth muscle.The enhanced production of cyclic GMP that results from activation of guanylate cyclase may result in: activation of GMP-dependent protein kinase or direct actions of cyclic GMP on ion channels or other second messenger systems may also be activated by NO (non-cGMP dependent NO effects).NO displaces nitrogen and increase the volume of gas in body cavities such as the middle ear, sinuses, pleural space, and GI tract 9 .NO and NO donors (e.g., sodium nitroprusside) cause relaxation of vascular smooth muscle through the accumulation of cyclic GMP or through the direct activation of K + channels.Several studies have suggested that NO might decrease the intracellular Ca 2+ level or reduce the Ca 2+ sensitivity of the contractile elements, which results in smooth muscle relaxation.Furthermore, the relaxing action of NO has been indicated indirectly by inhibiting the release of the neurotransmitters acetylcholine and substance P 10 .
We shall list a number of physiological functions of NO and we can distinguish the following: it is involved in the regulation of blood flow, maintenance of vascular tone, control of platelet aggregation, modulation of the activity of the mastocytes, as a neurotransmitter in the CNS and peripheral nervous system (NANC, neurons), in the nervous control of the cerebral blood flow and in the neuroendocrine regulation or synaptic plasticity [11][12][13][14][15] .
On the other hand, NO plays a role in memory formation.NO is a retrograde messenger at N-methyl-D-aspartate (NMDA) receptors mediated synapses.Inhibitors of NO synthesis in any case, block initiation of LTP which involves an NMDA receptor mediated intracellular cascade finishing in lasting modulation of synaptic morphology 16,17 .Inversely proportional relationship between NO and glutamate, is also described.In vivo and in vitro studies with NO donors, NO synthase inhibitors and glutamate receptor antagonists have shown that NO increases the release of glutamate in several regions of the brain (hippocampus, striatum, hypothalamus and locus ceruleus) and spinal cord 18,19 .
To maintain the postsynaptic activation, a retrograde communication with the presynaptic component must exist.It has been suggested that the retrograde NO molecule triggers the release of glutamate via cyclic GMP-dependent way.The effects of NO on glutamate release depends on the level of NO.Thus, when the concentration is low, NO reduces the release of glutamate in spite of elevated levels of cyclic GMP.But when NO increases the levels of cyclic GMP, an inhibitory effect on glutamate release reverses, indicating that cyclic GMP showed biphasic effects 20 .Morphineinduced impairment of memory formation can be prevented by NO donor 21 .

Nitric oxide in gastrointestinal smooth muscle
Opinions concerned with GI system is polarised as to whether or not NO causes nausea and vomiting.It can certainly increase intestinal and middle ear volumes, which may in turn lead to nausea 9 .On the other hand, ghrelin, a gastric peptide, which possesses orexigenic effects, is the endogenous ligand for the growth hormone with stimulating effects on growth hormone and GI motility.Gaskin et al. 22 , demonstrated that a sub-threshold dose of N(omega)-nitro-Larginine methyl ester (L-NAME) significantly blocked the ghrelin-induced increase in food intake.Ghrelin increased NO synthase levels in the hypothalamus-supporting the hypothesis that ghrelin's effects are NO dependent.
In GI smooth muscle, NO or NO donors evoke different responses, including relaxing, contractile effects, relaxations followed by contractions or contractions followed by relaxations, which depend on the compound, tissue and species [23][24][25] .The nerves whose transmitter function depends on the NO release are called "nitrergic" and such nerves are recognized to play major roles in the control of smooth muscle tone and motility 26 .NO is likely an inhibitory neurotransmitter in the human jejunal longitudinal smooth muscle, acting via mechanism mediated by guanylyl cyclase 27 .It was suggested that basal release of NO caused an oscillatory patterns of electrical and mechanical activities.NO is a vasodilator and mediates gastric blood flow and it is responsible for helping to maintain the integrity of the gastric epithelium and the mucus barrier 28 .
There are numerous data supporting the hypothesis that NO plays a pivotal role in NANC relaxation or inhibitory junction potential associated with electrical field stimulation or nicotinic agonists in the duodenum, jejunum, and ileum from a variety of mammals, including the human and rat 27,29 .It was reported that the ability of the nitrergic neurotransmitter to induce relaxation of the rat gastric fundus was influenced by the mechanism used to induce tone, and sarcoplasmic/endoplasmic reticulum Ca +2 ATPase appeared to play a role in nitrergic relaxation 30 .
Giant migrating contractions of the rat colon, possibly mediated by neuronal release of acetylcholine, appear to be partially suppressed by constitutive release of NO 31 .Nitrergic innervation also contributes to the regulation of the smooth muscle tone in the rat rectum 32 .
At the GI level, 2 constitutively expressed isoforms, namely endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS), are expressed basally at the vascular endothelium and the enteric nervous system of the GI tract 33 .
However, NO shows a dual behavior: at physiological concentrations, released through the constitutive synthase (cNOS), it regulates house-keeping functions and is responsible for production of NO in physiological context.In physiological conditions, NO acts as an endogenous mediator modulating both, the repairing and integrity of the tissues, and exhibits gastroprotective properties against different types of aggressive agents.In contrast, its overproduction by the inducible isoenzyme (iNOS) exhibits cytotoxic activity because interacting with reactive species producing peroxinitrites and other compounds, which are highly damaging for the tissues.iNOS produces NO in pathophysiological circumstances.High concentrations of NO are related to numerous pathological processes of GI tract including peptic ulcer, chronic gastritis, GI cancer, bacterial gastroenteritis, and celiac or chronic inflammatory bowel diseases.Indeed the adverse action of cigarette smoking on ulcer healing is largely dependent on the deficiency of cNOS and a subsequent depression of gastric blood flow and angiogenesis.To this end, NO may act as a crucial signal to promote endothelial cell differentiation into vascular tubes 34 .
Endogenous NO also contributes to the inhibition of acid secretion in the stomach.NO is implicated in mechanisms maintaining the integrity of the gastric epithelium.In this connection, it regulates gastric blood flow and directly stimulates gastric mucus secretion by activating soluble guanylate cyclase 35 .A blockade of NO production resulted in an impairment of the vascular response and the subsequent alkaline flux in the lumen.This would impair the restitution process.Intragastric administration of hydrochloric acid stimulated a subpopulation of nitrergic, but not cholinergic, myenteric plexus neurons, which might play a role in secretion, vasodilatation, and muscle relaxation 36 .Expression of nNOS in parietal cells suggests a participation of endogenous NO in the regulation of gastric acid secretion 37 .Helicobacter pylori increased pepsinogen secretion from dispersed human peptic cells through a Ca 2+ -and NO-mediated intracellular pathway 38 .
In the GI tract NO participates in the modulation of the smooth musculature tone, such as the regulation of intestinal peristaltism, gastric emptying and antral motor activity.It also regulates acid and gastric mucus secretion, alkaline production, and is involved in the maintenance of mucosal blood flow.NO biology can influence nutrition and be nutritionally modulated to affect mammalian (patho)physiology.NO as modulator of feeding behavior and mediator of GI homeostasis could be used for supplementation as a therapeutic modality for preserving GI health 39 .Presumed mechanisms of relaxation of NO in GIT tract are: -Cyclic GMP seems to be a key substance for nitrergic inhibitory responses in the most mammalian GI tracts, including that of humans.Cyclic GMP-dependent reduction of cellular free Ca 2+ without changing the membrane potential; -Reduce the Ca 2+ sensitivity of the contractile element 40 ; -Cyclic GMP-dependent opening of apamin-sensitive K + channels or other types of ion channels to produce hyperpolarization and relaxation; -Indirectly by inhibiting the release of the neurotransmitters acetylcholine and substance P 41 ; -Cyclic GMP-independent mechanisms, such as actions of NO on ion channels involved in muscle contractility, either directly or via membrane hyperpolarization.
Stimulation of these nerves, elicits hyperpolarization of postjunctional smooth muscle membranes referred to as inhibitory junction potentials and relaxation [42][43][44] .These neurons mediate the majority of inhibitory responses in the GI tract and regulate many important physiological reflexes, such as relaxation of the lower esophageal sphincter after swallowing, receptive relaxation of the proximal stomach during eating, and descending inhibition in response to distension 45 .
Clear species variations in the functioning of nitrergic nerves were also seen in the distal colon 46 .
L-NAME has been used by many investigators to determine the role of endogenous NO in various physiological and patophysiological conditions.In our experiments L-NAME shows increasing of the resting tone, amplitude and frequency of the contractions of the isolated duodenal segments.It has been shown that L-arginine reversed the action of L-NAME.These data confirm the evidence for the par-ticipation of the L-arginine-NO pathway in the relaxation of isolated rat duodenum 47 .

Nitric oxide signaling modulation by homocysteine in the gastrointestinal system
We wanted to examine the effects of D,L-homocysteine thiolactone (HCT) on duodenal motility, proved to have a prokinetic effect.Homocysteine is a sulfhydryl amino acid derived from catabolism of methionine.As GI smooth musculature is similar to blood vessel's muscles, we investigated how elevated homocysteine levels affect NO mediated neurotransmission in the gut.HCT leads to immediate increase in tone, amplitude and frequency of spontaneous movements of isolated rat duodenum 47 .In the presence of L-NAME, treatment with HCT caused significant increase of resting tone, amplitude and the frequency of the contractions.These results suggest that mechanism of acting of HCT on the duodenal segments contractions are based on the modulation of nitrergic neurotransmission in the gut 47 .We found that NANC relaxations induced by low frequencies of electrical field stimulation were significantly changed in duodenal preparations obtained from duodenal segments treated with HCT.These findings suggest that homocysteine causes an impotrant impairment on NANC innervation of the rat duodenum 47 .Our results show that HCT increases the motility of isolated rat duodenum.They are consistent with the results of Park et al. 48which suggest that sulfur-containing amino acids like D,L-homocysteine potentiates depolarisation of murine proximal colon cells.These effects include increasing the amplitude and frequency of spontaneous contractions of murine colonic stripes.
Choe et al. 49 in their study investigated the effects of methionine on the contractile activity of human colon smooth muscle in vitro.Methionine is a sulfur containing amino acid that is transformed into homocysteine during biometabolism.Their results indicate that methionine increases the amplitude contractions of colonic muscle strips, which supports our results 49 .

Nitric oxide-mediated neurotransmission in the central nervous system: focus on hyperexcitability and epileptogenesis
Disorders of the CNS are one of the primary categories in health care system funds expenditures 50,51 .With the prevalence of 1-2% in the general world population, epilepsy is among the leading neurological disorders 52 .Epilepsy is characterized by paroxysmal occurrence of motor seizures in behavior (different and specific semiology: from tonic-clonic via myoclonic to atonic seizurs) and ictal activity in electroencephalography (EEG) (different forms of spiking activity from isolated spikes to generalized bursts/trains of spikes or spike-and-wave discharges depending on the type of epileptic acitivity).Imbalance between excitatory and inhibitory phenomena within the CNS is thought to be the primary mechanisms involved in the process of epileptogenesis 53 .However, many other mechanisms are recognized as potential parts of epileptogenesis mosaic.Recently, inflammation, as well as gasotransmitters-mediated signaling processes have been identified as one of the important pathways in modulation of epileptic activity [54][55][56][57] .Our understanding of the process of epileptogenesis relies on adequate experimental models of epileptic disorder.Therefore, experimental models are unequivocal tools for investigations in epileptogenesis.It should be pointed out that no single model system could be useful for all types of epilepsy 58 .Having that in mind, a number of very useful experimental models of epilepsy were developed up to now.Recently, Stanojlović et al. 59 showed that acute administration of HCT to adult rats significantly alters neuronal circuits, leading to epileptogenic activity in the EEG with characteristic spikes-and-wave discharges (SWD), and convulsive episodes (manifested through 5grade descriptive rating scale addapted by Stanojlović et al. 59 ) in animal behavior.HCT-induced seizures are accepted as a suitable model of generalized epileptic seizures in which coexistence of convulsive and absence-like seizures were proven 60,61 .This is one of the advantages of this particular epilepsy model which allowed reliable investigations of variety seizure activity modifications, like paradoxical sleep deprivation 62 .
NO displays pleiotropic effects in the CNS.All the three NO synthase isoforms have been expressed in the brain.Neurons produce NO mostly by activation of nNOS 63 .Constitutive isoforms of NO synthase are responsible for the synthesis of physiologically vital amounts of NO 64 , while inducible NO synthase (iNOS) produces high amounts of NO lasting hours or days 65 .nNOS is found to be mostly expressed in the hippocampus, cerebral cortex, corpus striatum and cerebellum, as well as in some cells of the autonomic nervous system 66 .iNOS has been found to be a major contributor to initiation/exacerbation of the CNS inflammatory/degenerative conditions through the production of excessive NO 67 .iNOS is reported to be highly expressed in brains of humans with epilepsy.In some spontaneously epileptic mouse overexpression of iNOS is also found 68,69 .
The role of NO-mediated neurotransmission in the process of epileptogenesis is highly unpredictable and contradictor in the existing scientific literature, since numerous evidences exists for both its proconvulsive and anticonvulsive activity 6 .The results on the role of NO in epileptogenesis have been recognized to depend, among other factors, on the source of NO production [70][71][72] .Moreover, different NOS modulation upon generalized seizures along the anterioposterior axis of the brain have been recently proved, showing dependence on vicinity of original epileptic focus 73 .Recently, we showed that the systemic administration of increasing doses of L-arginine in a dose-dependent manner significantly decreased seizure incidence and the number of seizure episodes and the prolonged latency time to the first seizure elicited by the convulsive dose of HCT 74 .On the other hand, pretreatment with L-NAME, in a dosedependent manner, increased seizure incidence and severity and shortened latency time to the first seizure following the injection with the subconvulsive dose of HCT.In the same study, L-arginine decreased, while L-NAME increased the median number of SWD per rat, while duration of individual SWD was not modified.These results showed the functional involvement of NO in the HCT-induced epileptic activity 74 .
Further studies have been undertaken in order to investigate the involvement of nNOS in HCT -induced seizures.With this aim, pharmacological inhibition of nNOS by 7nitroindazole has been applied 75 .Systemic administration of 7-nitroindazole showed tendency to increase seizure incidence, decrease latency time to first seizure, increase number of seizure episodes per rat and increase severity of seizures induced by HCT in rats in a dose dependet manner.Therefore, these results were congruent with those obtained using non-selective NOS inhibition.Contribution of iNOS-derived NO in the process of epileptogenesis elicited by HCT was recently demonstrated in this model of seizures using aminoguanidine, as selective iNOS inhibitor 57 .The results of that study showed that pretreatment with aminoguanidine (applied in the three doses) increased convulsive properties, i.e. seizure incidence, the number of seizure episodes per rat and severity of HCT-induced seizures, as well as the number and duration of SWDs in EEG.Also, aminoguanidine decreased the latency time to the first seizure episode induced by HCT in the same dose-dependent manner.Quantitative analysis of ictal activity in EEG showed congruent results with those from behavioral assessment in that study.Namely, aminoguanidine pretreatment significantly increased the number and duration of SWD induced by HCT in that study 57 .
The interaction between NO and HCT is supposed to involve NMDA receptor complex, as well as interaction of both NO and HCT with glutamate and gamma-aminobutyric acid.These relationships, discussed in details in Hrnčić et al. 74 , could contribute to obtained results on NO effects in HCTinduced seizures.

Conclusion
Nitric oxide in low concentration derived from constitutive nitric oxide synthase is cytoprotective by directly acting as an inducer of defense responses in the gastrointestinal tract.However, higher concentrations of nitric oxide from inducible nitric oxide synthase exhibit toxic effects through nitrosative and oxidative stress.These findings suggest that the cholinergic and nonadrenergic noncholinergic inhibitory nerves play important roles in regulating contraction and relaxation of the gut, and nitric oxide plays an important role in nonadrenergic noncholinergic inhibitory nerves of the digestive tract.In addition, a decrease of the action of cholinergic nerves and an increase of the action of nonadrenergic noncholinergic inhibitory nerves by nitric oxide may be largely related to the low pressure in some part of the gut.However, the findings related to the nitrergic innervation may provide us a new way of understanding gastrointestinal tract physiology and pathophysiology and might result in the development of new therapies of gastrointestinal diseases.
We have shown that nitric oxide causes anticonvulsive effects in the experimental model of epilepsy induced by D, L-homocysteine thiolactone.Further studies are needed to elucidate all nitric oxide effects on the central nervous system, since new clues for dissolving the role of nitric oxide in the central nervous system and especially its disorders, like epilepsy, is of significant importance from physiological, pathophysiological and pharmacological viewpoint.