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Current Pharmaceutical Design, 2001, 7, 881-908 881

New Issues about Nitric Oxide and its Effects on the Gastrointestinal Tract

M.J. Martn*, M.D. Jimnez and V. Motilva

Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain

Abstract: Over the last years the important role of nitric oxide (NO) as endogenous modulator of
numerous physiological functions has been shown. NO is involved in the regulation of blood flow,
maintenance of vascular tone, control of platelet aggregation, and modulation of the activity of the
mastocytes. It also plays a key role as neurotransmitter in the central and peripheric nervous system (non
adrenergic non colinergic, NANC, neurons), in the nervous control of the cerebral blood flow and in the
neuroendocrine regulation or synaptic plasticity. However, NO shows a dual behavior: at physiological
concentrations, released through the constitutive synthase (cNOS), it regulates house-keeping functions,
whereas its overproduction by the inducible isoenzyme (iNOS) exhibits cytotoxic activity because
interacting with reactive species producing peroxinitrites (ONOO ) and other compounds, which are
highly damaging for the tissues.

In the gastrointestinal tract (GIT) 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. 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. However, high concentrations of NO are related to numerous pathological processes of
GIT including peptic ulcer, chronic gastritis, gastrointestinal cancer, bacterial gastroenteritis, celiac or
chronic inflammatory bowel diseases. Recently, this hypothesis that cNOS is always beneficial and iNOS
is always deleterious, has been questioned, since that a series of data suggest that the increase of cNOS
activity could be responsible for the derived pathological changes and, by contrast, NO liberated by the
inducible isoenzyme might play a repairing effect in certain pathological disorders.

The pharmaceutical industry is really interested in proving the clinical benefits of the mediator.
Numerous NO-donor drugs, nitrate derivatives, have been frequently used in the cardiovascular diseases
due to their vasodilating properties, which allow an enhancement of coronary blood flow. More recently,
the protective effect of NO against non steroidal antiinflammatory drugs (NSAID)-gastroenteropathy has
been shown, because its vasodilating and antioxidant properties render it a potentially useful agent.
Different NSAID, including acetyl salicylic acid, diclofenac or naproxen, have been formulated by
attaching a NO releasing-moiety. These NO-NSAID, antiinflammatories combined with precursors of the
mediator, or with inhibitors of the inducible synthase, are currently being evaluated. However, although
the pharmacotherapeutical possibilities of NO are considerable, it is necessary to elucidate the exact
mechanisms derived from stimulation/inhibition of the isoenzymes in order to determine the clinical
utility of NO-donors.

INTRODUCTION Science, which entitled the front page of the issue


NO news is good news. The Nobel prize for
In 1992, nitric oxide was considered the Medicine in 1998, was concurrently awarded to
molecule of the year by the prestigious journal the Northamerican investigators Robert Furchgott,
Louis Ignarro and Ferid Murad in
*Address correspondence to this author at the Dept. of Pharmacology,
Faculty of Pharmacy. University of Seville, P. Garca Gonzlez s/n., acknowledgement of their independent discoveries
41012-Seville, Spain, Tph. 34-95-4551666, Fax: 34-95-4233765, e-mail: which led to the identification of NO as a key
calero@fafar.us.es
molecule with important cardiovascular functions,

1381-6128/01 $28.00+.00 2001 Bentham Science Publishers Ltd.


882 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

especially in regulating blood pressure. The search NO and EDRF were the same molecule. In order to
in MEDLINE data base reveals that from 1990 on, verify this, their pharmacological properties were
over 30,000 studies related to this mediator have compared in smooth vascular muscle and in
been published. A gross body of these platelets and identical profiles were obtained. Both
investigations has been carried out by Spanish substances relaxed rabbit aortas deprived of
groups. These data confirm the interest of the endothelium. This effect was enhanced with SOD,
scientific society for a gas which until recently was whereas it was abolished in the presence of
considered an environmental toxic contaminant and hemoglobin and Fe2+ [4].
currently is arrogated with essential functions of
the organism. Further studies confirmed the identity of NO as
the knowledge about its functions grew. Most of
In 1980, Fuchgott and Zawadki (both NO functions are regulated by proteins and very
cardiovascular pharmacologists of the Brooklyn complex macrocompounds, which is striking in
Bownstate Hospital), proved that the relaxation of case of such a simple molecule. The first indication
the isolated rabbit aorta, induced by acetylcholine, that this gas could be related to the nervous
was dependent on the liberation of a substance system, goes back to 1982. Takeo Deguchi
which in the presence of vascular endothelium, observed that its synthesis in the brain required
diffused to the next smooth muscle cells. This gas the aminoacid arginine. The use of
was later called endothelium-derived relaxing immunohistochemical techniques made it possible
factor (EDRF) [1,2]. to localize the enzyme which synthesizes NO in
neurons of the hypophysis and of the cerebral
Further studies showed that this mediator had a cortex [6]. Later, Moncada confirmed the role of
short halflife of 6-10 seconds and its action was the aminoacid in the central nervous system (CNS)
inhibited by hemoglobin/methylene blue. It was and its relationship with the formation of NO [4,
also found that in the smooth muscle, its action 7, 8].
implicated the stimulation of the enzyme soluble
guanylyl cyclase (GC), increasing the levels of In the beginning of 1989, the possible
cyclic guanidin monophosphate (cGMP). involvement in synaptic transmission was
Bioassays proved that superoxide anions (O2-) investigated by searching the brain region where
provoked the instability of EDRF in such a way glutamate might influence cGMP [4]. James
that its action was prolonged in the presence of Ferrendel observed a pronounced and rapid rise in
superoxide dismutase (SOD) and was inhibited the breakdown product when glutamate was added
with Fe 2+ [3,4]. to cerebellum sections. Similarly, measuring the
activity of the enzyme nitric oxide synthase
In 1986, Furchgott hypothesized that EDRF (NOS), it could be demonstrated that N-methyl-D-
might be nitric oxide (NO), which coincided with aspartate (NMDA) produced a considerable
the proposal of Ignarro (University of California, increase in the levels of cGMP. After adding
Los Angeles), who had suggested that it was an certain inhibitors of NOS, specifically methyl-
oxide of nitrogen or a derivative of it [5]. Both arginine, the formation of the nucleotide was
scientists based their ideas on the pharmacological inhibited by the same concentrations which
similarities shared by the two molecules: inhibited the enzyme [9].
instability, relaxing effect by stimulation of GC,
inhibition by hemoglobin and stabilization by Numerous ongoing studies confirm the
superoxide dismutase [3]. important, varied and sometimes even
controversial role of this mediator in the different
Moncada and his colleagues (Wellcome physiological and pathological processes.
Research Laboratories of Beckenhan, UK)
provoked the liberation of EDRF in endothelial
cells and registered the relaxing effect on the SYNTHESIS, ENZYMATIC DIFFERENCES
smooth muscle. They also measured the amount of AND GENE REGULATION
liberated NO, and it happened to be sufficient for
completely explaining the relaxation in the adjacent Two facts sustained the hypothesis that L-
muscle cells. It could therefore be concluded that arginine (L-arg) might be the precursor of NO: on
Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 883

Fig. (1). Synthesis reaction of nitric oxide: substrats, cofactors and implicated agents (reduced nicotinamide adeninedinucleotide
phosphate, NADPH; flavin adenin denucleotide, FAD; flavin mononucleotide, FMN; tetrahydrobiopterin, BH4, calmoduline, CaM).

one hand, it was confirmed that the formation of flavin adenine dinucleotide (FAD), flavin
nitrates and nitrites in activated macrophages mononucleotide (FMN), calmoduline (CaM), heme
started from this aminoacid [6]. On the other hand, and tetrathydrobiopterin (BH4) [4, 15]. The latter
it was observed that endothelial cells cultured in cofactor plays a crucial role in the interaction of
the absence of L-arg, increased dramatically the the subunits of the enzyme and in the formation of
production of nitrogen oxides when the aminoacid the active center. Its synthesis is a restrictive
was added [4]. Mass spectroscopy using the process linked to the formation of NO and it acts
isotope 15N-L-arginine confirmed the formation of as a modulator in the gene expression of NOS
15NO precisely starting from the nitrogen atom of mRNA and of its proteins [16].
the guanidino end of the aminoacid [8].
NOS is a flavoprotein which has some
The biological meaning of NO synthesis was homology with cytochrome P-450. A prime
investigated using the compound NG-monomethyl- classification distinguishes basically two isoforms:
L-arginine (L-NMMA), whose inhibitory action in constitutive nitric oxide synthase (cNOS), and
the formation of nitrites and nitrates in inducible nitric oxide synthase (iNOS). Both
macrophages was wellknown [4]. L-NMMA share structural analogies [17] Fig.(2) and they
inhibited NO release in specifically cultured can be regulated by a process of negative feed-back
endothelial cells and this action was reversed in the mediated by NO itself. The enzymatically
presence of L-arg. Furthermore, L-NMMA generated NO as well as the endogenous NO
increased the tone of aortic tissues and inhibited inhibit the protein NOS by a mechanism related to
the endothelium-dependent relaxation induced by the heme-dependent oxidation of L-arg [18].
acetylcholine. Again, these effects were annuled by
L-arg. The antagonist L-NMMA was also shown cNOS releases small quantities (pmol) of the
to abolish the reduced nicotinamide gas in an intermittent way over short periods
adenindinucleotide phosphate (NADPH)- (seconds-minutes) [19]. Two varieties of cNOS
dependent conversion of the aminoacid into with similar characteristics, but different location,
citrulline, which is a by-product in the formation exist:
of NO. This biosynthetic route has been proven in
many tissues and cells, such as macrophages, Constitutive endothelial nitric oxide synthase
neutrophils, nervous tissue, adrenal glands or (eNOS or isoform III) appears primarily in
Kupffers cells, etc [8, 10]. endothelial cells, platelets and mesangial renal cells
and is mainly involved in the regulation of vascular
The enzyme responsible for the synthesis of homeostasis [20, 21, 22]. Neuronal constitutive
NO is nitric oxide synthase (NOS) which oxidizes nitric oxide synthase (nNOS or isoform I) is
the terminal nitrogen atom of L-arg, leading to the located in the neurons of the central and peripheral
equimolar production of NO and L-citrulline nervous system and it produces NO which acts as
Fig.(1) [6, 9, 11, 12]. Then, it is converted again neurotransmitter [23, 24, 25].
into L-arg by means of the incorporation of an
amino group [13, 14]. In order to exert its action, Both isoforms can undergo phosphorylation.
the enzyme requires the presence of two The phosphorylation of nNOS occurs due to the
cosubstrates, NADPH and O2 and five cofactors, action of protein kinase II, which is calcium- and
884 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

Fig. (2). Structure of the domains of cNOS (eNOS, nNOS) and iNOS. Domains for NADPH, FAD and FMN in the terminal carboxyl
group. Binding sites for the CaM and heme/L-arg complex are located in the terminal amino group, where phosphorylation and
myristoylation occur (From Marn and Rodrguez-Martnez [17]).

CaM-dependent, protein kinase C (PKC) and The isoform eNOS plays a key role
cyclic AMP (cAMP)-dependent protein kinase coordinating different extracellular stimuli which
[26, 27]. The phosphorylation of eNOS regulates modulate blood pressure and platelet aggregation.
its cellular translocation starting off the liberation Its synthesis is regulated at transcriptional, post-
of hormones and vasodilating agents from transcriptional and post-transductional levels. The
endothelial cells [9]. eNOS gene promoting region contains zones
controlled by agents responding to changes related
The activities of both NOS isoforms are strictly to stress or sexual hormones [31]. The levels of
controlled by the calcium levels. So, when the cells mRNA of eNOS are subjected to a dynamic
containing these enzymes receive the adequate regulation performed by cytokines [20].
stimulus (acetylcholine in case of endothelial cells Moreover, the enzyme is modified at post-
and glutamate in case of neuronal cells) the tranductional level by myristoylation in the
activation of the receptors located on the terminal N atom [32]. This reaction is necessary in
membrane produces a rise in the cytosolic calcium order to guarantee its interaction with the
levels. This pathway leads to the activation of the endothelial membrane of the cell.
enzyme and finally to the synthesis of NO [28].
The increase in the intracellular Ca2+ ion results On the other hand, NO liberated via nNOS
from the Na+ /Ca2+ exchange processes in the plays an important role as neurotransmitter in the
membrane and also from the movilysation out of neuronal development and regeneration and also in
the intracellular deposits (endoplasmatic numerous neurological disorders where an
reticulum). Calcium becomes linked to the protein excessive production is detected. Nevertheless,
calmoduline originating the CaM complex, which nNOS expression is not only limited to these cells,
regulates the activity of proteins with enzymatic as trancripts have been found in non neuronal cells,
function (protein kinases, adenylylcyclases, being dynamically regulated by different
phosphodiesterases, NOS and Ca2+ -Mg+ /ATPase) physiological or pathological conditions as well as
as well as proteins of structural function [29, 30]. physico-chemical, biological or electrical stimuli.
CaM exhibits four calcium ligand sites allowing Its increment is also associated to the co-induction
conformational changes which facilitate its binding of trancriptional factors similar to c-jun [33, 34]
to proteins. and c-fos [35, 36]. In the cerebellum, drastic
inhibition of the glutaminergic neurotransmission
Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 885

enhances the expression of nNOS [37], which is promoter of the iNOS gene, hence triggering the
often located next to NMDA-type receptors [38, transcription. The influence of antioxidants and
39]. In addition, some hormones like estradiol can protease inhibitors on the expression of iNOS after
induce its formation [40,41], whereas activation by LPS of macrophages has been
corticosterone decreases its transcription [35, 36]. extensively studied. In these circumstances, the
liberated NO acts as a proinflammatory agent and
The inducible isoform (iNOS or isoform II) is a therefore its inhibition elicits antiinflammatory
calcium-independent enzyme. It is located in responses. Transcriptional expression of iNOS
macrophages, hepatocytes, smooth vascular implicates ways which also affect the inducible
muscle, neutrophils or endothelial cells. Although expression of genes codifying for proteins that are
it requires a certain time to be expressed (hours) responsible for the immune response and
[19, 42], it is capable of generating NO in a inflammation.
continuous way (hours-days) in massive amounts
(nmol). It is transcripted in response to In addition, the possibility that non steroidal
immunological stimuli like certain endotoxins antiinflammatory drugs (NSAIDs) may interfere
(lipopolysaccharids, LPS) [10, 43]. Similarly to with the production of NO mediated by iNOS in
cNOS, NO can also feed-back the activity of macrophages has been evaluated [48]. Aspirine,
iNOS. sodium salicylate, ibuprofen, indomethacin,
diclofenac and naproxen inhibit the formation of
Its regulation is undertaken by transcriptional nitrates produced by LPS-stimulation in these
factors belonging to the rel family, among which cells. In spite of not directly altering the catalytic
the nuclear factor kappa B (NF-B) is included. activity of iNOS, NSAIDs certainly impair the
This factor is determinant in the regulation of synthesizing capacity of this enzyme in intact
many genes involved in the immune function and cells. Precisely, expression analyses reveal that
in the inflammatory response. Initially, NF-B NSAIDs impair transcription by annuling the
was described as a transcription factor which was synthesis of iNOS proteins [48].
able to bind to the gene of the light chain of B
lymphocytes. Later, binding sites were found in Rats treated with LPS have been demonstrated
other immunoreceptor genes or in cytokines. Its to increase their blood concentration of nitrates up
activity has been observed in epithelial cells of the to ten-fold. Moreover, it has been proven that
intestinal crypts, macrophages and epithelial cells nitrate production can be prevented by
close to small vessels of the inflamed mucosa [44] administrating NSAIDs one hour before the
and a correlation between the number of cells inductor (LPS) [50]. All studies suggest that
presenting activated NF-B and the inflammation activation of NF-B plays a remarkable role in the
grade can be established [45]. transcriptional activation of iNOS.

One of the most important mechanisms in the


regulation process of this nuclear factor when it FUNCTIONS OF NITRIC OXIDE
appears in the nuclear cytoplasm, implicates its
inactivation by binding to inhibitory proteins (I- NO shows a dual behavior: at low doses it
B). As a result of this binding, the complex I- regulates homeostasic functions, whereas at high
B is generated which then acts as a doses it acts as a cytotoxic molecule which lesions
transcription finisher [46, 47]. the tissue and affects the immune system.
Nevertheless, NO activity depends on its
Among the extracellular signals which induce a synthesis site and on the protein which takes part
rapid activation of NF-B, figure oxygen free in its formation.
radicals that stimulate the mRNA expression of
iNOS. These reactive species, start
phosphorylation and proteolytic reactions causing 1. Activity of Endothelial Constitutive Nitric
dissociation of the transcriptional factor and Oxide Synthase
inhibition of the I-B complex in the cytoplasm.
NO generated in endothelial cells by eNOS acts
The free translocated factor diffuses to the nucleus
as a mediator in the transmission of cell-cell stimuli
and activates NF-B which in turn stimulates the
and it plays a key role in the regulation of blood
886 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

Fig. (3). Actions related with NOS activity.

flow and maintenance of vascular tone Fig.(3). cGMP is a second messenger abundant in many
The target protein for the NO released from the cells of the organism: endothelial and epithelial
endothelium is GC. This is a membrane receptor cells (vessels, bronchi, intestinal wall), cones,
with own enzymatic activity. The extracellular sticks, cerebellar cells and cells that fix the
portion of the receptor exhibits a domain with the natriuretic peptide. This molecule behaves as an
binding site for the ligand. The presence of the intracellular signal and among others it can act
ligand leads to a conformational change enabling upon kinases, phosphodiesterases or ionic
the intracellular portion , which is enzymatically channels. The increment in the intracellular cGMP
active, to act on specific substrats. levels has different consequences: relaxation of the
smooth musculature of the vascular wall,
Two different types of GC are known: one is vasodilation and inhibition of the proliferation of
located on the plasmatic membrane and its smooth muscle cells. Furthermore, it provokes
structure is a monomeric protein with a single inhibition of platelet aggregation and of leucocyte
transmembrane portion.The domain which acts as adhesion to the vascular endothelium and also
a receptor, capable of identifying and binding to modulation of the activity of the mastocytes
different substrats, is located in its external N- Fig.(4) [52].
terminus. The intracellular portion exhibits the
domain with tyrosin-kinase protein activity and Frequently, the cells containing the enzyme
the domain with GC activity is located next to the NOS are not the same which produce cGMP. This
C-terminus. The other form of GC is located in the fact proves the capacity of NO to diffuse from the
soluble cellular fraction and it adopts a dimeric producing cells to the effecting cells.
conformation consisting of two subunits: (82
kD) and (70 kD). Each of them has a domain
with GC activity in C-terminal situation. The 2. Activity of Neuronal Constitutive Nitric
combined action of both subunits is necessary in Oxide Synthase
order to fully express GC. A main characteristic of
this enzyme is that it can be activated by NO as NO formed in the nervous tissue by the ncNOS
well as by other nitrates. NO is capable of isoform, acts as a neurotransmitter in the central
stimulating it after binding to the heme group, and peripheric nervous system (non adrenergic non
located in the active site of the enzyme. Inside the cholinergic fibres, NANC). It also plays an
heme group, NO binds to the Fe2+ ion giving rise important role in the control of the relaxation of
the smooth muscles along the gastrointestinal,
to a change in the structure of the enzyme, which
respiratory and genitourinary tract.
after activation increases the production of cGMP
[4, 51].
Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 887

Fig. (4). Nitric oxide is a resonance hybrid.

The brain exhibits the highest NOS activity of mediator with a non-heme Fe2+ atom which is
the organism and the ubiquitous distribution of the conjugated to a sulfur atom, resulting in the
enzyme suggests that NO may be implicated in inhibition of enzymes linked to the glucidic
numerous functions of the CNS: synaptic metabolism (aldolase), Krebs cycle (aconitase),
plasticity, establishment and refining of axonal electron transport respiratory chain (cytochrome
projections during the late stages of development, oxidase) and of the DNA synthesis (ribonucleotide
memory, nervous control of the cerebral blood reductase) [54].
flow, neuroendocrine regulation, autoregulation of
neuronal activity and production of other Another mechanism which accounts for the
neurotransmitters. NO diffuses back from the cytotoxic activity of NO, is its capacity of
postsynaptic neuron, and after binding to the heme interacting with reactive species like the
group of GC located in the presynaptic neuron, it superoxide radical (O2.), giving rise to
is supposed to activate the production of cGMP peroxinitrites (ONOO.) [55, 56] and to other
and other phosphorylated compounds which can compounds like trioxide of dinitrogen (N2O3),
increase and maintain the release of presynaptic which are highly damaging for the organism [57,
neurotransmitters [7, 8]. 58, 59]. The reactive character of NO is due to the
position of its eleven electrons of valency. Seven
Glutamate acts as an excitatory of them are placed in the most external orbital and
neurotransmitter in the CNS, as it provokes one electron is unpaired, which renders this
stimulating responses after activating the molecule a free radical Fig.(4) [60].
corresponding receptor protein. One of these
proteins is the NMDA receptor. It displays The radical ONNO. is generated when O2.
inotropic features and is associated to a channel radicals produced by neutrophils and macrophages
that allows the flux of Na+ , K+ and Ca2+ ions. This in inflammatory processes reach intracellular levels
way, different amplifying responses are stimulated similar to the ones of NO and both species react
by means of second and third messengers, like the together very rapidly Fig.(5) [56, 61]. Apart
activation of calcium-calmoduline-kinase II from oxidizing a wide range of biological molecules
complex, PKC, phospholipase A2, NOS activation including proteins, lipids and nucleic acids,
or synthesis of eicosanoids. ONNO. radicals can react with thiols and tyrosin
residues [52, 62, 63] damaging not only the cell but
3. Activity of Inducible Nitric Oxide Synthase also the genetic material. Moreover, ONNO. can
also conjugate with ONOOH acid, diffusing
NO produced by the activation of the iNOS immediately through the membranes and damaging
isoform is essential in inflammatory processes, cells that are far from its origin [64].
repairing of tissues and unspecific mechanisms of
host defence against the aggression of infectious The intermediate N2O3 has been profoundly
agents [53]. Several consequences result from the investigated and it has been proven that different
increase in tissular NO produced by this substrates can be nitrosilated by this oxidizing
enzymatic route Fig.(3). agent [53, 54], hence contributing to a chronic
inflammation and malignant transformations. The
NO produced in macrophages by this formation of S-nitrosilated complexes (RSNO)
isoenzyme contributes to the intra- and during the autooxidation of NO and further
extracellular activity of these cells. This anti- interaction with N2O3 and certain thiols [49, 54],
microbial activity is due to the interaction of the suggests that these compounds play an important
888 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

Fig. (5). The polymorphonuclear (PMN) leucocytes and macrophages release superoxide radicals (O2) in inflammatory processes
which react quickly with NO allowing the formation of peroxinitrites (ONOO) which can attack other cells or the leucocyte itself.

role in NO transport, in transduction signals and in continuous liberation of NO by the endothelial


regulation of gene expression [62] Fig.(6). cells prevents neutrophil adherence to vascular
walls and intervenes in the maintenance of cellular
From these reactions it might be inferred that integrity. In addition, its efficacy as pro- or
NO released via iNOS exerts a mainly cytotoxic antiinflammatory is related to the relative presence
and proinflammatory effect. However, other of other oxidizing molecules.
studies reveal its protective properties. The

Fig. (6). Physiological reactions of NO. The formation of S-nitrosilated (RSNO) complexes during the processes of NO autooxidation
induces oxidation and nitration reactions which contribute to cellular damage.
Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 889

In summary:

*Similar concentrations of NO and O2 peroxinitrites

oxidizing processes

*Excess of NO limited superoxide formation which prevents tissue lesion

4. Role of Nitric Oxide on Immunological and concentration: picomolar amounts of


Apoptotic Processes constitutively produced NO suffice in order to set
off certain intracellular signals and to induce
NO is also an important mediator of the physiological effects. On the other hand,
immune response. High levels are implicated in the micromolar concentrations synthesized by the
activation and proliferation of lymphocytes, inducible isoform, may lead to alterations in some
stimulation of tumoral necrosis factor (TNF-) tissues [67, 68]. In this way, NO has been related
production via NF-B and tyrosin kinase p56 to the etiopathogenesis of numerous immune
activity, which is involved in certain lymphocyte diseases: diabetes [70], rheumatoid arthritis [71],
signals [65, 66]. However, low levels suppress the lupus eritematosus [72] or multiple sclerosis [73].
activity of the antigen-presenting cells and
proliferation of T cells [67, 68]. There is evidence Apoptosis is another mechanism related to this
to show that NO exerts different effects on the gas. It is generally accepted that cell death occurs
subpopulations of these cells. So, it inhibits the in two different ways: apoptosis or programmed
secretion of interleukin-2 (IL-2) by Th1 cells, death and necrosis or uncontrolled death [74]. The
while it increases the production of interleukin-4 apoptotic process is genetically controlled and it is
(IL-4) by Th2 cells [69]. important for cell proliferation as well as for
maintaining homeostasis. Alteration of the
In relation to the non specific immunity, the regulation mechanism can give rise to numerous
cytotoxic effect depends also on the local lesions. In the beginning of this process plenty of

Fig. (7). Cellular mechanisms responsible for apoptosis in the enterocytes. Pro-caspase-8 is an endogenous protease implicated in the
process. It turns into the active form (caspase-8), by various signals: Fas ligation, viral infections, growth factors, DNA damage, etc.
Bcl-2 inactivates this route.
890 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

physiological and non physiological stimuli take its subsequent liberation. Once again, NO plays a
part Fig.(7), provoking the activation of certain dual role in the regulation of the process. On one
enzymatic routes. Among these enzymes are the hand, it allows the formation of ONNO. by
caspases, specific cysteinic proteins for aspartic reacting with superoxide anions and it triggers the
residues, which catalyze the repairing of DNA by increase in the membrane permeability and also in
ADP-ribose polymerase enzymes [75, 76, 77]. the calcium concentration, stimulating apoptosis
Caspases are in part responsible for the beginning, by means of these mechanisms [88, 89]. On the
amplification and fulfilment of apoptosis [78]. The other hand, NO activates Bcl-2/Bcl-xl resulting in
principal markers of this process are the increase the inhibition of the Bax/Bak pathway and thereby
in intracellular calcium and the descent in cytosolic blocking the caspases cascade [87, 89, 90].
pH [79] which produces stimulation of Cytochrome C intervenes in the cellular
endonucleases and DNA fragmentation. Although destruction, but it also promotes the release of
gene regulation is not completely clarified, several some anti-apoptotic substances which block the
implicated genes upon which NO acts, are known. caspases [78]. Another mechanism by which NO
The family Bcl-2 represents one of the principal prevents the apoptotic process is by modulating
controlling factors. Cells expressing this protein transcriptional factors AP-1, NF-B and
are totally resistant to apoptosis and malignant extracellular signals similar to TNF [91, 92]. In
transformations. The suppressing gene p 53 also summary, NO is a pleiotropic molecule, which
seems to play an important role in the regulation depending on its local concentration, either
of the dynamic cycle [80]. protects cells against apoptosis, or on the
contrary, induces apoptosis. Low concentrations
NO promotes apoptosis in macrophages, protect B lymphocytes against viral infections,
CD4+/CD8+ thymocytes , condriocytes and whereas high concentrations induce apoptosis in
pancreatic B cells [81, 82]. Caspases can be macrophages, hepatocytes, glial cells, neurons and
activated by the release of cytochrome C and other cells of the immune system.
possibly by other factors diffusing from the
mitochondrial intermembrane space into the
cytoplasm [83, 84, 85, 86]. Some antiapoptotic 5. Non Enzymatic Nitric Oxide Production
proteins like Bcl-2 and Bcl-x1 hinder this release,
whereas it can be activated by proapoptotic Bax Given the important physiological role of NO,
proteins [87]. One of the most defended numerous studies have been performed in order to
mechanisms is that cytochrome C itself induces an elucidate if nitrogenated compounds of the diet
increase in its own permeability, transition and in could be its precursors. So, a non enzymatic

Fig. (8). In vivo reduction routes of nitrites to give NO. Bacteria present in the oral cavity or urinary infections reduce the nitrates to
nitrites with the intervention of the enzyme nitrate reductase. The reduction to NO occurs in acidic conditions, as is the case of the
stomach.
Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 891

production has been detected, which results from by the salivary glands and are secreted in the
the reduction of nitrites Fig. (8). The properties saliva, where the concentration of nitrates is in the
of NO are constant irrespective of its origin. range of 200-600 M and the one of nitrates
reaches 30-210 M [97]. Part of these nitrates are
Two steps of the NO cycle should be explained reduced by the bacterial flora of the oral cavity to
in more detail, in first place the reaction catalyzed nitrites. Consequently, NO is synthesized in the
by NOS enzyme, and second, the reduction of stomach after the reduction of the salivary nitrites
nitrite-reductase, catalyzed by an electron- [98, 99].The intragastric production depends
donating system with the participation of fundamentally on the pH, oxygen content and
cofactors, such as NADH, NADPH and bacterial flora [100]. In the stomach, nitrites are
flavoproteins. The result of this enzymatic/non acidified and subsequently undergo dimerization
enzymatic cycle is the oxidation of NO itself [93]. and dehydration to N2O3, which can be converted
into NO and NO2 [99, 100].
Indeed, this agent and other nitrogen
compounds, including nitrous acid, are generated While the lack of nitrated compounds in the diet
from nitrites in a reaction of acid reduction (pKa can lead to untoward effects [101, 102], in the
3.2-3.4) Fig. (9). Ascorbate and ascorbic acid stomach, the nitrogen oxides stemming from
figure among the reducing agents. The metabolic nitrites can react with amines and originate toxic
routes of NO have been studied by monitoring the nitrosoamines.
excretion of 15N in urine and feces. Yoshida and
cols. [94], proved that 15N either inhaled or *NO Metabolism in the Renal System
injected, rapidly entered the blood stream, was
oxidized and was excreted in urine without any of Nitrates coming either from the vegetable intake
its metabolites appearing in high degree in the or produced by enzymatic synthesis, are
tissues. 93% of the inhaled 15N was converted into eliminated principally by urine [95], contrarily to
nitrate in the kidney. Comparable reactions were nitrites which only appear in minute amounts.
observed after treatment with 15NO 2 -15NO 3 -, as Filtration of these derivatives takes place in the
well as after oral administration of ( 15NH 3)-N [95, glomerulus and then they are reabsorbed again in
96]. the renal tubuli. The concentration of nitrates in
urine is 250-2000 M, whereas nitrites and NO
*Metabolism of NO in the Digestive Tract are hardly detected [98]. This fact is profitable in
the clinical diagnostic of urinary infections.
The nitrogen derivatives of the diet stem Pacients treated with antibiotics for a long time,
principally from pulses and vegetables. Over 25% exhibit an inhibition in the NO synthesis at renal
of the circulating nitrates are actively incorporated scale [103], which points to the participation of

Fig. (9). Biosynthetic route of NO starting from nitrogen compounds of the diet. Production of dehydroascorbic acid in the presence of
NO 2 and vitamin C (From Weitzberg and Lungberg [99]).
892 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

Fig. (10). Generation routes and antibacterial effects of NO. Due to the acidic reduction conditions, nitrites yield NO and other
nitrogen intermediate reactives with antimicrobial effect (From Weitzberg and Lungberg [99]).

renal microorganisms in this process. Acidification Similar to GC, the enzyme COX also contains a
of urine and vitamin C intake are prophilactic heme-Fe2+ group as active site, hence rendering it a
treatments against such infections, because in these target for NO. Some studies reveal that the
conditions a certain amount of non enzymatic NO increase in the mediator activates the enzyme by
is liberated and this could have germ-killing effects cGMP-independent mechanisms and this way
Fig.(10). induces the production of eicosanoids. It has been
further proven that NO and PGI2 or PGE2 act
synergistically amplifying physiological or
RELATIONSHIP BETWEEN NITRIC OXIDE
pathological responses [104, 105] Fig. (11).
AND THE ENZYME CYCLOOXYGENASE
It is known that NO is implicated in COX and NOS are distinct enzymes encoded
maintaining homeostasic integrity via by different genes. However, they share certain
cyclooxygenase (COX). In some conditions, it can biochemical and molecular characteristics. Both
activate this pathway and thereby stimulate the enzymes are hemeproteins and, in their native
synthesis of COX-derived prostaglandins (PGs). state, are homodimers which possess bifunctional

Fig. (11). Regulation model of the cyclooxygenase activity by nitric oxide.


Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 893

Fig. (12). Interactions of the cyclooxygenase (COX) and nitric oxide synthase (NOS) isoforms in physiological and pathological
conditions.

catalytic activities. In addition, both enzymes been obtained in epithelial colonic cells, where an
exhibit constitutive and inducible isoforms. important induction of COX-2 takes place when
NO production is stimulated. This finding suggests
The constitutive enzymes, COX-1 and cNOS, that NO modulates the expression of COX-2 and
give rise to products which play important roles in PG synthesis derived from this enzyme [109].
physiological conditions. At least in the vascular
system, they catalyze the synthesis of two potent
molecules, PGI2 and NO, which act synergistically ROLE OF NITRIC OXIDE IN THE
on controlling platelet, monocyte and smooth GASTROINTESTINAL TRACT
muscle cell activities, thereby maintaining vascular
integrity and blood fluidity [106]. Over the last years, numerous studies have been
conducted which reflect the regulation exerted by
On the other hand, the inducible proteins, NO on the homeostasic functions of the GI tract
COX-2 and iNOS, are concurrently expressed in [110,111,112]. In comparison with other tissues, it
inflammatory tissues. They catalyze the synthesis is known that NOS activity is particularly
of prostanoids, principally PGE2, NO and augmented in the gut [113]. In the muscular layer,
oxygenated intermediates such as peroxinitrite NO secretion is to a large extent of neuronal origin.
anions, which cause severe damage [105]. This In fact, a high expression of the constitutive
way, both enzymes act in a cooperative manner synthase has been located in the Auerbach plexus
contributing to the pathogenesis of inflammatory [114]. On the contrary, in the mucosa it is mostly
processes and tissue injury [104] Fig. (12). synthesized in epithelial cells and there is only
minute endothelial production of the mediator.
At gastrointestinal (GI) level, evidence
supporting the idea of a COX/NOS cooperation,
exists. In the presence of LPS-induced NO or of 1. Participation of Nitric Oxide as Endogenous
NO-donors, like sodium nitroprusside, an Modulator
increment in PGs occurs, which suffices in order to
explain the reduced injury when irritating agents NO acts together with vasoactive intestinal
are administrated [107, 108]. Similar results have peptide (VIP) as a neurotransmitter in the NANC
894 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

nervous system, which innervates the whole GIT. Apart from controlling of the motor activity of
These neurons of inhibitory character play a the musculature, NO intervenes in much other
notable role in the peristaltic reflex circuit acting functions of the GIT, such as acid secretion,
directly on the smooth muscle. In physiological regulation of mucosal blood flow as well as
conditions and after being liberated from these maintenance of the mucus-bicarbonate barrier and
structures, NO participates in the modulation of of the tissular integrity Fig. (13).
the digestive musculature tone and of the vessels
which irrigate it [110]. Although the intervention The mechanisms by which NO regulates the
of NO in the gastric reflex is not questioned, some activity of the parietal cell are not completely
authors suggest that the synthesis of the mediator clarified, but the available data suggest a
is stimulated by VIP in postjunctional cells and in multifactorial process. In 1994, Barrachina et al
nervous endings. This hypothesis is not in [119] found that the administration of NO-donors
accordance with the studies of Bayguinod et al did not modify the response to acid which was
[115], which in spite of confirming the effected by direct stimulators of the parietal cell,
participation of both neurotransmitters in the like histamine or pentagastrin. But on the contrary,
relaxing effect of the gastric fundus via cGMP and NO-donors antagonized the acidity induced by
cAMP, failed to provide evidence about the NO- gastric distension in rats. These results suggest
dependent responses being consequence of the that NO inhibits neuronal acid secretion. Acid
cascade stimulated by VIP. production following distension of gastric walls is
started by mechanical stimulation of sensory
NO is involved in the regulation of intestinal neurons and principally through the
peristaltism [116] of gastric emptying [117] and of vagocholinergic pathway. Acid inhibition, due to
the antral motor activity. Konturek et al [118] NO derived from vagal neurons, would be the
proved that endogenous NO delayed these consequence of the suppression exerted by the
parameters without affecting myoelectric gastric neurotransmitter on the liberation of histamine
activity. Their study showed that postprandial from histaminergic cells [120].
antral contractility, expressed as motility index, as
well as emptying of the stomach, increased It has been suggested that the changes induced
significantly in healthy volunteers receiving a on NO synthesis by endogenous secretagoges
treatment of L-NMMA (inhibitor of NOS might modulate the activity of the parietal cell
activity). On the contrary, this rise was not through a calcium-dependent process. Carbacol
observed in the group treated with the combination and pentagastrin increase NO production, while
of the inhibitor and L-arg (precursor in the histamine fails to modify it. From this it might be
synthesis of NO), or with the aminoacid alone. At infered that the effect exerted by NO on acid
the same time, none of the treatments modified secretion occurs through the cholinergic pathway
electrogastrographic results. [121]. NO also regulates the levels of acidity
originated by bile acids [122].

Fig. (13). Main physiological actions of NO in the gastrointestinal tract.


Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 895

In healthy individuals, L-NMMA NO plays a key role in maintaining the integrity


administration delays the recovery of intragastric of the microvasculature, as the enhancement of
pH from the fasting values and suppresses the physiological activity goes hand in hand with an
postprandial release of gastrin, whereas it increment in blood supply, this way guaranteeing
increments the plasmatic levels of somatostatin. the provision of necessary agents and the removal
These effects were reverted by L-arg, therefore of waste products. The inhibition of NO synthesis
these changes might be mechanisms used by NO at gastric level decreases the mucosal blood flow
for the regulation of postprandial acid secretion (MBF), and the enhancement of the gastric
[118]. Recent studies suggest that NO modulates circulation due to acetylcholine or bradykinin, is
the activity of the proton pump [123] which NO-dependent [130,131,132]. NOS inhibitors
undoubtedly constitutes a fundamental factor in attenuate the increment in the blood flow induced
the control of acidity. by pentagastrin. The compensatory capacity of
this molecule against the vasoconstrictive effects
Gastric mucus secretion increases in vivo [124] of noradrenalin, neuropeptide K or endothelins,
and in vitro [125] after administrating NO-donors. this way maintaining an adequate vascularization,
The mechanism might be related with an increment has also been reported [133]. In summary, these
in the activity of the calcium-dependent synthase, data reveal the important modulating role of NO in
which consequently would stimulate cGMP the vascular homeostasic control of the GI tract.
production through the activation of the GC
system. Contrarily, the inhibition of cNOS
activity diminishes the ability of mucosal cells to 2. Cytoprotective Effect on Gastric Mucosa
synthesize and secrete the protective gel. It is also
correlated with a descent in the concentration of In physiological conditions, NO acts as an
hexosamines and in the viscosity of the gel [126]. endogenous mediator, together with PGs and
It has been proposed that agents, which induce a gastric neuropeptides modulating both, the
rise in the calcium levels, might activate the repairing of the tissue and its integrity [134].
constitutive enzyme cNOS, hence favoring mucus Endogenous synthesis of NO mediates the
secretion [124]. protection against gastrolesive drugs (NSAIDs)
[135], necrotizing agents [136], ischemic processes
The effects of NO on alkaline production of the [137, 138], platelet activating factor (PAF) [139],
mucosa have also been extensively studied. endotoxines [140], stress [141] or pylorus ligature
Lambrecht and Peskar [127] and Kitagawa et [142]. In most of the assayed models the
al.[128] observed that the cytoprotection afforded administration of NOS inhibitors was
by some mild irritants was mitigated by demonstrated to augment the damage. In contrast,
pretreatment with inhibitors of the synthase or it has been proven that NO-donors or precursors
GC, suggesting that NO is involved in adaptative improve the beneficial effects of certain antiulcer
cytoprotection. Later, Takeuchi et al.[129] proved drugs like sucralfate [143], carbenoxolone [144] or
that the gastric alkaline response after the epidermal growth factors [145].
administration of mild irritants is mediated by PGs
as well as by NO. The authors propose that the Although the mechanisms for the protective
stimulation provoked by a hypertonic NaCl action of this mediator are not completely clarified,
solution on the mucosa releases NO, which it is known that several factors are involved. On
prevents acid secretion and allows the luminal one side, its vasodilating effect permits the
alkalinization as a consequence of the HCO3- flux maintenance of blood flow in adverse conditions in
in the lesioned area. This phenomenon is regulated which acid secretion and retrodiffusion of protons
locally and represents an adaptative response of are stimulated, or in situations of hypoxia, but the
the gastric mucosa in adverse conditions, because protecting action of NO results also from its
the liberation of the alkali affords a beneficial antioxidant and antiinflammatory activity.
microclimate for epithelial restitution. In these Currently, it is known that the amelioration of the
circumstances, NO contributes to the repairing of damage induced by ischemia-reperfusion processes
tissular damage and to modulate mucosal integrity. or by NSAIDs consumption, is connected with the
capacity of NO to inactivate the reactive species
896 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

produced during these processes, including the secretion, the tissular production of the nucleotide
radicals O2, OH and H2O2 [146], and is also cGMP as well as mucosal blood flow [155, 156,
related to its ability to prevent the peroxidation of 157].
lipids in the cellular membranes [147].
Furthermore, it has been proven that the The maintenance of the mucosa after
abolishment of NO production promotes administration of irritants or other sorts of
leucocyte adhesion to the endothelium, elicits aggressive agents, depends on its capacity of
vasoconstriction and suppresses hyperemia in activating either the pre-epithelial (mucus-alkaline
areas close to ulcers, hence delaying the healing of secretion) or the post-epithelial (increase in
the lesions. All these mechanisms are favored by microcirculation) defensive mechanisms. The
the simultaneous inactivation of the antioxidant mucosa responds quickly to the injury by means
properties. of cytoprotective adaptation, consisting in the
onset of processes which include the stimulation
Some authors have proven a modification in the of sensorial neurons, cell proliferation and local
resistance of the mucosa after vagal stimulation synthesis of PGs and NO [134, 158, 159]. As
[148] or after administration of capsaicin, already mentioned, NO promotes alkaline
papaverin, pentagastrin or gastrin [135, 149]. In a secretion in mucosas subjected to mild irritants,
model of NSAIDs-induced ulcers, specifically but its involvement has also been demonstrated in
ibuprofen, the severity of the lesions was other aggressive conditions. Such is the case of
compared after pretreatment with inhibitory prolonged aspirine consumption, which in spite of
molecules, aminoguanidine (AG, preferent iNOS producing a logical drop in PG levels, is
inhibitor), L-nitroarginine (L-NNA, preferent accompanied by a significant increment in eNOS
cNOS inhibitor) or L-NAME (non selective expression and blood flow [160]. It stands to
inhibitor of both isoenzymes). AG, though not reason that the mucosal increase in the endothelial
significantly, was shown to diminish the severity constitutive enzyme may compensate for the
of the lesions, while the inhibition of the reduction of PGs and that this increase may partly
constitutive form aggravated the damage [150]. account for the adaptative response of the mucosa
These results provide strong evidence for the against NSAIDs administration.
importance of the constitutively released NO in
the maintenance of GI homeostasia.
3. Involvement of Nitric Oxide in Gastric
Paradoxically, although the mediator is Disorders
synthesized starting from L-arg, the role of this
aminoacid in gastroprotection has not been This background might support the idea tha NO
thoroughly investigated. Recently, it has been is only related to protective mechanisms.
proven that pretreatment with this molecule dose- However, through its synthesis via iNOS it is
dependently decreases the lesions induced by HCl implicated in numerous pathological settings,
[151], ethanol [152] or stress [153]. This decrease including the pathologies associated with
goes hand in hand with a gradual rise in infections by Helicobacter pylori (Hp). This
angiogenesis and gastric MBF. Similarly, bacteria represents one of the most important
concurrent administration of L-arg and ibuprofen, etiopathogenic factors of diseases like peptic ulcer,
diminishes significantly NSAIDs-induced chronic gastritis or gastric cancer [161, 162, 163,
damage.This antiulcer protection of L-arg is in the 164]. The damage caused by Hp has been related
same range as the one afforded by equipotent to the increase in reactive nitrogenous species
doses of misoprostil (PGI2 analog) or H2 inhibitors including the formation of peroxinitrites.
like ranitidine and roxatidine [154]. These Expression studies evidenced a significant rise in
discoveries have led to the commercialization of mRNA of iNOS and COX-2 in gastric mucosa of
drugs consisting of NSAID combined with L-arg, patients suffering from chronic Hp(+) gastritis.
whose protective efficacy is similar to that of NO- Immunohistochemical analysis revealed the
releasing molecules, such as isosorbide presence of both proteins in different cellular
mononitrate. These associations provide not only populations of the lamina propria, suggesting that
the uncoupling of all the oxidative stress-related apart from being induced, they modulate
mechanisms, but also enhance the gastric mucus inflammation and alterations occurring in the
Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 897

epithelium. The high amounts of iNOS and COX-2 4. Implications of Nitric Oxide in
detected in the gastric antrum of these patients, Physiological an Pathophysiological Intestinal
where bacterial density is elevated, indicate that Processes
the expression of genes which modulate these
proteins is a direct response to the infection by The dual effect of NO is also reflected in the
Hp [165]. intestine. The gas liberated via eNOS might
contribute to the maintenance of mucosal integrity
Individuals suffering from Hp(+) peptic ulcer in adverse situations of hypoxia and inflammation
frequently exhibit an increased expression of iNOS where severe damage is produced. In fact, its
and nitrotyrosin (biological indicator of beneficial effect has been proven in different
peroxinitrites formation). In addition, accumulation models of experimental colitis [169, 170] or in case
of macrophages in the active ulcer margins and of intraabdominal surgery or organ manipulation,
sometimes in superficial cells, in cells of the lamina circumstances in which a strong increment in
propria and in areas distant from the lesion has vascular permeability (VP) takes place, which
also been detected in these patients. On the reverts after administrating the precursor or NO-
contrary, in samples of Hp(-) ulcerated mucosas, donors [171]. NO is an important endogenous
although the presence of iNOS and nitrotyrosin is modulator of the barrier functions and its
frequent in the ulcer margins, no immunoreactivity inhibition causes many of the hallmark features of
has been found in remote zones. In these patients, intestinal inflammation including neutrophil
eradication of the bacteria provokes a remarkable recruitment, oxidative stress, mast cell
descent in the concentration of the synthase, in the degranulation, and enhancement of microvascular
number of macrophages and in the concentration of and epithelial permeability [172, 173]. Moreover,
peroxinitrites, which suggests that this bacteria chronic administration of NOS inhibitors also
contributes to the stress related to the NO route. causes intestinal inflammation [174].

Goto et al [164] were studying the mechanisms By contrast, it has been observed that elevated
implicated in carcinogenesis associated with amounts of the mediator liberated after inducing
chronic Hp infection, when they discovered that iNOS expression by endotoxin, promote
Hp(+) mucosas which had developed gastric extravasation of plasma in GI tissues with a
cancer also showed high iNOS and nitrotyrosin worsening of the injury [175], while the inhibition
expression in comparison with healthy individuals. of the enzyme protects the microvasculature by
This fact suggests that the high production of decreasing permeability [176]. With the help of
these proteins may contribute to the process. In chemiluminiscence techniques, important increases
fact, the concentration of the inducible enzyme is in the intraluminal NO levels in the rectum of
clearly related to the intensity of metastasis, patients suffering from bacterial gastroenteritis
therefore a role in tumor extension is arrogated to [177] as well as higher concentrations of nitrates
this enzyme [166]. Nevertheless, it has not been and nitrites in urine and plasma [178] have been
possible to prove that iNOS expression might be detected. Similar results have been observed in
induced by inflammatory cytokines like TNF or jejunum of individuals suffering from celiac
IL-1. Modifications in cNOS expression among disease, which suggests that the excess of NO
healthy and affected individuals have neither been involved in the intestinal inflammation is liberated
observed. by the inducible synthase [179].

The analysis of Hp(+) biopsies reveals that Recent data support the hypothesis that NO
besides enhanced iNOS expression, an increment in constitutes an important etiological factor in
DNA fragmentation due to stimulation of the chronic inflammatory bowel disease (IBD) and
caspase-3 cascade (marker of the apoptotic ulcerative colitis (UC). The beginning of the
process) also takes place. Bacterial eradication and process is consequence of the toxic action of
a diet supplemented with vitamin C and - infectious agents and toxins which weaken the
carotenes (antioxidant compounds) prevent not mucosal barrier and set in the immune response.
only the inducible protein expression, but also Alteration of this response implies the abnormal
carcinogenesis and the apoptotic process [167, activation of the different cell types, muscular,
168]. epithelial and endothelial, and also the production-
898 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

release of inflammatory mediators such as (TNBS) diminishing injury and inflammation


cytokines, PGs, leukotrienes (LTs), PAF, growth degree and accelerating the process of cell
factors, which promote leukocyte adhesion from proliferation [189]. Nevertheless, the currently
the endothelium and subsequent migration to the available data are not concluding because other
inflamed zone [180,181,182]. authors, using the same experimental model, did
not accomplish a reduction in the severity of
In mucosal biopsies of patients suffering from colonic inflammation in animals treated with
IBD, an increase in NO concentration as well as selective (AG) and non selective (L-NAME)
iNOS overexpression, which may play a key role inhibitors of the synthases [187].
in aggravating the process and exacerbating the
immune response, has been observed [183, 184,
185]. Although no significant differences have been 5. Dual Action of Nitric Oxide from cNOS and
confirmed in the renal excretion of nitrates and iNOS on Gut
nitrites, the plasmatic concentration of these NO
metabolites in affected patients is significantly Although the dual beneficial or deleterious
higher than in healthy individuals [186]. The same effect, of NO on GIT has been frequently
applies to patients who develop colorectal associated respectively with its constitutive or
adenocarcinoma [187]. inducible production route, some ongoing studies
question this division. In a recent review Kubes
In models of experimental colitis induced by [190] profoundly analyzes the effects derived
trinitrobenzenesulfonic acid (TNBS), elevated from both synthases and concludes that the
amounts of nitrotyrosins, derivatives of hypothesis that cNOS is good and iNOS is bad is
malonyldialdehyde (MDA), myeloperoxidase far too simplistic and no longer explains the
(MPO) activity and expression of adhesive majority of data generated by the scientific
integrines have been detected. These changes are community. The results obtained by Mndez et al
related to an increment in the production of NO [191] confirm that in gastric alterations induced by
via iNOS. It has been demonstrated that genetic uremia, the increase in cNOS activity is
ablation of iNOS gene conferred to mice a responsible for the overproduction of NO and this
significant resistance to TNBS induced lethality mediator is possibly responsible for the derived
and colonic damage, and notably reduced pathological changes.
nitrotyrosin formation and concentrations of
MDA. This suggests that activation of NOS is Numerous studies have reported small amounts
required for nitrosative and oxidative damage in of mRNA of iNOS and protein under normal
experimental colitis [188]. In a series of studies conditions in the gut, thus a role for this isoform
using endotoxin, Boughton-Smith et al [176] cannot be excluded [190]. The presence of
showed that although inhibition of nitric oxide inducible synthase in normal intestine should not
within the first few hours of endotoxin be surprising since that the epithelium of this
administration was detrimental to the intestine, mucosal surface is always exposed to foreign
inhibition of NOS activity for a long time resulted antigens and the bacteria and bacterial products,
in protection against endotoxin damage. The normal milieau in the colon, are a potent stimulus
authors proposed that the administration of an for iNOS induction in tissues [192]. However, a
NOS inhibitor in the early phase of injury series of data suggest that NO liberated by this
inhibited NO produced by cNOS and hence isoenzyme might play a repairing role in certain
exacerbated the deleterious effect. By contrast, the physiopathological circumstances. After the
same NOS inhibitor in the second phase of inhibition of NOS in the model of TNBS induced
endotoxin injury inhibited the inducible synthase colitis, contradictory results have been obtained:
and functioned in a protective form. complete protection [193], partial protection
[170], decrease of lesions in a determinate area
These studies provide new opportunities in the [194], even no protection at all [195] or
pharmacological therapy oriented towards the use exacerbation of damage [196]. These data support
of selective iNOS inhibitors which fail to the possibility that iNOS may be produced only in
negatively affect the constitutive forms. The use of determinate areas, that its toxicity may be
AG has shown good results in experimental colitis restricted to determinate zones or that it may even
Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 899

participate in the healing process. In acetic acid The acid character of most of these molecules
induced colitis, Akiba et al [197] demonstrated renders them irritating agents directly damaging the
that the inhibition of iNOs increased the number of epithelium. This topical action derives from the
inflammatory cells in the crater margin and delayed biochemical changes induced by NSAIDs at the
ulcer healing. These observations suggest that NO absorption site and it is due to the uncoupling of
generated from iNOS not only participates in the the mitochondrial oxidative phosphorylation and
lesion but also plays a beneficial role in ulcer inhibition of electron transport [205, 206] which is
healing, in part by the exclusion of iNOS-positive related to an increment in the oxigen reactive
inflammatory cells from the regenerating mucosa. species (ORS) production, alteration of the
osmotic balance and loss of cellular control. The
In iNOS deficient (iNOS-/-) mice, some authors injury is also provoked by the systemic action of
reported that the lethal dose of Listeria and other NSAIDs in which different mechanisms take part.
infectious agents resulted less damaging than in The most important is the drop in mucosal PGs
wild-type mice [198]. In acetic acid induced colitis, synthesis via inhibition of the COX enzyme,
Mc Cafferty et al [199)] found a twofold increase leading to a decrease in the mucosal defences. The
in macroscopic damage in the iNOs-/- vs iNOS+/+ ulcerogenic capacity of NSAIDs is closely
mice. Although inflammation in wild-type mice connected with their COX-inhibitory potency, as
had resolved by seven days, a sevenfold increase in PGs take part in the modulation/regulation of the
damage score and elevated MPO levels were still mechanisms involved in the control of gastric
evident in transgenic mice. In addition, a striking homeostasis, like mucus and bicarbonate secretion,
enhancement in mRNA for iNOS was observed in maintenance of blood flow, cellular regeneration
normal animals at 24 hours and was still present at and restitution processes as well as in the mucosal
72 hours, but no message was found in iNOS- immune function.
deficient mice. The authors conclude that induction
of inducible synthase seems to be a critical Concurrently, other alternative mechanisms
protective response to injury in intestinal related to changes in the digestive microvasculature
inflammation. However, the results for responses occur. By this means, neutrophil adhesion to the
to LPS were different. While in some cases a higher endothelium happens hindering the
resistance to develop the damage was observed in microcirculation and provoking ischemic situations
mutant mice [200], other studies failed to find any [207, 208], which can lead to hemorrhagic erosions
difference between the responses of iNOS-/- and in those areas where blood flow is significantly
iNOS+/+ animals [201]. diminished. Moreover, after their activation,
neutrophils release ORS and proteases which are
In summary, it is evidenced by these data that in large measure responsible for lesion formation.
the equivocal role of NO in the gut must be In this way, an increase in neutrophilic activation
carefully evaluated. and in oxidative stress has been associated with
intake of NSAIDs, such as indomethacin [209],
NITRIC OXIDE IN THE PREVENTION OF piroxicam [210] and diclofenac [211]. TNF
NSAID-INDUCED GASTROPATHY seems to play an important role in the process of
leucocyte adhesion to the endothelium in response
Although NSAIDs are frequently used in the to NSAIDs. Several investigators have
treatment of arthritis and other osteoarticular demonstrated significant rises in the plasmatic
diseases, they often produce untoward effects in concentrations of this cytokine after NSAIDs
the GI tract, which include dispeptic symptoms, administration.They also showed how
erosions, ulcers and even severe complications like pretreatment with pentoxifiline, which produces a
bleeding and perforation [202]. Recent calculations dose-dependent decrease in TNF abolishes the
estimate that around 20-50% of the patients accumulation of neutrophils thereby preventing
consuming NSAIDs develop digestive the damage [212, 213].
complications and 1-2% of these require
hospitalization [203, 204]. The lack of energy supply in the cellular
environment as a consequence of the decrease in
Pathogenesis of the digestive toxicity associated local flow, in turn increases adenosine
with these drugs is consequence of several factors. triphosphate (ATP) consumption. During its
900 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

degradation process, ATP provokes an and inhibition of neutrophil adhesion processes


intracellular increment of the purine nucleotides, [221]. Recent results published by Saha et al [222]
especially of adenosine, inosine and hypoxanthine. about the possibilities of S-nitroso glutathione in
The enzymatic systems in charge of maintaining acute and chronic experimental models have shown
the ionic gradient throughout the membrane are how this compound, when administrated together
also affected, giving rise to a considerable increase with piroxicam, diminishes the lesions by 70%. In
in the intracellular calcium levels. All these factors an effort to antagonyze the gastrolesive effects,
provoke the activation of proteases capable of different NSAIDs, including acetyl salicylic acid,
converting xanthine dehydrogenase (XDH) into diclofenac, naproxen and flurbiprofen, have been
xanthine oxidase (XO) [214], which catalyzes the formulated by attaching a NO releasing-moiety
reaction from xanthine to uric acid. XO may also [223-225].These NO-NSAIDs keep their COX-
use hypoxanthine as reaction substrat this way inhibitory properties and therefore their
generating oxidative metabolites like O radicals, antiinflammatory and antithrombotic efficacy, but
which react rapidly with organic molecules and contribute to protecting gastrica mucosa.
produce damage by reductive reactions as the
spare electron is liberated. O radicals can interact The increment in gastric motility detected after
with H2O2 giving rise to the hydroxyl radical NSAIDs intake, contributes to the
(OH ), and even react with NO producing ONOO, microcirculatory alterations, therefore the muscular
which is highly toxic due to its capacity to relaxation mediated by NO via cGMP represents a
generate again OH [215]. Among the deleterious gastroprotective mechanism [226, 227]. In
effects arrogated to the reactive species, the addition, the capacity of the mediator to stimulate
following may be pointed out: inactivation of the production-liberation of mucus [144, 125]
enzymes, attack on genetic material and might also increment its cytoprotective capacity.
peroxidation of membrane lipids, which provoke
profound alterations in the properties of the It is likely, that the beneficial effect of L-arg on
membranes with serious repercussions for cellular the gastropathy induced by ibuprofen is afforded
funcionalism [216, 217]. by NO-dependent mechanisms, because the
sistemic administration of the non active isomer D-
Currently, it is accepted that the protective role arginine (D-arg) fails to protect against NSAIDs-
exerted by NO on the gastric mucosa is induced damage. On the other hand, the aminoacid
consequence of its vasodilating effect and of its is capable of reverting the deleterious effects,
hyperemic response against noxious agents. which NOS inhibitors like L-NAME or L-NNA
Moreover, it has also been demonstrated that the provoke on the gastric mucosa [150]. Different
reduction of damage in the ischemia/reperfusion hypothesis explain how L-arg annuls those effects:
process or after NSAIDs intake, both processes in amelioration of the ischemic conditions after
which numerous oxygenated metabolites are NSAIDs administration and previous to the
released, is connected with its capacity to appearing of the lesions, feed-back mechanisms or
inactivate these metabolites [146]. Simultaneously, independent beneficial effects which are capable of
the mediator inhibits the peroxidation of the counteracting the detrimental conditions due to the
membrane lipids [147] and promotes restitution presence of the NSAID. In this sense, it has been
processes in the mucosa [218]. Furthermore, it has demonstrated that the aminoacid in the presence of
been proven that the inhibition of NO synthesis ibuprofen, augments significantly the tissular
elicits leucocyte adherence, vasoconstriction and concentration of cGMP and inhibits the activity
decrease in hyperemia in areas close to ulcers, and iNOS expression [156], suggesting the
hence delaying lesion healing. All these participation of vasodilating mechanisms via
mechanisms are favored by the simultaneous NO/GC and by means of blocking the detrimental
inactivation of the antioxidant properties. On the effects derived from the overproduction of the
contrary, substances known as NO-donors, such mediator by the inducible synthase.
as glycerine trinitrate or nitroprusside, protect
gastric mucosa against different types of Given that in gastric and intestinal mucosas
aggression and accelerate the healing [219, 220]. damaged by NSAIDs an increment in NO
The mechanisms through which these agents exert production via iNOS is observed [229-231], to
their beneficial effect, include maintenance of MBF inhibit this enzyme might be another possibility in
Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 901

order to reduce gastroenteropathy. The use of acetyl penicillamine) are donors which act as
specific inhibitors of the inducible synthase has prodrugs exerting their effects according to their
been assayed in numerous experiments, ability to yield NO. Due to their vasodilating
accomplishing contradictory results. Although it qualities allowing an enhancement of coronary
has been demonstrated that pretreatment with AG blood flow, they are very useful in different
diminishes intestinal permeability and attenuates cardiovascular diseases, such as angina pectoris,
inflammation [230, 232, 233], it has been already heart infarct or coronary vasospasm.
mentioned that under some circumstances,
inhibition of the synthase produces detrimental Concerning the prevention of NSAIDs-induced
effects. Taking into account that the majority of gastroenteropathy, numerous strategies have been
available data proceed from experimental studies, designed including administration of PGs analogs,
more profound and long-term studies are required histamine H2 receptors antagonists or proton
in order to materialize this therapeutical pump inhibitors. More recently, the development
alternative. of the new selective inhibitors of the inducible
isoform of cyclooxygenase COX-2 have
contributed to minimizing the problem Fig. (14).
CLINICAL PROSPECTS Nevertheless, although these drugs statistically
produce less damage than classical NSAIDs, they
As can be infered from the adduced data, the are not completely safe in inflamed mucosas [234,
pharmacotherapeutical possibilities of NO are 235].
considerably wide because its vasodilating and
antioxidant properties render it a potentially useful These considerations encourage the synthesis of
agent in numerous pathologies. In fact, the NO-NSAIDs or antiinflammatories combined with
pharmaceutical industry and numerous precursors of the mediator or with inhibitors of the
investigators are really interested in proving the inducible synthase. Some of these molecules are
clinical benefit of the mediator. undergoing clinical phase trials, and in other cases
like the association of ibuprofen/L-arg, have
Nitrated derivatives have been frequently used already been commercialized. The results obtained
in the cardiovascular pharmacotherapy. Organic prove the gastroprotective effect of these drugs,
nitrates and nitrites (glyceryl trinitrate, isosorbide which might represent a therapeutical alternative in
mononitrate or amyle nitrite), inorganic nitrates the prevention and treatment of NSAIDs-induced
(sodium nitroprusside), sydoniomines gastroenteropathy. However, until the mechanisms
(molsydomine or its metabolite 3-morpholino- derived from stimulation/inhibition of the
sydoniomine) and S-nitroso thioles (nitroso-N- isoenzymes and the exact role of nitric oxide in

Fig. (14). Traditional (A) and new strategies (B) in the prevention of NSAIDs-induced gastropathy.
902 Current Pharmaceutical Design, 2001, Vol. 7, No. 10 Martn et al.

physiological and physiopathological processes of L-NNA = L-nitroarginine


the gut are not profoundly elucidated, it will not be
possible to totally evaluate the clinical utility of LPS = Lipopolysaccharide
NO-donating agents.
LT = Leukotrienes

ABBREVIATIONS MBF = Mucosal blood flow

AG = Aminoguanidine MDA = Malonyldialdehyde

ATP = Adenosine triphosphate MPO = Myeloperoxidase

AMP = Adenosine monophosphate NANC = Non adrenergic non cholinergic


fibres
cAMP = Cyclic adenosine-monophosphate
NF-B = Nuclear factor kappa
BH4 = Tetrahydrobiopterin
NMDA = N-methyl-D-aspartate
CaM = Calmoduline
NO = Nitric oxide
CNS = Central nervous system
cNOS = Constitutive nitric oxide synthase
COX = Enzyme cyclooxygenase
eNOS = Endothelial nitric oxide synthase
D-arg = D-arginine
iNOS = Inducible nitric oxide synthase
DNA = Desoxiribonucleic acid
NSAID = Non steroidal antiinflammatory
EDRF = Endothelium-derived releasing drugs
factor
ORS = Oxygen reactive species
FAD = Flavin adenin dinucleotide
PAF = Platelet activating factor
FMN = Flavin mononucleotide
PG = Prostaglandins
GC = Guanylyl ciclase
PKC = Protein-kinase C
GIT = Gastrointestinal tract
mRNA = Messenger ribonucleic acid
cGMP = Cyclic guanidin monophosphate
RSNO = S-nitrosilated complexes
GSH-px = Glutathione peroxidase
SOD = Superoxide dismutase
Hp = Helicobacter pylori
Th = T helper cells
IBD = Inflammatory bowel disease
TNBS = Trinitrobencensulfonic acid
I-B = Inhibitory protein kappa
TNF- = Tumoral necrosis factor
IL = Interleukin
VIP = Vasoactive intestinal peptide
L-arg = L-arginine
XDH = Xanthine deshydrogenase
L-NMMA = NG-monomethyl-L-arginine
XO = Xanthine oxidase
Nitric Oxide and its Effects Current Pharmaceutical Design, 2001, Vol. 7, No. 10 903

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