Indian Journal of Experimental Biology Vol. 38, March 2000, pp. 201-210

Review Article

Nitric oxide: A molecule of the millennium

U A Shinde, A A Mehta & R K Goyal*

Department of Pharmacology, L M College of Pharmacy, Navrangpura, Ahmedabad 380 009, Indi a

Recognition of Nitri c oxide (NO) as the chemical entity of endothelium-derived relaxing factor (EDRF) has renewed the interest o f the scientific community in the last decade. The outcome of research the world over is that the dreaded environmental pollutant is fo und to be a fundamental physiological mediator and effector. NO is synthesized endogenously by enzymes nitric ox ide synthase (NOS) in speciali zed tissues from its precursor L-argininc. The L-arginine-NO biosyntheti c pathway is involved in physiol ogical processes such as vasodilation, memory, neuroprotection , peristal sis, penile erection, immune defense, various endocrine and exocrine secretions in various sys tems such as cardiovascular, CNS ,. reproducti ve and immune system. Small quantities of NO produced by const itutive enzymes med iate these physiological effects. The expression of inducible enzyme or overstimul ation of constitutive enzymes leading to prod uction of large quantities of NO is implicated in the cytotoxic effects observed in various disorders like AIDS, cancer, Alzheimer' s, arthriti s ~tc. In conclu sion , NO is a 'double edged sword' and the challenge before the scientifi c community is to develop strategies for using it to our advantage.

Ce ll signali ng is a phenomenon that is ubiquitous and is an essential requirement in multicellular organisms for interce llular and intracellular communi cations. Many proteins and non-proteinaceous molecules have been identified that take part in cell signaling. Nitric oxide (NO) is a unique signaling molecule which was labeled as the " Molecule of the Year" in 199i. The 1998 Nobe l Prize in Medicine for the di scovery of NO was the third to be awarded in the area of cell signaling in the present decade. T he ro le of endothe lium as the source of a novel 'relax ing fac tor', endothelium-derived relaxing factor (EDRF), was first advanced by Furchgott and Zadawaski 2 when they demonstrated that endogenous vasodi lators such as acetylcholine do not act directly on vascul ar smooth muscle but instead on endothe lial cells, causing them to release a labile fac tor that diffu ses into the overlay ing smooth muscle. In 1986, it was proposed by Robert Furchgott and Louis Ignarro that EDRF was NO J.4 _ In 1987, a positive identification of EDRF as NO was made independently by two groups of scientists : Richard Palmer and Salvador Moncada with the ir colleagues and also by Louis Ignarro and his coll aborators 5

·6

. Pri or to 1987, the bio logical properties of NO were associated with bacteria l function, and wi th environmental concerns regarding NO and other nitric oxides present in car ex haust fumes and cigarette smoke. Discoveries in the 1980s, however, led to the realization that NO served a

*Correspondent author: Fax:91-79-6304865 Phone: 9 1-79-63027 46

number of important phys io logical roles in hi gher animals . Although biological effects of NO had been known for some time it , nonethe less , came as a surprise that NO could be synthesized in the body and played such important roles in the regul ation of myriad phys iological functions. Nitroglyceri n sublingual tablets frequently used to give relief from angina have now been fou nd to be medi ated through the release of NO. Recent introduction in the cure of

impotency- Sildenafil (V iagra®, Pfizer Inc.) is an indirect modulator of NO acti vity. In the present artic le we present some of the basic physiol ogical and pathophysio logical aspects of nitric oxide.

Production and regulation of NO in the body Bacteria generate NO from nitrite or oxidation of

ammonia. NO is biosynthesized in mammals by a modified urea cycle7 that has two important function s - a secretory ro le to regenerate L-argin ine for NO synthesis and an excretory role to eliminate excess nitrogen c reated by cell's metabolism. The initial step in the biosynthes is of NO is cata lyzed by nitric oxide synthase (NOS). NOS are NADPH-dependent oxygenases which require tetrahydrobiopterine, FAD, and FMN as cofactors (Fig. 1) . The normal level of L­argini ne in the body is suffici ent for a continuous secretory NO biosynthes is and at present two distinct types of enzymes that catalyse NO production are known , a) a Ca2

+ requiring constitutive enzyme or eNOS and b) a Ca2

+ independent inducible enzymes or iNOS . At least two isoforms of eNOS exist -

202 INDIAN J EXP BIOL, MARCH 2000

NADPH

0

0

L-arginine NG - OH- L-arginine

H2N YO

o· JNH /l

---- NO [Nitric Oxide]

YNH/ 0

L-cit ru lli ne

Fig. I- Biosynthetic pathway of Nitric oxide

eNOS, present in endothelial cells and nNOS , present in neuronal cell s. There is, however, some evidence that iNOS from different cell types (macrophages versus liver cell s) demonstrate variable Ca2+ sensitivity.8 Some of the established properties of NOS isoforms are summarized in Table I .

Molecular cloning of eDNA belonging to all the three forms of NOS has been successfull y achieved in recent years, using either the DNA probes/primers representing the conserved sequences or through the use of antibodies9

. All the three enzymes have been shown to have binding s:~-.;s for cofactors FAD, FMN, NADPH and calmodulin . They show close homology with cytochrome P-450 reductase (CPR) having consensus '>equences for the binding of NADPH , FAD and FMN. The close homology of NOS with CPR suggests that electrons follow the same path through NOS as they do through CRP, that NADPH reduces FAD, which in tum reduces FMN. Each of the three NOSs has - 36% homology to CPR in its C­terminal half containing the NOS reductase domain , w'hich contains the binding sites for NADPH, FAD and FMN . Thi s homology reflects the oxidative mechani sms of 0 biosynthesi s. The th ree NOSs also have considerable (> 50%) homology among themselves .10

Regulation and inhibition of NOS NOS is one of the most regulated enzymes in

biology and the structure of NOS reveals numerous regu latory mechani sms .

Regulation of nNOS and eNOS by Ca2+

NOS enzymes are regui ated by Ca2+. In the brain, stimulus such as glutamate acts at N-methyi-D­aspartate (NMDA) receptor and tri ggers Ca2+ influx which binds to calmodulin and ac tivates nNOS to form NO. This formation of NO is invol ved in neurotransmission. Similarly. in blood vessels, acetylcholine acts on acetylcholine receptors on endothel ial cells and act ivates pho phoinositide cycle to generate Ca2+, which activates NOS to form NO. Thus, the activity of NOS is regulated through Ca2+/calmodulin, both in neurotransmiss ion and vasod ilation and is in hibited by calmodulin antagoni sts like trifluoperazine 10

.

Regulation of inducible macNOS (iNOS) and immune response

Cal modulin has been shown to be tightl y bound to inducible macNOS (iNOS), but th is bi nd ing is unaffected by Ca2+. Synthes is of inducible OS is ac tua lly induced by st imuli such as in terferon-y and

SHINDE et al.: NITRIC OXIDE 203

Features

Table !-Properties of NOS isoforms

eNOS iN OS (inducible) (constitutive)

Other nomenclature

Molecular weight

Calcium dependency Source

nNOS (neuronal)

Type I bNOS (brain)

168 KDa

Yes NANC neurons Skeletal muscle White blood cells

eNOS (endothelial)

Type 3 ecNOS

135 KDa

Yes Endothelial cells Some neurones

Type 2 MacNOS (macrophage) Immunological

130 KDa

Respiratory and GIT epithelia

No Macrophages Neutrophils Hepatocytes Fibroblasts Chondrocytes Epithelial cells

Kidney macula densa cells Pancreatic islet cells

Stiumulus for activation I induction Glutamate HIV gp-120 b-amyloid peptide

Amount released Small, pulses

Function Regul atory

lipopolysaccharides (LPS) and this inducible synthesis is not restricted to macrophages and has been reported in tissues lacking macrophages. The ubiquitous distribution of this form of inducible NOS reflects a primitive form of immune response, where the NO system must have sufficed to repel the invading microorganisms without the aid of antibodies and/or T-cell receptors 10

.

Regulation of NOS by phosphotylation Consensus sequences for phosphorylation by

cAMP-dependent protein kinases have been shown to be present in nNOS and eNOS, but not in iNOS, leading to decrease in their activity. Other kinases including protein kinase C , cGMP-dependent protein kinase, and Ca2+/calmodulin dependent protein kinase, have been implicated in phosphorylation of nNOS. Phosphorylation also regulates sub-cellular distribution of NOS. 10

Inhibition of NOS Endogenous inhibitors of NOS, such as methyl

arginines, have been suggested to exist and, recently, it was reported that there is a I 0 KDa protein, named 'PIN' that is able to destabilize nNOS dimers, and hence inhibit enzyme activity, in vitro .11

Acetylcholine Bradykinin Thrombin Shear stress

Small, pulses

Regul atory

Lipopolysaccharide (LPL) Interferon y Cytokines Oxidised LDL

Large, continuous

Host defense

Many of the available synthetic NOS inhibitors are analogues of the substrate, L-arginine, which act by competing with L-arginine at the active site of the NOS. These inhibitors generally demonstrate little selectivity between three NOS isoforms and have the potential to interfere with other enzymes that utilize L-arginine, and affect its uptake into cells. The most widely used of these are NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester 12

.

Aminoguanidine has traditionally been used to inhibit iNOS selectively, but it also reduced eNOS and nNOS activity. Recently , the compound N-(3-(aminomethyl)benzyl) acetamidine has been shown to be a potent and selective inhibitor of iNOS in vitro and in vivo. 13 The compound 7-nitroimidazole is a non-selective inhibitors of nNOS and eNOS in vitro. [t acts by reducing the affinity of the enzymes for tetrahydrobiopterin and L-arginine 14

• It is claimed to have some selectivity for nNOS in vivo and has been widely used for this purpose. More recently 1-(2-trifluoromethylphenyl) imidazole has been identified as a competitive inhibitor of nNOS and iNOS 15

• The truly selective nNOS inhibitor is still eagerly awaited .

Interestingly, the induction of iNOS, but not the expressed enzyme eNOS, can be inhibited by

204 INDIAN J EXP BIOL, MARCH 2000

glucocorticoids and this may be an expl anation for the anti -infl ammatory effects of such drugs 16.

Molecular mechanisms involved in NO fu nction

NO as a signaling molecule is complete ly different from class ical medi ators 17

• Unlike class ical mediators, NO, a lipophilic free radica l gas diffuses freely through the pl asma membrane and does not need either the ves icles for secreti on from signaling cell or any cell surface receptors in the target ce ll s for triggering the signal. It passes readil y to the underlying smooth musc le and stimulate vasorelaxati on18

• The molecul ar targets of NO in victim cell s are Cu-Fe prote ins, releas ing free Cu2

+

and Fe2+ and generating 0 2. and highl y toxic hydroxyl

radicals, thus leading to large scale ox idative injury . Schematic representation of constituti ve and induced NO re lease and its signa l transducti on pathway is given in Figs 2a and b.

Severa l mechani sms of action of NO in vari ous tissues have been proposed. The one establi shed target, o r " receptor", for NO is the cytoso lic enzyme, solubl e guanylyl cyc lase (sGC). It is a heme­conta ining heterodimer that converts guanoc ine-5'­triphosphate (GTP) into the second messenger cyc lic guanosine monophosphate (cGMP) 19

. Thi s reacti on is Mg2

+ dependent and is dramaticall y stimul ated by the binding of NO to the sGC pros thetic group The acti vity of sGC is increased I 00-200-fo ld by NO and the enzyme displays a high degree of selecti vity for NO. cGMP has range of cellul ar targets, inc luding cyclic nuc leotide-gated ion channe ls and cGMP dependent prote in kinases. It can also contro l the breakdown of another nucl eotide, cAMP, by st imulating or inhibiting phosphod iasterases.

NO acts onl y locally, due to its short half li fe of about 5- l 0 sec in ext race llul ar space, where it is converted to nitrates by 0 2 and water. It mostly acts as a covale nt ly reacti ve redox type mediator. When NO is re leased by constituti ve pathway in small amounts it preferenti ally binds to haem moieties in the environment. Excess of NO is mopped up by binding to thi o ls or by nitrosation reaction.

itrosothiol s such as nitrosocys te ine are as acti ve as NO itself and act by re leas ing N020

"21

. It is al so suggested that NO c ircul ates in mammalian plasma bound to SH groups of a lbumin2 1

• Therefore intrace llular and extracellul ar thi o ls may act as stores prolonging the action of re leased NO.

Physiology and Pathophysiology of NO

Central nervous system

NO has been implicated in many areas of CNS funct ion . Biochemi ca l experiment s have indicated that exogenously applied NO is able to influence the release of several neurotransmitters such as gluatamate, GABA, dopamine, acetylcholine, adrena li ne.22 In bra in NO acts as a messenger and modul ates the ac ti on of excitatory neurotransmitter, glutamate. The hi ghest concentration of cGMP occurs in the cerebe llum, where g lutamate is the majo r neurotransmitter. Thi s is achieved by NMDA

h. h . I ?' 24 S I . receptor, w IC IS g uta mate receptor-·· . e ect1 ve inhibiti on of NOS by L-nitroargini ne blocks NM DA­induced elevati on of cGMP, suggesti ng the ro le of NO in this process.

In brain NOS occurs in all neurons of basket and granule cell s of cerebe llum while it is present onl y in 1-2% of the neurons of cerebral cortex, hi ppocampu s and corpus stri atum. Further the dis tri bution of NOS neurons often matches that of the neurons staining fo r NADPH-diphorase (NDP) and the purifi ed NOS has been shown to have NDP acti vity. T herefore, it has been suggested that NOS catalyti c ac ti vi ty requiring NADPH, is a lso responsible for NDP hi stochemical act ivity. NDP pos itive neurons are known for thei r resistance to destruction in neurodegenerative diseases. For instance in Huntington's disease, cerebral ischemi a and excitotox icity, overstimulation of NMDA receptors probabl y selecti ve ly protects N.ADPH-diphorase staining neurones 17

. It is poss ible that the presence of NO or the diphorase acti vity protec ts neurons from ox idative injury as it is the NOS activity that accounts for the diphorase staining.

Overstimul ati on of NM DA receptors can lead to excessive Ca2

+ entry and production of tox ic levels of NO and neurotox ic ity25

. Neuronal damage caused by overstimulati on of EAA receptors is assoc iated with a variety of neuropathological conditions includ ing stroke, epilepsy, and head trauma, as we ll as neurodegenerative di seases such as Huntington's Chorea and amyotroph ic lateral sc lerosis (ALS or Lou Gehrig's di sease). A ro le of NO has been implicated in the neurona l damage and dysfunction associated with Parkinsonism, mult iple sclerosis and the dementia assoc iated with Alzhe imer's and acquired immunodefi ciency syndrome. Whether there is dysfunction in the regul ati on of bNOS or eNOS ac tivity is increased in microglial cell s is not clear26

.

(A)

(B)

SHIN DE eta/.: NITRIC OXIDE

Acetylcholine, bradykinin, 5-HT, glutamate, ADP, ET-1

Shear stress C 2+ / a

/

GT~ cGMP s

+ cGMP-PK ----+ relaxation

Endotoxin, cytokines

02- stimulation of gene ~ expression of iNOS

Nuclear activating factor

L-Argin~ iNOS

L-Citrullinf~ NO

Cu2+ proteins

Fe2+ proteins

peroXY,nitrite

ox;datl ;,,-u

Stimulus

Generator cells Neurons,

endothelial cells

Target cells Neurons, smooth muslces, platelets

Stimulus cells

Killer cells rnacrphages, kupffer cells

Victim cells cancer cells, parasites

Fig. 2-Schematic representation of (A) constitutive NO release and (B) induced nitric oxide release and their signal transudation pathways

205

206 INDIAN 1 EXP BIOL, MARCH 2000

NO has been suggested to play a role in synaptic plasticity and act as a 'retrograde messenger' in the neurophysiological phenomena underlying memory 17

'27

. Long term potentiation (LTP) is an enhancement of synaptic efficacy lasting hours to weeks. The · subclass of EAA-receptor, NMDA recl!ptor ionophore complex trigger the L TP formation to let Ca2

+ into cell. The intracellular Ca2+

causes the potentiation in the postsynaptic response enhancing their sensitivity/efficacy. It is suggested that NO is produced in post-synaptic neurons and is diffused back to presynaptic terminals, spreading to the neighbouring synapses, thus functioning as a retrograde messenger in LTP induction28

.

Gastrointestinal system In all regions of the gastrointestinal tract, NOS

. h . I f 29 10 J I 'fh occurs m t e myentnc p exus o neurons ·· ·· . ese neurons mediate the physiologic relaxation of the gut and participate in the normal peristaltic activities of the gut. In the NANC nerves of gastrointestinal and urogenital tracts NO acts as a neurotransmitter25·n and can mimic the effects of nerve st imulation. Blocking the formation or action of NO inhibits the effect of nerve stimulation. The term 'nitrergic transmission' has been proposed for all neuro-effector transmission effected by NO in the gastrointestinal and urogenital tract. It has also been speculated that NO release in gastrointestinal tract is mediated through the activation of 5-HT4 receptors3

·' .

Reproductive system The physiology of penile erection IS thought to

involve parasympathetic neuronally mediated relaxation of blood vessels as well as of the trabecu lar mesh network of smooth musc les that comprise the corpora cavernosa leading to increased blood flow in to penile artery. The localization of NOS to neuronal fibres innervating blood vesse ls and the corpora cavernosa of the peni s suggested a possible role of NO as a neuromediator of penile erection. L­nitroarginine, a potent and selective inhibitor of NOS markedly diminished penile erection caused by electrical stimulation of intact rat cavernous nerves34

.

It has been estab lished that an insufficient production of NO by penile nerve terminals and/or vascular endothelium may result in an impaired erection or impotence35

. Yiagra®, chemically Sildenafil citrate, the new dmg for erecti le dysfunction introduced by Pfizer Inc. has no direct re laxant effect on isolated

human corpus cavornosa, but it enhances the smooth muscle relaxant effect of NO by inhibition of phosphodiesterase 5 which is responsible for the degradation of cGMP in the corpus cavornosum36

.

Immune system NO serves as a messenger mo lecule, when

macrophages and neutrophils exert their tumoricidal and bactericidal effects. NO formation may have originated as a first line defense for metazoan cells against intracellular parasitic fungi, helminths and mycobacteria. The fact that T-lymphocytes can synthesize N037 and that glucocorticoids inhib it iNOS production, as well as the ro le for NO syn thesis in suppressing graft rejection implicate the role NO plays in the immune response.

During bacterial sepsis, a dramatic loss of vascular tone occurs resulting in pronounced hypotension, a Joss of response to vasoconstrictor agonists and death as a consequence of circulatory collapse. When macrophages are activated by endotoxin in the form of LPS in bacterial cell wall , endogenous release of pro-inflammatory cytokines (interleukin I~ . tumor necrosis factor a and interferon y) is stimulated . These stimulate the expression of Ca2

+ independent NOS in the vascular wall. This results in a marked ly enhanced and continuous production of NO in blood vessels leading to hypotension. The synthesis can be prevented by inhibitors of protein synthesis (e.g. cycloheximide) or RNA transcr iption (e.g. dactinomycin) or by anti-inflammatory glucocorticoids 18

• Further more the resu ltant hyporeactivity to vasoconstrictors in isolated blood vessels can be actually reversed wi th inhibitors of NOS.

Many cytokines such as Interleukin- 1, LPS and gamma radiation can stimul ate macrophages for NO synthesis, which in turn produce cytotoxicity'8--

19• NO

ki lls tumor cells by its capacity to disrupt respiratory enzyme activ ity and DNA synthesis, inh ibi tion of tumor cell's ribonucleotide reductase or inactivation of cytoplasmic enzymes and proteins. L-arginine the precursor of NO enhances the natural kil ler (NK) and lymphoki ne activated killer (LAK) cell act ivit/0

. In vivo arginine supplementation (30 grn/day fo r 3 days) can increase the number of ci rcu lat ing CD56+ cells, thus suggesti ng the probable use of L-arginine in immuno-suppressive states like AIDS and cancer.

The pro-i nflammatory action of NO seem to play an important role in tissue damage that occur in

SHINDE eta/.: NITRIC OXIDE 207

diseases such as septic shock, heat stroke, ulcerative colitis, cerebral malaria and auto-immune diabetes mellitus. NO formation may play a vital role in the destruction of pancreatic-• cells leading to type I diabetes. Treatment with iNOS inhibitors reduces macrophage infiltration into the pancreas and prevents hyperglycemia41

•

A similar scenario is observed with respect to rheumatoid arthritis and osteoarthritis42

. NO seems to be essential for bone formation/resorption; however, nitrite levels are elevated in the synovial fluid of patients with both rheumatoid and osteoarthritis.

Respiratory system NO may be a physiological mediator in lungs. NOS

is present in lung epithelium and other pulmonary cells and has been suggested to be a mediator of nerve dependent bronchodilation43

. Reduced NO release may also be the underlying cause of hypoxic pulmonary vasoconstriction (HPV)44

. Inhalation of NO is known to abolish HPV in man44

. Encouraging reports have come from the use of inhaled NO in pulmonary hypertension associated with neonatal adult respiratory distress syndrome and cardiopulmaonary by-pass surger/5

-47

. Elevated levels of exhaled NO have been associated with asthmatics and this probably reflects the inflammatory component of asthma48

. As NOS inhibitors increase the pulmonary vascular resistance, enhance HPV, and cause hypoxia during spontaneous breathing, there exists a possibility of using these agents in treating asthma.

Cardiovascular system NO has been found to exert negative inotropic and

negative chronotopic effect on cardiac muscle cell s. There is also evidence that in cardiac muscle NO release and possibly other factors from the endocardium may be invol ved in the beat-to-beat regulation of cardiac function49

.

Studies have shown that endothelium is abnormal in persons sufferi ng from essential hypertension because acetylcholi ne causes less vasodilation in comparison to healthy persons . These studies should imply that persons with hypertension either produce less NOS or have eNOS with abnormal structure and regulation50

'5 1

.

The vascular endothelium has a primary regulatory role to inh ibit adhesion and aggression of platelets and other blood cells and to keep the blood vessels

dialated to maintain healthy blood flow. It is believed that NO is continuously released by vascular endothelial cells and regulates blood flow and pressure. NO is also a potent inhibitor of platelet aggression and the constant release of NO from endothelial cells is thus of importance in maintaining appropriate levels of platelet adhesiveness52

. NO has also been found inside the platelets53

. It reduces clotting by inhibiting the platelet aggregation54

-56 and

adhesion57-59

• The molecular basis of this action is unknown but possible mechanisms include, activation of platelet guanylate cyclase60

'61 and adenylate

cyclase, attaching ADP-ribose to platelet glyceraldehyde-3-P04-dehydrogenase62 and inhibiting phospholipase-C63

. Thus endothelial generation of NO not only regulates blood pressure but also clotting. Endothelial dysfunction may cause local deficiency of NO, which leads to platelet aggregation, and subsequent development of atherosclerosis. Oral and/or parenteral administration of L-arginine is known to increase NO synthesis from the endothelial cells which could be useful for therapeutic purposes 40

'64

'65

.

NO automatically regulates blood flow in response to local changes in some regions of the vasculature. Ischemia and reperfusion causes vasodilation only in affected tissue and this response is probably mediated by NO as shear stress and increase in blood flow through a vessel are the physical stimuli to which the endothelial cells respond by increasing the NO production66

'67

. Basal level of NO regulates blood flow in the brain68

-70

, heare 1--

7\ lungs74

,

. . I 75 d k'd 76-7s Th . . gastromtestma tract an 1 neys . us, mtnc oxide is an endogenous autoregulator of blood flow.

Conclusion The discovery that NO is responsible for so many

functions in the human body is of great interest. The expanding role of NO in inunune function, neurotransmjssion and cardiovascular homeostasis is an exciting development in biology. The formation of NO from L-arginine is fundamental to a wide range of physiological and pathological conditions. There are clearly many opportunities for manipulations of this pathway to mitigate the most dreaded diseases such as Alzheimer's, AIDS, cancer, arthri ti s etc. In general , the "low" concentrat ions of NO produced by the constitutive enzymes eNOS and bNOS, normally mediate the physiological effects, whereas "higher" levels of NO produced by iNOS. or dysregulation of

208 INDIAN 1 EXP BIOL, MARCH 2000

eNOS, result in the cytotoxic effects of NO. The drugs that provide targeted NO deli very will be beneficial in diseases characterized by hypofunct ion of L-arginine/NO system such as hypertens ion, atherosclerosis, genitourinary disorders etc. Furthermore, NO antagoni sts and NOS inhi bitors may be indicated in situati ons of induction of iNOS or overstimu lation of eNOS. The biggest challenge is to develop strategies to target the cytotoxic ac tion s of NO without altering its essenti al protective functions in a variety of biological systems. The challenge is to understand both the good and the bad abou t NO and to manipulate them to our advantage. In the years to come we can expect to see the development of drugs for use in di seases characterized by hypofunction of L-arginine/NO system as we ll as in d iseases a sociated with hyperactive L-arginine/NO system.

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