Melatonin and its metabolites as anti-nitrosating and anti-nitrating agents

Although basal and moderately elevated levels of nitric oxide are physiologically necessary and beneficial, excessive upregulations of this signaling molecule can be a cause of damage and cellular dysfunctions. In the presence of increased amounts of superoxide anions (•O2 ) and carbon dioxide, peroxynitrite (ONOO) and the peroxynitrite-CO2 adduct (ONOOCO2 ) generate hydroxyl (•OH), nitrogen dioxide (•NO2) and carbonate (•CO3 ) radicals, which damage biomolecules by oxidation/peroxidation, nitration and nitrosation reactions. Nitrosation also occurs with all three NO congeners (NO, •NO, and HNO = protonated NO), with •NO especially in combination with electron/hydrogen-abstracting compounds, or with N2O3. 3-Nitrotyrosine, found in low-density lipoprotein particles (LDL), atherosclerotic plaques, ion channels, receptors, transporters, enzymes and respirasomal subunits, is associated with numerous dysfunctions. Damage to the mitochondrial electron transport chain (ETC) is of particular significance and involves nitration, nitrosation and oxidation of proteins, cardiolipin peroxidation, and binding of •NO to ETC irons. Resulting bottlenecks of electron flux cause enhanced electron leakage which leads to elevated •O2 . In combination with high •NO, •O2 – initiates a vicious cycle by generating more peroxynitrite that leads to further blockades and electron dissipation. Mitochondrial dysfunction, as induced via the •NO/peroxynitrite pathway, is of utmost importance in inflammatory diseases, especially sepsis, but also relevant to neurodegenerative and various other disorders. It may contribute to processes of aging. Melatonin, hormone of the pineal gland and product of other organs, interacts directly with reactive nitrogen species, but, more importantly, has antiinflammatory properties and downregulates inducible and neuronal NO synthases (iNOS, nNOS). It does not block moderately elevated •NO formation, but rather blunts excessive rises as occurring in sepsis and breaks the vicious cycle of mitochondrial electron leakage. The melatonin metabolite N-acetyl-5-methoxykynuramine (AMK) forms stable nitrosation products and efficiently inhibits iNOS and nNOS, in conjunction with other antiinflammatory properties.


Introduction
The indoleamine melatonin (N-acetyl-5methoxytryptamine; Fig. 1), once discovered as a pineal hormone with skin lightening properties in fish and amphibia, later identified as a major regulator of seasonal and circadian rhythms, is now known to represent a highly pleiotropic compound produced in various tissues and exerting numerous, highly divergent effects [1,2].This remarkable diversity appears to be the consequence of a progressive gain of functions, from a primary, ancient protection against reactive intermediates [3,4] to the more recently acquired regulation of circadian, immunological, reproductive and neurobiological mechanisms [4] that have ended up, in vertebrates, in an orchestrating, highly versatile role [5].Therefore, it seems important to perceive melatonin neither only in its chronobiotic function nor simply as a radical scavenger, but rather as a compound acting in a complex manner at different levels of integration.
As will be outlined in this review, part of this complexity is reflected by melatonin's multiple interactions with reactive nitrogen species (RNS).This comprises chemical reactions with various nitrogen-containing compounds and regulatory influences as well, at the levels of organelles, especially mitochondria, of cells and tissues.These control mechanisms are also of considerable pathophysiological interest.Moreover, melatonin should be seen in relation to its metabolites.Especially one of its kynuramine derivatives, N 1acetyl-5-methoxykynuramine (AMK; Fig. 1), displays properties as a potent regulator and scavenger of nitric oxide (NO) [6].Structures of melatonin, its metabolite N 1 -acetyl-5-methoxykynuramine, their hydroxylamino intermediates, nitrosated and nitrated products.
The ensemble of modulatory effects exerted by melatonin and AMK seems to substantially contribute to the avoidance of dysfunctions at mitochondrial and cellular levels, including possible consequences for apoptosis, inflammationrelated necrosis and, presumably, also parthanatos and autophagy.Prevention of NO-dependent Ca 2+ overload and NO-promoted mitochondrial dysfunction by the indoleamine and its metabolite should be perceived in its significance for attenuation of free radical generation in more general terms, including the formation of organic, oxygen and carbonate radicals.From a pathophysiological point of view, the importance of radical avoidance [7][8][9] exceeds by far the solely biochemical aspects of damaged biomolecules to be replaced or repaired.Partial blockades within the mitochondrial electron transport chain (ETC), which lead to electron leakage and, thus, secondarily augmented radical formation, also cause ATP deficiency and, eventually, cytochrome c release that initiates apoptosis [9][10][11][12][13][14][15].Especially in neurons, ETC malfunction can change the mitochondrial fragmentation/fission balance, which ultimately leads to losses of peripheral mitochondria, synaptic function and connectivity [9,[14][15][16].Tyrosine nitration, another consequence of enhanced NO formation, is potentiated especially in conjunction with high CO 2 /HCO 3 -levels, which are normally present in mitochondria, but may become relevant in the cytosol or body fluids during hypoxia/hypercapnia because of impaired blood flow and also in ischemia/reperfusion [17].This type of protein modification can result, among other effects, in dysfunctional reuptake of neurotransmitters that further promotes neuronal overexcitation, or, in the circulation, to modified atherosclerotic plaque proteins which are more resistant to degradation.
These scenarios shed light on the potential value of compounds that allow NO synthases (NOS) to operate at low or moderately enhanced activities, but are, on the other hand, capable of preventing extreme, disadvantageous rises of NO with its deleterious consequences.Melatonin seems to fulfill these requirements.

Damage by reactive nitrogen species
Reactive nitrogen species (RNS) should not be regarded as just another subtype of free radicals and related compounds, which cause more or less the same damage to important biomolecules as known from reactive oxygen species (ROS).It seems necessary to perceive several peculiarities, namely, (i) the specific molecular differences in protein modification, (ii) the consequences of protein modification by RNS in functional and pathophysiological terms, (iii) redonation of RNS from scavengers, and (iv) the stimulation of ROS formation by RNS.The primary RNS is the NO radical (•NO) formed by NO synthases (NOS).Depending on the different regulation mechanisms controling the isoenzymes iNOS (inducible NOS), nNOS (neuronal NOS) and eNOS (endothelial NOS), including mitochondrially located variants, the production of this nitrogen radical is contextual.Other NO congeners do exist, but are of lower physiological and pathophysiological significance.The nitroxyl cation, NO + , can be generated in biological material, e.g., from nitrosothiol groups of S-nitrosocysteine (CysNO) or S-nitrosoglutathione (GSNO), but also by copper-or iron-containing metalloproteins, in particular, deoxygenated hemoglobin [18].The •NO metabolite nitrite is reduced by Hb(Fe II ), leading to the unstable complexes Hb(Fe III )•NO and Hb(Fe II )NO + , which are in equilibrium and release either •NO or NO + [19].The decomposition of Hb(Fe III )•NO to Hb(Fe II ) and NO + can be hardly distinguished from the decay of Hb(Fe II )NO + .NO + is reportedly formed in arterioles, capillaries and venules even when NO synthase is experimentally inhibited and despite the rapid reaction of NO + in water [20].Because of its favored combination with OH -, the half-life of NO + in aqueous solution at pH 7.4 is in the range of 10 -10 s and, thus, extremely short [21,22].Therefore, effects of NO + , as described by others (summarized in ref. [18]), seem to be of low physiological importance.
Another potential source of NO + is the manganese-dependent superoxide dismutase (MnSOD), which was found to catalyze the disproportionation of two •NO to NO + and NO - [23].The nitroxyl anion NO -is, however, immediately protonated at physiological pH to HNO [24,25].At first glance, one might assume that this NO congener, being a reduced compound, would not be involved in oxidative and nitrosative processes, but, in fact, HNO is an efficiently nitrosating agent [18].However, HNO is obviously not involved in nitration processes [26].
A chemically and pathophysiologically critical step in the various RNS interconversion reactions is the combination of •NO with the superoxide anion (•O 2 -).Since •O 2 -has a similar affinity to the free radical •NO as to the SODs, this reaction is practically unavoidable.The resulting product, peroxynitrite (ONOO -), represents a compound of high reactivity.Various interactions with biomolecules have been ascribed to this intermediate, but, in fact, direct reactions of peroxynitrite with organic metabolitesincluding melatonincan be hardly distinguished from others by peroxynitrite-derived free radicals [14].The non-radical RNS, peroxynitrite, undergoes, in particular, two combination reactions of high pathophysiological relevance.One of them, protonation of the anion, leads to the unstable peroxynitrous acid (ONOOH), which readily decomposes to a hydroxyl radical (•OH) and nitrogen dioxide (•NO 2 ).The other one consists of adduct formation with CO 2 , an abundantly available compound, especially in mitochondria.The resulting molecule, ONOOCO 2 -, decomposes correspondingly to a carbonate radical (•CO 3 -) and •NO 2 [9,14,15,17,[27][28][29].The devastating, mutagenic and carcinogenic properties of the extremely reactive hydroxyl radical are well-known [30].The less reactive carbonate radical is still capable of abstracting electrons or hydrogens from various partner molecules, as observed with •OH, but does not form comparable hydroxylated adducts [8,17,27,29,31,32].Frequently, oxidation reaction rates are strongly enhanced by addition of bicarbonate as a source of •CO 3 -, which can be generated either via CO 2 , or from HCO 3 -by interaction with •OH [17,31,32].These findings shed light on the damaging potential of •CO 3 -, which has a much longer lifetime and, therefore, a considerably larger radius of action than •OH, partially because of resonance stabilization.
The combination of either •OH or •CO 3 -with •NO 2 , however, gives rise to other, pathophysiologically highly relevant products.Although the classic nitration of aromates is of a non-radical nature, the combinations mentioned represent physiological nitration mixtures based on free radical reactions [17,28,29,31,32].Most frequently, tyrosine nitration is observed and measured under pathophysiological conditions.For instance, 3-nitrotyrosine is extensively present in atherosclerotic plaques [33][34][35][36][37][38].The roles of LDL tyrosine nitration and the contribution of iNOS of macrophages acting on the plaque areas have been well documented.Moreover, the nitrated proteins are relatively more resistant to proteolytic removal.Meanwhile, tyrosine nitration has been implicated in numerous diseases, in particular, aspects of inflammation including rheumatoid diseases, insults by ischemia/reperfusion, cancer, and various neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis [39][40][41].
Numerous biologically important proteins have been identified to be impaired by tyrosine nitration, under various pathological conditions and leading to a plethora of consequences.To mention a few examples, tyrosines are site-specifically nitrated in: L-type Ca 2+ channel hCa v 1.2b [42]; microvascular protein phosphatase 2A, an effect that prevents inactivation by phosphorylation and leads to endothelial barrier dysfunction [43]; α subunit of arterial voltage-gated potassium channel K v 1.2, especially observed in diabetic rats [44]; α, β and FXYD subunits of renal Na-K-ATPase, related to renal dysfunction [45]; glyceraldehyde 3-phosphate dehydrogenase, peripheral-type benzodiazepine receptor, MAP kinase Erk-1, and glutamine synthetase in astrocytes [46], and, perhaps, the synaptosomal glutamate transporter [47], presumably effects with consequences for extracellular glutamate concentrations; midbrain dopamine transporter and vesicular monoamine transporter-2 [48], modifications relevant to Parkinson's disease; α subunit of IκB, which leads to activation of the transcription factor NF-κB [49].
Proteomics and detailed analyses revealed that tyrosine nitration has a particular impact in mitochondria.After ischemia/reperfusion, 23 cardiac proteins were identified to be specifically tyrosine-nitrated, among which 10 were mitochondrially located [50].In this and other studies, several subunits of complexes I and III as well as the ATP synthase α subunit were affected [50,51].In other systems, the complex I subunit NDUFB8 [52], the Fp subunit of complex II [52,53], mitochondrial creatine kinase, dihydrolipoamide dehydrogenase, and the voltagedependent anion channel VDAC1 [52] were reported to be tyrosine-nitrated.
The mitochondrial effects of RNS by far exceed these nitrative protein modifications.On the one hand, the peroxynitrite-derived radicals •OH and •CO 3 -are also capable of causing oxidative damage to proteins and lipids [9,[14][15][16].Oxidative modification of ETC proteins likewise impairs the mitochondrial electron flux.Moreover, peroxidation of cardiolipin plays an additional, significant role in ETC dysfunction, as this specific lipid is required for the structural integrity of complexes III and IV [9,14,15,[54][55][56][57]. Additionally, •NO multiply interacts with components of the ETC.As a ligand of transitions metals, it can bind to protein-bound iron present in iron-sulfur clusters and to heme/hemin iron of cytochromes [9,14].•NO and its nitrosating derivatives can form S-nitrosothiols in respirasomal proteins.When synthesized at elevated levels leading to high peroxynitrite concentrations, the combination of •NO and •NO 2 causes the formation of N 2 O 3 , a strongly nitrosating agent [9,16].
In summary, all these effects by RNS and their derivatives described, i.e., binding of •NO to ETC irons, S-nitrosation, nitration and oxidation of respirasomal proteins, as well as peroxidation of lipids in the inner mitochondrial membrane, in particular, cardiolipin, can cause ETC dysfunction, since they block electron flux to a variable degree depending on the rates of •NO formation.Moderately elevated concentrations of •NO are not per se detrimental, but have, on the contrary, even been judged to be beneficial because of •OH scavenging, initiation of cGMP-mediated processes and stimulation of mitochondrial biogenesis [9,[60][61][62][63][64][65][66].However, secondary bottlenecks or regional blockades of the ETC cause interruption of electron flux, eventually electron backflow and electron leakage [9,[14][15][16].Anyway, the interactions of respirasomes and their mediating redox partners do not allow a steady, continual flux of electrons, as became apparent by the discovery of superoxide flashes [67,68].The highly dynamic system of electron flux even allows transient openings of the mitochondrial permeability transition pore (mtPTP) without causing immediate breakdown of the mitochondrial membrane potential (ΔΨ m ) and apoptosis induction [68,69], contrary to the former belief.However, persistent partial blockades of electron flux, as caused by an excess of nitrosating, nitrating and oxidizing RNS, represent a key feature of mitochondrial dysfunction.Electron leakage has especially been studied at complexes I and III.Previously, this had been mainly related to changes between respiration states, such as state 3 or 4 respiration.Although this is certainly of relevance, actual considerations should rather be more directed towards the appearance of bottlenecks within the ETC [9,14,15].
The sites at which •O 2 -is generated by electron transfer to O 2 have been recently summarized [9,14,15].Electrons dissipate from complex I mainly via the iron-sulfur cluster N2 of the amphipathic ramp, an extrusion of this respirasome to the matrix.Therefore, •O 2 -formed at this site directly enters the matrix.Electron leakage from complex III occurs especially at the Qo site, upon interruption of intramonomer electron transfer between the two b L hemes.In this case, •O 2 -is released to both sides of the inner mitochondrial membrane.Superoxide formation from complex IV has been rarely investigated directly, but the recently reported identity of the NADPH oxidase subform Nox4 with a subunit within this complex [83] would indicate a significant contribution of this respirasome to radical formation.
Transitory interruptions of electron flux, as caused by RNS and peroxynitrite-derived oxidizing radicals, lead to electron leakage.As soon as elevated levels of •NO and peroxynitrite are generated, rates of -, so that the ongoing process cannot be easily halted.This can be particularly dramatic under inflammatory conditions, especially in sepsis [10][11][12][71][72][73], in which •NO and its reactive metabolites are, in the extreme, capable of totally blocking respiration [15,73].Under such conditions, the relevance of the mitochondrial RNS/ROS loop has been impressively demonstrated by the relative maintenance of mitochondrial electron flux and ATP synthesis in iNOS double knockout mice [10,12,71,74].
It is a remarkable fact that melatonin is one of the few compounds that display the potential of efficiently antagonizing mitochondrial dysfunction, as initiated and maintained by elevated •NO synthesis, production of peroxynitrite and dramatically increasing electron leakage.

Direct interactions with reactive nitrogen species
Direct chemical interactions of melatonin with RNS do exist.Whether or not this is physiologically relevant with regard to melatonin concentrations may be questioned, but could become of interest in experiments using pharmacological doses.Nitrosation of melatonin has been observed under various conditions, but nitration was usually not observed, even not in the presence of peroxynitrite and hydrogen carbonate [75][76][77].Instead, peroxynitrite gave rise to nitrosation and oxidation products.The preferential site of nitrosation is the indolic nitrogen of melatonin, to give 1-nitrosomelatonin (= Nnitrosomelatonin; Fig. 1).Theto a certain degree surprisingnitrosation of melatonin by peroxynitrite, which contrasts with the nitration reactions observed with other aromates, was explained in different ways (Fig. 2).The peroxynitrite-derived free radicals •NO 2 or •CO 3 are capable of abstracting a hydrogen from melatonin, thereby forming a melatonyl radical.Additionally, an electron may be abstracted from peroxynitrite, e.g., by •NO 2 , to give a nitrosodioxyl radical (•ONOO; the radical analog of peroxynitrite) and NO 2 -.At C-atom 3, the melatonyl radical might undergo an intermediate addition reaction with the nitrosodioxyl radical, followed by its decomposition into •NO that is attached to ring atom 1 and O 2 which is released [77].Alternately, a reaction of melatonin with the nitrosodioxyl radical, formed from •NO and O 2 , was discussed [77].The nitrosodioxyl radical might directly attach to C3 of melatonin, thereby forming the indolic radical adduct, followed by decomposition to •NO, which interacts with ring atom 1, under release of a hydroperoxyl radical (•HO 2 ) (Fig. 2).Although these reactions are chemically possible, it should be kept in mind that, in systems generating both •NO and •NO 2 , the formation of the strongly nitrosating N 2 O 3 is also possible (Fig. 2), and also the sequential hydrogen abstraction by •NO 2 and addition of •NO.In fact, melatonin nitrosation was observed using the combination of •NO 2 and •NO [78].
Nitroxyl (HNO, the protonated NO -congener) was identified as another agent capable of nitrosating melatonin at the pyrrole nitrogen [79].In this case, HNO should be directly attached to the nitrogen, to give a melatonin N-hydroxylamine (= 1-hydroxylaminomelatonin, 1-OHAm-Mel; Fig. 1), which may either transfer two hydrogens to O 2 , to give H 2 O 2 and 1-nitrosomelatonin, or decompose into a melatonyl radical and •HNOH, which, upon transfer of 2 hydrogens to O 2 , would form •NO that combines with the melatonyl radical (Fig. 2).
All these reactions leading to the nitrosation of melatonin should not be misinterpreted in terms of scavenging, since 1-nitrosomelatonin readily redonates •NO [80][81][82].Although one might argue that a more dangerous compound, peroxynitrite, is exchanged in the respective nitrosation and redonation reactions by the less reactive •NO, the reformation of peroxynitrite is still possible, so that, in terms of protection, the balance may be poor.On the other hand, the use of 1-nitrosomelatonin as a pharmacologically suitable •NO donor was proposed, which might combine the property of releasing this gaseous regulator molecule with the protective potential of melatonin related to its antioxidant, antiinflammatory and antiexcitatory actions [81].
Direct interactions with RNS can be observed also with some melatonin metabolites.The indolic analogs are less of interest, compared to the kynuramine metabolite AMK.This compound displays a much higher reactivity than its precursor N 1 -acetyl-N 2 -formyl-5-methoxykynuramine (AFMK) [6,8,83,84].This difference was also obvious with nitrosating and nitrating agents [18].By contrast with melatonin, AMK is nitrated to N 1 -acetyl-3-nitro-5-methoxykynuramine (= 3-nitro-AMK = AMNK; Fig. 1) in reaction mixtures forming the peroxynitrite-CO 2 adduct and, thus, •CO 3 -and •NO 2 [29].In the absence of CO 2 /HCO 3 -, AMK was preferentially oxidized [29], reflecting is properties of a potent •OH scavenger [83,84].Another difference to melatonin concerns the formation of a stable nitrosation product [18,29,85].This compound was identified as 3-acetamidomethyl-6-methoxycinnolinone (AMMC; Fig. 1) [29].Contrary to 1-nitrosomelatonin, this metabolite does not easily re-donate •NO, because of the nitrogen incorporation into a second, resonance-stabilized ring [6,85].Moreover, AMK did not form, upon nitrosation, azo-adducts, toxic diazonium or carbenium anions [6,85] nor oxadiazoles or o-quinone diazides [6] known from other aromates including some kynuric compounds [86].AMMC is formed from AMK by interaction with all three NO congeners, which are efficiently scavenged by the methoxylated kynuramine [18].The mechanisms are similar to those mentioned for melatonin, but have to include water release in the cyclization reaction (Fig. 2).Nitrosation by NO + only requires deprotonation and water release.The corresponding reaction with •NO additionally requires hydrogen abstraction, which is, however, easily possible in systems containing reactive nitrogen species and in biological material as well.Hydrogen abstraction by •NO 2 followed by interaction with •NO is possible as much as nitrosation by N 2 O 3 , the •NO/•NO 2 adduct [6].Nitrosation by HNO should proceed via the respective N-hydroxylamine, followed by one of the two reaction pathways mentioned for 1nitrosomelatonin formation (2 H transfer to O 2 ; alternately, formation of AMKyl radical and •HNOH, which generates •NO that combines with the AMKyl radical) [18].Again, the N 2 -nitroso-AMK cyclizes under release of water.
As discussed for melatonin, the relevance of these reactions may largely be restricted to pharmacological concentrations, upon administration of AMK or its precursor, melatonin.Under experimental conditions, an additional type of reaction observed with AMK may interfere with RNS metabolism.Under oxidative conditions leading to N 2 -centered radicals, AMK forms adducts with aromates, in particular, tyrosine [87].Protein AMKylation may prevent not only phosphorylation or chlorination of tyrosine residues, but also 3-nitrotyrosine formation.However, this remains to be studied in quantitative terms.

Regulation of NO synthases by melatonin and AMK
The control of NOS subforms by melatonin and AMK is of particular relevance, especially as lower concentrations of these compounds are required.
Moreover, the consequences of limiting the formation of •NO and peroxynitrite seem to be more profound and of premier importance under many aspects of protection.
The three NOS isoenzymes are not only differently regulated by other stimuli, but also differently affected by melatonin.Effects of melatonin on eNOS, which is expressed in endothelial cells and some other cell types, have not been extensively studied under otherwise noncompromised conditions.Therefore, findings concerning a melatonin-mediated prevention of eNOS induction by H 2 O 2 [88], may be rather interpreted in terms counteraction against oxidative stress and may not allow conclusions on direct eNOS regulation by melatonin.Moreover, observations made in different systems including ischemia/reperfusion of various organs did not reveal consistent effects concerning up-or downregulation of eNOS and, similarly, the actions of melatonin on this isoenzyme were either stimulatory [89,90], preventive against decreases [91], or negligible [92].Contradictory findings on eNOS regulation by melatonin are not that much surprising, since the indoleamine displays regionally different effects in the vascular system, depending on the expression of melatonin receptor subtypes.Actions via MT 1 cause vasoconstriction by opening of BK Ca channels, whereas those via MT 2 result in vasodilation [1,2,[93][94][95][96].Therefore, downregulation of eNOS during MT 2 -mediated dilation responses would be functionally counterproductive and, if at all, the opposite should be expected.
Much more is known about the regulation of iNOS and nNOS by melatonin.iNOS is expressed in numerous tissues and cells.It is of utmost importance under inflammatory conditions, under which this isoenzyme is strongly upregulated by pro-inflammatory leukocytes, in particular, neutrophils and macrophages [97][98][99].This is the more important as, in the same situation, proinflammatory cytokines and other mediators such as prostaglandins are also induced, which further stimulate iNOS-related processes and, additionally, NOX subforms that generate considerable amounts of •O 2 -, so that the formation of peroxynitrite is promoted from two sides.It should be noted that melatonin as well as its metabolites AFMK and AMK downregulate COX-2, at least, at pharmacological concentrations [100], and that AMK is a COX inhibitor more potent than aspirin [101], perhaps, with some COX-2 specificity [96].Anti-nitrative and anti-nitrosative actions can, thus, be assumed for melatonin, AFMK and AMK because of their antiinflammatory actions.Direct effects of melatonin on NOX expression have not been studied in detail, although the oxidative stress induced by upregulation can be blunted by the indoleamine.
The induction of iNOS by inflammatory signals can be efficiently prevented by melatonin, what has been convincingly demonstrated in models of bacterial lipopolysaccharide exposure [102,103] and sepsis caused by cecal ligation and puncture [10-12, 71, 74, 104], effects that were consistently obtained in various organs such as diaphragm, heart, lung and liver.As will be discussed in the next section, this action of melatonin strongly attenuated mitochondrial damage.The relevance of iNOS suppression was supported by similar findings on prevention of mitochondrial dysfunction in homozygous iNOS knockouts [10,12,71,74].Moreover, the presence of a mitochondrial iNOS variant was shown, which may be of particular relevance in ETC dysfunction, which is also absent in the knockouts and whose upregulation is likewise prevented by melatonin [10-12, 71, 103, 105].Suppression of both cytosolic and mitochondrial iNOS variants was also obtained in a model of parkinsonism using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a neurotoxin specific for dopaminergic neurons with particular mitochondrial toxicity [106].Interestingly, this effect was not only obtained with melatonin, but also with AMK [106].
Negative regulation by melatonin was also shown in the case of nNOS, which is directly inhibited by the indoleamine [107][108][109][110]. Again, AMK displayed a similar action [109,110] and, despite a higher K i value, inhibitions by the kynuramine were demonstrable down to 10 -11 M [110].Generally, members of the kynurenine family were found to be more potent nNOS inhibitors than melatonin, provided that they are not substituted at N 2 [107].The contributions of AMK to the effects of melatonin are, however, uncertain as long as concentration measurements of the kynuric metabolite have not been provided.It may be difficult to obtain reliable data because of the transitory nature of AMK, which undergoes numerous reactions with ROS, RNS and other molecules [6].
The practical value of melatonin and, perhaps, AMK should be seen in the prevention of damage caused by excessive •NO production, especially in conjunction with high rates of •O 2 -formation, which lead to undesirable peroxynitrite levels.This would especially be the case under conditions of inflammation and in several neurodegenerative disorders.Although peroxynitrite and its products are responsible for a variety of diseases, basal or moderately enhanced •NO levels are obviously not detrimental, but rather beneficial [9,[60][61][62][63][64][65][66]111].Therefore, the intention should never be to totally suppress •NO formation, nor should the physiological role of melatonin be sought in a complete downregulation of melatonin-sensitive NOS subforms.This becomes particularly evident in the circadian rhythmicity of central nervous •NO production in nocturnal animals.In these species, the amount of •NO generated should largely reflect neuronal activity, and its maximum should be expected to coincide with the nocturnal melatonin peak [7].In fact, this was observed in rats, and even aged animals which can be assumed to produce less melatonin did not show dramatic differences in the •NO rhythm, compared to young animals [112].Nocturnal •NO maxima were also described in other organs of nocturnal rodents [7,113].
Nevertheless, melatonin should be regarded as potentially highly valuable pharmacological tool in treating diseases related to damage by peroxynitrite.

Significance to mitochondriarole in radical avoidance
In conjunction with the downregulation of melatonin-sensitive NOS subforms (Fig. 3), protection and improvements of ETC function were repeatedly observed.Melatonin was shown to prevent, in sepsis models, •NO/peroxynitritedependent decreases in the activities of complexes I and IV, sometimes also complexes II and III of the ETC of various organs [10,12,103,105,114], and it also protected against multi-organ failure [104].These effects were accompanied by the maintenance of ATP production and energy efficiency in the compromised animals [12,74,105,114], results that are insofar important, as they indicate the in vivo support of electron flux, whereas the respirasomal activities had been determined in submitochondrial particles.Similar results were also obtained for some of the parameters mentioned with AMK [105,106].Of course, the protection of the ETC comprises an additional antioxidant effect.This conclusion is in accordance with the observed suppression of lipid peroxidation [10,71,102], maintenance of reduced glutathione levels [71,114], of glutathione peroxidase and glutathione reductase activities [10,71].The effects on these enzymes and also on de novo synthesis of respirasomal subunits may reflect direct stimulatory actions of melatonin at the level of gene expression [15] and, in the balance, may be difficult to distinguish from the consequences of NOS downregulation.On the other hand, rises in peroxynitrite would also cause lipid peroxidation, damage to ETC proteins and, therefore, enhanced electron dissipation and oxidant formation, which leads to consumption of reduced glutathione.As outlined in the section on damage by RNS, nitrosative/nitrative and oxidative forms of stress are multiply interconnected and cannot be fully dissected, in a given pathophysiological situation.
Another important aspect concerning the maintenance of mitochondrial function is related to protection against neurodegenerative disorders.In these cases, both nNOS and iNOS can be involved, depending on an inflammatory component, which may be even atypical and lingering.Although these pathologies and beneficial effects of melatonin, as far as they exist, are much more complex than just described by nitrosative/nitrative and oxidative stress and protection thereof, a contribution of RNS and mitochondrial dysfunction to these diseases remains [115][116][117][118][119]. In several experimental studies, damage by the neurotoxin MPTP was shown to be counteracted by melatonin [120], but protection at the mitochondrial level as related to iNOS synthase suppression was not demonstrated before recently [106].In this investigation, complex I activity was shown to be normalized, in striatum and substantia nigra, by both melatonin and AMK.
The roles of RNS and mitochondrial dysfunction in aging, as well as the value of melatonin in antagonizing these processes have been extensively reviewed in the last years [9,[14][15][16].The studies summarized there clearly indicate a beneficial role of melatonin in maintaining electron flux capacity and ATP formation, reducing damage to the ETC, Figure 3.The vicious cycle of mitochondrial dysfunction started by extreme stimulations of •NO synthesis, followed by peroxynitrite formation and damage to the ETC, with the consequence of strongly enhanced •O2 -generation by electron leakage that drives further peroxynitrite production.Dotted lines: Blunting the vicious cycle by various antiinflammatory and antiexcitatory actions of melatonin and its metabolite AMK and by downregulation/inhibition of iNOS and nNOS.attenuating electron leakage and, thus, oxidant formation as well as other actions concerning mitochondrial biogenesis.Many of these effects could be related to the prevention of excessive formation of •NO, damage by peroxynitrite and its derivatives.Various of these studies have dealt with the senescence-accelerated mouse strain SAMP8, in comparison to the normally aging strain SAMR1, which carries the same genetic background.To mention a few potentially important findings, melatonin was shown to reduce losses of complex I activity in aging SAMP8 mice [121] and to substantially stimulate complex I and IV activities in both SAMP8 and SAMR1 old mice much above control levels [122].To which extent these rises are consequences of eliminating blockades by RNS remains to be studied in their mechanistic details.Improvements of complex I activity and reductions of electron leakage may well be supported by melatonin via additional pathways acting in a concerted way with the limitation of RNS formation.Removal of a bottleneck at complex I can include enhanced de novo synthesis of ETC subunits, generalized mitochondrial biogenesis and a newly discovered high affinity melatonin binding site (K d = 150 pM) located in the vicinity of the iron-sulfur cluster N2 in the amphipathic ramp of complex I (unpublished details cited in refs.[2,9,14,15]).
The removal of bottlenecks within the ETC seems to be a crucial requirement for appropriate mitochondrial function, whether exerted by blunting RNS formation and/or by other, additional means.Therefore, mechanisms supporting electron flux along with attenuations of electron leakage may be regarded as essential components within a regulatory network designed for radical avoidance [7][8][9].This new concept does not primarily focus on scavenging of radicals already formed, but rather on the control of processes leading to enhanced radical formation.Breaking the self-stimulatory loop of excessive •NO generation followed by •O 2 formation via electron dissipation and the appearance of deleterious peroxynitrite levels should be an important element in the system of radical avoidance (cf.Fig. 3).

Conclusion
Melatonin and •NO, this is not simply a story of antagonism.In a normal physiological range, moderate elevations of •NO are poorly affected by the indoleamine and certainly not entirely suppressed.The decisive property of melatonin in antagonizing undesired nitrosation and nitration as well as oxidative damage by peroxynitrite-derived free radicals consists in the limits set to excessive upregulation of iNOS and nNOS, in conjunction with antioxidative actions, e.g., by supporting the availability of reduced glutathione.The importance of these actions should be seen in the prevention of deleterious levels of peroxynitrite.At the mitochondrial level, this includes the possibility of interrupting the vicious cycle of •NO-and •O 2 -dependent inhibitions of electron flux, with its consequence of electron leakage and further oxidant formation.Whether direct modulation of electron flux and eventual prevention of electron backflow by controling complex I activity, at or close to ironsulfur cluster N2, may contribute to the attenuation of electron dissipation will be an intriguing question to be solved in the future.Additional effects of melatonin concerning enhanced expression of ETC subunits and mitochondrial biogenesis can be assumed to also support electron flux capacity and to avoid electron overflow.
Whether or not melatonin may be suitable for avoiding nitrative damage in cardiovascular diseases remains to be studied in detail.Previously, the focus had been on the antioxidant properties of melatonin and role of RNS had been poorly addressed.In experimental systems, protection of aortic smooth muscles from damage by peroxynitrite has been documented [123].In a mesenteric ischemia/reperfusion system, melatonin reduced nitrosative/nitrative stress in the intestine and lung of rats [124].However, it is still uncertain to what extent such findings could be generalized.Protective cardiovascular effects, as summarized elsewhere [125,126], may be also interpreted in different ways.In this context, antiinflammatory actions of melatonin [126], which comprise multiple mechanisms and are not restricted to the modulation of •NO formation, may be of relatively higher importance than downregulation of iNOS.Moreover, melatonin-induced rises in •NO production were also observed in some cases [127,128], which may reflect the regional distribution of MT 2 receptors within the vasculature.Melatonin was also reported to promote aortal atherosclerosis, however, in mice fed an atherogenic diet [129].Therefore, the suitability of melatonin may be conditional in cardiovascular pathologies and caution is due, before this can be judged on a reliable clinical basis.
With regard to the, sometimes widespread, peroxynitrite-induced damage frequently observed in various neurodegenerative diseases [9, 39-41, 48, 59, 60, 111, 118, 130, 131], melatonin may become an option of treatment.In these disorders, several factors can contribute to the overproduction of •NO, such as excitotoxic glutamate levels, Ca 2+ overload, and lingering processes of atypical inflammation with microglia activation.It seems relatively certain that mitochondrial dysfunction is involved in these pathologies [9,15,16,59,118].During normal aging, similar changes may occur, but proceed at much smaller rates.Inflammation and, in the extreme, sepsis provide the clearest examples for the importance of supranormal NOS upregulation, the deleterious potential of RNS, especially peroxynitrite and its products, and the involvement of mitochondria in nitrosative/nitrative stress, but also for the powerful counteractions by melatonin, which is capable of blunting these dangerous processes.
The respective actions of melatonin should be seen as a part of the orchestrating functions of this pleiotropic regulator molecule, which contributes at various levels to radical avoidance, from the temporal coordination of antioxidant mechanisms with prevalent phases of oxidant formation, to the antagonism of neuronal overexcitation, control of iNOS and nNOS, and direct as well as indirect mitochondrial modulation [7][8][9][14][15][16].