*2.2.2. The neuronal nNOS: a crucial element in neurotransmission*

nNOS, also called NOS-1, was the first isoform discovered in the neuronal tissue. It should be distinguished between two subfamilies: nNOSα and nNOSμ, thanks to their locations and functions. Indeed, nNOSα is found in central nervous system and plays a major role in the neurotransmission at neuronal synapses, whereas nNOSμ is found in skeletal and cardiac muscle where NO controls muscle contractility and local blood flow.

#### *2.2.2.1. nNOSα in the central nervous system*

and cofactors like BH4

their respective functions.

*2.2.1. The endothelial eNOS and vasodilatation*

26 Emerging Pollutants - Some Strategies for the Quality Preservation of Our Environment

platelet or leukocyte adhesion (**Figure 8**) [39, 40].

rosis and hypertension [43].

using O2

), this enzyme catalyzes the conversion of l-arginine into l-citrulline

− )

and leading to NO formation with a ratio of 1:1 [33]. However, if these parameters

are not available or if NOS remains monomeric, the enzyme only produces superoxide (O2

[32]. Even if NOS isoforms catalyze the same reaction, their distribution is largely related to

Endothelial nitric oxide synthase (eNOS) can be activated through calcium-dependent and independent pathways. On the one hand, through activation of specific receptors, such as acetylcholine muscarinic receptors, bradykinin receptors, and H1 histamine receptors, distributed in the endothelial cell membrane, agonists can trigger an increase in intracellular concentration of calcium (Ca2+) through the well-known polyphophoinosides pathway. Indeed, activation of those receptors stimulates membrane-associated PLCϒ and PI3K activation. This results into inositol trisphosphate (IP3) formation, which induces the release of intracellular calcium stock from endoplasmic reticulum [34]. Then, Ca2+ binds to calmodulin (CaM), which could later fix on the calmodulin-binding domain of eNOS controlling its enzymatic activity. On the other hand, in response to hormones or growth factors acting on their corresponding receptors, phosphate kinase A (PKA) or B (AKT) pathway can be induced mediating a phosphorylation cascade. The post-translational phosphorylation of eNOS on three specific sites (Ser617 and Ser1179 for AKt, Ser635 and Ser1177 for PKA) enhances the activity of the enzyme [35]. Moreover, the lipidation of eNOS (palmitoylation and myristoylation) could also enhance its activity [36]. This other post-translational modification promotes eNOS association with cell membrane and is stabilized by interaction with membrane chaperone proteins (caveolin-1 and HSP70/90). Both mechanisms are essential for linking upstream signal transduction pathway to eNOS activity in cells [37]. eNOS could then produce a large amount of NO, which latter diffuses freely inside smooth muscles. Herein, NO activates soluble guanylate cyclase (sGC) by reaction with the heme of the enzyme leading to the increase of cyclic guanosine monophosphate level (cGMP). cGMP subsequently activates phosphate kinase G (PKG) favoring a cytosolic Ca2+ reuptake into sarcoplasmic reticulum [38]. The decrease of intracellular level of Ca2+ leads to the relaxation of smooth muscle. Moreover, NO can inhibit caspases 3 and 8 and thus apoptosis through protein nitrosylation. NO released by endothelium is also reducing platelet aggregation and

Another source of NO inside endothelial cells was also described. Indeed, nitrite and nitrate reservoirs could be converted into NO by several enzymes such as cytochrome P450, hemoglobin, myoglobin, and others, under specific conditions [42]. However, several pathways could abolish NO production. Indeed, NO can be eliminated in combination with reactive oxygen species (ROS). eNOS could be inactivated by several phosphatases. The removal of eNOS membrane sequestration by interaction between NOS-interacting protein (NOSIP) and NOS-traffic inducer (NOSTRIN) complex favors eNOS recycling [36]. Since NO plays a central role in this phenomenon, any abnormality altering its production leads to pathogenesis and vascular disorders such as atheroscle-

Glutamate is the major excitatory neurotransmitter in the brain. When deliver into the synapse, this molecule is activating a specific receptor (NMDAR) located inside the postsynaptic membrane. Activated NMDAR thus allows the entry of calcium. Ca2+ are able to activate calmodulin by physical interaction [44]. Afterward calmodulin binds to nNOSα, which, in turn, is activated. Interestingly, nNOSα can indirectly interact with the NMDAR through its fixation on an adapter protein PSD95 by their PDZ interaction domain [45]. This allows the high speed of NO synthesis in response to NMDAR activation. Thereafter, NO can diffuse back to the presynaptic area leading to an increase glutamate release in the synaptic cleft and thus neurotransmission. CAPON, a chaperon protein of DexRas, was found altering the level of nNOSα present at the membrane [46]. Indeed, CAPON competes with the PDZ domain of nNOSα and thus promotes its detachment from the NMDAR-PSD95 complex. nNOSα interaction with CAPON-DexRas complex allows the activation of DexRas, a small protein G, through s-nitrosylation on its thiol residue [47]. Then, DexRas activates the MAPK cascade and the transcription of several genes [47]. Several shutdown systems are available to limit this process, which could dramatically affect the brain physiology by synapses hyperactivation. Indeed, kinases, such as PKC or PKII, can phosphorylate nNOSα leading to its loss of activity [48]. nNOSα can also be sequestrated by two membrane proteins (caveolins 1 and 3) preventing its interaction with calmodulin (**Figure 9**).

sources. This process is also enhanced by the activation of a voltage-dependent Ca2+ channel (CaCn) [53]. The increase of Ca2+ from exogenous origin activates ryanodine receptor present on the sarcoplasmic reticulum, which, in turn, delivers a large amount of Ca2+. When the Ca2+ threshold is reached, it is binding on the calmodulin protein. Calmodulin can then interact with the nNOSμ to activate the generation of NO. Interestingly, nNOSμ interacts through its PDZ domain with adapter proteins (syntrophins) linked to a membrane protein complex called dystrophin [54]. Produced NO could then diffuse back to the motor neuron to enhance the liberation of neurotransmitters or the vasodilatation of blood vessels, thus increasing the

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local blood flow to support muscle needs during contraction efforts (**Figure 10**) [55].

offer a potential avenue for therapy.

*2.2.3. The inducible iNOS and the innate immunity*

adapter between dystrophin and nNOSμ; Calp, calpain.

NO synthesis is also well regulated by calpain (Calp). Indeed, this protein interacts with the PDZ domain nNOSμ provoking its detachment from the dystrophin/syntrophin membrane complex [56]. Interestingly, in muscular Duchenne dystrophy, the damages caused by dystrophin absence are enhanced in case of simultaneous lack of nNOSμ [57]. Thus, NO donors may

NO also takes an important part in innate immunity. Indeed, NO and derived compounds are cytotoxic weapon against tumor cells, microorganisms, and viruses [58]. NO is generated by inducible nitric oxide synthases (iNOS and NOS2) within macrophages. However, more recently, studies have shown that this enzyme is also expressed in other cell line (vascular endothelial cells, hepatocytes, pulmonary, and colonic epithelial cells) [59]. Unlike nNOS or eNOS, its expression is stimulated by several pathways after activation of specific receptors by endotoxins (LPS) or cytokines (TNFα, IFNϒ, and IL1β). Briefly, the LPS endotoxins are carried by an LPS-binding (LPB) protein to its specific Toll-like receptor 4 (TLR4). Then, the transduction

**Figure 10.** nNOSμ activity at the neuromuscular junction (adapted from [49]). Ach, acetylcholine; CaCn, voltagedependent Ca2+ channel; AchR, acetylcholine receptor; yellow lightning symbols, membrane depolarization; Ca2+, calcium; RyR, ryanodine receptor; l-cit, l-citrulline; l-arg, l-arginine; CaM, calmodulin; A, syntrophin α1, β1 and β2

As mentioned above, any disorder affecting NO synthesis can lead to dramatic consequences in the brain. Indeed, a lack of NO could impair the neurotransmission and hence the neuronal plasticity, such as long-term potentiation (LTP) in the hippocampus or long-term depression (LTD) in the cerebellum [50]. NO ensures also the correct irrigation of brain by its vasodilator effect on peripheral blood vessel. The synthesis of NO can be altered by a deficiency or a loss of activity of nNOS. Other regulatory elements, such as the decrease of l-arginine precursor availability or cofactors crucial for the catalytic activity of NOS, contribute also to nNOS inactivation. Conversely, in some several brain pathologies (ischemia, strokes, neurodegenerative disorders including Parkinson's and Huntington's diseases, and amyotrophic lateral sclerosis), a large amount of NO is produced [51]. It is hypothesized that NO or derived nitrogen species interact with various proteins inducing several toxic effects. Thus, pharmacological regulation of NO synthesis offers important strategies for the treatment of neurodegenerative and muscle diseases.

### *2.2.2.2. nNOSμ in peripheral neurotransmission*

On the other hand, nNOSμ is located in skeletal and cardiac muscle where NO controls muscle contractility and local blood flow essential to support muscular effort. When the excitatory neurotransmitter acetylcholine (Ach) is released at the neuromuscular junction, it is activating specific acetylcholine nicotinic receptors present on muscle cells [52]. Activation of these receptors allows the release in the cell sarcoplasm of calcium from exogenous and/or endogenous

**Figure 9.** The neurotransmission mediated by NOSα in central nervous system (CNS) (adapted from [49]). Glu, glutamate neurotransmitter; NMDAR, N-methyl d-aspartate type-glutamate receptor; A, PSD95 protein adapter between nNOSα and NMDAR; CaM, calmodulin; PKC, phosphate kinase C; PKII, Ca2+/CaM-dependent protein kinase II; CaN, calcineurin; CAPON, C-terminal PDZ domain ligand of neuronal nitric oxide synthase; MAPK, mitogenactivated protein kinase.

sources. This process is also enhanced by the activation of a voltage-dependent Ca2+ channel (CaCn) [53]. The increase of Ca2+ from exogenous origin activates ryanodine receptor present on the sarcoplasmic reticulum, which, in turn, delivers a large amount of Ca2+. When the Ca2+ threshold is reached, it is binding on the calmodulin protein. Calmodulin can then interact with the nNOSμ to activate the generation of NO. Interestingly, nNOSμ interacts through its PDZ domain with adapter proteins (syntrophins) linked to a membrane protein complex called dystrophin [54]. Produced NO could then diffuse back to the motor neuron to enhance the liberation of neurotransmitters or the vasodilatation of blood vessels, thus increasing the local blood flow to support muscle needs during contraction efforts (**Figure 10**) [55].

NO synthesis is also well regulated by calpain (Calp). Indeed, this protein interacts with the PDZ domain nNOSμ provoking its detachment from the dystrophin/syntrophin membrane complex [56]. Interestingly, in muscular Duchenne dystrophy, the damages caused by dystrophin absence are enhanced in case of simultaneous lack of nNOSμ [57]. Thus, NO donors may offer a potential avenue for therapy.

#### *2.2.3. The inducible iNOS and the innate immunity*

of nNOSα present at the membrane [46]. Indeed, CAPON competes with the PDZ domain of nNOSα and thus promotes its detachment from the NMDAR-PSD95 complex. nNOSα interaction with CAPON-DexRas complex allows the activation of DexRas, a small protein G, through s-nitrosylation on its thiol residue [47]. Then, DexRas activates the MAPK cascade and the transcription of several genes [47]. Several shutdown systems are available to limit this process, which could dramatically affect the brain physiology by synapses hyperactivation. Indeed, kinases, such as PKC or PKII, can phosphorylate nNOSα leading to its loss of activity [48]. nNOSα can also be sequestrated by two membrane proteins (caveolins 1 and 3)

As mentioned above, any disorder affecting NO synthesis can lead to dramatic consequences in the brain. Indeed, a lack of NO could impair the neurotransmission and hence the neuronal plasticity, such as long-term potentiation (LTP) in the hippocampus or long-term depression (LTD) in the cerebellum [50]. NO ensures also the correct irrigation of brain by its vasodilator effect on peripheral blood vessel. The synthesis of NO can be altered by a deficiency or a loss of activity of nNOS. Other regulatory elements, such as the decrease of l-arginine precursor availability or cofactors crucial for the catalytic activity of NOS, contribute also to nNOS inactivation. Conversely, in some several brain pathologies (ischemia, strokes, neurodegenerative disorders including Parkinson's and Huntington's diseases, and amyotrophic lateral sclerosis), a large amount of NO is produced [51]. It is hypothesized that NO or derived nitrogen species interact with various proteins inducing several toxic effects. Thus, pharmacological regulation of NO synthesis offers important strategies for the treatment of neurodegenerative and muscle diseases.

On the other hand, nNOSμ is located in skeletal and cardiac muscle where NO controls muscle contractility and local blood flow essential to support muscular effort. When the excitatory neurotransmitter acetylcholine (Ach) is released at the neuromuscular junction, it is activating specific acetylcholine nicotinic receptors present on muscle cells [52]. Activation of these receptors allows the release in the cell sarcoplasm of calcium from exogenous and/or endogenous

**Figure 9.** The neurotransmission mediated by NOSα in central nervous system (CNS) (adapted from [49]). Glu, glutamate neurotransmitter; NMDAR, N-methyl d-aspartate type-glutamate receptor; A, PSD95 protein adapter between nNOSα and NMDAR; CaM, calmodulin; PKC, phosphate kinase C; PKII, Ca2+/CaM-dependent protein kinase II; CaN, calcineurin; CAPON, C-terminal PDZ domain ligand of neuronal nitric oxide synthase; MAPK, mitogen-

preventing its interaction with calmodulin (**Figure 9**).

28 Emerging Pollutants - Some Strategies for the Quality Preservation of Our Environment

*2.2.2.2. nNOSμ in peripheral neurotransmission*

activated protein kinase.

NO also takes an important part in innate immunity. Indeed, NO and derived compounds are cytotoxic weapon against tumor cells, microorganisms, and viruses [58]. NO is generated by inducible nitric oxide synthases (iNOS and NOS2) within macrophages. However, more recently, studies have shown that this enzyme is also expressed in other cell line (vascular endothelial cells, hepatocytes, pulmonary, and colonic epithelial cells) [59]. Unlike nNOS or eNOS, its expression is stimulated by several pathways after activation of specific receptors by endotoxins (LPS) or cytokines (TNFα, IFNϒ, and IL1β). Briefly, the LPS endotoxins are carried by an LPS-binding (LPB) protein to its specific Toll-like receptor 4 (TLR4). Then, the transduction

**Figure 10.** nNOSμ activity at the neuromuscular junction (adapted from [49]). Ach, acetylcholine; CaCn, voltagedependent Ca2+ channel; AchR, acetylcholine receptor; yellow lightning symbols, membrane depolarization; Ca2+, calcium; RyR, ryanodine receptor; l-cit, l-citrulline; l-arg, l-arginine; CaM, calmodulin; A, syntrophin α1, β1 and β2 adapter between dystrophin and nNOSμ; Calp, calpain.

of the signal leads to the transcription of NF-κB [60]. This transcription factor is essential for the expression of *iNOS* gene. On the other hand, cytokines produced by infected cells activate pathways responsible for the expression of several transcription factors (NF-κB, AP1, and IRF1) also involved in the transcription of iNOS [61]. Both pathways are necessary to fully upregulate iNOS expression. Moreover, iNOS is constitutively active and exhibits a high affinity for calcium explaining its Ca2+-independent activity. iNOS produces a large amount of NO until substrate depletion [62]. Then, NO acts directly or through derived compounds (mostly ONOO<sup>−</sup> and NO2 ) generated through the reaction of NO with ROS formed by NADPH oxidase. These molecules can be generated inside (phagosome) or outside the cells. Altogether, NO and derivatives could act in synergy on several components within the targeted hostile elements such as proteins (thiols, metal centers, and tyrosine residues), nucleic acids, and lipids [63, 64]. Altogether, these modifications lead to irreversible damages impairing cellular metabolism and ultimately inhibit growth and replication mechanisms and even cell death (**Figure 11**).

control in order to limit its tumorigenic potential. The development of iNOS inhibitors and NO-releasing agents offers interesting therapeutic strategies to struggle against cancer and

Several environmental, commensal, and pathogenic prokaryotes possess an arsenal of proteins dedicated to the nitrogen metabolism under anaerobic conditions [67]. Indeed, an enzymatic chain is involved in the denitrification pathway responsible for the production of NO through nitrite reductase (NIR) as an intermediate. The outcome of this mechanism is the

Interestingly, a shortened NOS-like protein called bNOS was discovered in Gram-positive bacteria [28]. This enzyme is deprived of the classical reductase domain already described in other NOS isoforms. However, bNOS is able to recruit other proteins exhibiting a reductase catalytic activity [69]. Similar to mammalian isoforms, bNOS catalyzes the production of NO using l-arginine precursor. However, NOx is key elements in the innate immunity to avoid infection. Indeed, similar to eukaryotic cells, prokaryotes are also sensitive to NOx activity. Thence, such compounds could alter DNA, proteins, and metal centers leading to severe alter-

As previously described, NO could be endogenously produced by bacteria but is also a powerful exogenous antimicrobial agent. This highlights the requirement of systems to manage a bearable threshold of NOx within the cell. First, bacteria have developed NO sensing system

−

, nitrate; NO<sup>2</sup>

−

, nitrite; NO, nitric oxide; N<sup>2</sup>

, ammonia; NAR/NAP, nitrate reductase; NIR, nitrite reductase; NOR,

O, nitrous oxide;

). This process is crucial for the growth of bacteria in the absence

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**3. NOx versus prokaryote: "Catch me if you can"**

**3.1. NOx at the center of prokaryotic life, virulence, and death**

of oxygen because it is coupled with ATP generation (**Figure 12**).

microbial infections.

formation of dinitrogen (N2

ations of bacterial growth and replication.

**Figure 12.** Bacterial nitrogen cycle (adapted from [68]). NO3

OH, hydroxylamine; NH<sup>4</sup>

OR, nitrous oxide reductase.

+

N2

, dinitrogen; NH<sup>2</sup>

nitric oxide reductase; N<sup>2</sup>

After infection, NO generation is abolished by neutralization of iNOS activity. Indeed, iNOS undergoes a posttranslational modification called polyubiquitination leading to its proteasomal degradation. Physiological aggresome, an alternative pathway for protein degradation, is also involved in iNOS inactivation [66]. The degradation of NO can also be modulated by its own interaction with ROS. The depletion of l-arginine precursor also shuts down iNOS activity.

Overexpression of iNOS is observed during chronic infection leading to an excessive level of NO and RNS. This phenomenon can impair the host physiology because these high reactive compounds represent mutagenic sources. Thus, the NO balance is a crucial point to

**Figure 11.** Implication of iNOS in the innate immunity (adapted from [65]). IFNϒ, interferon ϒ; IFNR1/2, IFNϒ receptors 1 and 2; JAK, Janus kinase; STAT, signal transducer and activator of transcription; IRF1, interferon response factor 1; IL1β, interleukin 1β; IL1R, IL1β receptor; MAPK, mitogen-activated protein kinase; JNK, C-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; AP1, activator protein 1; TNF-α, tumor necrosis factor α; TNFR, tumor necrosis factor receptor; NF-κB, nuclear factor κB; TLR4, Toll-like receptor 4; CD14, cluster of differentiation 14; LPS, lipopolysaccharide; LPB, LPS-binding protein; ROS, reactive oxygen species.

control in order to limit its tumorigenic potential. The development of iNOS inhibitors and NO-releasing agents offers interesting therapeutic strategies to struggle against cancer and microbial infections.
