Ca²⁺ and calmodulin control neuronal NOS (nNOS) activity. The subcellular distribution and function of nNOS were determined by the direct association of the PDZ domain of nNOS with other proteins [22]. PDZ domain brings up to an area that entails 80–120 amino acid residues that perform as components taking part in protein-protein interactions [22,23]. The canonical PDZ domain of nNOS connects to the nNOS adaptor protein NOS1AP, also well known as carboxy-terminal PSD-95-Dlg-ZO1 [PDZ] ligand of nNOS (CAPON). The ternary complex made by PDZ domain interactions between N-methyl-d-aspartate (NMDA) receptor, PSD-95, and nNOS performs the action of scaffolding nNOS to the NMDA receptor, and NMDA-encouraged Ca²⁺ influx is consequently well coupled to the activation of nNOS and the subsequent formation of NO [24]. The nNOS has been found in various parts and tissues of the body, including the brain, spinal cord, sympathetic ganglia, adrenal glands, peripheral nitrergic nerves of the nervous system, as well as epithelial cells of different tissues such as kidney macula densa cells, pancreatic islet cells, and vascular smooth muscle cells. The most abundant presence of nNOS has been determined in the mammal skeletal muscle [25].

nNOS does not seem to be involved in the acute neurotransmission in the central nervous system (CNS); however, it can mediate the long-term regulation of synaptic transmission (long-term potentiation, long-term inhibition). Furthermore, nNOS is involved in the central regulation of blood pressure. This has been shown by the induction of systemic hypertension while blocking nNOS activity in the medulla and hypothalamus [1]. The effects of nNOS on the regulation of vascular tone are independent of its CNS effects. The production of NO by nerves that contain nNOS (nitrergic nerves) stimulates NO-sensitive guanylyl cyclase and decreases the tone of the innervated smooth muscles in the periphery including blood vessels [25,26]. Ca²⁺/calmodulin (CaM)-dependent protein kinases, cyclic AMP-dependent protein kinase (PKA), cGMP-dependent protein kinase, and protein kinase C (PKC) participate in the phosphorylation of nNOS [27].

The effects on the smooth muscles of the corpus cavernosum and the relaxation of nitrergic nerves affects penile erection [28,29]. The relaxation of these smooth muscle cells is mediated by cyclic GMP, which can be degraded by phosphodiesterases (predominantly isoform 5). Therefore, a residual nNOS activity is needed for the selective PDE5 inhibitors to be effective [30].

iNOS enzymes are exclusively located in macrophages and their production is triggered when needed. The bacterial lipopolysaccharides and cytokines can influence the expression of iNOS [25,31]. When iNOS is expressed, it is activated continuously and is not affected by the intracellular Ca²⁺ concentrations. The iNOS in macrophages excretes large amounts of NO, which is important for them as a toxic defense molecule [32]. NO can disrupt the enzymes containing iron in their catalytic centers, such as iron-sulfur cluster-dependent enzymes (complexes I and II) entailed in mitochondrial electron transport, ribonucleotide reductase (the rate-limiting enzyme in DNA replication), and cis-aconitase (a key enzyme in the citric acid cycle) [32,33]. Recently, studies on ¹³C tracing and mitochondrial respiration revealed a new observation on the modulation in NO-mediated macrophage metabolic programming attributed to the regulation of the tricarboxylic acid (TCA) cycle and the accumulation of itaconate with a great fractional combination of glucose carbon at the overhead of glutamine carbon, which could link the suppression of pyruvate dehydrogenase (PDH) and aconitase to NO. NO has a functional impact on the accumulation of metabolite hallmarks of M1 polarization, including citrate, succinate, and itaconate. Indeed, during the stimulus, glucose uptake was elevated, immune-response gene-1 (Irg1) was upregulated, and citrate and itaconate were produced, incorporating glucose-derived carbon. [34,35,36]. At higher concentrations, NO can directly impede target cell DNA and affect strand breaks and fragmentation [37,38]. In addition to macrophages, other non-immune cells can produce NO by iNOS upon induction via cytokines and thus can affect other cells, i.e., endothelial cells exert NO-mediated lysing of tumor cells and cytokine-induced iNOS in hepatocytes synthesize NO to kill malaria sporozoites [39,40].

eNOS is the leading source of NO in vascular endothelium. Its promotor expression exists in various types of cells such as cardiomyocytes, platelets, specific brain neurons, in syncytiotrophoblasts of the human placenta, and in LLC-PK₁ kidney tubular epithelial cells [1,25]. NO production is regulated by Ca²⁺-activated calmodulin as the increase of intracellular Ca²⁺ induced binding of calmodulin subsequently increases the eNOS activity [19]. Other factors can also regulate the activity of eNOS, e.g., heat shock protein 90 (hsp90) acts as an allosteric modulator that promotes the eNOS’ (re)coupling and therefore its activity [41,42]. Another regulator of eNOS is caveolin-1 [43]. It is a tonic inhibitor interacting with the eNOS localized in caveolae [44]. The lack of caveolin-1 results in enhanced endothelium-dependent relaxation in mice [45]. The inhibitory effect of caveolin-1 can be blocked by the presence of calmodulin and hsp90 [46]. Fluid shear stress can activate eNOS independent of intracellular Ca²⁺ increase through phosphorylation of eNOS at several serine (Ser), threonine (Thr), and tyrosine (Tyr) residues [47,48]. During the regulation of eNOS activity by phosphorylation, the probable changes in phosphatase activity are able to have an impact on NO generation. Certainly, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) play diverse roles in the regulation of eNOS phosphorylation [48]. According to recent studies, aging is considered to be related to the greater protein acetylation levels and the defeat of sirtuin-1 deacetylation activity, which could subsequently cause the reduced eNOS activity [49,50]. Four lysine residues are found within the cAMP-response element-binding protein (CREB)-binding protein (CBP) of eNOS. The identification of two of these residues as a consequence of the acetylation suggests that SIRT1 may target more than a single residue within this domain, regulating enzymatic activity via the modulation of deacetylation of the targeted residues. Moreover, by knowing the recognized role of the phosphorylation in eNOS regulation, in a similar manner to other proteins, the acetylation and phosphorylation might both be responsible for the regulation of eNOS activity [51].

The impairment of the vascular function due to endothelial dysfunction is one of the main heart and vasculature alterations during cardiovascular aging. The endothelial dysfunction is characterized by the impaired vasodilator reaction to the flow or the agonists and one of its fundamental causes is the decreased bioavailability of NO under pathological conditions such as atherosclerosis, hypertension, and hypercholesterolemia [53,54,55,56]. The reduced abundance or activity of eNOS, the increased levels of endogenous NOS inhibitors, and the reduced supply of l-arginine, or the increased NO degradation or scavenging, can result in decreased NO bioavailability.

As part of the urea cycle, hydrolyzing l-arginine to ornithine and urea by arginase can affect the bioavailability of l-arginine. NO performance increases through the iNOS in the macrophages following the inhibition of arginase [57]. Arginase can also modulate the vascular tone by regulating l-arginine supply for eNOS activity. Berkowitz et al. (2003) have shown that arginase activity and expression are upregulated in the aorta rings of old rats while the NOS activity and cGMP levels and, consequently, the endothelial activity, were reduced. Moreover, arginase inhibition increased vasodilation in the aortic rings of old rats compared with young adult rats. Furthermore, arginase inhibition restored NOS activity and cyclic GMP levels in the old rat vessels compared with those of young rats. Arginase competitively controlled the bioavailability of l-arginine for eNOS by limited NO production despite the increased expression of eNOS or adding exogenous l-arginine. The pretreatment with arginase inhibitors was sufficient to restore l-arginine responsiveness in the old rat vascular ring preparations. This result indicates that the enzyme kinetics strongly favors arginase. During arginase inhibition, l-arginine can restore cytosolic NOS responsiveness and thus restore vasorelaxation. Arginase was in contact with NOS, contributing to impaired relaxation of the endothelium. In brief, Berkowitz et al.’s (2003) research showed that arginase expression and activity increases in the vasculature with advancing age and leads to endothelial dysfunction [58].

Chou et al. (1998) found lower expression and activity of eNOS in spontaneously hypertensive rats (SHR) compared with non-SHR at both young and old age. The SHR developed the early onset of the decrease in eNOS expression and activity. However, the eNOS decline did not progress by aging in a way that eNOS activity was lower in the old non-SHR rats compared to the old SHR rats. The iNOS expression and activity showed inversed correlation to eNOS in both SHR and non-SHR. The iNOS expression was increased further by lipopolysaccharide (LPS) stimulation only in the 14–17- and 63-week groups of SHR. Hypertension control could regulate the abnormal iNOS expression and activity in SHR but, a “cause and effect” relationship was not established. Besides, the increase in levels of the tumor necrosis factor-alpha (TNF-α) and NO₂⁻/NO₃⁻ induced by LPS was much higher in SHR than in non-SHR at both groups of 14–17- and 63-week [59]. In the studies of Wu et al. (1999), the significant elevation was observed in basal plasma and aorta levels of NO₂⁻ in SHR treated with LPS model. In vitro, the ACh-induced relaxation was decreased in the aortae isolated from SHR. However, this dissimilarity between the SHR and non-SHR was eliminated after the treatment of the rings with nitro-l-arginine methyl ester (l-NAME), and the damage of NO production was observed in the SHR. Expression of iNOS in both groups treated with LPS was augmented. These data supported the hypothesis that the raised plasma NO level in SHR might be the result of the NO release from protein-bound dinitrosyl nonheme iron complexes (DNIC) in the vascular bed, leading to the abolished hypertension [60]. Vaziri et al. (1998) concluded that the l-arginine/NO pathway was upregulated in young SHR before and after the onset of hypertension, which showed that NO formation was augmented in young SHR both before and after the commencement of the hypertension [61]. Further studies have demonstrated that the elevated production of NO in SHR, which was additionally improved by LPS treatment, is related to a primary expression of iNOS [62].

Cernadas et al. (1998) showed that aged rats had an abnormal hypotensive response to acetylcholine and bradykinin and a retained hypotensive response to nitroprusside sodium. Furthermore, the pressure effect of l-arginine antagonist (l-Nv-nitro-l-arginine) caused an increased sensitivity response and angiotensin II caused a decreased vasoconstrictor response in aged rats. The latter was improved after inhibition of NOS. Aged rats had higher plasma levels of nitrite and nitrate, higher cGMP content, and elevated eNOS and iNOS expression in the aortas. However, the activity of eNOS was significantly decreased in aged rats compared to the young ones, these results correlated with the iNOS activity. The locally released cytokines from endothelial injury sites in the vascular wall might have induced iNOS expression. The iNOS-related NO could decrease the action of the eNOS, thereby favoring impaired endothelium-dependent vasorelaxation. On the other hand, during aging, iNOS activity could play a significant function in retaining the vascular tone [63].

The reduction in endothelial vasoreactivity and excessive diastolic relaxation is linked with cardiovascular aging. Zeiman et al. (2001) showed that l-arginine increased ventricular relaxation in both young and aged rat hearts. The increased myocardial NOS-cGMP signaling was correlated with the elevated levels of eNOS protein in cardiac endothelial cells of aged rats. Furthermore, aged rats had elevated calcium-dependent NOS activity. However, in isolated myocytes, there was no difference between NOS activity and protein abundance. Administrating l-arginine decreased baseline isovolumic relaxation and left ventricular end-diastolic strain in both young and aged rat hearts. Sodium nitroprusside (a NO donor) promoted the NO/cGMP mediating pathway, which was attenuated by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one [64]. In the studies of the role of soluble guanylyl cyclase (sGC) in aging, it was found that sGC was a heterodimeric enzyme composed of one α1 and β1 subunit that shared a molecule of heme. It was observed that the existence of heme depended on the expression of enzymatic activity and rendered sGC sensitivity to NO. In numerous examples, the activation of sGC and production of cGMP mediated the impact of NO. Moreover, in the vasculature, NO-induced relaxation and repression of platelet aggregation happened through cGMP [65].

Regarding the preclinical findings, asymmetric dimethylarginine (ADMA) is an important endogenous NOS inhibitor in aging-associated cardiovascular risk. In aging-related biological conditions such as telomere shortening and cell senescence, ADMA is implicated [66]. Concentrations of vitamin D have reverse interactions with ADMA concentrations while h-sensitivity C-reactive protein (hs-CRP) has direct interactions with ADMA concentrations [67]. In a study by Scalera et al. (2006), it was observed that l-arginine could avert the commencement of endothelial aging in ADMA or the homocysteine-treated cells’ elevation of the production of NO and, subsequently, the induction of heme oxygenase (HO-1) enzyme activity and protein expression [68].

Vascular endothelial cells produce and release endothelin-1 (ET-1), a potent vasoconstrictor protein [69]. In stable, older, and sedentary adult humans, ET-1 signaling was enhanced, which can cause tonic vasoconstriction in peripheral arteries [70,71,72]. Furthermore, aortic endothelial cells of older individuals have shown a higher synthesis of ET-1 relative to young adult donors with distinct pathologies [73].

Donato et al. (2009) found an inverse relationship between decreased endothelium-dependent dilation and increased expression of the endothelial cell ET-1 protein. Moreover, the endothelium-dependent dilation was suppressed by the ET-1 signaling in old mice compared to the young. In endothelial cells collected from brachial arteries and peripheral veins of healthy young and old men, reduction in neither eNOS nor activated serine 1177 phosphorylated isoform (PeNOS) of eNOS was found. These data suggested the contribution of ET-1 expression and bioavailability, but not eNOS, to the vascular endothelial dysfunction in aging [74].

In vessels of aged animals, the decreased flow-induced dilation mainly depended on the compromised NO-mediated dilation [75,76]. Superoxide (O₂⁻) increases within the vascular wall with aging, which decreases the bioavailability of NO, leading to endothelial dysfunction in aging [76,77,78]. Production of superoxide could increase the interruption of NO resulting in considerable vasomotor dysfunction, which was attributed to vascular aging [79,80]. Collectively, it was proposed that reaction of NO with O₂⁻ led to accelerate the production of ONOO⁻, which attenuated the bioavailability of NO. Moreover, dysregulation of the eNOS caused impairment in the release of NO in the endothelium and increased superoxide formation [81]. This process of aging in the vascular wall can be worsened further as O₂⁻ formation can also be improved by a decline in SOD function [82]. Sun et al. (2004) showed a decrease in the shear stress-induced dilation and NO release following aging in rat mesenteric arterioles and arteries. Moreover, the shear stress stimulation of NO synthesis was decreased in aged vessels, evidenced by the decrease in the absolute perfused nitrite (NO₂⁻/NO₃⁻) compared to the young vessels. Furthermore, the aged rats’ vasculature had lower SOD levels due to the decreased extracellular superoxide dismutase (ecSOD) expression, but not the Cu/Zn-SOD and Mn-SOD expression. Suppressed ECSOD expression further decreased the NO bioavailability following the response to the shear stress in the aged vessels. In summary, isolated mesenteric arteries have decreased NO-mediated vasodilation response to the shear stress due to the lower NO bioavailability following the increased O₂⁻ production. The latter is caused by the suppressed ECSOD expression and consequently the lower SOD activities [83].

Caveolin, the intrinsic protein of caveolae, is quantitatively linked with the eNOS in ventricular myocytes and endothelial cells [84]. In the former, eNOS is linked with caveolin-3 and, to a lesser degree, caveolin-1, while, in endothelial cells, eNOS is associated only with caveolin-1 [85]. Arreche et al. (2012) found increased NOS activity, decreased eNOS, and caveolin-1 protein levels, as well as increased iNOS activity in the ventricle of the middle-aged rats [86]. The dissociation of caveolin-1 from eNOS correlated with aging. During the hemorrhage-induced hypovolemia, NOS activity and protein levels were increased in the myocardium while the caveolin-1 presence was decreased, which resulted in further dissociation of eNOS. The iNOS increased following blood loss in middle-aged rats. Thus, the modulatory roles of caveolin-1 on cardiac NOS have been demonstrated in the hypovolemic and aging states.

NO contributes to the regulation of water and electrolyte homeostasis in the cardiovascular system [87]. Arza et al. (2015) found that, following 1-month water restriction, both young and adult rats showed decreased NOS expression and activity related to the increased caveolin-1 levels. Interestingly, aging caused functional alterations in the cardiovascular system, evidenced by more remarkable ventricular NOS formation following water restriction and hypovolemia in adult rats. These findings demonstrate the roles of NO and its caveolin regulatory proteins in preserving cardiac physiological function during dehydration and aging [88].

Aging impairs the control of glucose metabolism, resulting in the decreased glucose tolerance and chronic hyperglycemia in the elderly. These changes can mimic accelerated aging and act as a risk factor for cardiovascular diseases [89,90,91]. Levels of NO and peroxynitrite might play a pivotal part in the progression of late impairment in the diabetic vasculature and kidney [92]. NO donor agents and the NO generated by iNOS persuaded insulin resistance via the S-nitrosation of proteins entailed in the primary stages of insulin action, for instance, insulin receptor β (IRβ), insulin receptor substrate 1 (IRS-1), and protein kinase B (Akt). Even though a number of studies have proposed that NO and iNOS might be important in numerous aging-associated complications, the contribution of NO pathways on aging-induced insulin resistance is indistinct [93]. Moreover, hyperglycemia along with aging correlated with endothelial dysfunction, vascular stiffening, and remodeling [94,95]. The basal activity of eNOS and endothelial dysfunction are evident at an advanced age and during hyperglycemia [96,97,98]. In addition, hyperglycemia has been shown to increase iNOS expression and activity, which correlates with excessive NO production and subsequent cellular damage [99].

Rogers et al. (2013) showed increased senescence markers following the glucose level fluctuations in human umbilical vein endothelial cells (HUVECs). The increase of aging markers was due to the elevated Akt’s transcription and expression and its downstream signaling targets. Moreover, eNOS production was decreased, which resulted in iNOS overexpression, decreased bioavailability of NO, and endothelial dysfunction [100].

Yu et al. (2011) reviewed the relationship between aging-induced heart failure and insulin resistance. The precise mechanism was not deceptive in this issue. The documented outcomes showed that insulin resistance might have adverse effects on bioenergetics and myocardial metabolism. Regarding the investigation of isolated perfused hearts obtained from aged rats and cardiomyocytes, the attenuation of contractile reaction and uptake of glucose to insulin, as well as the dampening of the Akt–eNOS–NO pathway due to the post-insulin receptor signaling, were perceived [101].

Classical pro-atherosclerotic stimuli such as oxidized low-density lipoprotein (oxLDL), proinflammatory cytokines, and reactive oxygen species (ROS) caused endothelial cell apoptosis and disrupted the endothelial monolayer integrity leading to vascular damage and atherosclerosis [102,103,104]. The endothelial NO production could inhibit apoptosis by suppressing caspases (a vital factor in apoptosis cascade) through the S-nitrosylation of its critical cysteine [105,106,107]. Hoffmann et al. (2001) reported a three-fold increase in levels of oxLDL, tumor necrosis factor-a-induced apoptosis, and caspase-3-like activity in aged HUVECs compared to the young cells. The substantially suppressed eNOS expression and decreased overall S-NO content implied that eNOS downregulation in aged HUVECs can lead to an age-dependent increase in vulnerability to apoptosis. This was confirmed as the knockdown of eNOS could elevate the apoptosis rate which was abolished by exogenous NO donors. Furthermore, the shear stress did not affect eNOS protein expression, S-NO content, and apoptosis rate in the aged HUVECs. Overexpression of wild-type eNOS restored the antiapoptotic effects of the shear stress but did not affect it when the shear stress was absent. Strikingly, apoptosis in aged HUVECs was further abrogated by the transfection of constitutively active phosphomimetic eNOS (S1177D) [108].

Oelze et al. (2104) showed that, in the absence of glutathione peroxidase-1 (GPx-1), the aging-associated production of oxidants, the activation of the redox system, and the presence of the dysfunctional and uncoupled eNOS are intensified. They found the increased phosphorylation of protein kinase C, protein tyrosine kinase, and eNOS S-glutathionylation associated with an uncoupling in aged peroxidase-1-deficient (GPx-1−/−) mice. The emerging role of GPx-1 ablation in aging animals has a considerable influence on the burden of oxidative damage, representing the experiential variation between vascular dysfunction in aged wild-type and GPx-1−/− mice [109]. GPx-1 is an intracellular antioxidant enzyme which has an important role in the reduction of H₂O₂ to H₂O in order to delimitate its adverse impacts. GPx-1 is a selenocysteine-comprising enzyme that has been involved in the prevention and progression of numerous usual, chronic, and complicated diseases containing cancer, diabetes, and cardiovascular disease [110,111].

Sirtuins are a family of protein deacetylases that regulate aging processes. They depend on NAD⁺ and thus control cell metabolism along with other cellular functions. Sirtuin 3 (SIRT3) acts mainly by regulating mitochondrial bioenergetics, an essential prerequisite for healthy aging [112,113]. Lu et al. (2020) found that SIRT3 mRNA and protein are decreased in aged human and rat veins. Ad-SIRT3 gene transfer increased the expression and concentration of SIRT3, MnSOD, catalase (CAT), eNOS, and NO. Furthermore, it reduced the neointimal thickness and neointimal area/media area ratio [114]. Mattagajasingh et al. (2007) showed that SIRT1 contributed to the endothelium-dependent vasodilation by activating eNOS in the endothelium of rat aortic rings through deacetylation in a caloric restriction animal model. SIRT1 targeted lysines 496 and 506 in the calmodulin-binding domain of eNOS. Moreover, SIRT1 inhibition decreased NO bioavailability and thus endothelium-dependent vasodilation in arteries [51].

Red blood cells (RBCs) affect NO generation and scavenging by hemoglobin-dependent nitrite. The changes in levels of NO₂⁻ could regulate the NO hemostasis as it can be reduced to NO and also can be oxidated into NO₃⁻ [115]. RBCs can affect vascular activity by modulating NO through suppressing NO₂⁻ oxidation by their antioxidants [116,117]. It has been shown that aged RBCs have lower antioxidant levels [118]. In a study by Owusu et al. (2013), regulatory roles of RBC on vascular hemostasis of NO vasodilatory mechanisms were investigated. Older RBCs had higher NO₂-oxidation kinetics and NO scavenging levels compared to young RBCs. The latter was reflected in the inhibited acetylcholine and NO-donor dependent vasodilation in isolated aortic rings. The similarity of NO₂⁻ reduction levels between young and old RBCs was confirmed by the inhibition of nitrite-dependent vasodilatation under oxygenated and deoxygenated conditions in old RBCs [119] (Figure 2).

The brain undergoes morphological and functional alterations during aging. The burden of oxidative stress on the CNS is more prominent due to the relative deficiency in the antioxidant defense mechanisms of CNS [120]. CNS aging is greatly affected by oxidative stress, which involves the production of free radicals including superoxide (O₂⁻), hydrogen peroxide (H₂O₂), NO, and peroxynitrite (ONOO⁻) [121,122]. Reactive oxygen species (ROS) containing O₂⁻, H₂O₂, and the OH⁻, and reactive nitrogen species (RNS) including ONOO⁻ and NO, are very important in the pathophysiology of neurodegenerative diseases. Due to the great reactive impact of ROS, they chemically interact with various biological molecules, causing significant alterations in cellular tasks, an increased probability of spreading to regions with high metal levels, and induced neuronal death [123,124,125]. Several functions of the CNS are related to NO, including neurotransmitter release, synaptic plasticity, and the regulation of neuronal electrical activity [126,127]. It has been shown that NO has a prominent role in aging processes and related disorders of CNS [128,129] (Figure 3).

Uttenthal et al. (1998) observed that in the cortex of the aged rats, nNOS-immunoreactive multipolar neurons were similar to those of the young rats in the terms of quantity, but had an abnormal morphology: varicose, vacuolated, and fragmented structure with an irregular outline and loss of spines. In addition, neurons that were weakly nNOS-positive with a ring of immunoreactive cytoplasm could be found only in the aged rat cortex. On the other hand, iNOS-immunopositive neurons were found only in the aged rat cortex. The old rat cortex had lower nitrotyrosine-positive cells (a biomarker of oxidative stress) compared to the total NOS-positive neurons, while the opposite was observed in the young rat cortex. The formation of nitrotyrosine-containing tissue proteins has the potential to modulate NO and O₂⁻ concentrations related to the introduction of protein tyrosyl residues to reaction. Other significant elements that might contribute to the greater protein nitrotyrosine levels identified could be an augmented production of O₂⁻ and an abridged rate of turnover of the related tissue proteins with age. According to the outcomes obtained in the study, the increase in nNOS and iNOS expression in the aged rat cortex is not essentially related to the proportional increase in NOS [130]. In a study by Hilbig et al. (2002), a decrease of nNOS expression was observed during the aging process in rats [131].

Brain aging has been linked with mitochondrial dysfunctions [132]. According to recent studies, NOS is not present in the mitochondrial matrix but rather makes part of the outer membrane. Lores-Arnaiz and Bustamante (2011) found a decrease in mitochondrial NO production in both synaptic and non-synaptic neurons of the aged rat cortex. Furthermore, the impairment of mitochondrial functions: ATP deficiency, H₂O₂ production at high levels, abnormal calcium homeostasis, and decreased levels of NO were observed in the aged rat compared to the young rat cortex neurons [133].

Synaptic mitochondria, which need to sustain the energy required for synaptic activity, showed present functional differences when compared to non-synaptic mitochondria [134]. However, although the aging-induced changes in the brain are thought to be mediated by the increased activity of NOS [135], the experimental data is rather controversial, as the demonstrated results range from an increase to a significant reduction in the nNOS activity in aged rat or mice models [136]. Mitochondrial investigations of NO effects support the hypothesis that NO levels could be increased during aging: endogenously-produced NO can inhibit mitochondrial respiration at cytochrome oxidase with the Ki ~50 nM (physiological range of NO concentrations is 0.1–100 nM) [137,138]. Moreover, NO can irreversibly suppress mitochondrial respiration, increase the production of ROS and RNS, and induce the mitochondrial permeability transition pore [137,138].

In the CNS, glutamate activates the N-methyl-d-aspartic acid (NMDA) receptor, which facilitates the influx of Ca²⁺ into the cell. The binding of Ca²⁺ to calmodulin activates the NOS production of NO which further activates the soluble guanylate cyclase and production of cGMP [139]. One of the main causes of dementia development is the aggregation of amyloid β (Aβ) peptides [140,141]. It has been shown that Aβ peptide interferes with the calmodulin-dependent NO synthesis. Chalimoniuk and Strosznajder (1998) showed that with advanced age, phosphodiesterase degradation of cGMP increased, which resulted in the decreased basal levels of cGMP. Moreover, the inhibition of cGMP-phosphodiesterase did not restore the NMDA-induced production of cGMP in the hippocampus and cerebellum of aged rats. However, the brain cortex in aged animals responded to the inhibition of cGMP-phosphodiesterase and showed a preserved production of cGMP. Interestingly, the NOS activity in the aged brain hippocampus and cerebellum was elevated by 175 and 160%, respectively. The treatment with Aβ fragment, peptide 25–35, reduced the Ca²⁺ transfer mediated by NMDA and thus calmodulin-dependent NO formation. The elevated NOS activity, despite disturbed cGMP-dependent signaling in the hippocampus and cerebellum of aged animals, might affect learning and memory function during aging, and the accumulation of Aβ during aging might be the cause of these disorders [142]. Activity-related Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and AMPA receptor phosphorylation were reported as having crucial implications for hippocampal dentate long-term potentiation. Interruption in these mechanisms could directly associate with Aβ-induced impairments in memory and hippocampal synaptic plasticity [143,144].

Under chronic neuroinflammation conditions such as brain aging or neurodegenerative diseases, excessive levels of NO can be found at the choroid plexus (CP) via iNOS mediation, an epithelial layer that forms the blood-cerebrospinal fluid barrier (BCSFB). Glial cells are the source for these NO when they are chronically activated under the conditions such as aging or neurodegeneration [145,146,147]. Leukocytes reduce the neuroinflammation by entering CSF through the CP gateway; however, leukocytes do not always enter the CSF optimally during neuroinflammatory conditions. Thus, increased levels of leukocytes in the CSF can act as a potential therapeutic strategy [148,149,150,151,152,153,154]. It was suggested that NO alone in pathologic levels did not lead to neuronal death and gliosis. NO together with other cytotoxic and pro-inflammatory factors could induce neurodegeneration [147].

Baruch et al. (2015) showed that NO suppressed the leukocyte trafficking in choroid plexus (CP) by inhibiting epithelial nuclear factor-κB (NF-κB) p65 nuclear translocation in AD-Tg mice. Furthermore, the administration of the NO scavenger, rutin, enhanced the levels of leukocytes in the brain [155].

The tolerance of the brain towards hypoxemic conditions reduces by aging [156]. One of the suggested mechanisms for this phenomenon was the increase of free radicals during hypoxia and even at the reoxygenation period [157]. Hypoxic/ischemic conditions, including stroke, aneurysm, trauma, and infection, are among the most common causes of CNS dysfunction [158]. The increased frequency of hypoxic conditions during aging, along with the dysfunctional activity of the aged brain during these situations, highlight the importance of understanding the underlying mechanism behind these malfunctions. It has been shown that, contrary to eNOS and nNOS, which are constitutively expressed, iNOS induction is enhanced under conditions such as hypoxia [159,160]. Molina et al. (2017) studied the onset of the changes in the gene expression of NOS isoforms and their protein levels, as well as NO level changes following striatum hypoxia/reoxygenation in aged rats. Rats were exposed to hypoxemic conditions for 20 min and then were exposed to reoxygenation for 0 h, 24 h, and 5 days, in which they were sacrificed immediately thereafter. NOS gene expression levels did not change at 0 h following hypoxia. After 24 h, eNOS gene expression and protein levels, along with nNOS protein levels, were increased in the aged rat striatum. On day 5 of the post-hypoxia, iNOS gene expression did not change significantly, while iNOS protein levels were increased. Moreover, eNOS gene expression was enhanced while nNOS gene expression was suppressed. The levels of NO did not change significantly in aged striatum after hypoxia and reoxygenation, despite the increased activities of NOS isoforms during this time span. Molina et al. (2017) suggested that aging might disrupt the production of NO by NOS via decreasing the availability of BH4 cofactor and the impairment of Ca²⁺ homeostasis. Moreover, reduced O₂ levels following hypoxia amplified the decrease in the NO production by NOS. Furthermore, the decreased NO in the hypoxic striatum could disturb the NO vasodilatory response in the brain vessels, which could further increase the severity of the hypoxic damage to the brain. The increased activity of NOS following hypoxia in the aged striatum was thought to be the compensatory mechanism of the brain to combat the effects of hypoxia on reducing O₂ levels in the brain. Due to the impaired production of NO by NOS in the aged brain, the O₂-independent production of NO was increased by denitrating the nitrated tyrosine residues to produce NO. The O₂-independent production of NO in the aged striatum was shown as a continuous decline in the nitrated proteins from 0 h until 5 days after hypoxia/reoxygenation [161]. Aging-related changes in gene expression and corticostriatal synaptic plasticity were examined by Chepkova et al. (2015) in the dorsal striatum of four mice groups, aged from young to old [162]. The findings revealed a substantial drop in transcripts encoding neuronal NOS and activation receptors (NR1 glutamate NMDA receptor subunit and D1 dopamine receptor) in aged mice (Figure 4).

Estrogen can upregulate the endothelium-derived vasodilator factors, including NO and prostacyclin [163,164]. Following the estrogen drop after menopause, women might have accelerated vascular dysfunction, which means that estrogen can be a trigger for these processes [165,166]. The overlap of menopause with aging brought up the debate of whether the increased risk of cardiovascular disease in this period results from aging or menopause or both [167].

A vasoconstrictor prostanoid, thromboxane A2 (TXA2), activates the TXA2 receptor (TP) and has a primary role in the hemostasis of normal systemic vasculature [168,169]. Cyclooxygenase (COX) activation and elevated TP receptor expression are implicated in age-associated vascular diseases [170]. In humans, the production of COX-derived, endothelium-derived contractile factors is a feature of the aged blood vessels resulting in an earlier onset and speeding up of endothelial dysfunction [171].

NO can suppress the prostanoid production through the regulation of the COX activity and, conversely, the prostanoids, such as TXA2, can decrease the NOS expression and activity [172,173]. During the aging processes, endothelium-derived TXA2 increases vasoconstriction and endothelial dysfunction, which is amplified by decreased NO activity [174].

Vidal-Gómez et al. (2016) showed that in the absence of estrogen in young and aged ovariectomized mice, TP activation decreased NO bioavailability in the aorta by stimulating COX and the production of superoxide. The administration of estradiol restored the decreased NO bioavailability in the aorta due to TP activation. COX upregulation contributed partially to TXA2 contractile functions, which could be enhanced by aging and the absence of estrogen, while it could be suppressed due to the inhibition of COX by indomethacin. The production of superoxide, and therefore decreased NO bioavailability by endothelium-derived prostanoid TXA2 and TP activation, was also age- and estrogen-dependent, and could be prevented by suppressing COX. Interestingly, when estrogen was absent, COX inhibition resulted in enhanced bioavailability of NO [175].

Novensà et al. (2011) studied how aging alters the estrogen affects the NO development in an accelerated senescence mouse model [176]. Aged animals were similar to young animals in terms of DAF-2 or plasma nitrite/nitrate (NO₂/NO₃) in NO production. Estrogen treatment improved the production of NO in young animals by increasing eNOS expression but did not affect NO production in aged animals. Estrogen inhibited NADPH-oxidase and its production of superoxide anion (O₂⁻). O₂⁻ catabolizes the NO and decreases its levels. Therefore, estrogen increased NO levels by decreasing NO catabolism via O₂⁻ through inhibiting the production of O₂⁻ by NADPH-oxidase. In Novensà et al.’s (2011) study, estrogen reduced the development of O₂⁻ in young female animals, while in the aged animals estrogen increased O₂⁻ [176]. The latter was due to the upregulated estrogen receptor (ERb/ERa) expression ratios in the aged female animals and thus modulated the antioxidant effects of estrogen, enhancing its pro-oxidant activity. The capacity of E2 to cause the modulation of eNOS and a decline of O₂⁻ was impaired by aging. Activation of ERα has been associated with augmented eNOS expression and NO formation, and its antioxidant mechanism of action. The recent studies on the ERα knockout mice demonstrated its importance in the regulation of eNOS. In the opposite, the knockout of ERβ caused the development of hypertension, regardless of its normal NO production. These outcomes suggested that ERα is the most important ER subtype accountable for NO formation by estrogen. Furthermore, augmented levels of ERβ could be related to cardiovascular risk, comprising coronary calcification and atherosclerosis [176].

Banerjee et al. (2012) showed that aromatase and ER-alpha were localized in Leydig cells of the testis and demonstrated a strong association between testicular aromatase and the level of circulating testosterone, implying that testicular steroidogenesis could be modulated by estrogen. The baseline level of NO during the reproductive activity cycle was shown in this study, although aged animals showed the correlation between decreased testicular activity and elevated serum NO. This research found that aromatase and NO levels were inversely associated. Furthermore, NO elevation downregulated steroidogenesis and germ cell survival. In summary, reduced estrogen enhances NO production in old age, which reduces testicular steroidogenesis and the apoptosis of germ cells [177] (Figure 5).

One of the causes of reproductive failure is the oocyte’s postovulatory aging [178,179]. NO is a critical intracellular and intercellular messenger adjusting numerous physiological components in the processes of folliculogenesis, ovulation, and oviductal travel [180,181,182,183]. Goud et al. (2005) showed that NO exposure to young and old mice oocytes slowed the aging processes and increased the stability of microtubular spindle apparatus. The underlying mechanisms for these effects of NO were the activation of guanylate cyclase, which led to increased cGMP production [184]. Previous studies have shown that GMP alone can alleviate meiosis in rat and hamster oocytes. According to the outcomes of the aforementioned research, the production of cGMP occurred in the cumulus cells and was possibly transported into the oocyte via gap junctions to impede meiosis [185,186]. Similarly, specific PDEs are also triggered by cGMP and contribute to cAMP decrease [187]. This might activate the M-phase-promoting factor (MPF), a core regulator of M II phase arrest [188,189]. During the fertilization of oocytes, cortical granule depletion has been associated with aging-like changes such as elevated cytosolic Ca²⁺ and activated protein kinase C; however, these changes can occur in unfertilized oocytes due to aging [190,191]. In Goud et al.’s (2005) study, the mechanism of anti-aging effects of NO in oocytes might be related to the increase in Ca²⁺ levels or the suppression of protein kinase C activation [184] (Figure 6).

In aged people, skin loses its ability to increase blood flow during heat stress, which can lead to the increased occurrence of heat-related diseases and even to death [192,193,194,195,196]. The sympathetic nervous system regulates the blood flow to the skin via the adrenergic vasoconstrictor system and an active vasodilator system [197].

An increase in body core temperature releases tonic adrenergic vasoconstrictor which elevates the skin blood flow. After a threshold, as temperature rises, sweating and cutaneous active vasodilation (AVD) occur [197]. In young individuals, about 30% of AVD is controlled by NO [198]. In older populations, NO-dependent vasodilation of skin is compromised [199]. Holowatz et al. (2003) showed that during passive whole-body heating, NO-dependent AVD was 23% and 60% in young and old individuals, respectively. Thus, the NO-mediated pathways were related more to the total vasodilatory response of the aged subjects at high core temperatures. The authors suggested that an unknown transmitter(s) could mediate the AVD during passive whole-body heating in aged individuals [200].

In another study, Bruning et al. (2012) showed that eNOS is primarily responsible for mediating cutaneous NO-dependent vasodilation during the local heating and perfusion of the endothelium-dependent agonist ACh in middle-aged skin. In addition, the expression of NOS isoforms or downstream vasodilatory molecular targets did not differ in the biopsy studies [201,202].

Reckelhoff et al. (1994) showed that, during aging, NO production decreases, which could be explained by the lack of the endogenous substrate in aged rats, l-arginine [203].

Age-dependent kidney diseases are more common in male humans. NO signaling dysfunction can cause age-dependent kidney damage. Erdely et al. (2003) found that renal NOS decreased by aging in male but not female rats [204]. These data suggested that the difference of NOS activity and NO function might be a determining factor for the gender gap in the rate of renal functional decline in advancing age. According to the study, a noticeable sexual dimorphism demonstrated that the females were protected both by the presence of estrogens and the deficiency of harmful androgens. The total NO formation, the activity of NOS and its abundance were important in the aging male rat kidney. Although arginine production was conserved, which showed elevation in the circulating NOS inhibitor, ADMA, in the aging male rat, these effects improved deteriorations in NO production [204,205].

Doleželová et al. (2016) showed that renal NO and CYP450-epoxygenase products, which counteract the development of hypertension and protect the kidney, were decreased in a genetic animal model of spontaneous hypertension (fawn-hooded hypertensive or FHH rat). Renal NOS activity was higher in adult animals with established hypertension but was similar between prehypertensive FHH rats and age-matched non-hypertensive controls [206]. Reduced vasodilator response in isolated renal vessels from adult rats has been shown in in vitro studies [207,208].

Stadler et al. (2003) demonstrated that before the occurrence of histopathological injuries in diabetes, NO and peroxynitrite are increased in streptozotocin-induced diabetic rats. Moreover, the ability of increased NO levels to suppress the formation of ROS in vessels and kidney tissues highlighted the possible roles of NO and peroxynitrite in late diabetic complications [92].

Impaired angiogenesis is believed to cause the age-related nephropathy, along with the gradual weakening of renal microvasculature. Satoh et al. (2013) showed that human umbilical vein endothelial cells (HUVECs) treated with NOS inhibitor had higher expression and activity of cathepsin D. Cathepsin D produces angiostatin, a potent in vivo angiogenesis inhibitor. Aged rats treated with l-NAME, had higher cathepsin D and angiostatin activity. On the other hand, treatment with a long-lasting NO-releasing vasodilator diminished the production and activity of cathepsin D and angiostatin. It was suggested that one of the most important steps to generate angiostatin would be related to the contribution of the proteolytic cleavage of plasminogen. Besides, the inhibition of cyclooxygenase (COX) and MMP-2 activities were also related to the angiostatin-generating mechanisms in vivo. In summary, cathepsin D-induced production of angiotensin was increased in the aging rat kidney. Decreased NO output triggered activity of cathepsin D. Increased development of angiostatin in the aged rat kidney could be due to the capillary loss and interstitial damage. [209]. However, in the aforementioned study, no direct interplay was observed between the generation of angiostatin in tubulointerstitial damage related to the peritubular capillary loss in the aged kidney.

The impairment of water reabsorption and improvements of aquaporin type 2 water channel (AQP2) were consistent with hypothyroid state and aging. AQP2 trafficking to the apical plasma membrane, in medullary duct collection cells, involves NO. Sarati et al. (2013) showed that the urine output and medullary NOS activity are correlated reversely in young and old rats, as the former was increased in the young and later was increased in the old rats. Elevated AQP2 in rats with hyperthyroidism was found in the plasma membrane of the young rats and at the cytosolic site in adult rats. These findings showed that medullary NO and AQP2 are implicated in maintaining water homeostasis [210]. Due to the role of the connection of NO-AQP2-hypothyroid damage in the regulation of water homeostasis, it was suggested that hypothyroidism may affect renal parameters in the aging processes.

The decrease of smooth muscle cells (SMCs) and collagen in the corpus cavernosum (CC) greatly contributes to erectile dysfunction (ED) during aging. One of the occurring phenomena during the aging process is tissue remodeling in the CC. The aforementioned tissue remodeling feasibly occurs by reason of phenotypic cellular alteration from SMCs to fibroblasts that latterly would cause an elevation in the content of collagen deposit [212]. According to anatomical localization, the corpora cavernosa are paired spongy cylinders that lay on the superior facet of the penis [213]. The accumulation of collagen is thought to be affected by ROS. The NO production by iNOS has been shown to suppress the ROS and thus decrease collagen accumulation [212]. To clearly describe the relationship between ROS formation and collagen accumulation, this process was described in Peyronie’s disease, which is accompanied by fibrosis and is characterized by an augmentation in collagen over the intracellular part. Fibrosis is related to the formation of profibrotic factors, including plasminogen activator inhibitor-1, transforming growth factor beta (TGF-β), and ROS throughout oxidative stress. This is accompanied by the stimulation of the iNOS, which plays a crucial part as an endogenous antifibrotic pathway in the case of exposure with profibrotic manners [214]. Inside the arterial media SMC, Ferrini et al. (2004) explored the possibility of related mechanisms that exist within aging and that share specific common physiological roles with the cavernosal SMC [215]. Male, brown Norwegian rats aged (22–24 months) were treated with an iNOS action inhibitor. Inhibition of iNOS activity was induced by l-N-(iminoethyl)-lysine acetate. Resistance arteries of the penis showed an increased SMC apoptosis, increased collagen amounts, and elevated ROS and iNOS levels. Administration of an iNOS action inhibitor worsened the SMC/collagen ratio and increased ROS levels. A prevalent etiology can be explained in the hypotheses of ED and arteriosclerosis in aging males, namely that the SMC’s production of iNOS is an effort to combat this fibrosis [215].