Nitric Oxide in the Mechanisms of Cell Death and Cytoprotection

1. Introduction

Nitric oxide. Nitric oxide (NO) is a unique molecule that, despite its extremely simple structure, plays a pivotal role in various physiological processes within the body. Its multifunctionality has spurred intensive research over the past decade [1,2,3]. NO serves as a crucial element in the cardiovascular system, facilitating vasodilation and regulating blood pressure. Additionally, it participates in signaling within both the central and peripheral nervous systems [4,5,6].

It has been demonstrated that NO is essential for exerting cytotoxic effects on tumor cells and cells affected by viruses. In this context, the mechanism of action of nitric oxide does not involve the activation of guanylate cyclase but is primarily attributed to the direct effects of NO itself. It is noteworthy that the free-radical nature of the nitric oxide molecule, characterized by the presence of an unpaired electron at the nitrogen atom, renders it highly reactive. In vivo, NO has an average lifetime of 5-30 seconds, during which it rapidly interacts with its targets, primarily thiols and transition metals, or undergoes oxidation to form inactive nitrate and nitrite, for instance, via cytochrome C oxidase [7,8] or NO may generate reactive oxygen species. Hence, the action of NO can occur through direct or indirect mechanisms. Direct effects result from the reactions of NO itself with its targets, such as the stimulation of guanylate cyclase or the formation of nitrosyl complexes with metals, often leading to the inactivation of enzymes containing these metal ions. Indirect effects of NO are defined as chemical reactions mediated by active forms of nitric oxide, which are generated through interactions with superoxide (O2-) or oxygen (O2). The action of these active forms of NO leads to the development of nitrosylation stress (resulting in the formation of nitrosoamines, S-nitrosothiols, and deamination of DNA bases) or oxidative stress [9]. Owing to its high lipophilicity, NO efficiently penetrates membranes, enabling it to diffuse from its source to distances several times the size of the cell, thus affecting its targets within this extended range [10,11].

2. Basic Mechanisms of Nitric Oxide (NO) Regulation

2.1. NO Synthesis in the Body

The investigation into the origin of endogenous NO has revealed that L-arginine is essential for its production by active macrophages. Subsequently, it was discovered that a family of enzymes called nitric oxide synthases (NOS) is responsible for NO production. These enzymes catalyze the conversion of L-arginine into NO and L-citrulline, simultaneously utilizing NADPH and reducing oxygen to water [12,13,14]. NOS enzymes are ubiquitously present in cells of almost all tissue types and are categorized into constitutive (cNOS) and inducible (iNOS) forms based on their expression patterns. The cNOS group typically includes neuronal (ncNOS or NOS1) and endothelial (ecNOS or NOS3) isoforms, with primary localization in neurons and endothelial cells, respectively, although they are also found in other cell types. iNOS is predominantly associated with macrophages and plays a key role in the immune system [15,16,17,18,19,20]. Its expression increases in response to activation by cytokines (such as IFN-g, IL-1b, and TNF-a) and other agents like lipopolysaccharides (LPS). This isoform is also expressed in the liver upon stimulation, which is associated with the barrier function of this organ [21,22,23]. A comprehensive study of NOS has revealed that they are among the most intricately structured and regulated enzymes, boasting an unusually high number of cofactors. NOS exist in the cell as dimers and are active only in this state. Within each subunit of the dimer, distinct domains such as reductase, calmodulin-binding, and oxygenase can be identified. The reductase domain contains the flavins FAD and FMN: FAD serves as the primary electron acceptor from NADPH, while FMN transfers electrons from FAD to the heme of the oxygenase domain. The oxygenase domain contains heme, arginine (L-Arg), and tetrahydrobiopterin (BH4) binding sites. Calmodulin-Ca2+ is believed to confer the enzyme with the necessary conformation for internal electron transfer [24,25,26,27]. It is the variations in the binding affinity of calmodulin to the NOS dimer that underlie the catalytic discrepancies between the isoforms: the activity of nNOS and eNOS is highly reliant on Ca2+ concentration, whereas calmodulin binds to iNOS so tightly that Ca2+ supplementation is unnecessary. Although the specific activities of all NOS isoforms are comparable, in vivo, it seems that cNOS produces small amounts of NO over brief intervals, while iNOS generates much larger quantities of NO over extended periods (up to several days). Thus, the expression and activity of a specific isoform may dictate NO’s role as either a physiological modulator or a cytotoxic agent [28,29,30,31].

In vitro studies of NO-mediated macrophage cytotoxicity have unequivocally demonstrated that the addition of NOS inhibitors, such as the substrate analogue NG-monomethyl-L-arginine (L-NMMA), to the medium suppresses the cytotoxic effect of macrophages on tumour cells. This supports the prevailing role of NO in mediating the macrophage’s effect on target cells. However, it is important to acknowledge the well-known phenomenon of the respiratory burst, which also plays a crucial role in pathogen destruction by phagocytes. Additionally, recent data have emerged that complicate the understanding of macrophage cytotoxicity induced by nitric oxide. Specifically, it has been discovered that wound macrophages capable of NO production do not exhibit cytotoxicity towards NO-sensitive cells of the P815 line. Thus, the question arises regarding the necessity and sufficiency of NO for the cytotoxicity of macrophages [32,33,34,35,36]. It should also be noted that NO production can have significant negative effects on macrophages that produce it. Studies have demonstrated that phagocytosis and the production of reactive oxygen species are markedly inhibited in rat or peritoneal macrophages cultured under conditions that allow NO production. Macrophages expressing iNOS or treated with nitric oxide exhibit nuclear and cytoplasmic condensation. Therefore, the release of NO by activated macrophages leads to their functional suppression, eventually resulting in apoptosis. These phenomena are clearly attributed to NO, as they can be prevented by the addition of NOS inhibitors [37,38].

2.2. Mechanisms of NO Cytotoxicity

Nitric oxide targets are currently under active investigation to determine whether NO itself is sufficiently cytotoxic or if its derivatives are more potent [39]. NO in target cells is known to generate active intermediates such as nitrosonium (NO+), nitroxyl (NO-), and peroxynitrite (ONOO-). Some researchers posit that most of the cytotoxic effects attributed to NO actually stem from ONOO-, which forms through reaction with superoxide (O2-). Indeed, peroxynitrite is significantly more reactive; it extensively nitrosylates proteins and can serve as a source of highly toxic hydroxyl radicals (-OH) through reactions [39,40,41,42].

NO· + O2- а ONOO- + H+ а ONOOH а ONO· + · OH

OH causes lipid peroxidation and other phenomena associated with oxidative stress. Another challenge encountered in the study of the mechanisms of nitrogen cytotoxicity is related to the NO-donors utilized for its generation, as described above. Specifically, S-nitrosothiols (mainly GSNO and SNAP), frequently employed in many studies, have the capability to engage in transnitrosylation reactions. These reactions involve the transfer of the NO+ group to thiols (such as glutathione and SH-groups of proteins), thereby disrupting their cellular functions [43,44,45,46]. However, it remains unclear whether such reactions should be attributed solely to the effects of NO itself. Some possible mechanisms of the cytotoxic action of nitric oxide will be discussed below, and some reactions can be induced by its derivatives. It is demonstrated that NO (from macrophages or exogenously administered) primarily inhibits oxidative phosphorylation in the mitochondria of target cells [47,48]. This inhibition occurs because NO reversibly binds to cytochrome-C-oxidase of the mitochondrial electron transport chain. However, the inhibition of electron transport in the mitochondrion leads to the generation of superoxide and subsequently the formation of peroxynitrite. The conversion of nitric oxide to peroxynitrite involves a reaction between two radicals: O2- and NO, resulting in the formation of ONOO-, a potent oxidant in the mitochondrial matrix. Normally, ONOO is reduced by mitochondrial reducing agents such as NADH2, ubiquinol UQH2, and glutathione GSH. However, when produced in excess due to loss of control (e.g., during ischemia/reperfusion or inflammation), it leads to tyrosine nitration and mitochondrial dysfunction. Its cumulative effect contributes to tissue aging. Another radical widely produced by both enzymatic and non-enzymatic processes is nitric oxide (NO), which serves as an intra- and intercellular signaling molecule [49,50]. NO and superoxide react in a diffusion-limited manner. This reaction halts the chain reaction initiated by superoxide, although peroxynitrite is generally considered a harmful molecule [51]. Peroxynitrite is a short-lived and highly reactive oxidant, thus representing another mechanism that imparts indirect toxicity to O2-, particularly targeting DNA, proteins, and lipids. Additionally, peroxynitrite has the capability to nitrate tyrosine or tryptophan residues or oxidize methionine residues [52,53]. This suppression of mitochondrial respiration leads to a decrease in the mitochondrial membrane potential, which can initiate the apoptotic process [54]. Conversely, it’s known that in the absence of glucose or when glycolysis is blocked, NO-induced suppression of respiration leads to necrosis rather than apoptosis [55]. There is also evidence of direct activation of giant pore opening by nitric oxide (NO), leading to the release of cytochrome C and initiation of the caspase cascade. However, this is also controversial, as other investigators have shown that low doses of NO (close to physiological doses) slow giant pore opening and apoptosis, whereas peroxynitrite (ONOO-) and nitrosothiols promote them. NO and its derivatives can cause peroxidation of phospholipids and oxidation of thiol groups of mitochondrial membrane proteins, which also lead to the release of apoptogenic factors into the cytosol [56,57,58].

Nitrosylation of proteins at tyrosine residues by peroxynitrite (ONOO-) can have profound functional consequences, as it inhibits Tyr phosphorylation, thus disrupting vital signal transduction pathways within the cell. Recent studies have revealed that peroxynitrite can also nitrosylate cytochrome C within mitochondria, altering its function significantly. This modification renders cytochrome C incapable of supporting electron transport in the respiratory chain and resistant to reduction by ascorbate. Concurrently, nitrated cytochrome C is released into the cytoplasm, suggesting potential involvement in signaling processes [59,60]. Emerging hypotheses propose that selective protein nitrosylation may function as a regulatory mechanism akin to phosphorylation [61,62]. Peroxynitrite’s activity extends beyond protein modification to include guanine nitrosylation and DNA strand breaks, which can instigate mutations or trigger apoptosis pathways [63]. Additionally, nitric oxide (NO) exerts effects on DNA repair enzymes by inhibiting their activity. Notably, different NO donors impact various enzymes responsible for DNA repair, such as alkyltransferase, formamidopyrimidine-DNA-glycosylase, and ligase, suggesting a multifaceted role for NO in genomic integrity maintenance and cellular signaling processes [9,64].

It is well-established that nitric oxide (NO) can activate Poly(ADP-ribose) polymerase (PARP) and induce ADP-ribosylation, potentially as a response to DNA damage. However, this activation tends to lead to necrosis due to the depletion of the NAD and ATP pools rather than triggering apoptosis. Regarding NO and its derivatives’ impact on DNA, investigations into their effect on p53 expression are particularly intriguing. p53 is a crucial protein involved in tumor suppression, genome maintenance, and the regulation of cell cycle progression or apoptosis. It is known that p53 can upregulate the expression of pro-apoptotic proteins such as Bax, Fas, and p53AIP (apoptosis-inducing protein) and other apoptogenic proteins [65,66,67,68]. Additionally, during apoptosis, p53 translocates into the mitochondrion, which may be one of the reasons for the production of ROS [69,70].

Under normal conditions, the cellular concentration of p53 remains low as it undergoes rapid degradation. However, DNA damage triggers the accumulation of p53. Experiments conducted on macrophages and RINm5F insulinoma cells have demonstrated p53 accumulation in NO-induced cell death scenarios [71,72]. Further research revealed that L-NMMA, a nitric oxide synthase (NOS) inhibitor, suppresses p53 accumulation induced by cytokines or lipopolysaccharides (LPS), indicating an active involvement of NO in this process. Some evidence suggests that NO’s effect on p53 accumulation may be linked to its ability to inhibit proteasome function, thus interfering with p53 degradation pathways [73,74]. Nevertheless, further experiments have uncovered the operation of p53-independent pathways in NO-induced apoptosis [75,76]. Additional studies have elucidated a negative feedback mechanism between the levels of NO and p53 in various human cell types: the accumulation of NO leading to DNA damage triggers the expression of p53, which in turn suppresses the human inducible nitric oxide synthase (iNOS) gene [77]. Additionally, NO represses iNOS expression by dampening NFκB activity in hepatocytes. Through these intricate pathways, precise regulation of NO synthesis is achieved, thus mitigating its deleterious effects on the tissue [78,79,80].

Figure 1. Nitric oxide synthesis.

Figure 1. Nitric oxide synthesis.

2.3. Interaction of NO with Mitochondria

Since the involvement of mitochondria and nitric oxide (NO) in apoptosis has been extensively elucidated in this study, it is pertinent to describe their combined role in its regulation. In experiments involving the transfection of the RAW264.7 macrophage line with human Bcl-2, the transfected cells exhibited protection against death induced by inducible nitric oxide synthase (iNOS) activation [81,82,83,84]. This suggests that Bcl-2 operates by nullifying the NO-induced upregulation of Bax protein expression. Additional experiments demonstrated that P815 tumor line cells transfected with Bcl-2 showed resistance to the effects of the NO donor SNAP (S-nitroso-N-acetylpenicillin-amine) and to NO-associated cytotoxicity from activated murine macrophages [85,86]. Furthermore, L929 cells overexpressing Bcl-2 were shielded from apoptosis triggered by iNOS activation. A multitude of other instances showcasing the interaction between NO and Bcl-2 are provided in articles [87,88,89,90,91].

The interaction of nitric oxide (NO) with members of the Bcl-2 superfamily is further evidenced by the significant reduction in intracellular Bcl-2 protein levels upon exposure to NO within the cell [92]. This reduction may occur through caspase-induced cleavage or p53-dependent suppression of its expression, although conflicting evidence exists regarding this mechanism [93,94]. Additionally, the proapoptotic effect of nitric oxide manifests through its inducible increase in Bax expression [95]. Apart from the aforementioned mitochondrial functions, recent studies have shed light on the involvement of mitochondria not only in the reception of apoptotic signals from NO but also in the production of NO itself. Notably, a constitutive form of nitric oxide synthase (NOS) has been identified within mitochondria [96,97].

The initial detection of NO production in rat liver mitochondria prompted further investigation into the purification of mitochondrial NOS and elucidation of its enzymatic characteristics [98]. This isoform of NOS appears to be localized within the mitochondrial membrane, particularly in the inner membrane [99]. It has been revealed that mitochondrial nitric oxide synthase (mtNOS) bears a striking resemblance to macrophage inducible NOS (iNOS) but is expressed constitutively. However, the classification of mtNOS as a distinct isoform remains uncertain, as it remains unclear whether it represents a separate isoform or if it is iNOS undergoing post-translational modifications that dictate its distinct subcellular localization. Notably, mtNOS exhibits independence from calmodulin and calcium addition, indicating a strong association with calmodulin. Purified mtNOS, when subjected to suboptimal concentrations of L-Arginine, demonstrates the capability to generate O2-, albeit at a relatively modest rate. This observation aligns with the documented homology of the C-terminal domain of NOS to NADPH: cytochrome P450-oxidoreductase, which also possesses NADPH-oxidase activity and generates O2- albeit at a rate approximately tenfold faster than mtNOS [100,101,102,103,104,105,106,107].

The identification of such a NOS within the mitochondrion raises numerous inquiries and suggests promising avenues for further investigation. Foremost among these is the inquiry into how the NO produced within mitochondria influences apoptosis. Considering the established role of non-mitochondrial NO, which acts directly on mitochondria, inducing a range of phenomena culminating in apoptosis, it is reasonable to speculate on the involvement of mtNOS in the regulation of apoptosis, although conclusive evidence is yet to be established [108,109]. Moreover, given its capacity to generate not only NO but also O2-, may be implicated in the production of reactive oxygen species (ROS), potentially contributing to various biological damages. In the context of apoptosis, intriguing insights have emerged from investigations into the release of cytochrome C from mitochondria upon mtNOS stimulation [110,111]. While elevated cytosolic Ca2+ levels have long been recognized as apoptosis inducers, recent findings have underscored the significant role of mtNOS in this process. It has been demonstrated that this form of apoptosis necessitates mitochondrial Ca2+ uptake, triggering mtNOS activation and subsequent cytochrome C release into the cytosol. Concurrently, there is an augmentation in lipid peroxidation (LPO). Notably, the release of cytochrome C and LPO are effectively inhibited by NOS inhibitors (such as L-NMMA), peroxynitrite scavengers (e.g., urate), and Bcl-2 expression. These findings suggest that upon Ca2+-induced activation of mtNOS, peroxynitrite is generated within the mitochondria, leading to LPO and cytochrome C release, ultimately leads to a pattern of typical apoptosis. Further elucidation of these mechanisms promises to enhance our comprehension of the roles played by mitochondria and NO in various pathways of cell death [112,113,114,115,116].

For instance, recent findings have demonstrated that inhibition of mitochondrial nitric oxide synthase (mtNOS) results in the accumulation of intramitochondrial Ca2+, indicating that NO produced by mtNOS impedes Ca2+ accumulation. Given that the elevation in matrix Ca2+ concentration is responsible for altering mitochondrial membrane permeability, it is deduced (contrary to the earlier assertion) that mitochondrial NO decelerates the opening of the mitochondrial permeability transition pore and the subsequent release of cytochrome C [117,118,119,120,121,122,123]. While there are indications that the synthesis of NO may be regulated through substrates of mtNOS (such as L-Arginine, NADPH) and its cofactors (including FMN, FAD, BH4), akin to other NOS isoforms, this aspect remains largely unexplored. Furthermore, this endogenous mitochondrial NO may play a crucial role in regulating mitochondrial activity by inhibiting cytochrome oxidase (Complex IV), as well as complexes I and II of the electron transport chain. Its reaction with oxygen could modulate mitochondrial respiration by altering the availability of oxygen for electron acceptance, thereby impacting cellular energy supply. Nevertheless, this facet of mitochondrial NO biology necessitates further investigation [124,125,126,127,128,129,130].

Summarizing all these data, we can say that the mitochondriа is the central link where many signaling pathways of apoptosis converge and are regulated. Within these pathways, mitochondria may assume a central role by initiating the pro-apoptotic cascade, particularly under stressors such as irradiation or NO action. Conversely, mitochondria can also amplify certain apoptogenic signaling cascades, such as those mediated by the Fas receptor or TNF receptor, through kinase-mediated mechanisms. Besides superoxide, both NO and its more aggressive derivative, peroxynitrite, are instrumental in the genesis of mitochondrial abnormalities and apoptosis [131,132,133,134,135,136]. Notably, neuronal mitochondria emerge as a significant source of NO, with evidence demonstrating the presence of a constitutive form of NOS localized in the inner mitochondrial membrane and NO production within the mitochondria of hippocampal neurons. Moreover, mitochondrial NOS is capable of generating superoxide at suboptimal concentrations of L-arginine. Importantly, mitochondrial NOS exhibits significant activation in response to the onset of glutamate excitotoxicity and mitochondrial calcium uptake. Additionally, cytokines such as IL-1β and TNF-α contribute to the activation of mitochondrial NOS [137,138,139,140,141,142].

This leads to the generation of peroxynitrite, which facilitates the opening of the mitochondrial permeability transition pore (mPTP). Additionally, peroxynitrite nitrosylates cytochrome C within mitochondria, inducing alterations in its functionality. Specifically, this modification renders cytochrome C incapable of supporting electron transfer within the respiratory chain and resistant to reduction by ascorbate. Concurrently, there is a concomitant release of cytochrome C, including nitrated forms, into the cytoplasm, suggesting the involvement of this nitrosylation process in various signaling pathways. Furthermore, peroxynitrite nitrosylates guanine, resulting in DNA strand breaks, mutations, or activation of apoptosis-related processes [138,143,144,145].

Excessive NO inhibits enzymes crucial for DNA repair, targeting alkyltransferase, formamidopyrimidine-DNA glycosylase, and ligase. Moreover, NO activates PARP and ADP-ribosylation, particularly under conditions of ATP depletion and accumulation of reduced pyridine nucleotides. Additionally, NO exerts a positive influence on the synthesis of the tumor suppressor protein p53. Enhanced p53 expression promotes the upregulation of pro-apoptotic proteins such as Bax, Fas, and p53AIP (apoptosis-inducing protein). Furthermore, NO translocates into the mitochondria during apoptosis, potentially contributing to the production of ROS and the reduction of transmembrane potential across the inner mitochondrial membrane [146,147,148,149,150,151]. Currently, there exists a widely recognized concept known as “mitochondrial dysfunction.” This represents a characteristic pathological process lacking etiological and nosological specificity.

The progression of mitochondrial dysfunction precipitates the disturbance of mediator reuptake (e.g., catecholamines, dopamine, serotonin), ion transport, impulse generation and conduction, de novo protein synthesis, as well as translation and transcription processes. Concurrently, “parasitic” energy-producing reactions are activated, resulting in a substantial depletion of neuronal cell energy reserves. Furthermore, the action of the hydroxyl radical triggers the opening of mitochondrial pores, leading to the expression and release of proapoptotic proteins into the cytosol. This pore opening occurs via the oxidation of thiol groups within the cysteine-dependent region of the mitochondrial inner membrane protein (specifically, the ATP/ADP antiporter), transforming it into a permeable nonspecific channel pore [152,153]. The opening of these pores transforms mitochondria from being “power plants” into “furnaces” for oxidation substrates, devoid of ATP production. Precise biochemical investigations have revealed that disturbances in tissue oxygenation, hyperproduction of excitotoxic amino acids, decreased “normal” calcium (Ca2+) accumulation by mitochondria, and damage to mitochondrial membranes ROS all contribute to the increased opening of these pores and subsequent release of apoptogenic proteins from damaged mitochondria. In this context, the pivotal role of the neurotrophic factor tumor necrosis factor-alpha (TNF-α) cannot be overstated, as it is intricately associated with the opening of mitochondrial pores, subsequent membrane disruption, and the onset of mitoptosis [138,154,155,156,157,158,159,160].

3. Effects of NO

3.1. Apoptosis and NO

Cu has been found to be repressed in neurons with evidence of apoptosis; while Zn-SOD is known to bind and facilitate the production of significant amounts of endogenous peroxynitrite (ONOO−) [161,162,163]. Low concentrations of NO donors have been shown to inhibit neuroapoptosis induced by growth factor deprivation or TNF-α addition, potentially through the activation of the heat shock protein HSP70 or the inhibition of caspase-3 [164,165]. Dinitrosyl iron complexes (DNICs) at concentrations ranging from 5 to 10 μM are believed to suppress IL-1β-induced neuroapoptosis by promoting NO synthesis. Conversely, elevating DNIC concentration (0.5 mM) has been associated with the induction of neuroapoptosis through the generation of ONOO− [166,167,168,169,170].

Conditions that lead to increased bioavailability of NO and reduced formation of ONOO- such as enhanced superoxide dismutase (SOD) activity and the presence of reduced thiol antioxidants render neurons resistant to Fas-induced apoptosis. Conversely, decreased bioavailability of NO and increased levels of cytotoxic forms of NO heighten cellular sensitivity to signals mediated through Fas receptors. Additionally, neuroapoptosis can be induced by the synergistic action of H2O2 and Fe2+, which convert NO to peroxynitrite, while depression of bcl-2 and induction of c-fos were simultaneously observed. In endothelial cells, H2O2 (125–1000 μM) stimulates the activity of NO synthase, contributing to oxidative cellular damage [171,172,173].

Enzymatic antioxidants such as catalase and glutathione peroxidase, along with α-tocopherol, exert inhibitory effects on apoptosis. However, ascorbic and gallic acids have been shown to enhance H2O2-induced neuroapoptosis. Notably, several antioxidants, including α-tocopherol, exhibit potent antiproliferative properties. In the presence of metal ions with varying valence states, ascorbic acid can display pro-oxidant characteristics and augment H2O2-induced neuroapoptosis [174,175,176]. Furthermore, it is noteworthy that NO production can have significant negative effects on macrophages producing it. Studies have demonstrated that phagocytosis and the production of reactive oxygen species are markedly inhibited in rat or peritoneal macrophages cultured under conditions conducive to NO production. Macrophages expressing inducible iNOS or treated with nitric oxide (NO) have condensed nucleus and cytoplasm. Consequently, the release of NO by activated macrophages results in their functional suppression and eventual apoptosis. These observations are directly linked to NO, as they are effectively prevented by the addition of NOS inhibitors. However, an additional challenge in investigating the mechanisms of NO cytotoxicity is related to the NO donors used for its generation, as described earlier. The utilization of S-nitrosothiols, particularly GSNO (S-nitrosoglutathione) and SNAP (S-nitroso-N-acetylpenicillamine), in numerous studies raises concerns regarding their potential involvement in transnitrosylation reactions. These reactions entail the transfer of NO⁺ groups to thiols, including glutathione and sulfhydryl groups of proteins, thereby disrupting their cellular functions. The attribution of such reactions to the effects of NO itself remains ambiguous [177,178,179,180,181,182,183] Importantly, studies have indicated that NO, whether produced endogenously by macrophages or administered exogenously, primarily inhibits oxidative phosphorylation in the mitochondria of neurocytes. This occurs because NO reversibly binds to cytochrome oxidase of the electron transport chain of mitochondria. Furthermore, there is evidence suggesting that NO can directly activate the opening of giant pores, resulting in the release of cytochrome C and the initiation of the caspase cascade, as previously described. Additionally, NO and its derivatives have the capacity to induce peroxidation of phospholipids and oxidation of thiol groups present on mitochondrial membrane proteins. These processes ultimately contribute to the release of apoptogenic factors into the cytosol [184,185,186,187,188].

3.2. Anti-Apoptotic Effects of NO

In addition to the extensively described cytotoxic effects of nitric oxide (NO), numerous studies in the literature have highlighted NO cytoprotective actions. However, upon comparing the evidence for the antiapoptotic effects of NO with the cytotoxic actions outlined earlier, it becomes evident that there are contradictions on many fronts [189,190]. This discrepancy underscores the highly ambiguous nature of NO’s actions, which are contingent upon various conditions.

The molecular mechanisms underlying the antiapoptotic effects of NO-mediated expression of HSP may involve two potential pathways [191]. The first possibility entails the direct suppression of apoptotic signal transduction pathways, involving the inhibition of caspase family protease activation. The second involves chaperone-mediated import of precursor proteins into mitochondria by HSP. This action controls mitochondrial function and membrane permeability, thereby preventing the release of cytochrome C, which is required for further activation of caspases. The relationship between Hsp70 and the induction of apoptosis in obstructive nephropathy was first discussed [192,193]. Other results have shown that Hsp70 can modulate the apoptosis cascade during renal obstruction [194]. Recently, we reported that nitric oxide prevents obstruction-induced cell death through the mitochondrial apoptotic pathway via the induction of heat shock protein 70 [195,196]. Our results demonstrated that the apoptotic effect induced by decreased levels of nitric oxide led to reduced expression of Hsp70. This was associated with a direct induction of apoptotic signal transduction involving caspase 3 activation by decreasing Bcl-2 stabilization. For some cell types, it has been shown that the protective effect of NO is mediated through the synthesis of cGMP [197,198]. Moreover, such an effect is produced by rather low doses of NO similar to those produced in vivo by NO synthases. It is assumed that cGMP generation can activate cGMP-dependent protein kinases, which in turn affect proteins of apoptotic cascades (e.g., caspases or Bcl-2) [199,200].

The protective effect of low doses of NO. Pretreatment of macrophage cells with low, nontoxic doses of GSNO (25-200 μM) induced resistance to higher doses of GSNO (1mM) upon repeated exposure [201,202]. Similarly, pretreatment of macrophages with LPS and IFN-γ in the presence of L-NMMA induced a comparable effect. Thus, the inducible defense mechanisms that suppress NO-induced apoptosis are activated by the action of NO-releasing substances as well as by pre-activation by lipopolysaccharides or cytokines. In other studies, low doses of NO were found to delay the opening of the giant pore and subsequent apoptotic events [203,204,205].

NO and defense proteins: Several studies have demonstrated enhanced expression of heat shock proteins (HSP) and Bcl-2 family proteins in response to NO. Heat shock proteins serve to protect the cell from various stressors, primarily temperature increases, but also oxidative stress and cytokine-induced cytotoxicity. The protective effect of NO was observed when hepatocytes were treated with tumor necrosis factor and when cells were deprived of serum [206,207,208]. In both cases, the anti-apoptotic action of NO correlated with an increase in Hsp70 synthesis. Hsp70 is characterized by its function as a molecular chaperone, assisting in protein folding and the removal of damaged proteins.

Hsp70 is known to play a crucial role in protecting the cell from ROS and mitochondrial damage by suppressing the interaction of proteins that transmit death signals to the mitochondria. Bcl-2, a proto-oncogene with anti-apoptotic properties, has already been described above. Increasing its expression upon NO treatment prevents apoptosis, most likely through the inhibition of giant pore opening [209,210,211]. A novel alternative anti-apoptotic mechanism of NO involves the induction of Hsp32 (hemoxygenase) and Hsp70 through NO-mediated modification of intracellular antioxidant levels [191,212]. The mechanism by which NO stimulates Hsp70 expression may involve the interaction of NO with thiol-containing molecules. There is ample evidence indicating that NO readily oxidizes low molecular weight thiols to form S-nitrosothiols and disulfides [213,214].

Among cellular low molecular weight thiols, glutathione is the most abundant and is also one of the intracellular targets of NO. NO can oxidize intracellular reduced glutathione, thereby altering antioxidant levels within the cell and leading to oxidative or nitrosative stress. This action stimulates the induction of the heat shock proteins Hsp32 (hemoxygenase) and Hsp70, which protect cells from apoptotic cell death induced by tumor necrosis factor (TNF) plus actinomycin D and oxidative or nitrosative stress . Pretreatment of hepatocytes with NO has been shown to alter the redox state accompanied by glutathione (GSH) oxidation and the formation of S-nitrosoglutathione [215,216,217]. GSH-oxidizing agents (diamide) and GSH-alkylating agents (N-ethylmaleimide) induced Hsp70 mRNA expression, whereas GSH synthesis inhibitor (buthionine sulfoximine) did not; this suggests that NO induces Hsp70 expression via GSH oxidation [218,219]. The aforementioned induction may occur via the activation of heat shock. Accumulation of misfolded proteins triggers the mobilization of HSP, leading to the formation of a free pool of Hsp70 and subsequent removal of the negative regulatory effect on HSF activation during heat shock or other stresses. The released HSF is phosphorylated and assembled into trimers, acquires DNA-binding activity, and leads to an increase in Hsp70 mRNA transcripts. During NO stimulation, multiple and complex pathophysiologic changes occur in the smooth muscle cells of blood vessels, including protein damage or modifications due to the cytotoxic action of NO [220,221].

5. Conclusions

Thus, despite intensive studies on apoptosis, a detailed understanding of the pathways regulating this process still requires further clarification. It is becoming increasingly apparent that various mechanisms regulating cell death are intricately intertwined, making it challenging to distinguish between pro- and anti-apoptotic components in the actions of signaling molecules. Nitric oxide serves as a prime example, with scientific literature presenting nearly equal evidence of both cytotoxic and protective effects of this compound. This complexity complicates the translation of theoretical advancements into practical applications of such substances, as it is challenging to predict their effects at the multicellular organism level based solely on in vitro studies. Therefore, a comprehensive understanding of the regulation of vital cell functions is constructed through focused investigations into the effects of specific compounds under particular conditions on the development of specific signaling pathways.