Nitric Oxide and Blood Pressure in Mice Lacking Extracellular-Superoxide Dismutase

Nitric oxide is a major vasorelaxant and regulator of the blood pressure. The blood vessels contain several active sources of the superoxide radical, which reacts avidly with nitric oxide to form noxious peroxynitrite. There are large amounts of extracellular-superoxide dismutase (EC-SOD) in the vascular wall. To evaluate the importance of EC-SOD for the physiology of nitric oxide, here we studied the blood pressure in mice lacking the enzyme. In chronically instrumented non-anaesthetized mice there was no difference in mean arterial blood pressure between wild-type controls and EC-SOD mutants. Extensive inhibition of nitric oxide synthases with N -monomethyl- l -arginine however resulted in a larger increase in blood pressure, and infusion of the nitric oxide donor nitrosoglutathione caused less reduction in blood pressure in the EC-SOD null mice. We interpret the alterations to be caused by a moderately increased consumption of nitric oxide by the superoxide radical in the EC-SOD null mice. One role of EC-SOD may be to preserve nitric oxide, a function that should be particularly important in vascular pathologies, in which large increases in superoxide formation have been documented.     

Nitric oxide in paraquat-mediated toxicity: A review

Paraquat, a cationic herbicide, produces degenerative lesions in the lung and in the nervous system after systemic administration to man and animals. Many cases of acute poisoning and death have been reported over the past few decades. Although a definitive mechanism of toxicity of paraquat has not been delineated, a cyclic single electron reduction/oxidation is a critical mechanistic event. The redox cycling of paraquat has two potentially important consequences relevant to the development of toxicity: the generation of the superoxide anion, which can lead to the formation of more toxic reactive oxygen species which are highly reactive to cellular macromolecules; and the oxidation of reducing equivalents (e.g., NADPH, reduced glutathione), which results in the disruption of important NADPH-requiring biochemical processes necessary for normal cell function. Nitric oxide is an important signaling molecule that reacts with superoxide derived from the paraquat redox cycle, to form the potent oxidant peroxynitrite, which causes serious cell damage. Although nitric oxide has been involved in the mechanism of paraquat-mediated toxicity, the role of nitric oxide has been controversial as both protective and harmful effects have been described. The present review summarizes recent findings in the field and describes new knowledge on the role of nitric oxide in the paraquat-mediated toxicity.     

Nitric oxide and oxidative stress in brain and heart of normal rats treated with doxorubicin: role of aminoguanidine

Doxorubicin (DOX) is a potent antitumor antibiotic drug known to cause severe cardiac toxicity. Moreover, its adverse effects were found to be extended to the cerebral tissue. Several mechanisms for this toxicity have been ascribed. Currently, one of the most accepted mechanisms is through free radicals; however, the exact role of nitric oxide (NO) is still unclear. Accordingly, a NO-synthase inhibitor with some antioxidant property, aminoguanidine (AG), was selected to examine its potential protective effect against DOX-induced toxicity. Male Wistar albino rats (150-200 g) were allocated into a normal control group, DOX-induced toxicity group, and DOX + AG-treated group. DOX was injected i.p. at a dose of 10 mg/kg divided into four equal injections over a period of 2 weeks. AG was injected i.p. at a dose of 100 mg/kg 1 h before each DOX injection. The animals were sacrificed 24 h after the last DOX injection and the following parameters were measured: serum lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activities, cardiac and cerebral contents of malondialdehyde (MDA), conjugated diene (CD), glutathione (GSH), NO, and cytosolic calcium, as well as superoxide dismutase (SOD) and glutathione peroxidase (GSHP(X)) activities. Cardiotoxicity was manifested by a marked increase in serum LDH and CPK in addition to the sharp increase in MDA reaching eightfolds the basal level. This was accompanied by significant increase in CD, NO, cytosolic calcium, SOD, and GSHP(X) content/activity by 69, 85, 76, 125, and 41% respectively as compared to normal control. On the other hand, GSH was significantly depressed. In brain, only significant increase in MDA and GSHP(X) and decrease in GSH were obtained but to a lesser extent than the cardiac tissue. AG treatment failed to prevent the excessive release of cardiac enzymes; however, it alleviated the adverse effects of DOX in heart. AG administration resulted in marked decrease in the elevated levels of MDA, NO, SOD, and GSHP(X), however, MDA level was still pathological. The altered parameters in brain were restored by AG. It is concluded that, AG could not provide complete protection against DOX-induced toxicity. Therefore, it is recommended that, maintenance of the endogenous antioxidant, GSH, and regulation of calcium homeostasis must be considered, rather than NO formation, to guard against DOX-induced toxicity.     

Mediation by nitric oxide of the stimulatory effects of ethanol on blood flow

The contribution of nitric oxide (NO) to the hemodynamic effects associated with alcohol oxidation was assessed in rats given either ethanol or water by gastric tube, with and without pre-treatment with either the NO synthase inhibitor N(omega)-monomethyl-L-arginine (L-NMMA; 15 mg/Kg i.p.) or the alcohol dehydrogenase inhibitor 4-methylpyrazole (4-MP; 82 mg/Kg i.p.). Alcohol increased plasma NO (measured with chemiluminescence) by 63%. This was prevented by either L-NMMA or 4-MP. Cardiac output and regional blood flows were determined with 57Cobalt-labeled microspheres. Alcohol markedly enhanced portal blood flow (130 +/- 6 ml/min/Kg vs. 62 +/- 4, in controls; p < 0.01) with no changes in the hepatic, splenic or pancreatic arterial blood flows, indicating that the vasodilatation is mainly mesenteric. In addition, it quadrupled the coronary blood flow, doubled the renal flow and increased cardiac output by 38%, with no significant changes in pulmonary, cerebral or testicular flows. All the stimulatory effects of ethanol on flow, as well as the rise in NO levels, were prevented by L-NMMA, incriminating NO as the mediator of the hemodynamic effects of ethanol oxidation, acting probably via acetate and adenosine.     

Enhanced alloxan-induced beta-cell damage and delayed recovery from hyperglycemia in mice lacking extracellular-superoxide dismutase.

Alloxan is a diabetogenic agent which apparently acts through formation of superoxide radicals formed by redox cycling. Superoxide radicals are also formed by a variety of mechanisms in hyperglycemia. We exposed extracellular-superoxide dismutase (EC-SOD) null mutant and wild-type mice to alloxan, and followed up both the initial diabetes induction and the long-term course of the hyperglycemia. The null mutant mice responded with a modestly enhanced hyperglycemia compared to the wild type controls. In the long-term follow-up all mice eventually regained glycemic control, although it took longer for individuals with higher initial hyperglycemia. This delaying effect of the hyperglycemia was much more pronounced in the null mutant mice. These data suggest that the difference in initial diabetes induction between the groups is due to interception by EC-SOD of extracellular superoxide radicals produced by alloxan. The delayed recovery in the null mutant mice suggests that superoxide radicals released as a result of hyperglycemia impair beta-cell regeneration and that EC-SOD provides some protection. Mouse islets were found to contain little EC-SOD, whereas the content of the cytosolic Cu- and Zn-containing SOD was very high. This low EC-SOD activity may contribute to the high alloxan susceptibility of beta-cells, and may also cause a high susceptibility to superoxide radicals produced by activated inflammatory leukocytes and in hyperglycemia.     

Peroxynitrite,a Stealthy Biological Oxidant

Peroxynitrite is the product of the diffusion-controlled reaction of nitric oxide and superoxide radicals. Peroxynitrite, a reactive short-lived peroxide with a pK_a of 6.8, is a good oxidant and nucleophile. It also yields secondary free radical intermediates such as nitrogen dioxide and carbonate radicals. Much of nitric oxide- and superoxide-dependent cytotoxicity resides on peroxynitrite, which affects mitochondrial function and triggers cell death via oxidation and nitration reactions. Peroxynitrite is an endogenous toxicant but is also a cytotoxic effector against invading pathogens. The biological chemistry of peroxynitrite is modulated by endogenous antioxidant mechanisms and neutralized by synthetic compounds with peroxynitrite-scavenging capacity.     

Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation

Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.     

Targeting superoxide dismutase to renal proximal tubule cells attenuates vancomycin-induced nephrotoxicity in rats

Vancomycin hydrochloride (VCM), a glycopeptide antibiotic, has a broad spectrum against methicillin-resistant Staphylococcus aureus (MRSA). As it is known to induce renal dysfunction, the dose and the duration of its administration are limited. Moreover, the mechanism of VCM-induced renal dysfunction remains to be unclear. To evaluate the involvement of free radical on VCM-induced renal dysfunction, we carried out analysis with a hexamethylenediamine-conjugated superoxide dismutase (AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. Male Wistar rats (weighing 200-210 g) were intraperitonealy administered with 200 mg/kg of VCM twice a day for 7 days. AH-SOD 5 mg/kg/day was subcutaneously injected 5 min before every VCM injection. VCM induced renal injury dose-dependently. Biochemical analyses revealed that plasma levels of blood urea nitrogen and creatinine significantly increased in the VCM-treated group by an AH-SOD-inhibitable mechanism. VCM simultaneously elicited an increase of 8-OHdG levels and chemiluminescence intensity of free radical generation in the kidney. Histological examination revealed that VCM also elicited a marked destruction of glomeruli and necrosis of proximal tubules. AH-SOD inhibited these phenomena in the kidney. These results suggested that oxidative stress might underlie the pathogenesis of VCM-induced nephrotoxicity and targeting SOD and/or related antioxidants to renal proximal tubules might permit the administration of higher doses of VCM sufficient for eradication of MRSA without causing renal injury.     

Distribution and Significance of Nitric Oxide Synthase in Brain Tissues of Rats Exposed to Deltamethrin Insecticide

The distribution of nitric oxide synthase(NOS)in brain tissues of rats exposed to deltamethrininsecticide has been examined by histochemical NADPH-diaphorase staining techniques on frozen sec-tions.After injection of deltamethrin(12.5mg/kg,i.p.),a reproducible sequence of toxic signs ofhyperexcitability were elicited.The observation and image analysis showed that,within brain sec-tions of rats exposed to deltamethrin,the numbers and the total staining areas of the NOS positiveneurons were greatly increased,especially in cerebral cortex,hippocampal formation and paraventric-ular nucleus.In addition,the density of single neuron and the processes were also increased.The re-sults suggested that deltamethrin may induce the NOS expression or activate the NOS activity.TheNOS activation may involve in the chains responsible for the excitatory neurotoxicities induced bydeltamethrin.     

TNP-470 inhibits oxidative stress, nitric oxide production and nuclear factor kappa B activation in a rat model of hepatocellular carcinoma

The objective of this study is to determine if treatment with the angiogenesis inhibitor TNP-470 results in impairment of oxidative stress, inhibition of nuclear factor kappa B (NF-κB) activation and decrease of nitric oxide production in an experimental model of rat hepatocarcinogenesis. Tumour was induced by diethylnitrosamine and promoted by two-thirds hepatectomy plus acetaminofluorene administration. Experiments were carried out at 28 weeks after initiating the treatment. TNP-470 was administered at 30 mg/kg, three times per week from 20 to 28 weeks. Carcinomatous tissue growing outside dysplastic nodules and a marked expression of placental glutathione S-transferase were detected in rats with induced carcinogenesis. Liver concentrations of thiobarbituric acid reactive substances, reduced glutathione (GSH) and glutathione disulfide (GSSG) were significantly higher than those of controls and there was a significant increase in the GSSG/GSH ratio. Tumour growth was accompanied by augmented expression of inducible nitric oxide synthase, activation of (NF-κB) and proteolysis of IkappaB. All these effects were absent in animals receiving TNP-470. Our results indicate that TNP-470 inhibits oxidative stress, nitric oxide production and NF-κB activation induced by experimental hepatocarcinogenesis. These changes would contribute to the beneficial effects of TNP-470 in cancer treatment.     

Nitric oxide induced oxidative changes in erythrocyte membrane components

The effects of NO in its environment may vary considerably depending on various factors. This study shows oxidative mechanism of cellular membrane alterations, which is not associated with triggering of ONOOH generation but is induced by pure NO. Our investigation examined the influence of low concentration of NO (0.1; 0.2 mmol/l) on the qualitative changes of structure and dynamics of erythrocyte membrane. NO causes a statistically significant increase in membrane fluidity on different depths of lipid bilayer that is correlated with increase of lipids peroxidation. Statistically significant changes in the conformational state of cytoskeleton proteins were also detected. NO can be considered as a molecule responsible for determining rheological properties of erythrocytes membrane. Therefore, we propose that NO acts as pro-oxidant molecule at concentrations for which membrane appeared to be the first target before it entered the cytosol.     

Endothelial argininosuccinate synthetase 1 regulates nitric oxide production and monocyte adhesion under static and laminar shear stress conditions

Laminar shear stress (LSS) is known to increase endothelial nitric oxide (NO) production, which is essential for vascular health, through expression and activation of nitric oxide synthase 3 (NOS3). Recent studies demonstrated that LSS also increases the expression of argininosuccinate synthetase 1 (ASS1) that regulates the provision of L-arginine, the substrate of NOS3. It was thus hypothesized that ASS1 might contribute to vascular health by enhancing NO production in response to LSS. This hypothesis was pursued in the present study by modulating NOS3 and ASS1 levels in cultured endothelial cells. Exogenous expression of either NOS3 or ASS1 in human umbilical vein endothelial cells increased NO production and decreased monocyte adhesion stimulated by tumor necrosis factor-α (TNF-α). The latter effect of overexpressed ASS1 was reduced when human umbilical vein endothelial cells were co-treated with small interfering RNAs (siRNAs) for ASS1 or NOS3. SiRNAs of NOS3 and ASS1 attenuated the increase of NO production in human aortic endothelial cells stimulated by LSS (12 dynes·cm(-2)) for 24 h. LSS inhibited monocyte adhesion to human aortic endothelial cells stimulated by TNF-α, but this effect of LSS was abrogated by siRNAs of NOS3 and ASS1 that recovered the expression of vascular cell adhesion molecule-1. The current study suggests that the expression of ASS1 harmonized with that of NOS3 may be important for the optimized endothelial NO production and the prevention of the inflammatory monocyte adhesion to endothelial cells.     

A Pivotal Role for Tryptophan 447 in Enzymatic Coupling of Human Endothelial Nitric Oxide Synthase (eNOS): EFFECTS ON TETRAHYDROBIOPTERIN-DEPENDENT CATALYSIS AND eNOS DIMERIZATION*

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.     

Nitric oxide diffusion to red blood cells limits extracellular,but not intraphagosomal,peroxynitrite formation by macrophages

Macrophage-derived nitric oxide (^•NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by ^•NO itself or ^•NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O₂^•−). In vivo, the facile extracellular diffusion of ^•NO as well as different competing consumption routes limit its bioavailability for the reaction with O₂^•− and, hence, peroxynitrite formation. In this work, we evaluated the extent by which ^•NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O₂^•− and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of ^•NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of ^•NO and O₂^•−-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of ^•NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O₂^•− reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of ^•NO and O₂^•--forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, ^•NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.     

Melatonin Prevents Cyclosporine-induced Nephrotoxicity in Isolated and Perfused Rat Kidney

Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. The precise mechanism by which CsA causes renal injury is not known. Reactive oxygen species (ROS) have been shown to play a role, since CsA-induced renal lipid peroxidation is attenuated in vivo and in vitro by the concomitant administration of antioxidants such as vitamin E. We show here the effect of the antioxidant melatonin (MLT), a hormone produced by the pineal gland during the dark phase of the circadian cycle, in a model of CsA nephrotoxicity in the isolated and perfused rat kidney. Kidneys isolated from rats were divided into seven groups. At the end of perfusion, malondialdehyde and 4-hydroxyalkenals (MDA+4-HDA), metabolites of nitric oxide N O 2 &#109 +N O 3 &#109 were measured and histopathological examination was performed. CsA treatment induced a significant increase in MDA+4-HDA while not affecting the nitric oxide metabolite level. MLT remarkably prevented glomerular collapse and tubular damage as revealed by morphometric analysis. Our study suggests that lipid peroxidation is an early important event in the pathogenesis of CsA nephrotoxicity and that MLT is able to protect kidneys from CsA at a relatively low concentration.     

Melatonin prevents cyclosporine-induced nephrotoxicity in isolated and perfused rat kidney

Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. The precise mechanism by which CsA causes renal injury is not known. Reactive oxygen species (ROS) have been shown to play a role, since CsA-induced renal lipid peroxidation is attenuated in vivo and in vitro by the concomitant administration of antioxidants such as vitamin E. We show here the effect of the antioxidant melatonin (MLT), a hormone produced by the pineal gland during the dark phase of the circadian cycle, in a model of CsA nephrotoxicity in the isolated and perfused rat kidney. Kidneys isolated from rats were divided into seven groups. At the end of perfusion, malondialdehyde and 4-hydroxyalkenals (MDA+4-HDA), metabolites of nitric oxide N O 2 - +N O 3 - were measured and histopathological examination was performed. CsA treatment induced a significant increase in MDA+4-HDA while not affecting the nitric oxide metabolite level. MLT remarkably prevented glomerular collapse and tubular damage as revealed by morphometric analysis. Our study suggests that lipid peroxidation is an early important event in the pathogenesis of CsA nephrotoxicity and that MLT is able to protect kidneys from CsA at a relatively low concentration.     

Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages

Nitric oxide (NO) reacts with superoxide to produce peroxynitrite, a potent oxidant and reportedly exerts cytotoxic action. Herein we validated the hypothesis that interaction of NO with superoxide exerts protection against superoxide toxicity using macrophages from mice with a knockout (KO) of inducible NO synthase (NOS2) and superoxide dismutase 1 (SOD1), either individually or both. While no difference was observed in viability between wild-type (WT) and NOS2KO macrophages, SOD1KO and SOD1-and NOS2-double knockout (DKO) macrophages were clearly vulnerable and cell death was observed within four days. A lipopolysaccharide (LPS) treatment induced the formation of NOS2, which resulted in NO production in WT and these levels were even higher in SOD1KO macrophages. The viability of the DKO macrophages but not SOD1KO macrophages were decreased by the LPS treatment. Supplementation of NOC18, a NO donor, improved the viability of SOD1KO and DKO macrophages both with and without the LPS treatment. The NOS2 inhibitor nitro-l-arginine methyl ester consistently decreased the viability of LPS-treated SOD1KO macrophages but not WT macrophages. Thus, in spite of the consequent production of peroxynitrite in LPS-stimulated macrophages, the coordinated elevation of NO appears to exert anti-oxidative affects by coping with superoxide cytotoxicity upon conditions of inflammatory stimuli.     

The chemical biology of nitric oxide: implications in cellular signaling

Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the past few years, the importance of steady-state NO concentrations has emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose five basic distinct concentration levels of NO activity: cGMP-mediated processes ([NO]<1-30 nM), Akt phosphorylation ([NO] = 30-100 nM), stabilization of HIF-1alpha ([NO] = 100-300 nM), phosphorylation of p53 ([NO]>400 nM), and nitrosative stress (1 microM). In general, lower NO concentrations promote cell survival and proliferation, whereas higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell-cycle arrest profile to a cell survival profile. The resulting reactive nitrogen species that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability, which are referred to as kinetic determinants for molecular target interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.