Unraveling the biological significance of nitric oxide

Independent investigations into the biochemical changes and cytostatic properties induced in immunostimulated macrophages and studies involving the identity and mechanism of action of endothelium-derived relaxing factor led to the finding of a new metabolic pathway which converts L-arginine to nitric oxide and citrulline. The pathway has since been reported in a number of additional cell types including cells in the central nervous system (CNS). In the endothelium and CNS nitric oxide is acting as a signaling agent with the evidence supporting activation of the enzyme guanylate cyclase in the target cell. Nitric oxide is toxic and evidence supports a cytostatic/cytotoxic function as the primary action of macrophage-derived nitric oxide.     

Nitric oxide and atherosclerosis: possible implications for therapy

Atherosclerosis and hypercholesterolaemia disturb the endothelium-dependent regulation of the vascular tone by the labile liposoluble radical nitric oxide. This defect predisposes to vasospasm and ischaemia, with anginal pain as a clinical manifestation. It is now appreciated that endothelial dysfunction is an early event in atherogenesis, possibly also involving the microcirculation, in which atherosclerosis does not develop. Furthermore, it is becoming clear that nitric oxide, in addition to regulating vasomotion, might also modulate the progression of the disease process. The latter notion could have therapeutic implications.     

Mechanisms and potential therapeutic targets for folic acid in cardiovascular disease

Folic acid (FA) is a member of the B-vitamin family with cardiovascular roles in homocysteine regulation and endothelial nitric oxide synthase (eNOS) activity. Its interaction with eNOS is thought to be due to the enhancement of tetrahydrobiopterin bioavailability, helping maintain eNOS in its coupled state to favor the generation of nitric oxide rather than oxygen free radicals. FA also plays a role in the prevention of several cardiac and noncardiac malformations, has potent direct antioxidant and antithrombotic effects, and can interfere with the production of the endothelial-derived hyperpolarizing factor. These multiple mechanisms of action have led to studies regarding the therapeutic potential of FA in cardiovascular disease. To date, studies have demonstrated that FA ameliorates endothelial dysfunction and nitrate tolerance and can improve pathological features of atherosclerosis. These effects appear to be homocysteine independent but rather related to their role in eNOS function. Given the growing evidence that nitric oxide synthase uncoupling plays a major role in many cardiovascular disorders, the potential of exogenous FA as an inexpensive and safe oral therapy is intriguing and is stimulating ongoing investigations.     

Nitric oxide modulation in neuroinflammation and the role of mesenchymal stem cells

Nitric oxide is a versatile mediator formed by enzymes called nitric oxide synthases. It has numerous homeostatic functions and important roles in inflammation. Within the inflamed brain, microglia and astrocytes produce large amounts of nitric oxide during inflammation. Excessive nitric oxide causes neuronal toxicity and death and mesenchymal stem cells can be used as an approach to limit the neuronal damage caused by neuroinflammation. Mesenchymal stem cell therapy ameliorates inflammation and neuronal damage in disease models of Alzheimer’s disease, Parkinson’s disease, and other neuroinflammatory disorders. Interestingly, we have reported that in vitro, mesenchymal stem cells themselves contribute to a rise in nitric oxide levels through microglial cues. This may be an undesirable effect and highlights a possible need to explore acellular approaches for mesenchymal stem cell therapy in the central nervous system.     

Heteroaromatic-aminomethyl quinolones: potent and selective iNOS inhibitors

The overproduction of nitric oxide during the biological response to inflammation by the nitric oxide synthase (NOS) enzymes have been implicated in the pathology of many diseases. By removal of the amide core from uHTS-derived quinolone 4, a new series highly potent heteroaromatic-aminomethyl quinolone iNOS inhibitors 8 were identified. SAR studies led to identification of piperazine 22 and pyrimidine 32, both of which reduced plasma nitrates following oral dosing in a mouse lipopolysaccharide challenge assay.     

Glutathione depletion is accompanied by increased neuronal nitric oxide synthase activity

The effect of glutathione depletion, in vivo, on rat brain nitric oxide synthase activity has been investigated and compared to the effect observed in vitro with cultured neurones. Using L-buthionine sulfoximine rat brain glutathione was depleted by 62%. This loss of glutathione was accompanied by a significant increase in brain nitric oxide synthase activity by up to 55%. Depletion of glutathione in cultured neurones, by approximately 90%, led to a significant 67% increase in nitric oxide synthase activity, as judged by nitrite formation, and cell death. It is concluded that depletion of neuronal glutathione results in increased nitric oxide synthase activity. These findings may have implications for our understanding of the pathogenesis of neurodegenerative disorders in which loss of brain glutathione is considered to be an early event.     

The influence of nitric oxide on the regulation of plasminogen activator inhibitor type 1 and tissue-type plasminogen activator expression

Nitric oxide produced in various human tissues by nitric oxide synthase is involved in the regulation of many physiological processes. Mechanism of its action is diverse. The most important physiological activity of nitric oxide is guanylate cyclase activation and an increase of cGMP synthesis. At low concentrations NO plays a pivotal role in vessel relaxation and possesses antithrombotic, antiproliferative and anti-inflammatory features as well. An excessive production of nitric oxide can disturb vascular hemostasis and contribute to development of cardiovascular diseases. Studies provide that NO also participate in fibrynolysis regulation by the influence on the PAI-1 and t-PA expression, what may have important clinical implications. The aim of this review is to present current knowledge about the role of nitric oxide in the regulation of these plasminogen activation system factors.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.     

Role of nitric oxide in insulin-dependent diabetes mellitus-related vascular complications.

Patients with insulin-dependent diabetes mellitus are at high risk for vascular disorders such as hypertension, nephropathy, and retinopathy. The most common cause of morbidity and mortality in patients with insulin-dependent diabetes is vascular disease. Despite ongoing research, the pathogenesis of vascular disease in diabetes remains unclear. In recent years, numerous investigators have examined the role of the endothelium-derived relaxing factor, nitric oxide, in the disease state of hypertension and its complications. We review the role of nitric oxide in the development of diabetes-related vascular disease and discuss findings suggesting that nitric oxide metabolism and vascular responsiveness to nitric oxide are altered in diabetes. Patients with diabetes may benefit from therapy that addresses this pathogenic deficiency.     

Interferon-γ and Nitric Oxide Down-Regulate Lipopolysaccharide-Induced Prostanoid Production in Cultured Rat Microglial Cells by Inhibiting Cyclooxygenase-2 Expression

Abstract: We have used purified microglial cultures obtained from neonatal rat brains to study some aspects of the regulation of prostanoid production in these cells. We found that nitric oxide, which is released into the culture medium along with prostanoids when the cells are exposed to lipopolysaccharide (1–100 ng/ml), can down-regulate prostanoid biosynthesis. Indeed, the abrogation of endogenous nitric oxide production, obtained by depleting the medium of the precursor l -arginine or by blocking the activity of nitric oxide synthase by the specific inhibitor N^G-monomethyl-l -arginine, led to a remarkable increase in lipopolysaccharide-induced prostanoid release. Moreover, the nitric oxide-generating compound 3-morpholinosydnonimine caused a substantial reduction of prostanoid formation, in the absence of endogenous nitric oxide, suggesting that both endogenous and exogenous nitric oxide may inhibit the induced prostanoid production. We also found that interferon-γ potentiated the effect of lipopolysaccharide on nitrite accumulation as previously described by others and depressed the lipopolysaccharide-evoked production of prostaglandin E₂, prostaglandin D₂, and thromboxane. It is interesting that the inhibitory effect of interferon-γ on prostanoid production did not appear to depend on the potentiation of NO release, as it was present also when the endogenous synthesis of nitric oxide was abrogated. Additional experiments showed that interferon-γ and nitric oxide act mainly by down-regulating the lipopolysaccharide-induced enzymatic activity and expression (analyzed by western blot) of cyclooxygenase-2. Our data indicate that microglial prostanoid biosynthesis induced by proinflammatory stimuli, such as lipopolysaccharide, is tightly regulated by nitric oxide. Interferon-γ appears to affect the balance between these local mediators by favoring nitric oxide production and inhibiting the prostanoid cascade and may thus contribute to the modulation of inflammation, local immune reactivity, and neuronal damage.     

Sickle cell disease and nitric oxide: a paradigm shift?

Traditionally the pathophysiology of sickle cell disease is thought to result from the polymerization of hemoglobin S in red cells, under hypoxic conditions, resulting in the occlusion of blood vessels. Adhesion of cells to the venular endothelium also appears to play a role. Recent studies have also suggested that in addition to the polymerization of hemoglobin S in the red blood cell, a deficiency of the endogenous vasodilator, nitric oxide may be involved. Hemoglobin released as a result of hemolysis rapidly consumes nitric oxide resulting in a whole program of events that inhibit blood flow. Therapies directed at decreasing the destruction of nitric oxide, increasing the production of nitric oxide, or amplifying the nitric oxide response may prove beneficial.     

Regulation of cytochrome c peroxidase activity by nitric oxide and laser irradiation

Apoptosis can be induced by activation of so-called "death receptors" (extrinsic pathway) or multiple apoptotic factors (intrinsic pathway), which leads to release of cytochrome c from mitochondria. This event is considered to be a point of no return in apoptosis. One of the most important events in the development of apoptosis is the enhancement of cytochrome c peroxidase activity upon its interaction with cardiolipin, which modifies the active center of cytochrome c. In the present work, we have investigated the effects of nitric oxide on the cytochrome c peroxidase activity when cytochrome c is bound to cardiolipin or sodium dodecyl sulfate. We have observed that cytochrome c peroxidase activity, distinctly increased due to the presence of anionic lipids, is completely suppressed by nitric oxide. At the same time, nitrosyl complexes of cytochrome c, produced in the interaction with nitric oxide, demonstrated sensitivity to laser irradiation (441 nm) and were photolyzed during irradiation. This decomposition led to partial restoration of cytochrome c peroxidase activity. Finally, we conclude that nitric oxide and laser irradiation may serve as effective instruments for regulating the peroxidase activity of cytochrome c, and, probably, apoptosis.     

Biological selectivity and functional aspects of protein tyrosine nitration

The formation of nitric oxide in biological systems has led to the discovery of a number of post-translational protein modifications that could regulate protein function or potentially be utilized as transducers of nitric oxide signaling. Principal among the nitric oxide-mediated protein modifications are: the nitric oxide-iron heme binding, the S-nitrosylation of reduced cysteine residues, and the C-nitration of tyrosine and tryptophan residues. With the exception of the nitric oxide binding to heme iron proteins, the other two modifications appear to require secondary reactions of nitric oxide and the formation of nitrogen oxides. The rapid development of analytical and immunological methodologies has allowed for the quantification of S-nitrosylated and C-nitrated proteins in vivo revealing an apparent selectivity and specificity of the proteins modified. This review is primarily focused upon the nitration of tyrosine residues discussing parameters that may govern the in vivo selectivity of protein nitration, and the potential biological significance and clinical relevance of this nitric oxide-mediated protein modification.     

Low density lipoprotein inhibits accumulation of nitrites in murine brain endothelial cell cultures.

Endothelial cells produce nitric oxide which is considered to serve as a major source of endothelial derived relaxing factor activity. It has been demonstrated that activation of mouse brain endothelium by TNF-alpha and IFN-gamma led to accumulation of nitrite which is presumably formed by oxidation of nitric oxide. A number of studies suggest that reactive oxygen species produced by cytokine-activated cells are involved in the conversion of nitric oxide to nitrites and nitrates. We investigated whether low density lipoprotein (LDL), acting as a radical scavenger, is able to inhibit nitrite accumulation in mouse brain endothelial cell cultures and in a cell-free system in which sodium nitroprusside was used as a source of nitric oxide. A comparison of these two models indicates the active involvement of LDL in suppressing nitrite accumulation in murine endothelial cultures.     

Simulated microgravity promotes nitric oxide-supported angiogenesis via the iNOS-cGMP-PKG pathway in macrovascular endothelial cells

Angiogenesis is a physiological process involving the growth of blood vessel in response to specific stimuli. The present study shows that limited microgravity treatments induce angiogenesis by activating macrovascular endothelial cells. Inhibition of nitric oxide production using pharmacological inhibitors and inducible nitric oxide synthase (iNOS) small interfering ribo nucleic acid (siRNA) abrogated microgravity induced nitric oxide production in macrovascular cells. The study further delineates that iNOS acts as a molecular switch for the heterogeneous effects of microgravity on macrovascular, endocardial and microvascular endothelial cells. Further dissection of nitric oxide downstream signaling confirms that simulated microgravity induces angiogenesis via the cyclic guanosine monophosphate (cGMP)-PKG dependent pathway.     

Nitric oxide production from hydroxyurea

Hydroxyurea is a relatively new treatment for sickle cell disease. A portion of hydroxyurea's beneficial effects may be mediated by nitric oxide, which has also drawn considerable interest as a sickle cell disease treatment. Patients taking hydroxyurea show a significant increase in iron nitrosyl hemoglobin and plasma nitrite and nitrate within 2 h of ingestion, providing evidence for the in vivo conversion of hydroxyurea to nitric oxide. Hydroxyurea reacts with hemoglobin to produce iron nitrosyl hemoglobin, nitrite, and nitrate, but these reactions do not occur fast enough to account for the observed increases in these species in patients taking hydroxyurea. This report reviews recent in vitro studies directed at better understanding the in vivo nitric oxide release from hydroxyurea in patients. Specifically, this report covers: (1) peroxidase-mediated formation of nitric oxide from hydroxyurea; (2) nitric oxide production after hydrolysis of hydroxyurea to hydroxylamine; and (3) the nitric oxide-producing structure-activity relationships of hydroxyurea. Results from these studies should provide a better understanding of the nitric oxide donor properties of hydroxyurea and guide the development of new hydroxyurea-derived nitric oxide donors as potential sickle cell disease therapies.     

Regulation of bone resorption and osteoclast survival by nitric oxide: possible involvement of NMDA-receptor

Nitric oxide has been shown to play an important role in regulation of bone resorption. However, the role of endogenous nitric oxide on osteoclast activity remains still controversial. In this work, using RT-PCR amplification, we demonstrated that rabbit mature osteoclasts express mRNA encoding for neuronal nitric oxide synthase suggesting that this enzyme could be involved in basal nitric oxide production in these cells. Then we assessed the effect of carboxy-PTIO, a nitric oxide scavenger, on in vitro bone resorption and osteoclast survival. Carboxy-PTIO (10-100 microM) inhibited osteoclastic bone resorption in a dose dependent manner and induced osteoclast apoptosis by a mechanism involving caspase 3 activation. These results suggest that basal concentration of endogenous nitric oxide may be essential for normal bone resorption by supporting osteoclast survival. Because osteoclasts express N-methyl-d-aspartate-receptor (NMDA-R), we hypothesized that in osteoclasts NMDA-R may be involved in nitric oxide production as in neuronal cells. We confirmed that blockade of NMDA-R with specific non-competitive antagonists, MK801 and DEP, strongly inhibited bone resorption. As for carboxy-PTIO, we showed that blockade of NMDA-R by both antagonists induced osteoclast apoptosis in a dose dependent manner by a mechanism dependent on caspase 3 activation. Intracellular calcium concentration in osteoclasts decreased within minutes in the presence of both antagonists. Finally, MK801-induced osteoclast apoptosis was partially reversed in the presence of small amount of SNAP (100 nM), a nitric oxide donor, suggesting that the effect of NMDA-R on osteoclast apoptotic cell death could be due to a decrease in nitric oxide production. Taken together, our results are consistent with the hypothesis that NMDA-R on osteoclasts could have a similar function as those in neuronal cells, i.e., to allow a calcium influx, which in turn activates a constitutive neuronal nitric oxide synthase. Nitric oxide generated by this pathway may be essential for osteoclast survival and hence for normal bone resorption.     

The nitric oxide response in plant-associated endosymbiotic bacteria

Nitric oxide (NO) is a gaseous signalling molecule which becomes very toxic due to its ability to react with multiple cellular targets in biological systems. Bacterial cells protect against NO through the expression of enzymes that detoxify this molecule by oxidizing it to nitrate or reducing it to nitrous oxide or ammonia. These enzymes are haemoglobins, c-type nitric oxide reductase, flavorubredoxins and the cytochrome c respiratory nitrite reductase. Expression of the genes encoding these enzymes is controlled by NO-sensitive regulatory proteins. The production of NO in rhizobia-legume symbiosis has been demonstrated recently. In functioning nodules, NO acts as a potent inhibitor of nitrogenase enzymes. These observations have led to the question of how rhizobia overcome the toxicity of NO. Several studies on the NO response have been undertaken in two non-dentrifying rhizobial species, Sinorhizobium meliloti and Rhizobium etli, and in a denitrifying species, Bradyrhizobium japonicum. In the present mini-review, current knowledge of the NO response in those legume-associated endosymbiotic bacteria is summarized.