Xanthine oxidoreductase (XOR) is generally known as the final enzyme in purine metabolism and as a source of reactive oxygen species (ROS). In addition, this enzyme has been suggested to mediate ...nitric oxide (NO) formation via reduction of inorganic nitrate and nitrite. This NO synthase (NOS)-independent pathway for NO generation is of particular importance during certain conditions when NO bioavailability is diminished due to reduced activity of endothelial NOS (eNOS) or increased oxidative stress, including aging and cardiovascular disease. The exact interplay between NOS- and XOR-derived NO generation is not fully elucidated yet. The aim of the present study was to investigate if eNOS deficiency is associated with changes in XOR expression and activity and the possible impact on nitrite, NO and ROS homeostasis. Plasma levels of nitrate and nitrite were similar between eNOS deficient (eNOS
) and wildtype (wt) mice. XOR activity was upregulated in eNOS
compared with wt, but not in nNOS
, iNOS
or wt mice treated with the non-selective NOS inhibitor L-NAME. Following an acute dose of nitrate, plasma nitrite increased more in eNOS
compared with wt, and this augmented response was abolished by the selective XOR inhibitor febuxostat. Livers from eNOS
displayed higher nitrite reducing capacity compared with wt, and this effect was attenuated by febuxostat. Dietary supplementation with nitrate increased XOR expression and activity, but concomitantly reduced superoxide generation. The latter effect was also seen in vitro after nitrite administration. Treatment with febuxostat elevated blood pressure in eNOS
, but not in wt mice. A high dose of dietary nitrate reduced blood pressure in naïve eNOS
mice, and again this effect was abolished by febuxostat. In conclusion, eNOS deficiency is associated with an upregulation of XOR facilitating the nitrate-nitrite-NO pathway and decreasing the generation of ROS. This interplay between XOR and eNOS is proposed to play a significant role in NO homeostasis and blood pressure regulation.
Nitric oxide (NO) is a ubiquitous gas with free radical groups that is soluble in water, and which is involved in numerous physiological functions including inflammatory and immune responses. ...However, the role of NO in tumor biology is controversial and misunderstood. NO has been shown to have both anti-cancer and carcinogenic effects, which are dependent on the time, location, and concentration of NO. This duality presents a double challenge to determine the net impact of NO on cancer and to define the therapeutic role of NO-centered anti-cancer strategies. Nevertheless, it is believed that a comprehensive and dynamic understanding of the cascade of molecular and cellular events underlying tumor biology that are affected by NO will allow researchers to exploit the potential anti-tumor properties of drugs that interfere with NO metabolism.
Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from l-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be ...characterized by a global model with three main rate-limiting steps. These are the rate of heme reduction by the flavin domain (kr), of dissociation of NO from the ferric heme-NO complex (kd), and of oxidation of the ferrous heme-NO complex (kox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus, enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with the NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outer-sphere mechanism, remained unclear. Here, we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound FeIII-N(O)OO− intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidine-coordinated heme.
A considerable number of human diseases have an inflammatory component, and a key mediator of immune activation and inflammation is inducible nitric oxide synthase (iNOS), which produces nitric oxide ...(NO) from l‐arginine. Overexpressed or dysregulated iNOS has been implicated in numerous pathologies including sepsis, cancer, neurodegeneration, and various types of pain. Extensive knowledge has been accumulated about the roles iNOS plays in different tissues and organs. Additionally, X‐ray crystal and cryogenic electron microscopy structures have shed new insights on the structure and regulation of this enzyme. Many potent iNOS inhibitors with high selectivity over related NOS isoforms, neuronal NOS, and endothelial NOS, have been discovered, and these drugs have shown promise in animal models of endotoxemia, inflammatory and neuropathic pain, arthritis, and other disorders. A major issue in iNOS inhibitor development is that promising results in animal studies have not translated to humans; there are no iNOS inhibitors approved for human use. In addition to assay limitations, both the dual modalities of iNOS and NO in disease states (ie, protective vs harmful effects) and the different roles and localizations of NOS isoforms create challenges for therapeutic intervention. This review summarizes the structure, function, and regulation of iNOS, with focus on the development of iNOS inhibitors (historical and recent). A better understanding of iNOS’ complex functions is necessary before specific drug candidates can be identified for classical indications such as sepsis, heart failure, and pain; however, newer promising indications for iNOS inhibition, such as depression, neurodegenerative disorders, and epilepsy, have been discovered.
Puberty is a crucial phase for the development of female sexual behavior. Growing evidence suggests that stress during this period may interfere with the development of sexual behavior. However, the ...neural circuits involved in this alteration remain elusive. Here, we demonstrated in mice that pubertal stress permanently disrupted sexual performance without affecting sexual preference. This was associated with a reduced expression and activation of neuronal nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Fiber photometry revealed that VMHvl nNOS neurons are strongly responsive to male olfactory cues with this activation being substantially reduced in pubertally stressed females. Finally, treatment with a NO donor partially restored sexual performance in pubertally stressed females. This study provides insights into the involvement of VMHvl nNOS in the processing of olfactory cues important for the expression of female sexual behavior. In addition, exposure to stress during puberty disrupts the integration of male olfactory cues leading to reduced sexual behavior.
Autophagy, a major degradation process for long-lived and aggregate-prone proteins, affects various human processes, such as development, immunity, cancer, and neurodegeneration. Several autophagy ...regulators have been identified in recent years. Here we show that nitric oxide (NO), a potent cellular messenger, inhibits autophagosome synthesis via a number of mechanisms. NO impairs autophagy by inhibiting the activity of S-nitrosylation substrates, JNK1 and IKKβ. Inhibition of JNK1 by NO reduces Bcl-2 phosphorylation and increases the Bcl-2–Beclin 1 interaction, thereby disrupting hVps34/Beclin 1 complex formation. Additionally, NO inhibits IKKβ and reduces AMPK phosphorylation, leading to mTORC1 activation via TSC2. Overexpression of nNOS, iNOS, or eNOS impairs autophagosome formation primarily via the JNK1–Bcl-2 pathway. Conversely, NOS inhibition enhances the clearance of autophagic substrates and reduces neurodegeneration in models of Huntington's disease. Our data suggest that nitrosative stress-mediated protein aggregation in neurodegenerative diseases may be, in part, due to autophagy inhibition.
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► NO inhibits autophagy by independently inhibiting JNK1 and IKKβ ► NO inhibits autophagic flux via mTOR and mTOR-independent routes ► NOS overexpression impairs autophagosome synthesis via JNK1–Bcl-2 pathway ► NOS inhibition induces autophagy and protects against neurodegeneration
Recent research has shown that nitric oxide (NO) produced by nitric oxide synthases (NOS) is an inhibitor of ion transporter activity and a modulator of epithelial ion transport in fish, but little ...is known on changes in the NOS/NO system during osmotic stress. We hypothesized that the NOS/NO system responds to salinity changes as an integrated part of the acclimation process. Expression and localization of nos1/Nos1 and nos2/Nos2 were investigated in gill, kidney, and intestine of freshwater (FW)- and seawater (SW)-transferred trout using quantitative PCR, Western blotting, and immunohistochemistry, along with expressional changes of major ion transporters in the gill. The classical branchial ion transporters showed expected expressional changes upon SW transfer, there among a rapid decrease in Slc26a6 mRNA, coding a branchial Cl
/Formula: see text exchanger. There was a major downregulation of nos1/ nos2/Nos2 expression in the gill during SW acclimation. A significant decrease in plasma nitrite supported an overall decreased Nos activity and NO production. In the middle intestine, Nos1 was upregulated during SW acclimation, whereas no changes in nos/Nos expression were observed in the posterior intestine and the kidney. Nos1 was localized along the longitudinal axis of the gill filament, beneath smooth muscle fibers of the intestine wall and in blood vessel walls of the kidney. Nos2 was localized within the epithelium adjacent to the gill filament axis and in hematopoietic tissues of the kidney. We conclude that downregulation of branchial NOS is integrated to the SW acclimation process likely to avoid the inhibitory effects of NO on active ion extrusion.
Sulfur dioxide (SO
2) is a common gaseous pollutant. It is also, however, endogenously generated from sulfur-containing amino acids. Recent studies have demonstrated that rat blood pressure can be ...lowered by SO
2-exposure in vivo and that vasodilation caused by SO
2 at low concentrations (<
450
μM) is endothelium-dependent in rat aorta. However, effects of SO
2 on nitric oxide synthase (NOS) and nitric oxide (NO) production have not been previously studied in rat aorta. The objective of the present study is to assess the effects of acute (10
min) and prolonged (2
h) stimulation with different concentrations of SO
2 on NO/cGMP pathway in isolated rat aorta. The results show that: (1) the acute and prolonged pretreatments with SO
2 produced an inhibition of vasoconstrictions induced by norepinephrine. (2) SO
2 potentiated activity of endothelial nitric oxide synthase (eNOS), but not of induced NOS (iNOS). (3) SO
2 could increase expression of eNOS gene on the transcription and translation levels in rat aorta. (4) SO
2 enhanced NO formation in aortic tissue. (5) The level of cGMP in rat aorta was increased by SO
2 and no change of cAMP. These findings led to the conclusion: there were acute and prolonged effects of SO
2 on the NO/cGMP signalling pathway; and SO
2 could upregulate the eNOS–NO–cGMP pathway and at least partly by which the SO
2 might cause vasodilation and inhibition to vasoconstriction.
Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant ...metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using N
-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.
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