Multimodality imaging of nitric oxide and nitric oxide synthases

Nitric oxide (NO) and NO synthases (NOSs) are crucial factors in many pathophysiological processes such as inflammation, vascular/neurological function, and many types of cancer. Noninvasive imaging of NO or NOS can provide new insights in understanding these diseases and facilitate the development of novel therapeutic strategies. In this review, we will summarize the current state-of-the-art multimodality imaging in detecting NO and NOSs, including optical (fluorescence, chemiluminescence, and bioluminescence), electron paramagnetic resonance (EPR), magnetic resonance (MR), and positron emission tomography (PET). With continued effort over the last several years, these noninvasive imaging techniques can now reveal the biodistribution of NO or NOS in living subjects with high fidelity which will greatly facilitate scientists/clinicians in the development of new drugs and/or patient management. Lastly, we will also discuss future directions/applications of NO/NOS imaging. Successful development of novel NO/NOS imaging agents with optimal in vivo stability and desirable pharmacokinetics for clinical translation will enable the maximum benefit in patient management.     

Aged garlic extract enhances production of nitric oxide

Nitric oxide (NO) controls several physiological functions of the cardiovascular system. Three kinds of NO synthases (NOSs), neuronal constitutive NOS (ncNOS), inducible NOS (iNOS) and endothelial constitutive NOS (ecNOS), were responsible for NO biosynthesis. This study investigated the effect of aged garlic extract (AGE) on NO production by measuring the NO metabolites nitrite and nitrate in the plasma of mice. AGE (2.86 g/kg, p.o.) temporarily increased NO production by 30-40% from 15 to 60 min after administration. The time course of the fluctuation in NO levels in the AGE-treated group was clearly different to that in a group of mice treated with lipopolysaccharides, a typical iNOS inducer. Arginine (63 mg/kg, p.o.) at the equivalent dose of AGE did not increase NO production. However diphenyleneiodonium chloride (1 mg/kg, i.p.), a selective cNOS inhibitor, administered prior to AGE, overcame the effect of AGE. These results indicate that AGE increased NO production by activating cNOS, but not iNOS. The arginine contained in AGE was not responsible for the effect. AGE may be a useful tool for the prevention of cardiovascular disease.     

Distribution and properties of arginase in the salivary glands of four species of laboratory mammals

Important progress in arginine metabolism includes the discovery of widespread expression of two isoforms of arginase, arginase I and II, not only in hepatic cells but also in non-hepatic cells, and the formation of nitric oxide, a widely distributed signal-transducing molecule, from arginine by nitric oxide synthase. Possible physiological roles of arginase may therefore include regulation of nitric oxide synthesis through arginine availability for nitric oxide synthase. In this paper, arginase was investigated in the submandibular, sublingual, and parotid glands of rat, mouse, guinea pig, and rabbit. From their arginase contents, the salivary glands of these species were divided into two groups. Variable levels of arginase activity were detected in the salivary glands of mouse and rat. However, salivary glands of rabbit and guinea pig had almost no arginase activity. The presence of nitric oxide synthase has been reported in all the salivary glands used in this study. Therefore, one of the important findings was the presence of species specificity in the co-localization of arginase and nitric oxide synthase in the salivary glands of the four species. The highest specific activity of arginase was found in mouse parotid gland. In rat, considerable arginase activity was detected in all three glands, at 3.6–7.3% of that in rat liver. In rat submandibular gland, arginase was detected in both cytosolic and particulate fractions. In addition, arginase was detected in isolated acinar cells, but not in duct cells. Experiments on the intracellular distribution and the effects of the arginase inhibitors ornithine and N-hydroxy-L-arginine (NOHA), suggested the presence of both arginase I and arginase II in rat submandibular gland.Abbreviations cGMP cyclic guanosine 3,5-monophosphate - NO nitric oxide - NOHA N-hydroxy-L-arginine - NOS nitric oxide synthase Communicated by I.D. Hume     

Nitric oxide and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

Time course of vascular arginase expression and activity in spontaneously hypertensive rats

There is growing evidence that vascular arginase plays a role in pathophysiology of vascular diseases. We recently reported high arginase activity/expression in young adult hypertensive spontaneously hypertensive rats (SHR). The aim of the present study was to characterize the time course of arginase pathway abnormalities in SHR and to explore the contributing role of hemodynamics and inflammation. Experiments were conducted on 5, 10, 19 and 26-week-old SHR and their age-matched control Wistar Kyoto (WKY) rats. Arginase activity as well as expression of arginase I, arginase II, endothelial and inducible NOS were determined in aortic tissue extracts. Levels of L-arginine, NO catabolites and IL-6 (a marker of inflammation) were measured in plasma. Arginase activity/expression was also measured in 10-week-old SHR previously treated with hydralazine (20 mg/kg/day, per os, for 5 weeks). As compared to WKY, SHR exhibited high vascular arginase I and II expression from prehypertensive to established stages of hypertension. However, a mismatch between expression and activity was observed at the prehypertensive stage. Arginase expression was not related either to plasma IL-6 levels or to expression of NOS. Prevention of hypertension by hydralazine significantly blunted arginase upregulation and restored arginase activity. Importantly, arginase activity and blood pressure (BP) correlated in SHR. In conclusion, our results demonstrate that arginase upregulation precedes blood pressure rising and identify elevated blood pressure as a contributing factor of arginase dysregulation in genetic hypertension. They also demonstrated a close relationship between arginase activity and BP, thus making arginase a promising target for antihypertensive therapy.     

Identification and structure activity relationship of novel flavone derivatives that inhibit the production of nitric oxide and PGE2 in LPS-induced RAW 264.7 cells

In an effort to identify novel anti-inflammatory compounds, a series of flavone derivatives were synthesized and biologically evaluated for their inhibitory effects on the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂), representative pro-inflammatory mediators, in LPS-induced RAW 264.7 cells. Their structure-activity relationship was also investigated. In particular, we found that compound 3g displayed more potent inhibitory activities on PGE₂ production, similar inhibitory activities on NO production and less weak cytotoxicity than luteolin, a natural flavone known as a potent anti-inflammatory agent.     

Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO)

Background

Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase.

Methods

The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo.

Results

MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling.

Conclusions

Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders.

General significance

MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be utilized for the development of a new candidate for treatment of the hyper-pigmentation disorders.     

Increased nitric oxide formation followed by increased arginase activity induces relative lack of arginine at the wound site and alters whole nutritional status in rats almost within the early healing period

Nitric oxide (NO) production and free amino acid fluxes at the wound side during the first 3 days following cutaneous wound were investigated. Experiments were performed on Albino Oxford rats (n = 18) underwent cutaneous implantation of polyvinyl sponges. Intact animals (n = 6) were controls. Nitrites, nitrates, free amino acids and urea were measured both in plasma and wound fluids. Inducible nitric oxide synthase (iNOS) gene expressions at wound site were analyzed, too. The highest levels of both iNOS gene expression and its activity (increased wound fluid citrulline and nitrites) were at the first day. Wound fluid nitrates were significantly above plasma levels throughout the whole period, while molar nitrate to nitrite ratio steadily increased. It was associated with gradual increase of both ornithine and urea as well as steadily decreases of arginine and increases of phenylalanine at the wound site. Gradual decrease in glycine to branched-chain molar ratio was observed both in plasma and wound fluids. In conclusion, an early locally induced alterations in Arg metabolism, due to increased NO formation followed by increased arginase activity, produces relative lack of Arg at the wound site and disturbs nutritional status of the whole body almost within early healing period following cutaneous wound in rats. It is likely that NO autoxidation at the wound side is influenced by substrate availability.     

Relationship between serum arginase I and l-arginine or exhaled nitric oxide in asthma

The relationship between serum arginase I and serum l-arginine or fractional exhaled nitric oxide (FENO) was evaluated cross-sectionally in asthmatic patients. No sex difference was observed in the serum mean levels of arginase I and l-arginine or FENO. Arginase I and FENO were higher in patients 60 or younger years than in those over 60 years. Asthmatic patients were divided into three groups: no steroid therapy, inhalation steroid therapy, and oral steroid therapy. Arginase I, FENO and high-sensitivity-C-reactive protein (hs-CRP) were significantly lower in the inhalation steroid therapy group than in the no steroid therapy group. Correlations were observed between arginase I and FENO, l-arginine, hs-CRP, WBC, and age, and also between FENO and IgE, WBC, and age. A logistic regression analysis revealed the positive association of arginase I with FENO, and the negative association of l-arginine. FENO was positively associated with arginase I and IgE. These results indicated that serum arginase I might influence serum levels of l-arginine and FENO, and that IgE might influence FENO in asthmatic patients.     

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.     

Relationship among managanese, arginase, and nitric oxide in childhood asthma

It has been demonstrated that the lowest intakes of manganese (Mn) were associated with more than a fivefold increased risk of bronchial reactivity. It was also known that nitric oxide (NO) production was found to be significantly higher in asthmatics. There is a reciprocal pathway between arginase and nitric oxide synthase (NOS) for NO production, and Mn is required for arginase activity and stability. We investigated plasma NO, arginase, and its cofactor Mn levels to evaluate this reciprocal pathway in patients with childhood asthma. Arginase activities and Mn and NO levels were measured in plasma from 31 patients with childhood asthma and 22 healthy control subjects. Plasma arginase activities and Mn concentrations were found to be significantly lower and NO levels were significantly higher found to be significantly lower and NO levels were significantly higher in patients with childhood asthma as compared to the control subjects. There was a significantly positive correlation between plasma Mn and arginase and negative correlations between arginase and NO values and Mn and NO values in patients with childhood asthma. These data indicate that the lower concentration of Mn could cause lower arginase activity and this could also upregulate NO production by increasingl-arginine content in patients with childhood asthma.     

Gold nanoparticle modifies nitric oxide release and vasodilation in rat aorta

Nitric oxide (NO) plays an important role on several biological functions. Recently, it has been reported the possibility of modifying the NO release profile from the NO donors through its coupling to gold nanoparticles (AuNPs). Thus, AuNPs were synthesized and they were exposed to the NO donor ruthenium complex Cis-[Ru(bpy)₂(NO)(4PySH)].(PF6)₃ termed (Ru-4PySH)—forming AuNPs-{Ru-4PySH}_n cluster. Our results indicate that AuNPs do not modify the maximum effect (ME) and potency (pD₂) in the vasodilation induced by Ru-4PySH. Both complexes induce similar vascular relaxation in concentration-dependent way. However, the NO released from the complex AuNPs-{Ru-4PySH}_n is lower than Ru-4PySH. Both complexes release only NO⁰ specie, but AuNPs-{Ru-4PySH}_n releases NO in constant way and exclusively in the extracellular medium. In time-course, Ru-4Py-SH was faster than AuNPs-{Ru-4PySH}_n in inducing the maximum vasodilation. Inhibition of soluble guanylyl cyclase (sGC) abolished the vasodilation induced by Ru-4PYSH, but not by AuNPs-{Ru-4PySH}_n. Non-selective potassium (K⁺) channel blocker TEA had no effect on the vasodilation induced by AuNPs-{Ru-4PySH}_n, but it reduced the potency to Ru-4PySH. In conclusion, our results suggest that AuNPs can reduce the permeability of NO donor Ru-4PySH due to AuNPs-{Ru-4PySH}_n cluster formation. AuNPs reduce NO release, but they do not impair the vasodilator effect induced by the NO donor. Ru-4PySH induces vasodilation by sGC and K⁺ channels activation, while AuNPs-{Ru-4PySH}_n activates mainly sGC. Taken together, these findings represent a new pharmacological strategy to control the NO release which could activate selective biological targets.     

低氧对高原鼠兔和大鼠血液NO 与ET-1的影响

将Wistar大鼠暴露于3 780 m低氧环境,分别于24 h、2 wk及3 wk后采用酶联免疫法和硝酸还原酶法测定血液中的ET~(-1)和NO的含量,计算NO/ET~(-1)值,并与高原鼠兔比较,探讨低氧条件下大鼠与高原鼠兔血液中NO与ET~(-1)含量的变化趋势。结果表明,低氧24 h后,大鼠血液中NO和ET~(-1)的含量显著高于同海拔的高原鼠兔(P<0·01),而NO/ET~(-1)值无显著差异(P>0·05)。随着大鼠在高海拔停留时间的延长,血液中NO含量呈减少趋势,而ET~(-1)则有上升趋势,二者呈显著的负相关(r2=0·2416,P<0·01)。高原鼠兔NO/ET~(-1)值约为大鼠低氧2 wk和3 wk的2倍(P<0·01)。说明不同低氧暴露时间,高原鼠兔和大鼠的NO、ET~(-1)及NO/ET~(-1)值有显著差异,提示NO/ET~(-1)值可以作为有机体是否适应高原低氧环境的一个指标。     

The role of L-arginine in toxic liver failure: interrelation of arginase,polyamine catabolic enzymes and nitric oxide synthase

Summary. The existing interrelation in metabolic pathways of L-arginine to polyamines, nitric oxide (NO) and urea synthesis could be affected in sepsis, inflammation, intoxication and other conditions. The role of polyamines and NO in the toxic effect of mercury chloride on rat liver function was studied. Administration of mercury chloride for 24 h led to significantly elevated plasma activities of Alanine transaminase (ALT) and Aspartate transaminase (AST). Malondyaldehyde (MDA) levels were unaffected (p > 0.05) and arginase activity was significantly decreased (p < 0.05) while nitrate/nitrite production was significantly elevated (p < 0.001) in liver tissue. Polyamine oxidase (PAO) and diamine oxidase (DAO) activities, enzymes involved in catabolism of polyamines, were decreased. L-arginine supplementation to intoxicated rats potentiated the effect of mercury chloride on NO production and it was ineffective on arginase activity. Results obtained in this study show that mercury chloride-induced toxicity leads to abnormally high levels of ALT and AST that may indicate liver damage with the involvement of polyamine catabolic enzymes and NO.     

EPR spin-trapping study of nitric oxide formation during bilateral carotid occlusion in the rat

The formation of nitric oxide (NO) radicals was demonstrated by electron paramagnetic resonance spectroscopy in the rat during varying degrees of brain ischemia. Diethyldithiocarbamate and Fe-citrate were used as in vivo spin-trapping reagents. The signal of NO spin adducts increased in accordance with the degree of ischemic insults. The formation of NO radicals was inhibited by N^G-nitro]l-arginine methyl ester.     

The role of nitric oxide in cancer

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including va-sodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases(NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interest-ingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting theetiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and hasbeen associated with tumour grade, proliferation rate and expression of important signaling componentsassociated with cancer development such as the oestrogen receptor. It appears that high levels of NOSexpression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumorcells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxicallytherefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic properties.Increased NO-generation in a cell may select mutant p53 cells and contribute to tumour angiogenesis byupregulating VEGF. In addition, NO may modulate tumour DNA repair mechanisms by upregulating p53,poly(ADP-ribose) polymerase (PARP) and the DNA-dependent protein kinase (DNA-PK). An understand-ing at the molecular level of the role of NO in cancer will have profound therapeutic implications for thediagnosis and treatment of disease.     

The role of nitric oxide in cancer

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including va-sodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic pro     

The role of nitric oxide in orofacial pain

Nitric oxide (NO) is a free radical gas that has been shown to be produced by nitric oxide synthase (NOS) in different cell types and recognized to act as a neurotransmitter or neuromodulator in the nervous system. NOS isoforms are expressed and/or can be induced in the related structures of trigeminal nerve system, in which the regulation of NOS biosynthesis at different levels of gene expression may allow for a fine control of NO production. Several lines of evidence suggest that NO may play a role through multiple mechanisms in orofacial pain processing. This report will review the latest evidence for the role of NO involved in orofacial pain and the potential cellular mechanisms are also discussed.