Nitric Oxide Abstracts 5



Effects of hesperidin on cyclic strain-induced endothelin-1 release in human umbilical vein endothelial cells.
            (Chiou et al., 2008) Download
1. Hesperidin, a member of the flavanone group of flavonoids, can be isolated in large amounts from the rinds of some citrus species and has been reported to have antihypotensive and vasodilator properties. However, the mechanism of action of hesperidin in the prevention and treatment of vascular diseases remains unclear. 2. The vascular endothelium can produce potent contracting factors, such as endothelin (ET)-1, and endothelium-derived relaxing factors, such as nitric oxide (NO). The aims of the present study were to test the hypothesis that hesperidin may alter strain-induced ET-1 secretion and NO production and to identify the putative underlying signalling pathways in human umbilical vein endothelial cells (HUVEC). 3. Hesperidin (10 and 100 micromol/L) inhibited strain-induced ET-1 secretion. Hesperidin also inhibited strain-induced increases in the formation of reactive oxygen species and extracellular signal-regulated kinase (ERK) phosphorylation. 4. Hesperidin treatment of HUVEC enhanced NO production, endothelial NO synthase (eNOS) activity and the phosphorylation of eNOS and Akt. Furthermore, hesperidin modulated strain-induced ET-1 release and suppressed ERK phosphorylation in part via the NO/protein kinase G pathway. 5. In summary, we have demonstrated that hesperidin inhibits strain-induced ET-1 secretion and enhances NO production in HUVEC.

Pomegranate juice reduces oxidized low-density lipoprotein downregulation of endothelial nitric oxide synthase in human coronary endothelial cells.
            (de Nigris et al., 2006) Download
We examined the hypothesis that pomegranate juice (PJ) can revert the potent downregulation of the expression of endothelial nitric-oxide synthase (NOSIII) induced by oxidized low-density liporotein (oxLDL) in human coronary endothelial cells. Western blot and Northern blot analyses showed a significant decrease of NOSIII expression after a 24-h treatment with oxLDL. Accordingly, we observed a significant dose-dependent reduction in nitric oxide bioactivity represented by both basal and bradykinin-stimulated cellular cGMP accumulation. These phenomena were corrected significantly by the concomitant treatment with PJ. Our data suggest that PJ can exert beneficial effects on the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in humans by enhancing the NOSIII bioactivity.

The role of the nitric oxide pathway in brain injury and its treatment--from bench to bedside.
            (Garry et al., 2015) Download
Nitric oxide (NO) is a key signalling molecule in the regulation of cerebral blood flow. This review summarises current evidence regarding the role of NO in the regulation of cerebral blood flow at rest, under physiological conditions, and after brain injury, focusing on subarachnoid haemorrhage, traumatic brain injury, and ischaemic stroke and following cardiac arrest. We also review the role of NO in the response to hypoxic insult in the developing brain. NO depletion in ischaemic brain tissue plays a pivotal role in the development of subsequent morbidity and mortality through microcirculatory disturbance and disordered blood flow regulation. NO derived from endothelial nitric oxide synthase (eNOS) appears to have neuroprotective properties. However NO derived from inducible nitric oxide synthase (iNOS) may have neurotoxic effects. Cerebral NO donor agents, for example sodium nitrite, appear to replicate the effects of eNOS derived NO, and therefore have neuroprotective properties. This is true in both the adult and immature brain. We conclude that these agents should be further investigated as targeted pharmacotherapy to protect against secondary brain injury.

Nitric oxide scavenging by hydroxocobalamin may account for its hemodynamic profile.
            (Gerth et al., 2006) Download
BACKGROUND: Antidotal doses of hydroxocobalamin are associated with transient increases in blood pressure in some animals and humans. These studies in anesthetized rabbits were undertaken to explore the possible mechanisms underlying the hemodynamic effects of hydroxocobalamin by investigating 1) possible hemodynamic effects of cyanocobalamin, which is formed on a molar-to-molar basis when hydroxocobalamin binds cyanide, and 2) the interference of hydroxocobalamin with the endothelial nitric oxide system. METHODS: Study 1 investigated the hemodynamic effects of cyanocobalamin. This study included two treatment arms: 1) cyanocobalamin (75 mg/kg, IV) followed by saline (n = 7) and 2) saline followed by cyanocobalamin (n = 7). Study 2 assessed the hemodynamic effects of hydroxocobalamin (75 mg/kg, IV) in the presence and absence of the nitric oxide synthase inhibitor L-Nomega-nitro-L-arginine methyl ester (L-NAME; 30 mg/kg, IV). Nitric oxide synthase inhibition itself increases blood pressure. Thus, as part of Study 2, the hemodynamic effects of hydroxocobalamin were also investigated in the presence of an equipressor dose of angiotensin II (ANGII; 0.05 microg/kg/min, IV) in order to determine whether elevated blood pressure per se could interfere with hydroxocobalamin's hemodynamic effects. This study included six treatment arms (designated as first treatment + second treatment): saline + saline (n = 5), L-NAME + saline (n = 7), saline + hydroxocobalamin (n = 7), L-NAME + hydroxocobalamin (n = 7), ANGII + hydroxocobalamin (n = 7), and ANGII + saline (n = 7). RESULTS: In Study 1, the effects of cyanocobalamin on hemodynamic parameters were indistinguishable from those of saline. In Study 2, hydroxocobalamin infusion was associated with moderate hemodynamic effects, including an increase in systemic vascular resistance, an increase in blood pressure, and a decrease in cardiac output. Administration of L-NAME abolished the effects of hydroxocobalamin on all hemodynamic parameters. ANGII at a dose producing a pressor response comparable to that of L-NAME did not influence the hydroxocobalamin-associated hemodynamic changes. CONCLUSION: These studies in anesthetized rabbits demonstrate that the moderate pressor effect of hydroxocobalamin is not related to the formation of cyanocobalamin but is very likely related to the scavenging of nitric oxide by hydroxocobalamin.

Is nitric oxide a hormone
            (Ghasemi and Zahediasl, 2011) Download
Nitric oxide (NO) is a simple ubiquitous signaling molecule and plays important roles in almost every biological system. Recent evidences suggest that NO may act as an endocrine molecule. The aim of this review is considering available literature on endocrine roles of NO and/or its metabolites, i.e. nitrite and nitrate. Existing data suggest the idea that NO is a hormone that after production in tissues, it is stabilized and transported as nitrite and/or S-nitrosothiols in the blood to target cells.

Pomegranate juice protects nitric oxide against oxidative destruction and enhances the biological actions of nitric oxide.
            (Ignarro et al., 2006) Download
Pomegranate juice (PJ), which is a rich source of potent flavonoid antioxidants, was tested for its capacity to protect nitric oxide (NO) against oxidative destruction and enhance the biological actions of NO. Employing chemiluminescence headspace analysis, PJ was found to be a potent inhibitor of superoxide anion-mediated disappearance of NO. PJ was much more potent than Concord grape juice, blueberry juice, red wine, ascorbic acid, and DL-alpha-tocopherol. As little as 3 microl of a 6-fold dilution of PJ, in a reaction volume of 5000 microl, produced a marked antioxidant effect, whereas 300 microl of undiluted blueberry juice or nearly 1000 microl of undiluted Concord grape juice were required to produce similar effects. PJ and other antioxidant-containing products were found to augment the anti-proliferative action of NO (DETA/NO) on vascular smooth muscle cell (rat aorta) proliferation. PJ was much more effective than the other products tested and elicited no effects when tested alone in the absence of added NO. Similarly, neither PJ nor the other products enhanced the anti-proliferative action of alpha-difluoromethylornithine, a stable substance that inhibits cell growth by NO-independent mechanisms. In order to determine whether PJ is capable of increasing the production of NO by vascular endothelial cells, PJ was tested for its capacity to upregulate and/or activate endothelial NO synthase (eNOS) in bovine pulmonary artery endothelial cells. PJ elicited no effects on eNOS protein expression or catalytic activity. Moreover, PJ did not enhance promoter activity in the eNOS gene (COS-7 cells transfected with eNOS). These observations indicate that PJ possesses potent antioxidant activity that results in marked protection of NO against oxidative destruction, thereby resulting in augmentation of the biological actions of NO.

Distinct effects of naringenin and hesperetin on nitric oxide production from endothelial cells.
            (Liu et al., 2008) Download
Diets rich in citrus and citrus-based products have been negatively correlated with the risk of cardiovascular disease, but so far no studies have been conducted to determine whether naringenin and hesperetin, two major flavanones in citrus plants, influence endothelium nitric oxide (NO) production. The aim of this study is to clarify estrogenic activities of naringenin and hesperetin and to examine whether they affect endothelial NO production via estrogen receptor (ER) activation. Both naringenin and hesperetin were observed to promote growth of MCF-7 cells under greatly reduced estrogen conditions and to suppress estrogen-induced response. Naringenin activated both ERalpha and ERbeta, whereas hesperetin exhibited stronger potential to activate ERalpha rather than ERbeta. Hesperetin, but not naringenin, increased NO releases from human umbilical vein endothelial cells in a dose-dependent manner. Hesperetin-induce responses were suppressed by ICI 182 780 and actinomycin D. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western-blotting analysis revealed that hesperetin up-regulated endothelium nitric oxide synthase (eNOS) expression. These results suggested that hesperetin exerts an antiatherogenic effect, in part, via ER-mediated eNOS expression and subsequent increase of endothelial NO production. Distinct effects of naringenin and hesperetin on NO production also imply that ERalpha might play the major role in estrogen-induced eNOS expression. However, the inefficacy of naringenin on NO production remains to be elaborately studied. Our findings add more proof to the molecular explanations for the health benefits of citrus used to prevent cardiovascular disease, especially for postmenopausal women.

Nitric oxide (NO) scavenging and NO protecting effects of quercetin and their biological significance in vascular smooth muscle.
            (Lopez-Lopez et al., 2004) Download
The flavonoid quercetin reduces blood pressure and endothelial dysfunction in animal models of hypertension. However, the results concerning the relationship between quercetin and NO present a complex picture. We have analyzed the mechanisms involved in the NO scavenging effects of quercetin and its repercussion on NO bioactivity in vascular smooth muscle. Quercetin scavenged NO with apparent zero-order kinetics with respect to NO. This effect was strongly dependent on the O(2) concentrations, so that NO decay at pH 7.4 could be fitted to the equation -d[NO]/dt = k x [O(2)] x [quercetin], where k was 0.15 M(-1) s(-1). The NO scavenger effects were prevented by superoxide dismutase (SOD), reduced by lowering pH, accompanied by O(2)(.) production and correlated with decreased NO bioactivity in rat aortic rings. However, under conditions of increased O(2)(.) concentrations, quercetin was a better scavenger of O(2)(.) than of NO. When NO scavenging by quercetin was prevented by addition of SOD, NO bioactivity was increased. Quercetin also prevented the inhibitory effects of the SOD inhibitor diethyldithiocarbamic acid (DETCA) on NO bioactivity. In the presence of DETCA, quercetin reduced tissue O(2)(.) as measured by nitro blue tetrazolium staining. In conclusion, quercetin exerts dual effects on O(2)(.) and NO. At physiological conditions of pH, O(2) concentrations and NO, quercetin effectively scavenged NO in the low micromolar range, and the rate-limiting step was the autooxidation of quercetin and the formation of O(2)(.). When the extracellular NO scavenging effect was prevented, quercetin increased the biological activity of NO, an effect related to its O(2)(.) scavenger properties and/or its inhibitory effect on tissue O(2)(.) generation.

The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761.
            (Marcocci et al., 1994) Download
Ginkgo biloba extract EGb 761 was found to be a scavenger of nitric oxide in in vitro acellular systems, under physiological conditions. EGb 761 competed with oxyhemoglobin for reaction with nitric oxide generated during the interaction of hydroxylamine with Complex I of catalase. An EGb 761 dose-dependent decrease in the amount of nitrite formed in the reaction of oxygen with nitric oxide produced from solution of 5 mM sodium nitroprusside was also observed. These data implicate it as a potential therapeutic agent in conditions of altered production of nitric oxide.


Interactions between hydroxocobalamin and nitric oxide (NO): evidence for a redox reaction between NO and reduced cobalamin and reversible NO binding to oxidized cobalamin.
            (Rochelle et al., 1995) Download
Interactions of nitric oxide (NO) with various cobalamin species have been examined, apparently for the first time, with both absorption and electron paramagnetic resonance spectroscopy. Only slight shifts in the absorption spectrum of hydroxocobalamin, B12a [Cb(III)], were produced by NO, but dramatic changes in the spectrum of B12r [Cb(III)] were found on addition of NO. The addition of NO shifted the spectrum of Cb(II) to one very similar to that of Cb(III), indicating the oxidation of Cb(II). The addition of NO to Cb(III) resulted in a novel, weak and previously undescribed electron paramagnetic resonance signal. Although it has not been fully characterized, this appears to represent a reversible complex in which NO is liganded to the Cb(III). When NO was added to Cb(II), its strong electron paramagnetic resonance spectrum was replaced by that of this novel species, consistent with oxidation of Cb(II) by NO and then binding of additional NO by the resulting Cb(III). Porcine, aortic endothelial cells were able to partially reduce Cb(III), and release to the supernatant a previously characterized superoxide cobalt(III) complex, but some Cb(II) remained with the cell fraction. These reactions of Cb species could play a role in altering intracellular and intratissue levels of NO.

Nitric oxide scavenging by curcuminoids.
            (Sreejayan and Rao, 1997) Download
Because curcumin, a compound with anti-inflammatory and anticancer activity, inhibits induction of nitric oxide synthase in activated macrophages and has been shown to be a potent scavenger of free radicals we have investigated whether it can scavenge nitric oxide directly. Curcumin reduced the amount of nitrite formed by the reaction between oxygen and nitric oxide generated from sodium nitroprusside. Other related compounds, e.g. demethoxycurcumin, bisdemethoxycurcumin and diacetylcurcumin were as active as curcumin, indicating that the methoxy and the phenolic groups are not essential for the scavenging activity. The results indicate curcumin to be a scavenger of nitric oxide. Because this compound is implicated in inflammation and cancer, the therapeutic properties of curcumin against these conditions might be at least partly explained by its free-radical scavenging properties, including those toward nitric oxide.


Hydroxocobalamin, a nitric oxide scavenger, in the prophylaxis of migraine: an open, pilot study.
            (van der Kuy et al., 2002) Download
Drugs which directly counteract nitric oxide (NO), such as endothelial receptor blockers, NO-synthase inhibitors, and NO-scavengers, may be effective in the acute treatment of migraine, but are also likely to be effective in migraine prophylaxis. In the underlying pilot study the prophylactic effect of the NO scavenger hydroxocobalamin after intranasal administration in migraine was evaluated. Twenty patients, with a history of migraine of > 1 year and with two to eight migraine attacks per month, were included in an open trial. A baseline period was followed by an active treatment period of 3 months with 1 mg intranasal hydroxocobalamin daily. Patients were instructed to complete a diary in which details of each attack were described. A reduction in migraine attack frequency of >/ or = 50% was seen in 10 of 19 patients, which corresponds to 53% of the patients (responders). A reduction of > or = 30% was noted in 63% of the patients. The mean attack frequency in the total study population showed a reduction from 4.7 +/- 1.7 attacks per month to 2.7 +/- 1.6 (P < 0.001). For the responders the migraine attack frequency was reduced from 5.2 +/- 1.9 (baseline) to 1.9 +/- 1.3 attacks per month (P < 0.005), while for those who did not respond a non-significant reduction was found: 4.1 +/- 1.4 to 3.7 +/- 1.5 (P > 0.1). A reduction was also observed for the total duration of the migraine attacks per month, the total number of migraine days per month and the number of medication doses for acute treatment used per month. This is the first prospective, open study indicating that intranasal hydroxocobalamin may have a prophylactic effect in migraine. As a percentage of responders in prophylactic trials of > 35-40% is unlikely to be a placebo effect, a double-blind study is warranted.


The return of the Scarlet Pimpernel: cobalamin in inflammation II - cobalamins can both selectively promote all three nitric oxide synthases (NOS), particularly iNOS and eNOS, and, as needed, selectively inhibit iNOS and nNOS.
            (Wheatley, 2007) Download
The up-regulation of transcobalamins [hitherto posited as indicating a central need for cobalamin (Cbl) in inflammation], whose expression, like inducible nitric oxide synthase (iNOS), is Sp1- and interferondependent, together with increased intracellular formation of glutathionylcobalamin (GSCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), may be essential for the timely promotion and later selective inhibition of iNOS and concordant regulation of endothelial and neuronal NOS (eNOS/nNOS.) Cbl may ensure controlled high output of nitric oxide (NO) and its safe deployment, because: (1) Cbl is ultimately responsible for the synthesis or availability of the NOS substrates and cofactors heme, arginine, BH(4) flavin adenine dinucleotide/flavin mononucleotide (FAD/FMN) and NADPH, via the far-reaching effects of the two Cbl coenzymes, methionine synthase (MS) and methylmalonyl CoA mutase (MCoAM) in, or on, the folate, glutathione, tricarboxylic acid (TCA) and urea cycles, oxidative phosphorylation, glycolysis and the pentose phosphate pathway. Deficiency of any of theNOS substrates and cofactors results in 'uncoupled' NOS reactions, decreasedNO production and increased or excessive O(2) (-), H(2)O(2), ONOO(-) and other reactive oxygen species (ROS), reactive nitric oxide species (RNIS) leading to pathology. (2) Cbl is also the overlooked ultimate determinant of positive glutathione status, which favours the formation of more benign NO species, s-nitrosothiols, the predominant form in which NO is safely deployed. Cbl status may consequently act as a 'back-up disc' that ensures the active status of antioxidant systems, as well as reversing and modulating the effects of nitrosylation in cell signal transduction.New evidence shows that GSCbl can significantly promote iNOS/ eNOS NO synthesis in the early stages of inflammation, thus lowering high levels of tumour necrosis factor-a that normally result in pathology, while existing evidence shows that in extreme nitrosative and oxidative stress, GSCbl can regenerate the activity of enzymes important for eventual resolution, such as glucose 6 phosphate dehydrogenase, which ensures NADPH supply, lactate dehydrogenase, and more; with human clinical case studies of OHCbl for cyanide poisoning, suggesting Cbl may regenerate aconitase and cytochrome c oxidase in the TCA cycle and oxidative phosphorylation. Thus, Cbl may simultaneously promote a strong inflammatory response and the means to resolve it.



Chiou, CS, et al. (2008), ‘Effects of hesperidin on cyclic strain-induced endothelin-1 release in human umbilical vein endothelial cells.’, Clin Exp Pharmacol Physiol, 35 (8), 938-43. PubMedID: 18430059
de Nigris, F, et al. (2006), ‘Pomegranate juice reduces oxidized low-density lipoprotein downregulation of endothelial nitric oxide synthase in human coronary endothelial cells.’, Nitric Oxide, 15 (3), 259-63. PubMedID: 16413211
Garry, PS, et al. (2015), ‘The role of the nitric oxide pathway in brain injury and its treatment--from bench to bedside.’, Exp Neurol, 263 235-43. PubMedID: 25447937
Gerth, K, et al. (2006), ‘Nitric oxide scavenging by hydroxocobalamin may account for its hemodynamic profile.’, Clin Toxicol (Phila), 44 Suppl 1 29-36. PubMedID: 16990191
Ghasemi, A and S Zahediasl (2011), ‘Is nitric oxide a hormone’, Iran Biomed J, 15 (3), 59-65. PubMedID: 21987110
Ignarro, LJ, et al. (2006), ‘Pomegranate juice protects nitric oxide against oxidative destruction and enhances the biological actions of nitric oxide.’, Nitric Oxide, 15 (2), 93-102. PubMedID: 16626982
Liu, L, DM Xu, and YY Cheng (2008), ‘Distinct effects of naringenin and hesperetin on nitric oxide production from endothelial cells.’, J Agric Food Chem, 56 (3), 824-29. PubMedID: 18197618
Lopez-Lopez, G, et al. (2004), ‘Nitric oxide (NO) scavenging and NO protecting effects of quercetin and their biological significance in vascular smooth muscle.’, Mol Pharmacol, 65 (4), 851-59. PubMedID: 15044614
Marcocci, L, et al. (1994), ‘The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761.’, Biochem Biophys Res Commun, 201 (2), 748-55. PubMedID: 8003011
Rochelle, LG, et al. (1995), ‘Interactions between hydroxocobalamin and nitric oxide (NO): evidence for a redox reaction between NO and reduced cobalamin and reversible NO binding to oxidized cobalamin.’, J Pharmacol Exp Ther, 275 (1), 48-52. PubMedID: 7562589
Sreejayan and MN Rao (1997), ‘Nitric oxide scavenging by curcuminoids.’, J Pharm Pharmacol, 49 (1), 105-7. PubMedID: 9120760
van der Kuy, PH, et al. (2002), ‘Hydroxocobalamin, a nitric oxide scavenger, in the prophylaxis of migraine: an open, pilot study.’, Cephalalgia, 22 (7), 513-19. PubMedID: 12230592
Wheatley, C (2007), ‘The return of the Scarlet Pimpernel: cobalamin in inflammation II - cobalamins can both selectively promote all three nitric oxide synthases (NOS), particularly iNOS and eNOS, and, as needed, selectively inhibit iNOS and nNOS.’, J Nutr Environ Med, 16 (3-4), 181-211. PubMedID: 18836533