Nitric Oxide Abstracts 1


Could thiamine pyrophosphate be a regulator of the nitric oxide synthesis in the endothelial cell of diabetic patients?

            (Alcazar-Leyva and Alvarado-Vasquez 2011) Download

Thiamine (Vitamin B1) is considered an essential micronutrient for humans; its deficient intake brings about the Wernicke-Korsakoff syndrome (encephalopathy and psychosis) or beriberi (a neurological and cardiovascular disease). Once thiamine enters the cells it is phosphorylated by thiamine pyrophosphokinase (TPPK), and converted into the coenzyme thiamine pyrophosphate (TPP), the active form of thiamine. TPP is a relevant cofactor for transketolase (TK), alpha-ketoglutarate dehydrogenase (alphaKDH), and pyruvate dehydrogenase (PDH), all these enzymes are fundamental for glucose metabolism. Diabetes mellitus (DM), however, is considered both a deficient thiamine and deficient energy state, as a consequence of the limited TPP synthesis. Recent evidences have shown that the administration of thiamine or lipid-soluble derivatives, such as benfotiamine (developed to improve the bioavailability of thiamine), has positive effects in the diabetic patient (after thiamine is transformed into TPP). For this reason, administration of supplements with TPP in the diabetic patients is recommended to avoid complications, like neuropathy and nephropathy. It has been suggested that these beneficial effects are a consequence of the activation of TK (pentose pathway) or the PDH complex in mitochondria. Nitric oxide (NO) is synthesized by the endothelial cell and is also an important element for the viability and functionality of this cell type. However, in the DM patient, a deficient synthesis of NO has been reported. It is relevant to mention that recent evidences have led to propose mitochondrial activity as an important regulator of nitric oxide synthesis (ON). We consider that the exogenous administration of TPP facilitates the utilization of this molecule, regulating some metabolic processes such as phosphorylation of thiamine by TPPK, energy consumption (ATP), as well as mitochondrial activity, inducing eventually NO synthesis. If this is confirmed, the administration of TPP to the diabetic patient would provide additional protection to endothelial cells, reducing the risk of vascular damage, to which the diabetic patient is highly susceptible.

Effect of Grape Seed Extract and Quercetin on Cardiovascular and Endothelial Parameters in High-Risk Subjects

            (Clifton 2004) Download

Grape seed extract (GSE) has in vitro antioxidant activity but whether or not it works in vivo is not clear. In a fully randomised, crossover trial with 4-week treatment periods on 36 men and women with above-average vascular risk, we aimed to demonstrate that 2 g/day of GSE (1 g of polyphenols) alone, or with 1 g/day of added quercetin in yoghurt, favourably alters vascular function, endothelial function, and degree of oxidative damage in comparison to a control yoghurt. GSE alone improved flow-mediated dilatation determined ultrasonically by an absolute $1.1$ % compared with control. There was no effect of the combination of GSE with quercetin. No other blood or urine measure was altered. Thus sufficient polyphenols from GSE appear to be absorbed to influence endothelial nitric oxide production, and GSE has the potential to favourably influence vascular function.

eNOS-uncoupling in age-related erectile dysfunction

            (Johnson, Bivalacqua et al. 2011) Download

Aging is associated with ED. Although age-related ED is attributed largely to increased oxidative stress and endothelial dysfunction in the penis, the molecular mechanisms underlying this effect are not fully defined. We evaluated whether endothelial nitric oxide synthase (eNOS) uncoupling in the aged rat penis is a contributing mechanism. Correlatively, we evaluated the effect of replacement with eNOS cofactor tetrahydrobiopterin (BH(4)) on erectile function in the aged rats. Male Fischer 344 'young' (4-month-old) and 'aged' (19-month-old) rats were treated with a BH(4) precursor sepiapterin (10 mg/kg intraperitoneally) or vehicle for 4 days. After 1-day washout, erectile function was assessed in response to electrical stimulation of the cavernous nerve. Endothelial dysfunction (eNOS uncoupling) and oxidative stress (thiobarbituric acid reactive substances, TBARS) were measured by conducting western blot in penes samples. Erectile response was significantly reduced in aged rats, whereas eNOS uncoupling and TBARS production were significantly increased in the aged rat penis compared with young rats. Sepiapterin significantly improved erectile response in aged rats and prevented increase in TBARS production, but did not affect eNOS uncoupling in the penis of aged rats. These findings suggest that aging induces eNOS uncoupling in the penis, resulting in increased oxidative stress and ED.

Inhibition of nitric oxide production by quercetin in endotoxin/cytokine-stimulated microglia

            (Kao, Ou et al. 2010) Download

AIMS: Flavonoids possess several biological and pharmacological activities. Quercetin, a naturally occurring flavonoid, has been shown to down-regulate inflammatory responses and provide neuroprotection. However, the mechanisms underlying the anti-inflammatory properties of quercetin are poorly understood. In the present study, we investigated the modulatory effect of quercetin against neuroinflammation. MAIN METHODS: We herein describe a potential regulatory mechanism by which quercetin suppresses nitric oxide (NO) production by lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-stimulated BV-2 microglial cells. The underlying regulatory cascades were approached by biochemical and pharmacological strategies. KEY FINDINGS: Quercetin produced an inhibitory effect on inducible nitric oxide synthase (iNOS) expression and NO production. Biochemical studies revealed that the anti-inflammatory effect of quercetin was accompanied by the down-regulation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, Akt, Src, Janus kinase-1, Tyk2, signal transducer and activator of transcription-1, and NF-kappaB. In addition, quercetin scavenged free radicals and produced inhibitory effects on serine/threonine and tyrosine phosphatase activities. Intriguingly, the accumulation of lipid rafts, which is the critical step for signaling, was disrupted by quercetin. SIGNIFICANCE: The data indicate that the anti-inflammatory action of quercetin may be attributable to its raft disrupting and anti-oxidant effects. These distinct mechanisms work in synergy to down-regulate iNOS expression and NO production.

Regulation of nitric oxide production in health and disease

            (Luiking, Engelen et al. 2010) Download

PURPOSE OF REVIEW: The purpose of this review is to highlight recent publications examining nitric oxide production in health and disease and its association with clinical nutrition and alterations in metabolism. RECENT FINDINGS: The role of the cofactor tetrahydrobiopterin in nitric oxide production and its relation with arginine availability is indicated as an important explanation for the arginine paradox. This offers potential for nitric oxide regulation by dietary factors such as arginine or its precursors and vitamin C. Because diets with a high saturated fat content induce high plasma fatty acid levels, endothelial nitric oxide production is often impaired due to a reduction in nitric oxide synthase 3 phosphorylation. Increasing the arginine availability by arginine therapy or arginase inhibition was, therefore, proposed as a potential therapy to treat hypertension. Recent studies in septic patients and transgenic mice models found that inadequate de-novo arginine production from citrulline reduces nitric oxide production. Citrulline supplementation may, therefore, be a novel therapeutic approach in conditions of arginine deficiency. SUMMARY: Both lack and excess of nitric oxide production in diseases can have various important implications in which dietary factors can play a modulating role. Future research is needed to expand our understanding of the regulation and adequate measurement of nitric oxide production at the organ level and by the different nitric oxide synthase isoforms, also in relation to clinical nutrition.

Is iNOS beginning to smoke?

            (Nathan 2011) Download

Inducible nitric oxide synthase (iNOS) helps drive numerous inflammatory disorders, but its inhibition has not had therapeutic success. Now Seimetz et al. (2011) make a case for inhibiting iNOS in an effort to treat one of the world's leading causes of death-chronic obstructive pulmonary disease.

Tetrahydrobiopterin: a novel antihypertensive therapy

            (Porkert, Sher et al. 2008) Download

Tetrahydrobiopterin (BH(4)) is a cofactor for the nitric oxide (NO) synthase enzymes, such that its insufficiency results in uncoupling of the enzyme, leading to release of superoxide rather than NO in disease states, including hypertension. We hypothesized that oral BH(4) will reduce arterial blood pressure (BP) and improve endothelial function in hypertensive subjects. Oral BH(4) was given to subjects with poorly controlled hypertension (BP >135/85 mm Hg) and weekly measurements of BP and endothelial function made. In Study 1, 5 or 10 mg kg(-1) day(-1) of BH(4) (n=8) was administered orally for 8 weeks, and in Study 2, 200 and 400 mg of BH(4) (n=16) was given in divided doses for 4 weeks. Study 1: significant reductions in systolic (P=0.005) and mean BP (P=0.01) were observed with both doses of BH(4). Systolic BP was 15+/-15 mm Hg (P=0.04) lower after 5 weeks and persisted for the 8-week study period. Study 2: subjects given 400 mg BH(4) had decreased systolic (P=0.03) and mean BP (P=0.04), with a peak decline of 16+/-19 mm Hg (P=0.04) at 3 weeks. BP returned to baseline 4 weeks after discontinuation. Significant improvement in endothelial function was observed in Study 1 subjects and those receiving 400 mg BH(4). There was no significant change in subjects given the 200 mg dose. This pilot investigation indicates that oral BH(4) at a daily dose of 400 mg or higher has a significant and sustained antihypertensive effect in subjects with poorly controlled hypertension, an effect that is associated with improved endothelial NO bioavailability.

Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by delta-tocotrienol and quercetin

            (Qureshi, Tan et al. 2011) Download

ABSTRACT: BACKGROUND: Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, delta-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1-/- and PPAR-alpha-/- knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (delta-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-alpha, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), delta-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-beta (IFN-beta), or LPS plus interferon-gamma (IFN-gamma), and inflammatory responses assessed as measured by production of NO and TNF-alpha, mRNA reduction for TNF-alpha, and iNOS genes, and microarray analysis. RESULTS: Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-alpha among these two groups. However, macrophages obtained after feeding with quercetin, delta-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, delta-tocotrienol, or dexamethasone were also treated with IFN-beta or IFN-gamma compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-alpha protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters. CONCLUSIONS: The present results demonstrated that quercetin and delta-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or delta-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1beta, IL-1alpha, IL-6, TNF-alpha, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-kappaB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and delta-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.

Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice

            (Seimetz, Parajuli et al. 2011) Download        

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD.

Nitric oxide inhibits aromatase activity: mechanisms of action

            (Snyder, Holmes et al. 1996) Download

NO synthase is present in human ovarian granulosa-luteal cells and NO inhibits estradiol secretion by granulosa cells in culture. These findings suggest that NO is an autocrine regulator of ovarian steroidogenesis. The purpose of this investigation was to explore the mechanisms through which NO exerts an inhibitory effect on cytochrome P450 aromatase activity. To examine the effect of NO on aromatase mRNA levels, human granulosa-luteal cells were cultured in the presence or absence of the NO donor SNAP for 16 h. Using a probe for human aromatase, Northern blots revealed a 26% decrease in aromatase mRNA in cells exposed to SNAP. Because this modest decrease in mRNA is unlikely to explain a rapid and profound reduction in estradiol secretion that we have observed, we looked for direct effects of NO on cytochrome P450 aromatase activity. Aromatase activity was assayed in placental microsomes and granulosa-luteal cells by measuring the release of 3H2O from [1 beta-3H] androstenedione. NO (10(-4)-10(-3)M), added as a saturated saline solution, reduced aromatase activity by as much as 90% in a concentration-dependent, non-competitive manner. In contrast, carbon monoxide (CO), a gas known to bind to the heme iron in aromatase, had no effect on aromatase activity when added alone nor could CO reverse the NO-induced inhibition of aromatase. These data suggest that NO binding to the heme is insufficient to inhibit aromatase activity. NO has been reported to alter protein function by reacting with the sulfhydryl group of cysteines, forming a nitrosothiol group. Because a cysteine sulfhydryl group is thought to participate in the catalytic mechanism of all P450 enzymes, experiments were designed to test whether NO might inhibit aromatase via such a mechanism. Addition of increasing amounts of mercaptoethanol, a chemical with free sulfhydryl groups, blocked the NO-induced inhibition of aromatase in microsomes. N-Ethylmaleimide, a chemical which covalently modifies sulfhydryl groups, reduced aromatase activity in a concentration-dependent manner. We conclude that NO inhibits aromatase both by decreasing mRNA for the enzyme and by an acute, direct inhibition of enzyme activity. We hypothesize that the direct inhibition occurs as a result of the formation of a nitrosothiol on the cysteine residue adjacent to the heme in aromatase.

Quercetin administration ameliorates pulmonary complications of cirrhosis in rats

            (Tieppo, Cuevas et al. 2009) Download

In the hepatopulmonary syndrome (HPS), a common complication of liver cirrhosis, pulmonary endothelial endothelin B (ETB) receptor overexpression, enhanced endothelial nitric oxide (NO) synthase (eNOS)-derived NO production, and increases in pulmonary inducible NO synthase (iNOS) and heme oxygenase (HO-1) are important factors in the development of vasodilatation. These changes may be influenced by redox-sensitive signaling pathways, including nuclear factor-kappaB (NF-kappaB). In this study, our aim was to evaluate the effects of the flavonoid antioxidant quercetin on the development of HPS in rats with common bile duct ligation (CBDL). Rats were divided into the following 4 groups: rats subjected to CBDL, Sham (rats subjected to simulated CBDL), quercetin-treated sham, and quercetin-treated CBDL. Quercetin (50 mg/kg) was administered for 2 wk starting on d 14 after surgery. Increased NO production, overexpression of iNOS, eNOS, HO-1, and ETB-receptor and activation of NF-kappaB were observed in lung of CBDL rats. Quercetin inhibited oxidative stress, NF-kappaB activation, and the expression of different pulmonary mediators involved in HPS. Quercetin also ameliorated liver injury and reduced the expression of hepatic endothelin-1 and HO-1 in untreated cirrhotic rats. Our findings suggest that quercetin administered after the onset of hepatic injury significantly ameliorates pulmonary complications in CBDL rats and that limitation of cirrhotic evolution contributes to this effect.

Tetrahydrobiopterin, superoxide, and vascular dysfunction

            (Vasquez-Vivar 2009) Download

(6R)-5,6,7,8-Tetrahydrobiopterin (BH(4)) is an endogenously produced pterin that is found widely distributed in mammalian tissues. BH(4) works as a cofactor of aromatic amino acid hydroxylases and nitric oxide synthases. In the vasculature a deficit of BH(4) is implicated in the mechanisms of several diseases including atherosclerosis, hypertension, diabetic vascular disease, and vascular complications from cigarette smoking and environmental pollution. These ill-effects are connected to the ability of BH(4) to regulate reactive oxygen species levels in the endothelium. The possibility of using BH(4) as a therapeutical agent in cardiovascular medicine is becoming more compelling and many biochemical and physiological aspects involved in this application are currently under investigation. This review summarizes our current understanding of BH(4) reactivity and some aspects of cellular production and regulation.

Antioxidants to enhance fertility: role of eNOS and potential benefits

            (Visioli and Hagen 2011) Download

The use of antioxidants is now often used as a pharmacological adjunct to limit infertility. Indeed, the lay public rightly perceives oxidative stress and, thus, antioxidant treatment as important modulators of infertility. While the direct effects of antioxidant treatment on the quality of semen and oocytes are still under investigation, a significant body of evidence points to loss of vascular tone as a root-cause of erectile dysfunction and, possibly, alterations to female reproduction. In this article, we will critically review the often neglected link between vascular dysfunction and infertility. A particular emphasis will be on the potential use of antioxidants to increase fertility and promote conception.

The role of arginine-nitric oxide pathway in patients with Alzheimer disease

            (Vural, Sirin et al. 2009) Download

There is a reciprocal regulation of arginase and nitric oxide synthase in L-arginine-metabolizing pathways. There are various evidences of the role of nitric oxide in several neuropsychiatric disorders including Alzheimer's disease. However, there is no study that has investigated the role of arginase as an important part of the arginine regulatory system affecting nitric oxide synthase activity in Alzheimer's disease. This study aims to investigate arginase, manganese (a cofactor of arginase), and total nitrite levels (a metabolite of NO) and their relationship to the arginine-NO pathway in patients with Alzheimer's disease. Arginase activities, Mn, and total nitrite levels were measured in plasma from 47 patients with Alzheimer's disease and 43 healthy control subjects. Plasma arginase activities and manganese were found to be significantly lower and total nitrite level higher in patients with Alzheimer's disease compared with controls. Our results suggest that the arginine-NO pathway is involved in the pathogenesis of Alzheimer's disease.

Grape seed proanthocyanidin extract attenuates airway inflammation and hyperresponsiveness in a murine model of asthma by downregulating inducible nitric oxide synthase

            (Zhou, Du et al. 2011) Download

Allergic asthma is characterized by hyperresponsiveness and inflammation of the airway with increased expression of inducible nitric oxide synthase (iNOS) and overproduction of nitric oxide (NO). Grape seed proanthocyanidin extract (GSPE) has been proved to have antioxidant, antitumor, anti-inflammatory, and other pharmacological effects. The purpose of this study was to examine the role of GSPE on airway inflammation and hyperresponsiveness in a mouse model of allergic asthma. BALB/c mice, sensitized and challenged with ovalbumin (OVA), were intraperitoneally injected with GSPE. Administration of GSPE remarkably suppressed airway resistance and reduced the total inflammatory cell and eosinophil counts in BALF. Treatment with GSPE significantly enhanced the interferon (IFN)- gamma level and decreased interleukin (IL)-4 and IL-13 levels in BALF and total IgE levels in serum. GSPE also attenuated allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells in the airway. The elevated iNOS expression observed in the OVA mice was significantly inhibited by GSPE. In conclusion, GSPE decreases the progression of airway inflammation and hyperresponsiveness by downregulating the iNOS expression, promising to have a potential in the treatment of allergic asthma.


Alcazar-Leyva, S. and N. Alvarado-Vasquez (2011). "Could thiamine pyrophosphate be a regulator of the nitric oxide synthesis in the endothelial cell of diabetic patients?" Med Hypotheses 76(5): 629-31.

Clifton, P. M. (2004). "Effect of Grape Seed Extract and Quercetin on Cardiovascular and Endothelial Parameters in High-Risk Subjects." J Biomed Biotechnol 2004(5): 272-278.

Johnson, J. M., T. J. Bivalacqua, et al. (2011). "eNOS-uncoupling in age-related erectile dysfunction." Int J Impot Res 23(2): 43-8.

Kao, T. K., Y. C. Ou, et al. (2010). "Inhibition of nitric oxide production by quercetin in endotoxin/cytokine-stimulated microglia." Life Sci 86(9-10): 315-21.

Luiking, Y. C., M. P. Engelen, et al. (2010). "Regulation of nitric oxide production in health and disease." Curr Opin Clin Nutr Metab Care 13(1): 97-104.

Nathan, C. (2011). "Is iNOS beginning to smoke?" Cell 147(2): 257-8.

Porkert, M., S. Sher, et al. (2008). "Tetrahydrobiopterin: a novel antihypertensive therapy." J Hum Hypertens 22(6): 401-7.

Qureshi, A. A., X. Tan, et al. (2011). "Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by delta-tocotrienol and quercetin." Lipids Health Dis 10(1): 239.

Seimetz, M., N. Parajuli, et al. (2011). "Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice." Cell 147(2): 293-305.

Snyder, G. D., R. W. Holmes, et al. (1996). "Nitric oxide inhibits aromatase activity: mechanisms of action." J Steroid Biochem Mol Biol 58(1): 63-9.

Tieppo, J., M. J. Cuevas, et al. (2009). "Quercetin administration ameliorates pulmonary complications of cirrhosis in rats." J Nutr 139(7): 1339-46.

Vasquez-Vivar, J. (2009). "Tetrahydrobiopterin, superoxide, and vascular dysfunction." Free Radic Biol Med 47(8): 1108-19.

Visioli, F. and T. M. Hagen (2011). "Antioxidants to enhance fertility: role of eNOS and potential benefits." Pharmacol Res 64(5): 431-7.

Vural, H., B. Sirin, et al. (2009). "The role of arginine-nitric oxide pathway in patients with Alzheimer disease." Biol Trace Elem Res 129(1-3): 58-64.

Zhou, D. Y., Q. Du, et al. (2011). "Grape seed proanthocyanidin extract attenuates airway inflammation and hyperresponsiveness in a murine model of asthma by downregulating inducible nitric oxide synthase." Planta Med 77(14): 1575-81.