Increasing Nitric Oxide Bioavailability Research Articles

Asymmetric dimethylarginine (ADMA) determines the improvement of hepatic endothelial dysfunction by vitamin E in cirrhotic rats

Hepatic endothelial dysfunction (HED), which is caused by decreased hepatic nitric oxide (NO) bioavailability and increased lipid peroxidation, contributes to portal hypertension, which is a characteristic of cirrhosis. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is involved in cirrhosis-related HED and portal hypertension. We evaluated the effect of vitamin E treatment on the lipid peroxidation, HED and portal hypertension in cirrhotic rats. The common bile duct ligation (BDL)-induced cirrhotic rats were treated orally either with vehicle or with vitamin E for 1month immediately after BDL. Systemic and portal haemodynamics, the magnitude of the increase in portal pressure induced by volume expansion, HED, oxidative stress, levels of ADMA, various proteins and mRNAs were then measured. In the vitamin E-treated BDL rats, a decrease in portal pressure was associated with an attenuation of the increased portal pressure induced by volume expansion. In isolated and perfused BDL rat livers, the vitamin E treatment significantly inhibited the (paradoxical) vasoconstriction response to methoxamine and acetylcholine (HED), and this was abolished by the presence of NOS. Vitamin E decreased ADMA synthesizing enzyme PRMT1 expression and the level of thiobarbituric acid-reactive substances (TBARS) in the liver, while increasing the levels of hepatic ADMA metabolizing enzyme DDAH2, eNOS, phosphor-eNOS, ADMA level and superoxide dismutase activity. The administration of vitamin E suppressed hepatic ADMA and oxidative stress in the cirrhotic liver circulation, and therefore increases NO bioavailability, which improved HED and portal hypertension.

Effects of training and nitric oxide on diabetic nephropathy progression in type I diabetic rats

The aim of the paper is to assess nitric oxide (NO) production during aerobic training and its role on the progression of diabetic nephropathy in rats. Induction of diabetes mellitus (DM) was achieved in adult male Wistar rats with streptozotocin. Half of the animals underwent training on a treadmill and the others (sedentary) stayed on a turned-off treadmill for the same period according to the following groups: sedentary control (CTL + SE); training control (CTL + EX); sedentary diabetic (DM + SE); and training diabetic (DM + EX) (n = 9 for all groups). The training on treadmill was carried out at a work rate of 16 m/min, 60 min/d, 5 d/week for eight weeks. Before and after the exercises, rats were placed in individual metabolic cages with standard chow and water ad libitum, for 24-h urine collection, followed by three hours' fasting blood sample withdrawal from the retro-orbital plexus, under anesthesia. Diabetic animals showed reduction of body weight, creatinine and urea depurations and NO excretion, increased blood glucose concentrations, albuminuria and thiobarbituric acid reactive substance (TBARS) excretion, when compared with the respective controls. All these alterations induced by DM were attenuated in the DM + EX versus DM + SE group. Analysis of insulin concentrations at the end of the protocol showed no significant change between the DM + SE and DM + EX groups. In conclusion, our data show that a routine physical exercise resulted in a better control of glycemia with an increased NO bioavailability and oxidative stress control, associated with an amelioration of renal function. We suggest aerobic training and the control of oxidative and nitrosative stress as useful non-pharmacological tools to delay the progression of diabetic nephropathy.

Simvastatin enhances the antihypertensive effect of ramipril in hypertensive hypercholesterolemic animals and patients. Possible role of nitric oxide, oxidative stress, and high sensitivity C‐reactive protein

In this study, the effects of simvastatin on the blood pressure and on the antihypertensive activity of ramipril in hypertensive hypercholesterolemic animals and patients were evaluated. In hypertensive hypercholesterolemic animals, repeated administration of simvastatin slightly but significantly decreased the systolic blood pressure, enhanced its progressive reductions induced by repeated administration of ramipril and corrected the compromised lipid profile. Concomitantly, repeated administration of simvastatin, ramipril or simvastatin in combination with ramipril to these animals, increased nitric oxide (NO) production and decreased the elevated serum malondialdehyde (MDA) and high sensitivity C-reactive protein (hs-CRP) levels. The effects of combined treatment were greater than those of simvastatin or ramipril alone. In hypertensive hypercholesterolemic patients, repeated administration of ramipril decreased systolic and diastolic blood pressure, increased NO production, and decreased the elevated serum MDA and hs-CRP levels. Addition of simvastatin to ramipril therapy enhanced these effects and corrected the compromised lipid profile. Simvastatin but not ramipril inhibited the contractile responses of isolated aortic rings induced by angiotensin 11. l-arginine and acetylcholine enhanced, while l-NAME inhibited effects of simvastatin, and simvastatin in combination with ramipril on these contractile responses. These findings suggest that simvastatin exerts antihypertensive effect and enhances the antihypertensive effect of ramipril in hypertensive hypercholesterolemic animals and patients. In addition to its cholesterol-lowering effect, the ability of simvastatin to ameliorate endothelial dysfunction through increasing NO bioavailability and through suppression of oxidative stress and vascular inflammation and its ability to enhance the effect of ramipril on these parameters may play a pivotal role in these effects.

Endothelial nitric oxide synthase enhancer reduces oxidative stress and restores endothelial function in db/db mice

Endothelial dysfunction is caused by reduced nitric oxide (NO) bioavailability and/or over-produced reactive oxygen species (ROS). The present study investigated a vascular benefit of AVE3085, an endothelial nitric oxide synthase (eNOS) enhancer, in preserving endothelial function in diabetic mice and the mechanisms involved. Male db/db and db/m(+) mice were orally administered AVE3085 for 7 days (10 mg kg(-1) day(-1)). Vascular reactivity of arteries was studied via myography under both isometric and isobaric conditions. ROS levels in aortas were determined using dihydroethidium fluorescence dye and electron paramagnetic resonance spin trapping. Chronic treatment with AVE3085 reduced blood pressure, enhanced endothelium-dependent relaxations (EDR) to acetylcholine in aortas, mesenteric, and renal arteries, lowered oxidative stress, and augmented the attenuated flow-dependent dilatation in mesenteric resistance arteries from db/db mice. Incubation of aortas from C57BL/6J mice in high glucose (30 mmol L(-1)) culture medium for 48 h impaired EDR and elevated ROS generation, and these effects were reversed by co-treatment with AVE3085 (1 µmol L(-1)). Benefits of AVE3085 were abolished by the transcription inhibitor actinomycin D, the NOS inhibitor N(G)-nitro-L-arginine methyl ester, and in eNOS(-/-) mice. NO production in primary endothelial cells from mouse aortas was detected with a NO-sensitive fluorescence dye. Protein expression was assayed by western blotting. Treatment with AVE3085 enhanced NO production in endothelial cells and eNOS expression in aortas. AVE3085 ameliorates endothelial dysfunction in db/db mice through increased NO bioavailability, which reduces oxidative stress in the vascular wall. Targeting eNOS and NO production may be a promising approach to combat diabetic vasculopathy.

Folic acid may be a potential addition to diabetic foot ulcer treatment - a hypothesis

Delayed wound healing in diabetes is a challenging medical and societal problem for which there is currently no efficacious treatment. One of the major contributors of this problem is nitric oxide (NO) deficiency. NO is a critical signalling molecule essential for normal wound repair. Sustained hyperglycaemia in diabetes leads to increased vascular superoxide production, which inactivates NO and causes vascular dysfunction. New therapeutic regiments and strategies to enhance endothelial NO production are a new hope to improve impaired diabetic wound healing. One of the agents that have the ability to improve endothelial NO generation in diabetic patients is folic acid. Folic acid ability to conserve NO bioactivity may be due to homocysteine-lowering effects of folates, antioxidant actions and effects on cofactor availability. Considering these data, we hypothesised that folic acid supplementation may ameliorate delayed diabetic wound healing by increasing NO bioavailability. The potential of exogenous folic acid as an inexpensive and safe oral therapy stimulates ongoing investigations.

Blockade of NADPH Oxidase Restores Vasoreparative Function in Diabetic CD34+Cells

The vasodegenerative phase of diabetic retinopathy is likely caused by endothelial dysfunction and reduced endothelial repair. Migration of endothelial progenitor cells (EPCs) into areas of vascular injury is critical to vascular repair. This key function, often defective in diabetes, is largely mediated by nitric oxide (NO), which is known to be inactivated by superoxide produced by NADPH oxidase. The authors tested the hypothesis that either increasing eNOS expression or inhibiting NADPH oxidase would restore the reparative function in diabetic EPCs. Peripheral blood was obtained from healthy (n = 27) and diabetic (n = 31) persons, and CD34(+) cells were isolated. Expression and activation of eNOS and NADPH oxidase and intracellular levels of NO, superoxide, and peroxynitrite were evaluated. cGMP production and migration to SDF-1α were also determined. Reparative function was evaluated in a mouse model of retinal ischemia-reperfusion injury. Diabetic EPCs demonstrate reduced eNOS expression and decreased NO bioavailability and migration in response to SDF-1α. Increasing eNOS expression in diabetic cells by AVE3085 resulted in increased peroxynitrite levels and, therefore, did not enhance NO-mediated functions in vitro and in vivo. Expression of Nox2, NADPH oxidase activity, and superoxide levels were higher in diabetic than in nondiabetic EPCs. Pretreatment with apocynin or gp91ds-tat increased NO bioavailability without increasing eNOS activity in response to SDF-1α. Ex vivo NADPH oxidase inhibition in diabetic cells restored migratory function in vitro and enhanced their homing to ischemic retinal vasculature in vivo. The NADPH oxidase system is a promising target for correcting vasoreparative dysfunction in diabetic EPCs.

Protection by Red Wine Polyphenols against Metabolic and Cardiovascular Alterations Associated with Obesity: A Possible Link with Estrogen Alpha Receptor

Epidemiological studies report an inverse association between dietary flavonoid consumption and mortality from cardiovascular and metabolic diseases. Red wine contains a wide variety of polyphenols (RWPC), which mainly derive from grape solids (skin and seeds) and can be divided into two classes, flavonoids and non-flavonoids. RWPC exert numerous effects including antioxidant and free radical properties, anti-platelet aggregating and anti-thrombotic activities. Moreover, RWPC are powerful vasodilators and contribute to the preservation of the integrity of the endothelium and to the inhibition of smooth muscle cell proliferation and migration. All these effects of red wine may interfere with atherosclerotic plaque development and stability, vascular thrombosis and occlusion and explain the prevention of ischemic heart disease, stroke and metabolic diseases observed in different experimental models. With regard to obesity, we studied the effects of dietary supplementation of RWPC in Zucker fatty rats (ZF). We show that RWPC lead to an improvement of glucose and lipid metabolism in ZF rats, enhances cardiac function and decreases peripheral resistance. In addition, RWPC improve endothelium-dependent relaxation in both aorta and small mesenteric arteries from ZF rats, an effect accompanied by an increase of vascular nitric oxide (NO) production and a decrease of superoxide anions release, resulting in an increase of NO bioavailability. Thus, RWPC lead to an overall improvement in obesity-associated alterations, including glucose and lipid metabolism, as well as endothelial and cardiac functions. A molecular basis of the effect of RWPC has been provided on the endothelium, indentifying the alpha isoform of the estrogen receptor (ERα) as the key receptor, or at least one of those transducing vascular effects exerted by these compounds, particularly delphinidin with respect to NO production. These results are of importance inasmuch as RWPC, by interacting with ERα, can activate other targets such the sirtuin-1-adenosine monophosphate protein kinase network, both of which are amongst the most potent regulators of cellular metabolism, including vascular cells, adipocytes and hepatocytes. Altogether, the available evidence indicates that RWPC might be of therapeutic benefit for cardiovascular and metabolic protections, although prospective controlled clinical trials are still needed.

Role of endothelial dysfunction in modulating the plasma redox homeostasis in visceral leishmaniasis

BackgroundEvidence in the literature suggests that down-regulation of nitric oxide (NO) is associated with the pathophysiological conditions during visceral leishmaniasis (VL). Here we have investigated the mechanism that leads to the down regulation of systemic NO in the infected condition. Moreover, we have determined whether down regulation of NO is associated with increased generation of reactive oxygen species (ROS) during this disease. Therapeutic strategy targeting signaling molecules of these events was evaluated. MethodsPlasma protein-nitrotyrosine was examined by ELISA kit. Generation of superoxides and peroxynitrites was investigated by flow cytometry. NO bioavailability in endothelial cells was evaluated using DAF-2DA fluorescence. Ceramide contents were evaluated using FACS analysis, HPTLC and HPLC. ResultsL. donovani infected reticulo-endothelial cells regulated the activity of eNOS and NAD(P)H oxidase in the endothelial cells through the generation of intercellular messenger, ceramide. Activation of SMases played an important role in the generation of ceramide in animals during chronic infection. These events led to generation of ROS within endothelial cells. Modulation of redox status of plasma and accumulation of ROS in endothelial cells were critically involved in the regulation of NO bioavailability in plasma of the infected animal. Endothelial dysfunction and decline of NO were resulted from an increased production of superoxide where upregulation of eNOS expression appeared as an ineffective compensatory event. Inhibition of ceramide generation increased NO bioavailability, prevented endothelial dysfunction and concomitant oxidative stress. Conclusion and general significanceDecreased NO bioavailability and endothelial dysfunction were the downstream of ceramide signaling cascade. ROS accumulation promoted peroxynitrite generation and reduced NO bioavailability. Inhibition of ceramide generation may be a potential therapeutic option in preventing the co-morbidity associated with VL.

Voluntary running and caloric restriction reverse cerebrovascular endothelial dysfunction in old mice by restoring nitric oxide bioavailability

We tested the hypothesis that age-associated cerebrovascular endothelial dysfunction can be reversed by exercise- and dietary-related lifestyle interventions. Maximal ex vivo endothelium-dependent dilation (EDD) to acetylcholine (ACh) in isolated middle cerebral arteries (MCA) was impaired in old (O, 29–31 mo, n=12) vs. young (Y, 6–8 mo, n=12) cage-control (C) B6D2F1 mice (30±7 vs. 73±7%, P<0.01). Inhibition of nitric oxide (NO) using L-NAME resulted in 73% (YC) and 12% (OC) reductions in EDD (P<0.05), abolishing age group differences. Voluntary wheel running (VR, 9–11 weeks: YVR, n=9, 10.1±0.8 km/day; OVR, n=5, 2.4±0.8) had no effect on EDD in Y mice (66±8%), but restored EDD in O mice to levels (63±9%) not different from YC. The improvement in EDD in OVR was mediated by enhanced NO bioavailability. In a separate group of O mice (n=7), lifelong caloric restriction (40% < ad libitum) improved (P<0.05) MCA EDD (to 52±8%) and increased sensitivity to ACh (IC50) and NO bioavailability. MCA endothelium-independent dilation to sodium nitroprusside was not influenced by age or interventions. VR and caloric restriction improve MCA EDD in O mice by increasing NO bioavailability. This preclinical evidence supports the efficacy of voluntary aerobic exercise and reductions in energy intake for treatment of cerebrovascular dysfunction and its clinical sequelae with aging in humans. NIH AG013038, AG029337, AG033196

Autophagy‐enhancing therapy reduces oxidative stress and restores vascular endothelial function in old mice

Aging results in vascular endothelial dysfunction caused by oxidative stress and reduced nitric oxide (NO) bioavailability. Autophagy (process of cell recycling of damaged biomolecules) is a key mediator of oxidative stress. We assessed ex vivo acetylcholine (ACh)‐induced endothelium‐dependent dilation (EDD) in adult C57/Bl6 mice: young (Y: 5 mo, n=5), old (O: 27 mo, n=5) and old treated with the autophagy enhancer trehalose (OT: 27 mo, n=5). Aging was associated with a ~40% reduction (P&lt;0.05) in aortic expression of the autophagy markers beclin‐1 and LAMP‐2a, and impaired carotid artery EDD (Max EDD: Y: 96 ± 2%, O: 73 ± 3%, P&lt;0.01) mediated by reduced NO bioavailability (ACh + L‐NAME). Trehalose (20 g/L drinking water, 4 weeks) increased aortic beclin‐1 and LAMP‐2a and restored EDD (OT: 95 ± 2%). TEMPOL, a superoxide scavenger, improved EDD in O (P&lt;0.05), but not Y or OT. Consistent with this, aortic superoxide production (electron paramagnetic resonance) was ~150% greater in O vs. Y (P&lt;0.01), but was similar to Y in OT. Compared with O mice, OT had ~30% greater expression of endothelial NO synthase (eNOS) and ~80% higher levels of SIRT1 and AMPK, key modulators of autophagy and eNOS activity (all P&lt;0.05). Impaired autophagy may contribute to vascular endothelial dysfunction with age. Autophagy‐enhancing therapy restores endothelial function in old mice by reducing oxidative stress and increasing NO bioavailability.

Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging

We tested the hypothesis that short-term nitrite therapy reverses vascular endothelial dysfunction and large elastic artery stiffening with aging, and reduces arterial oxidative stress and inflammation. Nitrite concentrations were lower (P < 0.05) in arteries, heart, and plasma of old (26-28 month) male C57BL6 control mice, and 3 weeks of sodium nitrite (50 mg L(-1) in drinking water) restored nitrite levels to or above young (4-6 month) controls. Isolated carotid arteries of old control mice had lower acetylcholine (ACh)-induced endothelium-dependent dilation (EDD) (71.7 ± 6.1% vs. 93.0 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (P < 0.05 vs. young), and sodium nitrite restored EDD (95.5 ± 1.6%) by increasing NO bioavailability. 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), a superoxide dismutase (SOD) mimetic, apocynin, a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, and sepiapterin (exogenous tetrahydrobiopterin) each restored EDD to ACh in old control, but had no effect in old nitrite-supplemented mice. Old control mice had increased aortic pulse wave velocity (478 ± 16 vs. 332 ± 12 AU, P < 0.05 vs. young), which nitrite supplementation lowered (384 ± 27 AU). Nitrotyrosine, superoxide production, and expression of NADPH oxidase were ∼100-300% greater and SOD activity was ∼50% lower in old control mice (all P < 0.05 vs. young), but were ameliorated by sodium nitrite treatment. Inflammatory cytokines were markedly increased in old control mice (P < 0.05), but reduced to levels of young controls with nitrite supplementation. Short-term nitrite therapy reverses age-associated vascular endothelial dysfunction, large elastic artery stiffness, oxidative stress, and inflammation. Sodium nitrite may be a novel therapy for treating arterial aging in humans.

Nitric oxide delays atrial tachycardia-induced electrical remodelling in a sheep model

Rapid atrial pacing for 1 week leads to decreased expression of endocardial nitric oxide (NO)-synthase and decreased NO concentrations. We hypothesized that increasing NO bioavailability may reduce electrical remodelling induced by atrial tachycardia. We examined the effect of molsidomine, a NO donor, and N(ω)-nitro-l-arginine methylester (l-NAME), a NO-synthase inhibitor, on electrical remodelling occurring during 4 h of rapid atrial pacing in sheep. Haemodynamic and electrophysiological parameters were measured at baseline, 1 h after the start of the infusion and before the start of pacing, and 2 and 4 h after pacing. We measured the effect of molsidomine on atrial monophasic action potentials (MAPs) in non-instrumented sheep and on l-type Ca(2+) currents and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients in right atrial cells, isolated from control sheep. In control sheep, rapid atrial pacing shortened the atrial effective refractory period (AERP) by 12 ± 0.18% after 4 h, an effect that was unaffected by l-NAME. Infusion of molsidomine increased AERP at baseline (+13.4 ± 1.04%) and transiently attenuated pacing-induced AERP shortening (13.6 ± 0.1% at 2 h). Molsidomine tended to increase MAP duration by 20.7 ± 13.4 ms. Incubation of isolated atrial myocytes with NO donor 3-morpholino-sydnonimine (SIN-1) increased significantly l-type Ca(2+) current and [Ca(2+)](i) transients. Infusion of molsidomine, a NO donor, delayed shortening of the action potential during short-term rapid atrial pacing, by increasing [Ca(2+)](i). Whereas the former could be protective against repetitive short episodes of atrial fibrillation, the latter might be detrimental in the long term.

Acute Modulation of Vasoconstrictor Responses by Pravastatin in Small Vessels

Statins have been shown to inhibit conduit vessel constrictor responses via the endothelial nitric oxide (NO) pathway. Clinical studies have implicated an effect in microvascular resistance vessels; however, direct effects of therapeutically relevant statin concentrations have not been examined. We examined the effect of acute pravastatin pretreatment on vasoconstrictor responsiveness of isolated rat mesenteric small vessels. Pravastatin (112 nmol/L) pretreatment for 60 min reduced both the potency and maximal constrictor responses to phenylephrine, thromboxane (U46619) and serotonin in small vessels. This effect was abolished by endothelial denudation, NO synthase (NOS) inhibition with N-ω-nitro-L-arginine methyl ester (L-NAME 300 µmol/L) and Akt inhibition (Akt1/2 kinase inhibitor 500 nmol/L), confirming an endothelium-dependent mechanism and implicating a NO-mediated effect via the Akt pathway. Maximal superoxide scavenging with polyethylene glycol-superoxide dismutase (PEG-SOD), 150 U/ml did not influence phenylephrine constrictor responses but potentiated pravastatin's effect, suggesting that the statin did not increase NO bioavailability merely via an antioxidant mechanism. In contrast, pravastatin did not affect endothelin-1 (ET-1) constrictor responses. However, after pre-incubation with a selective endothelin-B (ET(B)) receptor antagonist (BQ788 3 µmol/L) pravastatin inhibited ET-1 constriction, suggesting that its effect is via the same mechanistic pathway as the ET(B) receptor. In small vessels, pravastatin inhibits constrictor responses by increasing endothelial NO bioavailability via the Akt pathway. Furthermore, ET(B) receptor blockade unmasks this effect in ET-1 constrictor responses.

Low intensity light stimulates nitrite-dependent nitric oxide synthesis but not oxygen consumption by cytochrome c oxidase: Implications for phototherapy

Cytochrome c oxidase (Cco) has been reported to be a receptor for some of the beneficial effects of low intensity visible and near-infrared light on cells and tissues. Here, we have explored the role of low intensity light in affecting a newly described function of Cco, its ability to catalyze nitrite–dependent nitric oxide (NO) synthesis (Cco/NO). Using a new assay for Cco/NO we have found that both yeast and mouse brain mitochondrial Cco produce NO over a wide range of oxygen concentrations and that the rate of NO synthesis increases as the oxygen concentration decreases, becoming optimal under hypoxic conditions. Low intensity broad-spectrum light increases Cco/NO activity in an intensity-dependent fashion but has no effect on oxygen consumption by Cco. By using a series of bandpass filters and light emitting devices (LEDs) we have determined that maximal stimulation of Cco/NO activity is achieved by exposure to light whose central wavelength is 590 ± 14 nm. This wavelength of light stimulates Cco/NO synthesis at physiological nitrite concentrations. These findings raise the interesting possibility that low intensity light exerts a beneficial effect on cells and tissues by increasing NO synthesis catalyzed by Cco and offer a new explanation for the increase in NO bioavailability experienced by tissue exposed to light.

Coordination Between Nitric Oxide and Superoxide Anion Radical During Progressive Exercise in Elite Soccer Players

Background:Exercise increases production of reactive oxygen and nitrogen species (RONS) via several mechanisms. Inter alia, increased blood flow during exercise exposes endothelial cells to shear stress, resulting in increased nitric oxide (NO) production. Increased oxygen consumption or hypoxia during exercise induces increased production of superoxide anion radical (O2-). Objective:This study investigates the effects of maximal progressive treadmill exercise test on time-course of peripheral blood NO and O2- production, as well as the effect of long-term training on NO bioavailability. Methods:Blood samples of 19 elite soccer players were gathered immediately before the test, during last 10 sec of every test stage, and during active recovery phases. Results:Significant increase (p<0.05) in NO production (estimated through nitrites (NO2-)), found between stage I (5.69 ± 1.32 nmol/ml) and basal values (5.36 ± 1.25 nmol/ml), was followed by the decrease in stage II (4.21 ± 0.42 nmol/ml) and production lower than basal to the end of the test. Significant increase (p<0.05) in O2- values was found between stage I (4.18 ± 0.77 nmol/ml) and resting values (4.01 ± 0.69 nmol/ml), and at stages V (4.24 ± 0.85 nmol/ml) and 1st phase of recovery (4.39 ± 0.92 nmol/ml). Conclusion:The regression lines of NO2- and O2- crossed at the level of anaerobic threshold, suggesting that anaerobic threshold could be of a crucial importance not only in the anaerobic and aerobic metabolism but in mechanisms of signal transductions as well. Long-term exercise increases NO bioavailability, and there is positive correlation between NO bioavailability and maximal oxygen uptake (VO2max).

Synergistic Enhancement of Sickle Red Blood Cell Adhesion to Endothelium by Hypoxia and Low Nitric Oxide Bioavailability

AbstractAbstract 1637The mechanisms underlying sickle red blood cell (RBC) adhesion to the endothelium, which constitutes a major pathologic event in sickle cell disease (SCD), are not fully understood. Adhesion of sickle RBCs to endothelial cells is believed to be regulated by multiple hematologic and physiologic factors including fetal hemoglobin levels, leukocyte numbers, oxygen tension, inflammatory cytokines, and nitric oxide (NO) bioavailability, but the extent to which each parameter contributes to sickle RBC adhesion remains unclear. Here we studied the effects of hypoxia and NO bioavailability on sickle RBC adhesion using mice deficient for endothelial nitric oxide synthase (eNOS). We found that these mice had NO metabolite levels comparable to those of SCD UAB mice (Ryan et al. Science 278:873, 1997). Sickle RBCs obtained from SCD UAB mice or control RBCs from control mice (C57BL/6J) were injected into eNOS-deficient or control mice. Sickle RBC adhesion in the skull bone marrow venules was studied by intravital microscopy. Control RBCs did not show any adherence to the endothelium in either control mice or eNOS-/- mice and only sickle RBCs adhered to the endothelium in eNOS-/- mice. Sickle RBCs adhered to endothelial cells in control mice under normoxia at an adhesion score of 0.6, which was enhanced to 2.7 by hypoxia, suggesting that hypoxia increases sickle RBC adhesion. In contrast, in eNOS-/- mice, the adhesion score of sickle RBCs was 2.5 under normoxia and increased to 13.7 under hypoxia, indicating a synergistic enhancement of sickle RBC adhesion under hypoxia and low NO availability. To examine whether increasing NO bioavailability in eNOS-deficient mice is sufficient for inhibiting sickle RBC adhesion, we treated eNOS-/- mice under hypoxia with low-dose NO (20 ppm). Inhalation of low-dose NO (20 ppm) restored reduced NO metabolite levels in eNOS-/- mice and decreased sickle RBC adhesion scores under hypoxia to levels which were statistically indistinguishable from those of eNOS-/- mice under normoxia and comparable to those of control mice under hypoxia. These results suggest that NO inhalation disrupted sickle RBC adhesion by hypoxia and low NO bioavailability. Moreover, we showed that inhaled NO induced only a marginal change in wall shear rates which exert effects on blood cell-endothelial cell interactions, indicating that inhaled NO inhibits sickle RBC adhesion under hypoxic conditions by both wall shear rate-dependent and -independent mechanisms. These in vivo studies demonstrate that sickle RBC adhesion is regulated in a synergistic manner by hypoxia and NO bioavailability, and that inhaled NO inhibits sickle RBC adhesion in part through modulation of wall shear rates. Inhalation of NO may alleviate the clinical severity of SCD patients by interrupting the synergy created by hypoxia and reduced NO availability for sickle RBC adhesion. Disclosures:No relevant conflicts of interest to declare.

Pravastatin normalises peripheral cardiac sympathetic hyperactivity in the spontaneously hypertensive rat

Hypertension is associated with heightened cardiac sympathetic drive whilst statins reduce angiotensin II (ATII) signalling, superoxide anion production and increase nitric oxide bioavailability, events that can potentially reduce peripheral cardiac sympathetic neurotransmission. We therefore investigated whether pravastatin alters peripheral cardiac sympathetic control in the spontaneously hypertensive rat (SHR). SHRs (16–18weeks) had significantly (p<0.05) enhanced atrial 3H-norepinephrine (3H-NE) release to field stimulation compared to normotensive WKYs. 2-week pravastatin supplementation significantly reduced 3H-NE release to levels observed in the WKY. In-vivo, pravastatin lowered resting heart rate (HR) in the SHR despite not affecting arterial blood pressure or serum cholesterol. In SHR atria/right stellate ganglion preparations, the HR response to stellate stimulation (1, 3, and 5Hz) was also significantly reduced by pravastatin whilst the HR response to exogenous NE (0.025–5μmol) remained similar. The nitric oxide synthase (NOS) inhibitor l-NAME (1mmol/l) increased 3H-NE release by similar amounts in atria from supplemented and non-supplemented SHRs, whilst Western blotting showed no difference in protein levels of nNOS, eNOS, guanylyl cyclase, or the NADPH oxidase subunits Gp91 and P40phox. Pravastatin significantly reduced cardiac ATII levels and angiotensin converting enzyme 1 and 2 expressions whilst protein levels of the ATII receptor (ATR1) remained unchanged in the SHR. Immunohistochemistry co-localised ATR1 with tyrosine hydroxylase positive neurons in the stellate ganglion. The ATR1 antagonist Losartan (5μmol) equalised release of 3H-NE to comparable levels in supplemented and non-supplemented SHRs. These results suggest 2-week pravastatin treatment reduces cardiac ATII, and prevents its facilitatory effect on NE release thus normalising cardiac sympathetic hyper-responsiveness in SHRs.

Melatonin and vitamin C increase umbilical blood flow via nitric oxide‐dependent mechanisms

Inadequate umbilical blood flow leads to intrauterine growth restriction, a major killer in perinatal medicine today. Nitric oxide (NO) is important in the maintenance of umbilical blood flow, and antioxidants increase NO bioavailability. What remains unknown is whether antioxidants can increase umbilical blood flow. Melatonin participates in circadian, seasonal, and reproductive physiology, but has also been reported to act as a potent endogenous antioxidant. We tested the hypothesis that treatment during pregnancy with melatonin increases umbilical blood flow via NO-dependent mechanisms. This was tested in pregnant sheep by investigating in vivo the effects on continuous measurement of umbilical blood flow of melatonin before and after NO blockade with a NO clamp. These effects of melatonin were compared with those of the traditional antioxidant, vitamin C. Under anesthesia, 12 pregnant sheep and their fetuses (0.8 of gestation) were fitted with catheters and a Transonic probe around an umbilical artery, inside the fetal abdomen. Following 5 days of recovery, cardiovascular variables were recorded during fetal i.v. treatment with either melatonin (n=6, 0.5±0.1 μg/kg/min) or vitamin C (n=6, 8.9±0.4 mg/kg/min) before and after fetal NO blockade with the NO clamp. Fetal treatment with melatonin or vitamin C increased umbilical blood flow, independent of changes in fetal arterial blood pressure. Fetal NO blockade prevented the increase in umbilical blood flow induced by melatonin or vitamin C. Antioxidant treatment could be a useful clinical tool to increase or maintain umbilical blood flow in complicated pregnancy.

Nitric oxide bioavailability modulates the dynamics of microvascular oxygen exchange during recovery from contractions

lowered microvascular PO(2) (PO(2) mv) during the exercise off-transient likely impairs muscle metabolic recovery and limits the capacity to perform repetitive tasks. The current investigation explored the impact of altered nitric oxide (NO) bioavailability on PO(2) mv during recovery from contractions in healthy skeletal muscle. We hypothesized that increased NO bioavailability (sodium nitroprusside: SNP) would enhance PO(2) mv and speed its recovery kinetics while decreased NO bioavailability (l-nitro arginine methyl ester: l-NAME) would reduce PO(2) mv and slow its recovery kinetics. PO(2) mv was measured by phosphorescence quenching during transitions (rest-1 Hz twitch-contractions for 3 min-recovery) in the spinotrapezius muscle of Sprague-Dawley rats under SNP (300 microm), Krebs-Henseleit (CONTROL) and l-NAME (1.5 mm) superfusion conditions. relative to recovery in CONTROL, SNP resulted in greater overall microvascular oxygenation as assessed by the area under the PO(2) mv curve (PO(2 AREA) ; 3471 ± 292 mmHg s; SNP: 4307 ± 282 mmHg s; P < 0.05) and faster off-kinetics as evidenced by the mean response time (MRToff; 60.2 ± 6.9 s; SNP: 34.8 ± 5.7 s; P < 0.05), whereas l-NAME produced lower PO(2 AREA) (2339 ± 444 mmHg s; P < 0.05) and slower MRToff (86.6 ± 14.5s; P < 0.05). no bioavailability plays a key role in determining the matching of O(2) delivery-to-O(2) uptake and thus the upstream O(2) pressure driving capillary-myocyte O(2) flux (i.e. PO(2) mv) following cessation of contractions in healthy skeletal muscle. Additionally, these data support a mechanistic link between reduced NO bioavailability and prolonged muscle metabolic recovery commonly observed in ageing and diseased populations.

Flavonoids: Antioxidants Against Atherosclerosis

Oxidative stress results from an imbalance between excessive formation of reactive oxygen species (ROS) and/or reactive nitrogen species and limited antioxidant defences. Endothelium and nitric oxide (NO) are key regulators of vascular health. NO bioavailability is modulated by ROS that degrade NO, uncouple NO synthase, and inhibit synthesis. Cardiovascular risk conditions contribute to oxidative stress, causing an imbalance between NO and ROS, with a relative decrease in NO bioavailability. Dietary flavonoids represent a range of polyphenolic compounds naturally occurring in plant foods. Flavonoids are potentially involved in cardiovascular prevention mainly by decreasing oxidative stress and increasing NO bioavailability.