Marker Of Nitric Oxide Production Research Articles

NITRATE-RICH BEETROOT JUICE ALONE OR IN COMBINATION WITH FOLIC ACID REDUCES BLOOD PRESSURE IN HYPERTENSIVE TANZANIAN ADULTS: A DOUBLE-BLIND RANDOMIZED CONTROLLED TRIAL

Objective: In Sub-Saharan Africa, current management strategies are struggling to control the burgeoning hypertension epidemic. Dietary interventions such as inorganic nitrate and folate could represent alternative strategies for reducing blood pressure in this setting. This study aims to explore the effects of dietary inorganic nitrate supplementation alone or in combination with folic acid supplementation on blood pressure in a group of adult individuals with elevated blood pressure in Tanzania. Design and method: A placebo-controlled, double blind, randomized controlled trial (RCT) was conducted. Forty seven participants were randomly assigned to three conditions for a period of 60 days: 1) high-nitrate beetroot juice (∼400 mg nitrate) and folic acid (∼5 mg folate), 2) high-nitrate beetroot juice and placebo, 3) nitrate-depleted beetroot juice and placebo. Clinic and 24-hour ambulatory blood pressure, markers of nitric oxide production, and measurements of intervention compliance were obtained prior to and following the intervention period. Results: Resting clinic-based systolic blood pressure was significantly lower after 60 days of nitrate supplementation alone (p = 0.01). Both alone and combined nitrate + folate interventions had a significant effect on 24-Hour systolic blood pressure. After 60 days systolic blood pressure dropped by −10.8 ± 9.8 mmHg (p < 0.001) and −6.1 ± 13.2 mmHg (p = 0.03) in the nitrate alone and nitrate + folate groups, respectively. There was a significant decrease in 24-hour diastolic blood pressure in the nitrate alone group (−5.4 ± 5.0 mmHg, p = 0.04). Conclusions: Dietary inorganic nitrate, alone or in combination with folic acid, represents a potential nutritional strategy to lessen the hypertension epidemic in Sub-Saharan Africa. These findings support the rationale for future long-term investigations exploring the efficacy of dietary nitrate for lowering blood pressure and attenuating CVD risk in this setting.

The pathophysiology of acute gastric ulcer development in normotensive and hypertensive rats: A comparative study

Although gastric ulcers and hypertension are diseases that affect a large part of the population, the association of these comorbidities is still poorly studied. Therefore, the present study investigated the response of normotensive (NTR) and spontaneously hypertensive (SHR) rats to gastric ulcers induced by indomethacin or ethanol. For that, adult male and female NTR and SHR received indomethacin (100 mg/kg, p.o) or ethanol P.A (5 ml/kg, p.o) to induce gastric ulcer, after the pre-treatment with prostaglandin E2 (PGE2) and carbenoxolone (CBX), respectively. The results revealed that, when compared to NTR, the SHR, both male and female, showed lower lesion area indexes when exposed to indomethacin. On the other hand, ethanol caused an area of lesion approximately 60% larger in the male and female SHR in comparison with the NTR. Significantly, the pre-treatment with PGE2 or CBX prevented the gastric ulcer damage promoted by indomethacin or ethanol, respectively. The histological analyses of the gastric mucosa from ethanol-induced ulcer revealed severe disruption of gastric architecture and bleeding points, that have been exacerbated in the SHR group. The gastric tissue from the SHR group also showed high levels of nitrite, a marker of nitric oxide production, which was accompanied by an increase in lipid hydroperoxide levels, an important biomarker of oxidative damage, in comparison with NTR. Taking together, the results of the present study showed important differences in the development of gastric ulcer between NTR and SHR. Further studies are needed for an in-depth analysis of the pathophysiological mechanisms involved in these responses.

1109-P: Blood Pressure (BP) Lowering Effect of Empagliflozin Is Related to Decreased L-Arginine Clearance in Diabetic Rats

Introduction: The availability of L-arginine is the rate-limiting factor in Nitric Oxide (NO) synthesis, a potent vasodilatory factor. L-arginine plasma levels are blunted in diabetes, thus L-arginine supplementation decreases BP in animal diabetic models, and type 2 diabetic patients. Renal reabsorption of filtered L-arginine in the proximal tubule is mainly sodium dependent. Higher filtered glucose load in diabetes may alter sodium gradient via higher glucose reabsorption by sodium-glucose cotransporters (SGLTs). Purpose: The mechanism by which SGLT2 inhibitors, such as Empagliflozin, decrease BP in type 2 diabetic patients, is still unknown. We hypothesize that SGLT2 inhibitors could decrease the urinary excretion of L-arginine, by restoring sodium gradient necessary for L-arginine reabsorption in the proximal tubule. Methods: Male obese Zucker diabetic fatty/Spontaneously hypertensive heart failure hybrid (ZSF1) rats and their lean littermates (CTR) received either Empagliflozin or vehicle for 6 weeks. Levels of L-arginine in urine and plasma were quantified via Liquid Chromatography-Mass Spectrometry. Plasmatic cGMP level, a marker of NO production and urinary 8-hydroxydeoxyguanosine (8-OHdG) level, a marker of oxidative stress, were measured. Results: The urinary level of L-arginine was higher in ZSF-1 rats compared to CTR ones, and plasmatic concentrations of L-arginine were lower in ZSF-1 rats compared to CTR ones. Both parameters were significantly reversed by Empagliflozin. Empagliflozin treatment increased plasmatic cGMP and decreased urinary 8-OHdG in diabetic rats. Conclusion: Empagliflozin decreased urinary excretion of L-arginine, associated with an increased plasmatic concentration of L-arginine and cGMP, and decreased oxidative stress in diabetic rats. This suggests that Empagliflozin increased NO production and bioavailability. Such effects might contribute to the BP lowering effect of Empagliflozin in diabetics. Disclosure S. Khemais-Benkhiat: Employee; Self; Boehringer Ingelheim International GmbH. D. Veyel: Employee; Self; Boehringer Ingelheim Pharmaceuticals, Inc. V. Schini-Kerth: Research Support; Self; Boehringer Ingelheim International GmbH. B. Krawczyk: Employee; Self; Boehringer Ingelheim Pharmaceuticals, Inc. M. Pieper: Employee; Self; Boehringer Ingelheim Pharmaceuticals, Inc. M. Mark: Employee; Self; Boehringer Ingelheim International GmbH. E. Mayoux: Employee; Self; Boehringer Ingelheim Pharmaceuticals, Inc.

Maternal undernutrition aggravates renal tubular necrosis and interstitial fibrosis after unilateral ureteral obstruction in male rat offspring

Maternal undernutrition is known to reduce glomerular number but it may also affect tubulointerstitium, capillary density, and response to oxidative stress. To investigate whether the latter elements are affected, we examined the response to unilateral ureteral obstruction (UUO), an established model of renal tubulointerstitial fibrosis, in the kidney of offspring from control and nutrient restricted rats. Six-week old male offspring from rats given food ad libitum (CON) and those subjected to 50% food restriction throughout pregnancy (NR) were subjected to UUO for 7 days. Body weight was significantly lower in NR. Systolic blood pressure and blood urea nitrogen increased similarly in CON and NR after UUO. Tubular necrosis in the obstructed kidney, on the other hand, was more extensive in NR. Also, the collagen area, a marker of fibrosis, of the obstructed kidney was significantly increased compared with the contralateral kidney only in NR. Capillary density was decreased similarly in the obstructed kidney of CON and NR compared with the contralateral kidney. Urine nitrate/nitrite, a marker of nitric oxide production, from the obstructed kidney was significantly increased in NR compared with CON. Nitrotyrosine, a marker of nitric oxide-mediated free radical injury, was increased in the obstructed kidney compared with the contralateral kidney in both CON and NR, but the extent was significantly greater in NR.In conclusion, more severe tubular necrosis and fibrosis after UUO was observed in NR, which was thought to be due to increased nitrosative stress.

Antidepressant and Antiaging Effects of Açaí (Euterpe oleracea Mart.) in Mice

Depression is a mental disorder that affects 300 million people of all ages worldwide, but fewer than half of those with the condition receive adequate treatment. In addition, the high pharmacological refractoriness (affecting 30%-50% of patients) and toxicity of some classical antidepressants support the pursuit of new therapies. People with this condition show depressed mood, loss of pleasure, high levels of oxidative stress, and accelerated biological aging (decreased telomere length and expression of the telomerase reverse transcriptase (TERT), the enzyme responsible for telomere maintenance). Because of the close relationship between depression and oxidative stress, nutraceuticals with antioxidant properties are excellent candidates for therapy. This study represents the first investigation of the possible antidepressant and antiaging effects of commercial samples of clarified açaí (Euterpe oleracea) juice (EO). This fruit is rich in antioxidants and widely consumed. In this study, mice were treated with saline or EO (10 μL/g, oral) for 4 days and then with saline or lipopolysaccharide (0.5 mg/kg, i.p.) to induce depressive-like behavior. Only four doses of EO were enough to abolish the despair-like and anhedonia behaviors and alterations observed in electromyographic measurements. The antidepression effect of EO was similar to that of imipramine and associated with antioxidant and antiaging effects (preventing lipid peroxidation and increasing TERT mRNA expression, respectively) in three major brain regions involved in depression (hippocampus, striatum, and prefrontal cortex). Additionally, EO significantly protected hippocampal cells, preventing neuronal loss associated with the depressive-like state and nitrite level increases (an indirect marker of nitric oxide production). Moreover, EO alone significantly increased TERT mRNA expression, revealing for the first time a potent antiaging action in the brain that suggests neuroprotection against long-term age-related consequences.

The Cerebral Brain-Derived Neurotrophic Factor Pathway, Either Neuronal or Endothelial, Is Impaired in Rats with Adjuvant-Induced Arthritis. Connection with Endothelial Dysfunction.

Cognitive abilities are largely dependent on activation of cerebral tropomyosin-related kinase B receptors (TrkB) by brain-derived neurotrophic factor (BDNF) that is secreted under a bioactive form by both neurons and endothelial cells. In addition, there is mounting evidence for a link between endothelial function and cognition even though the underlying mechanisms are not well known. Therefore, we investigated the cerebral BDNF pathway, either neuronal or endothelial, in rheumatoid arthritis (RA) that combines both endothelial dysfunction (ED) and impaired cognition. Adjuvant-induced arthritis (AIA) in rats was used as a model of RA. Clinical inflammatory symptoms were evaluated from an arthritis score and brains were collected at day 31 ± 2 post-immunization. Neuronal expression of BDNF and TrkB phosphorylated at tyrosine 816 (p-TrkB) was examined in brain slices. Endothelial BDNF and p-TrkB expression was examined on both brain slices (hippocampal arterioles) and isolated cerebral microvessels-enriched fractions (vessels downstream to arterioles). The connection between endothelial nitric oxide (NO) and BDNF production was explored on the cerebrovascular fractions using endothelial NO synthase (eNOS) levels as a marker of NO production, Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) as a NOS inhibitor and glyceryl-trinitrate as a slow releasing NO donor. Brain slices displayed lower BDNF and p-TrkB staining in both neurons and arteriolar endothelial cells in AIA than in control rats. For endothelial cells but not neurons, a strong correlation was observed between BDNF and p-TrkB staining. Of note, a strong correlation was also observed between neuronal p-TrkB and endothelial BDNF staining. In cerebral microvessels-enriched fractions, AIA led to decreased BDNF and eNOS levels with a positive association between the 2 parameters. These effects coincided with decreased BDNF and p-TrkB staining in endothelial cells. The exposure of AIA cerebrovascular fractions to GTN increased BDNF levels while the exposure of control fractions to L-NAME decreased BDNF levels. Changes in the cerebral BDNF pathway were not associated with arthritis score. The present study reveals that AIA impairs the endothelial and neuronal BDNF/TrkB pathway, irrespective of the severity of inflammatory symptoms but dependent on endothelial NO production. These results open new perspectives for the understanding of the link between ED and impaired cognition.

Prolonged diuretic and saluretic effect of nothofagin isolated from Leandra dasytricha (A. Gray) Cogn. leaves in normotensive and hypertensive rats: Role of antioxidant system and renal protection

Although the acute diuretic effect of nothofagin has been recently demonstrated, its effects after dose-repeated treatment have not yet been explored. For that, male Wistar normotensive (NTR) and spontaneously hypertensive rats (SHR) were orally treated, once a day, with vehicle (VEH: distilled water; 1 ml/kg), hydrochlorothiazide (HCTZ; 10 mg/kg) or nothofagin (NOT; 1 mg/kg). The cumulative diuretic index and urinary electrolytes excretion were measured each 24 h. On the last day of the experiment (7th day), urine, blood and kidney samples were collected for biochemical and molecular analyzes. The urinary volume of both NTR and SHR were significantly increased with the treatment with NOT (from the second to the seventh day of treatment), with final values reaching an increase of 56% and 82%, respectively, when compared with VEH-treated group. This effect was associated with increased levels of urinary excretion of Na+ and Cl−, without any changes on K+ excretion. None of the treatments modified urinary pH or density values. Importantly, neither the NOT nor the HCTZ caused any change in body weight following the dose-repeated treatment, and also did not provoke an electrolytic disturbance. Regarding the renal analyzes, when compared with the vehicle-treated NTR group, the activity of superoxide dismutase (SOD) and the reduced glutathione (GSH) levels in kidney homogenates of the SHR group were decreased, while the generation of lipid hydroperoxides were significantly increased. The daily treatment with NOT was able to restore the GSH levels and SOD activity, as well as reduced the lipoperoxidation in the kidney homogenates obtained from SHR animals. Finally, NOT significantly augmented the levels of nitrite, a marker of nitric oxide production, in the plasma obtained from SHR group when compared with the vehicle-treated only NTR. This study revealed the prolonged diuretic and saluretic effect of nothofagin in normotensive and hypertensive rats. Our data also showed the renal protective effects of nothofagin by the improvement of antioxidative capacity, as well as by the augmented bioavailability of plasma nitric oxide in the hypertensive group.

Relationship between asymmetric dimethylarginine, nitrite and genetic polymorphisms: Impact on erectile dysfunction therapy

Sildenafil is the most used treatment of erectile dysfunction, however a large part of patients do not respond to therapy. This drug enhances nitric oxide (NO) signaling, and therefore factors that alter NO production may impact this drug responsiveness. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of all NO synthases, and is metabolized by Dimethylarginine Dimethilaminohydrolase (DDAH) 1 and 2. Here we aimed to assess the relationship between plasma levels of ADMA and nitrite (marker of nitric oxide production) with Sildenafil responsiveness. We also studied genetic polymorphisms in DDAH1 and DDAH2 genes and their relation with biochemical and clinical data. Were included here 140 patients, divided in Clinical Erectile Dysfunction (CED) or Post-Prostatectomy Erectile Dysfunction (PPED) groups. Erectile function was evaluated before and after Sildenafil on-demand treatment using the International Index for Erectile Function Questionnaire. We have found that nitrite was associated with worse response to Sildenafil (r = − 0.25, P = 0.040). rs1554597 and rs18582 DDAH1 polymorphisms were associated with changes in ADMA levels in CED (B = − 0.23, P = 0.002; B = − 0.15, P = 0.017 for both variant genotypes, respectively). Finally, DDAH2 polymorphisms were associated with altered responsiveness to Sildenafil in PPED (B = +0.19, P = 0.027).

Involvement of hypothalamic nitric oxide signaling in the modulation of a rat's exercise capacity.

The aim of this study was to explore the effect of nitric oxide (NO) in some regions of the hypothalamus on exercise capacity. To assess the role of central NO in exercise capacity, L-arginine (L-Arg, a precursor of NO synthesis), NG-nitro-L-arginine methyl ester (L-NAME, a NO synthase inhibitor), or placebo saline was injected into the lateral cerebral ventricle of rats once a day for 4 consecutive days. Thereafter, an one-time exhaustive treadmill exercise was performed, and the levels of nitrate/nitrite, as a marker of NO production, in blood plasma and hypothalamus were assayed. Neuronal nitric oxide synthase (nNOS)-expressing cells were immunohistochemically stained and analyzed in the paraventricular nucleus (PVN), the dorsomedial hypothalamus (DMH), and the ventromedial hypothalamus. Exercise time to exhaustion and total workload were determined. Compared with the rats in the saline group, the exercise time to exhaustion and total workload increased 50% in the L-Arg group and decreased 50% in the L-NAME group. The nitrate/nitrite level of hypothalamus in the L-Arg group increased 50% and decreased 29.4% in the L-NAME group. The number of nNOS-positive cells was significantly increased, 56.5%, in PVN and, 119%, in DMH, but not in ventromedial hypothalamus. No significant changes in nNOS-positive cells were found in L-NAME-treated rats. These results show that the modulation of hypothalamic NO signaling can affect the rat's running performance during a treadmill exercise and that enhanced NO signaling by induction of nNOS in PVN and DMH plays a role in improving exercise capacity after central administration of L-Arg. NO signaling in PVN and DMH may be a useful target for the pharmacological intervention of exercise performance or capacity.

Acute effects of quercetin-3-O-glucoside on endothelial function and blood pressure: a randomized dose-response study

Epidemiologic studies have suggested that a flavonoid-rich diet can reduce the risk of developing cardiovascular disease. Certain flavonoids, in particular quercetin, have been shown to ameliorate endothelial dysfunction and reduce blood pressure (BP), possibly by increasing the bioavailability of the potent vasodilator nitric oxide (NO). Several studies have indicated that improvements in measures of cardiovascular health do not occur linearly, but rather, plateau or decrease with an increasing dose of flavonoids. We determined whether the acute administration of increasing doses of a common quercetin glycoside (quercetin-3-O-glucoside) improves endothelial function and reduces BP in a dose-dependent manner. We also explored whether any effects were correlated with changes in plasma NO production. A randomized, controlled, crossover study was performed in 15 healthy volunteers who each completed 5 visits with a minimum washout period of 1 wk between testing days. Participants received each of the following 5 interventions in a random order: 1) 0, 2) 50, 3) 100, 4) 200, or 5) 400 mg quercetin-3-O-glucoside. Endothelial function and BP were assessed before and 60 min after intervention. A blood sample was taken before and 90 min after intervention for the analysis of plasma nitrate and nitrite as markers of NO production as well as of plasma quercetin metabolites. Although we observed a significant correlation between the dose of quercetin-3-O-glucoside and plasma concentrations of total quercetin (R(2) = 0.52, P < 0.001) and isorhamnetin (R(2) = 0.12, P = 0.005), we showed no improvements in endothelial function or BP and no changes in NO production after any dose. From these results, we conclude that there are no acute changes in BP or the NO-mediated endothelium-dependent relaxation of the brachial artery with doses of quercetin ranging from 50 to 400 mg in healthy men and women. This trial was registered at www.anzctr.org.au as ACTRN12615001338550.

Source memory in rats is impaired by an NMDA receptor antagonist but not by PSD95-nNOS protein–protein interaction inhibitors

Limitations of preclinical models of human memory contribute to the pervasive view that rodent models do not adequately predict therapeutic efficacy in producing cognitive impairments or improvements in humans. We used a source-memory model (i.e., a representation of the origin of information) we developed for use in rats to evaluate possible drug-induced impairments of both spatial memory and higher order memory functions in the same task. Memory impairment represents a major barrier to use of NMDAR antagonists as pharmacotherapies. The scaffolding protein postsynaptic density 95kDa (PSD95) links NMDARs to the neuronal enzyme nitric oxide synthase (nNOS), which catalyzes production of the signaling molecule nitric oxide (NO). Therefore, interrupting PSD95-nNOS protein–protein interactions downstream of NMDARs represents a novel therapeutic strategy to interrupt NMDAR-dependent NO signaling while bypassing unwanted side effects of NMDAR antagonists. We hypothesized that the NMDAR antagonist MK-801 would impair source memory. We also hypothesized that PSD95-nNOS inhibitors (IC87201 and ZL006) would lack the profile of cognitive impairment associated with global NMDAR antagonists. IC87201 and ZL006 suppressed NMDA-stimulated formation of cGMP, a marker of NO production, in cultured hippocampal neurons. MK-801, at doses that did not impair motor function, impaired source memory under conditions in which spatial memory was spared. Thus, source memory was more vulnerable than spatial memory to impairment. By contrast, PSD95-nNOS inhibitors, IC87201 and ZL006, administered at doses that are behaviorally effective in rats, spared source memory, spatial memory, and motor function. Thus, PSD95-nNOS inhibitors are likely to exhibit favorable therapeutic ratios compared to NMDAR antagonists.

The red-vine-leaf extract AS195 increases nitric oxide synthase-dependent nitric oxide generation and decreases oxidative stress in endothelial and red blood cells.

The red‐vine‐leaf extract AS195 improves cutaneous oxygen supply and the microcirculation in patients suffering from chronic venous insufficiency. Regulation of blood flow was associated to nitric oxide synthase (NOS)‐dependent NO (nitric oxide) production, and endothelial and red blood cells (RBC) have been shown to possess respective NOS isoforms. It was hypothesized that AS195 positively affects NOS activation in human umbilical vein endothelial cells (HUVECs) and RBC. Because patients with microvascular disorders show increased oxidative stress which limits NO bioavailability, it was further hypothesized that AS195 increases NO bioavailability by decreasing the content of reactive oxygen species (ROS) and increasing antioxidant capacity. Cultured HUVECs and RBCs from healthy volunteers were incubated with AS195 (100 μmol/L), tert‐butylhydroperoxide (TBHP, 1 mmol/L) to induce oxidative stress and with both AS195 and TBHP. Endothelial and red blood cell–nitric oxide synthase (RBC‐NOS) activation significantly increased after AS195 incubation. Nitrite concentration, a marker for NO production, increased in HUVEC but decreased in RBC after AS195 application possibly due to nitrite scavenging potential of flavonoids. S‐nitrosylation of RBC cytoskeletal spectrins and RBC deformability were increased after AS195 incubation. TBHP‐induced ROS were decreased by AS195, and antioxidative capacity was significantly increased in AS195‐treated cells. TBHP also reduced RBC deformability, but reduction was attenuated by parallel incubation with AS195. Adhesion of HUVEC was also reduced after AS195 treatment. Red‐vine‐leaf extract AS195 increases NOS activation and decreases oxidative stress. Both mechanisms increase NO bioavailability, improve cell function, and may thus account for enhanced microcirculation in both health and disease.

Hypotensive effects of omentin-1 related to increased adiponectin and decreased interleukin-6 in intra-thoracic pericardial adipose tissue

BackgroundOmentin is an adipokine expressed in visceral adipose tissue (VAT). In vitro studies demonstrated that omentin induces vasorelaxation in isolated rat mesenteric arteries, and in vivo studies showed inhibition of agonist-induced increases in blood pressure, possibly mediated by nitric oxide (NO)-dependent mechanisms. MethodsWe investigated, in normotensive rats, the effects of subacute omentin-1 administration [8μg/kg, intraperitoneally (ip), once daily for 14 days] on cardiac activity, blood pressure, plasma concentration of l-citrulline (as a marker of NO production from l-arginine), and the gene expression of adiponectin, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in intra-thoracic pericardial adipose tissue (PAT). Electrocardiography (ECG), heart rate (HR), mean blood pressure (MBP), pulse pressure (PP) were monitored before and after treatment with omentin-1 or vehicle. ResultsWith respect to baseline and vehicle, we found a significant decrease of MBP (p<0.005) and PP (p<0.05) after treatment with omentin-1, while ECG and HR were not modified. Omentin-1 significantly increased l-citrulline levels in plasma (p<0.05), and the gene expression of adiponectin in PAT (p<0.05). On the other hand, we found decreased gene expression of IL-6 (p<0.005), while TNF-α mRNA in PAT was not affected. ConclusionWe conclude that the hypotensive effects of omentin-1 could be driven by stimulated production of NO in the vascular system, possibly related to increased adiponectin and decreased IL-6 mRNA in PAT.

Correlation between asymmetric dimethylarginine and cerebral vasospasm secondary to subarachnoid hemorrhage

Objective To investigate the correlation between asymmetric dimethylarginine (ADMA) and cerebral vasospasm (CVS) secondary to subarachnoid hemorrhage (SAH).Methods Seventy-eight healthy male adult Sprague-Dawley rats were randomly allocated into control group (n =6),sham group (n =36) and SAH group (n =36).In SAH group,a SAH model was established by double autologous blood injection into cisterna magna.Arterial blood,cerebrospinal fluid (CSF) and basilar artery samples of each group were collected at the preconcerted time.ADMA levels in the plasma and CSF samples were assessed using enzyme-linked immuno sorbent assay (ELISA).Nitrite and nitrate concentrations were used as a marker of nitric oxide production in plasma and CSF samples and determined by using nitrate reductase method.Pathomorphological changes were observed with optical microscope and inner diameter of basilar artery was measured by Micrometer after hematoxylin-eosin staining was performed.Results (1) In SAH group,ADMA levels in CSF samples were significantly increased at 1st,3rd,5th and 7th day (P ＜0.05),and NO levels in CSF samples were significantly decreased at 3rd,5th,and 7th day as compared with sham group (P＜0.01).(2) The ratio of thickness and inner diameter of basilar artery was used to indicate the degree of CVS.The level of ADMA in CSF samples was strongly correlated with the degree of CVS (r =0.641,P ＜0.01).Nitrite levels in CSF samples were negatively correlated with ADMA levels in CSF samples (r =-0.687,P ＜0.01).(3) Plasma ADMA levels in sham group and SAH group were similar and remained essentially unchanged during the several days after SAH.Plasma ADMA levels were neither correlated with NO levels (r =0.019,P ＞ 0.05) nor with the degree of vasospasm (r =-0.046,P ＞ 0.05).Conclusion Asymmetric dimethylarginine reduces the level of nitric oxide in CSF by inhibiting nitric oxide synthase and may be involved in the progression of cerebral vasospasm.The level of ADMA in CSF samples was strongly correlated with the degree of CVS in SAH rats.There is no evident correlation between the level of ADMA in plasma and the degree of CVS. Key words: Subarachnoid hemorrhage; Cerebral vasospasm ; Asymmetric dimethylarginine ; Nitric oxide

Development of the Reliable Fluorometric Assay for the Sensitive Determination of Nitric Oxide Metabolites in Biological Fluids

In this study, we developed a fluorescence measurements in microplates for high-throughput determination of nitric oxide (NO) metabolites, which is used as a reliable and sensitive marker of NO production, in biological fluids using a pH-independent fluorescence increasing probe, DAMBO-PH. The sensitivity of this method is lower than that of the other detection methods reported in the literature. This method was demonstrated to be useful for the rapid screening of large numbers of biological and clinical samples, and should be an important tool for further clarifying the physiology, pathology and pharmacology roles of NO.

Postprandial effects of a high salt meal on serum sodium, arterial stiffness, markers of nitric oxide production and markers of endothelial function

AimThe aim of the study was to determine if a high salt meal containing 65 mmol Na causes a rise in sodium concentrations and a reduction in plasma nitrate/nitrite concentrations (an index of nitric oxide production). Secondary aims were to determine the effects of a high salt meal on augmentation index (AIx) a measure of arterial stiffness and markers of endothelial function. Methods and resultsIn a randomised cross-over study 16 healthy normotensive adults consumed a low sodium soup containing 5 mmol Na and a high sodium soup containing 65 mmol Na. Sodium, plasma nitrate/nitrite, endothelin-1 (ET-1), C-reactive protein (CRP), vasopressin (AVP) and atrial natriuretic peptide (ANP) concentrations before and every 30 min after the soup for 2 h. Blood pressure (BP) and AI were also measured at these time points.There were significant increases in serum sodium, osmolality and chloride in response to the high sodium meal. However plasma nitrate/nitrite concentrations were not different between meals (meal p = 0.812; time p = 0.45; meal × time interaction p = 0.50). Plasma ANP, AVP and ET-1 were not different between meals. AI was significantly increased following the high sodium meal (p = 0.02) but there was no effect on BP. ConclusionsA meal containing 65 mmol Na increases serum sodium and arterial stiffness but does not alter postprandial nitrate/nitrite concentration in healthy normotensive individuals. Further research is needed to explore the mechanism by which salt affects vascular function in the postprandial period.This trial was registered with the Australian and New Zealand Clinical Trials Registry Unique Identifier: ACTRN12611000583943http://www.anzctr.org.au/trial_view.aspx?ID=343019.

Rolipram Improves Renal Perfusion and Function during Sepsis in the Mouse

Microcirculatory dysfunction is correlated with increased mortality among septic patients and is believed to be a major contributor to the development of acute kidney injury (AKI). Rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, has been shown to reduce microvascular permeability and in the kidney, increase renal blood flow (RBF). This led us to investigate its potential to improve the renal microcirculation and preserve renal function during sepsis using a murine cecal ligation and puncture (CLP) model to induce sepsis. Rolipram, tested at doses of 0.3-10 mg/kg i.p., acutely restored capillary perfusion in a bell-shaped dose-response effect with 1 mg/kg being the lowest most efficacious dose. This dose also acutely increased RBF despite transiently decreasing mean arterial pressure. Rolipram also reduced renal microvascular permeability. It is noteworthy that delayed treatment with rolipram at 6 hours after CLP restored the renal microcirculation, reduced blood urea nitrogen and serum creatinine, and increased glomerular filtration rate at 18 hours. However, delayed treatment with rolipram did not reduce serum nitrate/nitrite levels, a marker of nitric oxide production, nor reactive nitrogen species generation in renal tubules. These data show that restoring the microcirculation with rolipram, even with delayed treatment, is enough to improve renal function during sepsis despite the generation of oxidants and suggest that PDE4 inhibitors should be evaluated further for their ability to treat septic-induced AKI.

L-Arginine Supplementation and Nitric Oxide Production:No Additional Effect When Associated to Exercise

L-arginine is an amino acid semiessencial considered a precursor of nitric oxide, a gas mainly produced in endothelial cells. Nutritional supplements based on the amino acid L-arginine have been broadly marketed in order to increase vasodilation and the blood supply to muscle in order to optimize metabolic responses induced by exercise. The main objective of this study was to evaluate the effect of L-arginine supplementation on nitric oxide production in response to exercise. Furthermore, the biochemical parameters of muscle fatigue were assessed. Fourteen trained runners were divided in two groups, supplemented with L-arginine (ARG) and placebo (PLA). Blood samples were collected before supplementation (T0), immediately after the first exercise session (T1), immediately after the second exercise session (T2), and after 20 minutes of rest (T3). Plasma cyclic guanosine monophosphate was assessed as a marker of nitric oxide production. The biochemical parameters of muscle fatigue analyzed were plasma lactate and ammonia. There was significant increase in plasma cyclic guanosine monophosphate in both groups in response to exercise: ARG (T0: 3.6 ± 1.4; T1: 17.9 ± 5.8; T2: 15.9 ± 5.3; T3: 7.3 ± 2.5 pmol/mL) and PLA (T0: 4.1 ± 1.1; T1: 18.8 ± 9.9; T2: 16.1 ± 3.5; T3: 9.3 ± 3.7 pmol/mL). A significant reduction in plasma lactate and ammonia were observed in the recovery period after exercise (T3). However, no significant difference was observed between groups in the variables studied. Therefore, L-arginine supplementation was unable to increase the effects of exercise on nitric oxide production and did not improve the metabolic responses to exercise in runners.

Measuring exhaled nitric oxide in animal models: methods and clinical implications

Animal models such as rats and mice are useful for studying the multiple roles of nitric oxide (NO) in various respiratory disorders. The production of NO is catalyzed by the three isoforms of the enzymes (NO synthases; NOS). Indirect assessment of NOS gene or protein expression only provides partial information on the role of NO in health and lung disease. NO can also be measured in exhaled air by invasive or non-invasive approaches as a direct and quantitative marker of NO production in animal models. Whilst addressing the different methods of exhaled NO analysis in small animals (rats and mice), this review also focuses on the possible clinical implications, and discusses the advantages and limitations of these methods.

Nanoparticles disrupt lung function either directly or by inflammatory induction

Nanoparticle interactions with lung lining fluid are dependent upon aggregation and agglomeration of the particles, which is determined by particle size, shape, and surface chemistry. In this study, we examined how silver composition altered particle interaction. C57-6 Black male mice were dosed intratracheally with either carbon black or silver nanospheres (1 or 10 mg/kg) and compared to HBSS instillation as control. 24 h after exposure, mechanical data of the lung were acquired via Flexivent. Following ventilation, bronchoalveolar lavage fluid was sampled for cell count/differentials, analysis of inflammatory markers, markers of nitric oxide production, and to determine phospholipid content and/or changes in surfactant protein ratios/structure. Mechanical data demonstrated that silver particles as well as high dose carbon black mediate an increase in low frequency resistance and increased tissue stiffness. The low frequency resistance was not dependent upon PEEP, whereas the initial tissue stiffness resolved by increasing PEEP. All treatment groups except 10 mg carbon black demonstrated little neutrophilia, as well as no significant increase in inflammatory markers, as compared to the control, and no major differences in NO production. High dose carbon black treated lung lining fluid, however, contained increased neutrophilia, increased expression of IL1b, IL12p40, IL-6, and CCL2, as well as an increased level of nitrates. Additionally, 1 μg and 10 μg silver and 10 μg carbon black treated lungs displayed decreased surfactant protein-D (SP-D) content. This study demonstrates that silver nanoparticles disrupt lung function, possibly via surfactant disruption, while carbon black induces a classic inflammatory response.