Index Of Nitric Oxide Production Research Articles

S-11-5: CLINICAL IMPLICATION OF FMD

A healthy endothelium maintains vascular tone and structure by regulating the balance between vasodilation and vasoconstriction, growth inhibition and growth promotion, antithrombosis and prothrombosis, anti-inflammation and proinflammation, and also antioxidation and pro-oxidation. The vascular endothelium is involved in the release of various vasodilators, including nitric oxide (NO), prostaglandins, and endothelium-derived hyperpolarizing factor as well as vasoconstrictors. NO plays an important role in the regulation of vascular tone, the inhibition of platelet aggregation, and the suppression of smooth muscle cell proliferation. Cardiovascular diseases are associated with endothelial dysfunction. In addition, endothelial dysfunction is an early feature of atherosclerosis and vascular diseases in humans. Therefore, it is clinically important to estimate the degree of endothelial dysfunction. Several methods have been used to assess endothelial function in humans. Measurement of flow-mediated vasodilation (FMD) using an ultrasound is noninvasive and is an accurate indicator of NO production. Measurement of FMD is widely performed as an index of conduit artery endothelial function. Several investigators have shown that FMD is an independent predictor for cardiovascular outcomes. It is thought that FMD is useful for surrogate endpoint of cardiovascular outcomes and index of atherosclerotic treatment. However, the evaluation technique by the FMD measurement system still requires skills in measurement of the vessel diameter by an ultrasound system, and is difficult to handle. In addition, there is a problem in that the technique requires a large-scale apparatus and lacks in simplicity. In this session, we would like to explain in detail concerning the methods of assessing and clinical importance of endothelial function, especially FMD.

Vitamin D3 mediates spatial memory improvement through nitric oxide mechanism in demyelinated hippocampus of rat

Studies have revealed beneficial role of vitamin D3 in neuro-cognitive function. There is also supporting evidence on the involvement of nitric oxide (NO) in the neuro-protective action. However, its over production could contribute to brain disorders. In this study, demyelination was induced by ethidium bromide (EB) injection into the right side of the hippocampus area of male rats. Vitamin D3 was administered to rats for 7 and 28 days prior to behavioral experiments using Morris water maze (MWM). Travelled distance, time spent to reach the platform, and time spent in target zone, were considered for learning and spatial memory evaluation. Nitrite oxide (NO2-) concentration was measured as an indicator for nitric oxide production. The time spent to reach the platform and the travelled distance were decreased significantly by 28 days of vitamin D3 administration (compared to 7 days experiment). Time spent in target quadrant was significantly lowered by administered vitamin on day 28. Therefore, considering a number of studies that have shown the effect of vitamin D3 on cognition, these findings could support their potential effect. Besides, nitric oxide concentration significantly differed in 28 days of vitamin D3 treated group compared with the groups treated with EB or 7 days of vitamin D3.

THE VASCULAR ENDOTHELIUM FUNCTION INDICATORS IN ORAL LIQUID OF IQOS SMOKING ADOLESCENTS

Heated tobacco products (HTP) were introduced to the world market as an alternative to conventional tobacco cigarettes. Active advertising, readily available to purchase and the public's perception that HTP are a safer alternative to conventional tobacco cigarettes lead to increasing their consumption by young age group in Ukraine. Despite the claims by the tobacco corporations about reduction of adverse effects the HTP on the activity of cardiovascular system, the published studies of potential vascular endothelial consequences were not found. Therefore, the aim of our study is to assess the HTP effect on vascular endothelial cells by determination of the nitrate, nitrite, S-nitrosothiols, and endothelin-1 levels in saliva of smoking adolescents. The object and research methods. Thirty adolescents aged 14-17 years old were divided to three groups. Group I was introduced by ten healthy non-smoking adolescents (control group). Group II consists of ten smoking adolescents have smoked the IQOS for two years. Group III is formed by ten smoking adolescents who have smoked the IQOS for three years. The salivary nitrate and nitrite levels were measured using the Griess assay such as an indicator of NO production. The salivary S-nitrosothiols were measured spectrophotometrically. The salivary endothelin-1 level was measured with ELISA kit. Statistical analysis of the data was carried out using the STATISTICA 7.0 program. The data were presented as Median values (Me), lower and upper quartile, i.e. Me [Lq; Uq]. Statistical significance was accepted if p < 0.05. Results. The salivary nitrate concentrations in groups II (Me = 14.11 [13.98; 14.52] μmol/l) and III (Me = 12.06 [11.44; 12.31] μmol/l) were significantly decreased compared to group I (Me = 15.89 [14.98; 16.89] μmol/l, KW test: H = 23.59, p=0.000. The values of nitrite in groups II and III were the following: Me = 2.32 [1.47; 2.48] μmol/l and 1.37 [1.25; 1.44] μmol/l, respectively. The indices were lower in 1.41 and 2.36 times than in group I: Me = 3. 24 [3.05; 3.78] μmol/l, KW test: Н = 22.92, p = 0.000. The levels of salivary S-nitrosothiols in group II are: Me = 1.02 [0.84; 1.27] μmol/l and group III Me = 0.36 [0.25; 0.62] μmol/l were reduced in 1.75 and 4.97 times compared to group I: Me = 1.79 [1.67; 1.97] μmol/l, KW test: Н = 24.46, р = 0.000. We assume that the reduction of salivary nitrate and nitrite concentrations is associated with a low level of S-nitrosothiols which serve as deposited and released nitric oxide (NO). Probably, the decrease of nitrates and nitrites is associated with the inhibition of endothelial NO synthase. The salivary endothelin-1 level was significantly higher in group II: Ме=0.58 [0.53; 0.73] pmol/ml and in group III: Ме = 0.82 [0.79; 0.93] pmol/ml than in group I: Ме = 0.17 [0.14; 0.26] pmol/ml, KW test: Н = 23.94, р = 0.000. It should be noted, the significant differences between data of nitrate, nitrite, S-nitrosothiol and endothelin-1 content in groups II and III were found (Mann-Whitney U test, nitrates - Z = 3.62, p = 0.000; nitrites - Z = 2.91 , p = 0.003; S-nitrosothiols - Z = 3.74, p = 0.00; endothelin-1 - Z = 3.36, p = 0.000). These results showed interrelation between adverse IQOS effect on vascular endothelium of adolescents duration of IQOS smoke. Conclusions. The adverse effect of heated tobacco product (IQOS) on the biochemical indicators of the vascular endothelium function of adolescents was found in our experimental study. The salivary nitrate, nitrite and S-nitrosothiols levels were significantly decreased in IQOS smoking adolescents. The salivary endothelin-1 content was significantly elevated in IQOS smoking adolescents. The revealed changes indicate the endothelial dysfunction in young smokers. The development of pathological processes in the vascular endothelium depends on the smoking duration.

Female rats express heroin-induced and -conditioned suppression of peripheral nitric oxide production in response to endotoxin challenge

Opioids and opioid-conditioned stimuli (CS) negatively alter host immunity, impairing the response to pathogens during opioid use and following drug cessation. Using male rats, our laboratory has determined that heroin or heroin-CS exposure preceding a lipopolysaccharide (LPS) challenge markedly suppresses normal induction of peripheral pro-inflammatory biomarkers. Presently, it is unknown if these heroin-induced and -conditioned effects extend to the female immune response. To begin this venture, the current study tested the direct effects of heroin and heroin-CS on LPS-induced peripheral nitric oxide (NO) production in female rats. We focused investigations on peripheral NO as it is a critical pro-inflammatory molecule necessary for pathogen resistance. In Experiment 1, male and female Lewis rats were administered 0 (Saline), 1, or 3 mg/kg heroin subcutaneously (s.c). Sixty minutes later, animals were injected with LPS (1 mg/kg, s.c.). Spleen and plasma samples were collected 6 h later to examine NO production through inducible NO synthase (iNOS) expression and nitrate/nitrite concentration, respectively. In Experiment 2, female Lewis rats underwent five, 60-minute context conditioning sessions with heroin (1 mg/kg, s.c.) or saline. On test day, CS-exposed and control (home cage) animals were injected with LPS (1 mg/kg, s.c.). Tissue was collected 6 h later to examine splenic iNOS expression and plasma nitrate/nitrite concentration. Both heroin administration alone and exposure to heroin-CS suppressed LPS-induced indices of NO production in spleen and plasma. Our results are the first to indicate that, similar to males, female rats express heroin-induced and -conditioned immunomodulation to a LPS challenge.

Circulating Concentrations of Key Regulators of Nitric Oxide Production in Undernourished Sheep Carrying Single and Multiple Fetuses.

Simple SummaryThe present study aimed to determine the blood concentrations of L-arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and L-homoarginine, modulating nitric oxide (NO) synthesis, in single, twin. and triplet pregnancies in ewes undergoing either dietary energy restriction or receiving 100% of their energy requirements. Blood concentrations of L-arginine, of its metabolites. and the ratio between NO synthesis boosters and inhibitors are altered in energy-restricted ewes, these alterations being higher in ewes carrying multiple fetuses.The aim of this study was to investigate the blood concentrations of L-arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and L-homoarginine, which are regulators of nitric oxide (NO) synthesis, in single, twin, and triplet pregnancies in ewes undergoing either a dietary energy restriction or receiving 100% of their energy requirements. From day 24 to 100 of pregnancy, the ewes were fed ryegrass hay and two different iso-proteic concentrates fulfilling either 100% of ewes’ energy requirements (control group; n = 30, 14 singleton pregnancies, 12 twin pregnancies, and 4 triplet pregnancies) or only 45% (feed-restricted group; n = 29; 11 singleton pregnancies, 15 twin pregnancies, and 3 triplet pregnancies). Blood samples were collected monthly to measure, by capillary electrophoresis, the circulating concentrations of arginine, ADMA, homoarginine, SDMA, and of other amino acids not involved in NO synthesis to rule out possible direct effects of diet restriction on their concentrations. No differences between groups were observed in the circulating concentrations of most of the amino acids investigated. L-homoarginine increased markedly in both groups during pregnancy (p < 0.001). SDMA (p < 0.01), L-arginine, and ADMA concentrations were higher in feed-restricted ewes than in controls. The L-arginine/ADMA ratio, an indicator of NO production by NOS, decreased towards term without differences between groups. The ADMA/SDMA ratio, an index of the ADMA degrading enzyme activity, was higher in controls than in feed-restricted ewes (p < 0.001). Obtained results show that circulating concentrations of L-arginine, of its metabolites, and the ratio between NO synthesis boosters and inhibitors are altered in energy-restricted ewes, and that these alterations are more marked in ewes carrying multiple fetuses.

Convolvulus aucheri’nin In vitro Antioksidan Özelliği ve LPS-aktif NSCLC Hücrelerinde NO Engelleyici Potansiyeli

Latterly, scientists have turned into progressively curious about therapeutic herbs. A lot of Convolvulus taxa display different biologic activities. In this study, we aimed to evaluate nitric oxide (NO) inhibitive effect of Convolvulus aucheri in LPS-induced non-small cell lung cancer (NSCLC) cells, such as HCC78 and H1975 and determine its antioxidant capacity. While Griess assay was applied to determine the nitrite level as an index of NO production, β-carotene/linoleic acid test was used for the determining antioxidant effect. Cytotoxic activity of C. aucheri extract was detected by using CellTiter-Glo assay. The extract induced a bigger cytotoxic activity on H1975 cells than the HCC78 cells and decreased NO production in concentration-dependent manner. The highest inhibitory potential against NO formation was observed to be 113.06 μM nitrite at a concentration of 40 μg/mL in HCC78 cells. As for the antioxidant activity, C. aucheri showed the highest antioxidant activity of 70.89%. These data bring to mind that C. aucheri may be a useful source for exploration new anticancer compounds.

Optimization of a microchip electrophoresis method with electrochemical detection for the determination of nitrite in macrophage cells as an indicator of nitric oxide production.

Nitric oxide (NO) is involved in many biological functions, including blood pressure regulation, the immune response, and neurotransmission. However, excess production of NO can lead to the generation of reactive nitrogen species and nitrosative stress and has been linked to several neurodegenerative diseases and cardiovascular disorders. Because NO is short-lived and generally difficult to detect, its primary stable degradation product, nitrite, is frequently monitored in its place. In this paper, an improved method using microchip electrophoresis with electrochemical detection (ME-EC) was developed for the separation and detection of nitrite in cell lysates. A separation of nitrite from several electroactive cell constituents and interferences was optimized, and the effect of sample and buffer conductivity on peak efficiency was explored. It was found that the addition of 10 mM NaCl to the run buffer caused stacking of the nitrite peak and improved limits of detection. A platinum black working electrode was also evaluated for the detection of nitrite and other electroactive cellular species after electrophoretic separation. The use of a modified platinum working electrode resulted in 2.5-, 1.7-, and 7.2-fold signal enhancement for nitrite, ascorbic acid, and hydrogen peroxide, respectively, and increased the sensitivity of the method for nitrite twofold. The optimized ME-EC method was used to compare nitrite production by native and lipopolysaccharide-stimulated RAW 264.7 macrophage cells.

Immunological alterations in frail older adults: A cross sectional study.

Frailty is a progressive physiologic decline in multiple body systems, characterized by loss of function, loss of physiologic reserve, and increased vulnerability to disease and death. This condition is induced by a complex and multifactorial interaction between genetic, biological, physical, psychological and environmental factors. To understand the interplay between the age-related decline of the immune response, and the upregulation of the inflammatory response, the so called inflammaging, we investigated the role of different inflammatory mediators on frailty status in the elderly. The study was performed in a population of 180 older adults (≥65 years), who were classified according to Fried's frailty phenotype. Plasma concentrations of neopterin, tryptophan, kynurenine, phenylalanine, tyrosine as well as kynurenine/tryptophan (Kyn/Trp) and phenylalanine/tyrosine (Phe/Tyr) ratios were analyzed as immune stimulation biomarkers. In addition, nitrite and C-reactive protein levels were measured as indicators of nitric oxide production and acute inflammation, respectively. Significant increases in neopterin, C-reactive protein and Kyn/Trp ratio, and decreases in tryptophan and nitrite concentrations in frail individuals compared with non-frail group were found. Both Kyn/Trp and Phe/Tyr ratios were significantly and positively correlated with neopterin. A positive correlation between kynurenine and tryptophan was also observed. Four parameters, i.e., neopterin, tryptophan, nitrite and C-reactive protein, were found to be strongly related to frailty status, although only nitrite confirmed its role of predictor after multiple regression analysis, supporting its use as a potential biomarker of frailty. Further investigation is required to strengthen the consistence and reproducibility of these findings, and to establish this parameter as a clinical biomarker of frailty.

Consequences of advanced aging on renal function in chronic hyperandrogenemic female rat model: implications for aging women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine and reproductive disorder in premenopausal women, characterized by hyperandrogenemia, metabolic syndrome, and inflammation. Women who had PCOS during their reproductive years remain hyperandrogenemic after menopause. The consequence of chronic hyperandrogenemia with advanced aging has not been studied to our knowledge. We have characterized a model of hyperandrogenemia in female rats and have aged them to 22–25 months to mimic advanced aging in hyperandrogenemic women, and tested the hypothesis that chronic exposure to hyperandrogenemia with aging has a deleterious effect on renal function. Female rats were chronically implanted with dihydrotestosterone pellets (DHT 7.5 mg/90 days) that were changed every 85 days or placebo pellets, and renal function was measured by clearance methods. Aging DHT‐treated females had a threefold higher level of DHT with significantly higher body weight, mean arterial pressure, left kidney weight, proteinuria, and kidney injury molecule‐1 (KIM‐1), than did age‐matched controls. In addition, DHT‐treated‐old females had a 60% reduction in glomerular filtration rate, 40% reduction in renal plasma flow, and significant reduction in urinary nitrate and nitrite excretion (UNOxV), an index of nitric oxide production. Morphological examination of kidneys showed that old DHT‐treated females had significant focal segmental glomerulosclerosis, global sclerosis, and interstitial fibrosis compared to controls. Thus chronic hyperandrogenemia that persists into old age in females is associated with renal injury. These data suggest that women with chronic hyperandrogenemia such as in PCOS may be at increased risk for development of chronic kidney disease with advanced age.

The effects of 100% wild blueberry (Vaccinium angustifolium) juice consumption on cardiometablic biomarkers: a randomized, placebo-controlled, crossover trial in adults with increased risk for type 2 diabetes

BackgroundWild blueberries have a high content of polyphenols, but there is limited data evaluating their health benefits in adults at risk for type 2 diabetes. The objective of the study was to investigate whether consumption of 100% wild blueberry juice improves cardiometabolic biomarkers associated with type 2 diabetes risk.MethodsA single-blind, randomized, placebo-controlled, crossover design trial was conducted in which adults (women, n = 19, ages 39–64 y) at risk for type 2 diabetes consumed 240 mL of wild blueberry juice or a placebo beverage as part of their free-living diet for 7 days. Blood was collected to determine various biomarkers such as fasting plasma glucose, fasting serum insulin, surrogate markers of insulin sensitivity, triglycerides, inflammation (interleukin-6, interleukin-10, high-sensitivity C-reactive protein, tumor necrosis factor-alpha, serum amyloid A), adhesion molecules (soluble intercellular adhesion molecule-1, soluble vascular adhesion molecule-1), oxidative stress (LDL-oxidation, total 8-isoprostanes), and nitric oxide. Endothelial function and blood pressure were also assessed.ResultsWild blueberry juice consumption for 7 days produced no significant changes in glucose, insulin, insulin sensitivity, triglycerides, inflammatory markers, adhesion molecules, oxidative stress, endothelial function or blood pressure. However, wild blueberry juice consumption showed a trend for lowering systolic blood pressure: 120.8 ± 2.2 mmHg in the placebo group vs 116.0 ± 2.2 mmHg in the blueberry juice group (P = 0.088). Serum concentrations of nitrates and nitrites, an index of nitric oxide production, increased from 2.9 ± 0.4 μM after placebo drink to 4.1 ± 0.4 μM after drinking wild blueberry juice (P = 0.039).ConclusionsShort-term consumption of wild blueberry juice may promote cardioprotective effects, by improving systolic blood pressure, possibly through nitric oxide production, in adults at risk for type 2 diabetes. This outcome warrants longer-term human studies of blueberries, including defined amounts of either the whole fruit or juice, to clarify whether polyphenol-rich foods can be efficacious for improving cardiometabolic biomarkers in adults at risk for type 2 diabetes.Trial registrationNCT02139878, clinicaltrials.gov; date of registration: May 4, 2014.

Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase

Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthine-guanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention.

P32 Effects of intermittent vs. constant hyperglycaemia on saphenous vein endothelial cells

Type 2 diabetes mellitus (T2DM) presents an increased risk of cardiovascular disease, and cardiovascular disease is a major cause of death in patients with diabetes. These vascular complications are thought to occur as a result of endothelial dysfunction, a pathological state in which endothelial cells shift towards a pro-contractile and pro-thrombotic phenotype, including a decrease in the bioavailability of the vasodilator nitric oxide (NO). Hyperglycaemia is thought to play a key role in the development of endothelial dysfunction. Research suggests that fluctuating hyperglycaemia, as experienced post-prandially, may be more damaging to the endothelium than hyperglycaemia per se. In order to assess the effects of fluctuating glucose on endothelial cell function, human saphenous vein endothelial cells (hSVECs) were exposed to either normoglycaemia (5 mM glucose), hyperglycaemia (25 mM glucose), or fluctuating glucose (cells alternately exposed to 5 mM and 25 mM glucose; 2 hour intervals) for 10 hours. Following this, cells were stimulated with 100 nM insulin, and phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), as an indicator of NO production, were assessed by western blotting. In addition, RNA was extracted from cells exposed to the different glucose conditions in order to determine any changes in gene expression of eNOS, cyclooxygenase-2 and the adhesion molecules VCAM-1 and ICAM-1. Being able to closely mimic diverse fluctuations in blood glucose experienced in vivo in patients with T2DM is vital for determining how this leads to detrimental changes in endothelial cell function, exploring the underlying mechanisms and revealing targets for therapeutic intervention.

Inhibitory effects of Kaempferia parviflora extract on monocyte adhesion and cellular reactive oxygen species production in human umbilical vein endothelial cells.

The rhizome of Kaempferia parviflora (KP) is used in traditional Thai medicine. In this study, we investigated the effects of an ethanol KP extract and two of its components [5,7-dimethoxyflavone (DMF) and 5-hydroxy-3,7,3',4'-tetramethoxyflavone (TMF)] on monocyte adhesion and cellular reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVECs), which provide an in vitro model of events relevant to the development and progression of atherosclerosis. RAW264.7 mouse macrophage-like cells were incubated with various concentrations of KP extract or polymethoxyflavonoids and stimulated with lipopolysaccharide prior to measuring nitrite levels in the culture media. Monocyte adhesion was evaluated by measuring the fluorescently labeled human monocytic leukemia THP-1 cells that is attached to tumor necrosis factor-α (TNF-α)-stimulated HUVECs. Cellular ROS production was assessed by measuring cellular antioxidant activity using pyocyanin-stimulated HUVECs. KP extract and DMF reduced nitrite levels (as indicator of nitric oxide production) in LPS-stimulated RAW264.7 cells and also inhibited THP-1 cell adhesion to HUVECs. These treatments induced mRNA expression of endothelial nitric oxide synthase in TNF-α-stimulated HUVECs and downregulated that of various cell adhesion molecules, inflammatory mediators, and endothelial function-related genes. Angiotensin-converting enzyme activity was inhibited by KP extract in vitro. Furthermore, KP extract, DMF, and TMF inhibited the production of cellular ROS in pyocyanin-stimulated HUVECs. KP extract, DMF, and TMF showed potential anti-inflammatory and antioxidant effects in these in vitro models, properties that would inhibit the development and progression of atherosclerosis.

PP.13.06

Objective: Increased arterial stiffness (AS) is an independent risk factor for cardiovascular disease. Endothelial nitric oxide (NO) is vasoprotective by decreasing not only vascular tone in resistance arteries, but also stiffness in conductance arteries. Asymmetric (ADMA) and symmetric (SDMA) dimethylarginines, endogenous inhibitors of NO synthesis from the precursor L-arginine (ARN), increase in conditions of oxidative stress and contribute to progression of endothelial dysfunction and arteriosclerosis. In essential hypertensives (EH) we investigated the relationship between systemic AS, as assessed by the ratio of pulse pressure (PP) to stroke volume (SV), with circulating levels of ADMA, SDMA, ARN and the activity of the renin-aldosterone system in resting conditions. Design and method: In 25 untreated grade I-II EH (age 50 ± 12 yrs, range 19–71, M/F = 15/10) blood pressure (BP, sphygmomanometer), heart rate (HR, EKG), stroke volume (SV, impedance cardiography) were measured after supine rest; venous blood was simultaneously sampled for plasma ADMA, SDMA, ARN (by HPLC), plasma renin activity (PRA) and aldosterone (Aldo)(by RIA). Results: Table shows means ± sd:Supine BP was 142/96 ± 15/8.4 mmHg, HR = 70.0 ± 9.5 bt/min; PRA was 0.3 ± 0.05 ng/ml/h (range 0.1–0.9) and Aldo 9.8 ± 0.7 ng/dl (range 3.7–17). AS (range 0.89–2.21 mmHg/ml) was directly related to age (r = 0.61, p < 0.05), to SDMA (r = 0.83, p < 0.01) and inversely to ARN/ADMA ratio, an indicator of NO production (r = -0.64, p < 0.05); AS was not related to PRA or Aldo. In a multiple regression analysis, SDMA was the best predictor of AS (p < 0.05). Conclusions: Systemic AS in EH was directly related to circulating levels of endogenous inhibitors of NO production. These data are compatible with an impaired activity of endothelial NO associated with increased stiffness of conductance arteries, a condition at higher cardiovascular risk in uncomplicated hypertensives.

In vitro effect of nanosilver on gene expression of superoxide dismutases and nitric oxide synthases in chicken sertoli cells

To evaluate effects of different concentrations of nanosilver colloid on the cell culture of Sertoli cells, the proportion of lipid peroxidation, antioxidant capacity, nitric oxide (NO) production and genes expression of superoxide dismutases (SOD1 and SOD2) and nitric oxide synthases (eNOS and iNOS) were measured. Sertoli cells were incubated at concentrations of 25, 75 and 125 ppm nanosilver for 48 h. There was progressive lipid peroxidation in treatments according to increasing of nanosilver. Lipid peroxidation, as indicated by malondialdehyde levels, was significantly elevated by the highest concentration of silver colloid (125 ppm), although antioxidant capacity, as measured by ferric ion reduction, was unaffected. Nitrite, as an index of NO production was reduced only in 125 ppm of nanosilver. Expression of SOD1 gene was reduced in nanosilver-treated cells at all concentrations, whereas expression of SOD2 gene was reduced only in cells treated with 125 ppm nanosilver. Expression of iNOS gene was progressively increased with higher concentrations of nanosilver. Expression of eNOS gene was also increased in 125 ppm of nanosilver. In conclusion, toxic effects of nanosilver could be due to high lipid peroxidation and suppression of antioxidant mechanisms via reduced expression of SOD genes and increased expression of NOS genes.

Abstract 647: Histone Deacetylase 1 Reduces No Production In Endothelial Cells Via Lysine Deacetylation Of No Synthase 3

The lysine acetylation state of non-histone proteins may be regulated through histone deacetylases (HDACs). Evidence suggests that NO synthase 3 (NOS3; eNOS) is post-translationally lysine acetylated leading to increased NO production in the endothelium. The purpose of this study was to test the hypothesis that NOS3 is lysine-acetylated and that upregulated HDAC1-mediated deacetylation leads to reduced NO production in endothelial cells. We determined that NOS3 is basally lysine acetylated in cultured bovine aortic endothelial cells (BAECs). In BAECs, HDAC1 is expressed in the nucleus and cytosol, and forms a novel protein-protein interaction with NOS3. Overexpression of HDAC1 in BAECs resulted in a significant reduction in NOS3 lysine acetylation (control = 1.0 ± 0.1, HDAC1 = 0.59 ± 0.08 A.U., P &lt; 0.01), while HDAC1 knockdown with siRNA resulted in no significant change in NOS3 acetylation level. Moreover, overexpression or knockdown of HDAC1 resulted in no significant effect on NOS3 protein expression or on expression levels of NOS3 phosphorylation sites T497, S635, S1179. HDAC1 overexpression in BAECs significantly blunted basal nitrite production (an index of nitric oxide production; control 287.7 ± 29.1 pmol/mg p/h, HDAC1 172.4 ± 31.7 pmol/mg p/h, P &lt; 0.05) as well as attenuating endothelin-1 stimulated nitrite production (control 481.8 ± 50.3 pmol/mg/h, HDAC1 243.1 ± 48.2 pmol/mg/h, P&lt; 0.05). On the contrary, HDAC1 knockdown increased basal nitrite production (730.6 ± 99.1 pmol/mg/h), and further exaggerated increases in endothelin-1 stimulated nitrite production (1276.9 ± 288.2 pmol/mg/h). Thus, these data indicate that up-regulated HDAC1 decreases NOS3 activity, most likely, through direct lysine deacetylation of NOS3. We propose that HDAC1-mediated deacetylation of NOS3 may represent a novel target for endothelial dysfunction.

Metabolites of polyphenols preserve indices of endothelial cell nitric oxide bioavailability under glucotoxic conditions (372.6)

We hypothesized that biological metabolites of quercetin, resveratrol, and grape seed extract previously identified in human plasma can prevent impairment of nitric oxide (NO) bioavailability due to glucotoxic conditions (e.g. Type 1 or 2 diabetes).Human aortic endothelial cells were treated for 24 h with 2μΜ Quercetin‐3‐O‐glucoronide, 5μΜ Piceatannol, or 1μΜ 3‐Hydroproponoic acid. Cells were next exposed to normal (5mM) or high (25mM) glucose for 48h, then treated with insulin (100nM, 10 min) to stimulate NO production.In the absence of polyphenols, insulin stimulation increased (P&lt;0.05) indices of NO production, phosphorylated to total Akt (p‐AktSer473:Akt), and endothelial nitric oxide synthase (p‐eNOSser1177:eNOS) in cells grown in 5mM but not 25mM glucose. Pretreatment of cells with polyphenol metabolites prior to 25mM glucose exposure preserved insulin stimulated increases (P&lt;0.05) in NO, p‐AktSer473:Akt and p‐eNOSser1177:eNOS. These effects may be secondary to oxidative stress as elevations (P&lt;0.05) in reactive oxygen and nitrogen species in cells treated with 25mM glucose were completely prevented by all polyphenol metabolites.These data indicate that biological metabolites of quercetin, resveratrol, and grape seed extract protected against gluocotoxic impairment of insulin dependent NO bioavailability, and preserved the insulin ‐ Akt ‐ eNOS signaling axis.

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.

The role of the neuropeptide somatostatin on methamphetamine and glutamate-induced neurotoxicity in the striatum of mice

A large body of evidence shows that methamphetamine (METH) causes sustained damage to the brain in animal models and human METH users. In chronic users there are indications of cognitive and motor deficits. Striatal neuropeptides are in a position to modulate the neurochemical effects of METH and consequently striatal neural damage. Somatostatin (SST) is an intrinsic striatal neuropeptide that has been shown to inhibit glutamate transmission; glutamate is integral to METH toxicity and contributes to nitric oxide (NO) synthesis. We hypothesize that SST will protect from METH by inhibition of NO synthesis and thus reducing oxidative stress. To this end, the SST analogue octreotide (OCT) was microinjected into the striatum prior to a systemic injection of METH (30mg/kg). We then assessed 3-nitrotyrosine (3-NT), an indirect index of NO production, tyrosine hydroxylase (TH) protein levels (dopamine terminal marker) and Fluoro-Jade C positive cells (degenerating cells). The SST agonist OCT dose dependently attenuated the METH-induced accumulation of striatal 3-NT. Moreover, pretreatment with OCT effectively mitigated cell death but failed to protect dopamine terminals. Next we co-infused OCT and NMDA and measured 3-NT and Fluoro-Jade C staining. Treatment with OCT had no effect on these parameters. The data demonstrate that SST attenuates the METH-induced production of NO protecting the striatum from the METH-induced cell loss. However, SST failed to prevent the toxicity of the dopamine terminals suggesting that pre- and post-synaptic striatal damage occur via independent mechanisms.

Abstract WP331: Diverse Microglia Morphologies Induced By Ischemic Stroke And Reperfusion Are Not Accompanied By Altered Brain Inducible Nitric Oxide Synthase Expression.

Microglia cells continuously survey the healthy brain in a ramified morphology and, in response to injury, undergo progressive morphological and functional changes that encompass microglia activation. Activated microglia may contribute towards secondary injury during ischemic stroke and reperfusion (ISR) through pro-inflammatory mechanisms and increased oxidative stress. Nitric oxide (NO), a product of microglia inducible NO synthase (iNOS) expression, contributes toward increased oxidative stress after brain injury. Although ideally positioned for an immediate response, microglia morphological responses have not been characterized after ISR. In addition it is not well understood if microglia morphology correlate to microglia function relative to oxidative stress. Our aim was to quantify microglia morphology and iNOS expression, an indicator of NO production, during the evolution of cerebral injury after ISR. Anesthetized mice were subjected to focal ischemic stroke accompanied by varied lengths of reperfusion (ischemia only, 8hr and 24hr reperfusion). Microglia process length/cell and number of endpoints/cell was quantified from immunofluorescent confocal images of brain regions spatially related to the necrotic core using a skeleton analysis method developed for this study. Live cell microglia morphology and process movement was measured from movies acquired in acute brain slices from GFP-CX3CR1 transgenic mice after IS and 24hr reperfusion. iNOS expression was measured at all time-points and similar brain regions using western blot techniques. We show a significant spatiotemporal relationship between microglia process length/cell and number of process endpoints/cell and evolving brain lesion after ISR (F=4.17, p&lt;0.001, F=3.57, p&lt;0.01). After 24hr of reperfusion, process activity was significantly decreased the peri-infarct region vs. contralateral (p&lt;0.05). However, iNOS protein expression remained unchanged in all ipsilateral regions vs. matching contralateral regions after 60min ischemia and first 24hr of reperfusion. While microglia morphological responses to ISR are immediate and diverse, we have yet to confirm microglial sources of NO are increased within the first 24hr of ISR.