Plasma Nitrate Concentrations Research Articles

GW24-e1289 Atorvastatin decreases nitric oxide production and restore vascular responsiveness impaired by LPS in rats

ObjectivesLipopolysaccharides (LPS) were found in the outer membrane of Gram-negative bacteria and be used to induce experimental endotoxic shock, which could decrease vasoconstrictor response, produce excessive nitric oxide and decrease...

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (Nω-nitro-L-arginine methyl ester; 100mg/kg; po)-induced hypertension treated with TEMPOL (18mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.

P28: Mechanisms underlying nitrite reduction in hypertension: Role for erythrocytic xanthine oxidoreductase

Within the human circulation physiological levels of nitrite can act as a source of NO and as such exert important vasodilator effects (Cosby et al., Nat. Med. 9 (2003) 1498–1505). It is now accepted that the red blood cell (RBC) is an important site for nitrite reduction and it has been proposed that deoxyhaemoglobin is the primary nitrite reductase within this cell type under physiological conditions. In addition, our previous studies have shown that purified RBCs collected from healthy volunteers facilitate nitrite reduction that is dependent upon activity of endothelial nitric oxide synthase (eNOS) at pH 7.4, and xanthine oxidoreductase (XOR) at more acidic pH (6.8) (Webb et al., Circ Res 103 (2008) 957–964). Whether such a profile might also be evident in hypertension is unknown. We investigated RBC nitrite reductase activity in the spontaneously hypertensive rat (SHR) and Wistar Kyoto (WKY) strain controls and in patients with hypertension. Male SHR (SBP 153.4 ± 3.9, DBP 101.1 ± 2.7 mm Hg) and WKY (SBP 119.3 ± 2.6, DBP 74.4 ± 3.8 mm Hg) (14–16 weeks of age, Charles River, UK) were anaesthetized for blood collection. Hypertensive patients (SBP 151.5 ± 1.8, DBP 89.7 ± 2.2 mm Hg) were recruited and blood samples collected. Plasma and erythrocyte nitrite and nitrate concentration were measured by liquid phase ozone chemiluminescence. The nitrite reductase activity of blood vessel homogenates or purified RBCs was determined using gas phase ozone chemiluminescence. To ascertain the role of XOR in any activity seen samples were incubated with the XOR inhibitor allopurinol (100 μmol/L) for 30 min. XOR protein expression was quantified using Western blotting. There were no differences in nitrite and nitrate concentrations in the plasma (n = 18) and aortic homogenates (n = 12–15) between SHRs and WKY controls. Levels of RBC nitrite (WKY 53.4 ± 10.0 nmol/g; SHR 7.8 ± 1.3 nmol/g, p < 0.001) and nitrate (WKY 706.0 ± 187.2 nmol/g; SHR 268.4 ± 45.1 nmol/g, p < 0.05) were significantly lower in SHR (n = 8) compared to WKY (n = 4), respectively. Nitrite-derived (300 μmol/L) NO formation was similar in blood vessel homogenates of SHR (n = 5) and WKY (n = 5) at pH 7.4 and 6.8, however erythrocytic nitrite reductase activity of SHRs (n = 8) was substantially enhanced compared to WKY (9i = 4) at pH 7.4 (p < 0.05). Furthermore, allopurinol (100 μmol/L) virtually abolished this activity in SHRs at pH 7.4 (n = 8, p < 0.001) and pH 6.8 (n = 8, p < 0.001). In contrast, no effect of allopurinol was evident in erythrocytic nitrite reductase activity of WKY (n = 4). Western blotting for erthrocytic XOR demonstrated a doubling of expression in SHRs. Erythrocytes isolated from the blood of grade 1 hypertensives (n = 11) caused concentration-dependent NO generation that was attenuated by allopurinol at pH 7.4 (p < 0.001) and pH 6.8 (p < 0.001). This enhanced activity in hypertensive volunteers was associated with an approximate doubling of erythrocytic XOR expression. Our findings suggest that in hypertension the RBC is an important site for nitrite reduction within the circulation and that XOR acts as a major nitrite reductase. SMG is funded by an MRC MRes Ph.D. and this work was supported by the BHF. MRC MRes PhD students hiPBHF support.

Even and carbon dioxide independent distribution of nitrite between plasma and erythrocytes of healthy humans at rest

In the literature, the distribution of nitrite and nitrate, the major metabolites of nitric oxide (NO), between plasma and erythrocytes and its dependency on partial CO2 pressure (pCO2) in mammalian blood are uncertain. By means of a previously reported fully validated stable-isotope dilution gas chromatography-mass spectrometry (GC–MS) method, we measured nitrite and nitrate concentrations in heparinized plasma from venous, arterial and arterialized blood donated by five healthy non-exercising volunteers at three different time points (0, 15, 30min). pCO2, pH and oxygen saturation were measured by standard techniques. The nitrite and nitrate concentrations and the nitrite-to-nitrate ratio in plasma did not correlate with pCO2 (r=−0.272, P=0.07). Nitrite was found to be almost evenly distributed between plasma and erythrocytes of another eleven healthy non-exercising subjects. In a rabbit model of ARDS, no differences were found in the plasma nitrite and nitrate concentrations comparing normoventilation with hypercapnia. Our studies suggest that the distribution of nitrite between plasma and erythrocytes at rest is largely even and independent of pCO2 in blood of healthy humans and rabbits with ARDS.

Effects of glycine, pyruvate, resveratrol, and nitrite on tissue injury and cytokine response in endotoxemic rats

BackgroundGlycine, pyruvate, resveratrol, and nitrite are well-known protective compounds among others in ischemic tissue injury. Here, we compared their effects in acute lipopolysaccharide (LPS)-induced shock in rats to assess whether inhibition of the proinflammatory cytokine response is a prerequisite for their protective actions. Materials and methodsRats (six or eight per group) were anesthetized, received LPS as an intravenous bolus (2.5 mg/kg), and were observed for 5 h. Glycine, sodium pyruvate, resveratrol, and sodium nitrite were continuously infused starting 30 min before LPS administration. Parameters included histopathologic changes, organ-specific cytokine levels, plasma nitrite and nitrate concentrations, and time courses of biomonitoring parameters, marker enzyme activities, and plasma cytokine concentrations. ResultsGlycine, pyruvate, resveratrol, and nitrite enhanced arterial blood pressure after LPS-induced shock. Also, parameters reflecting tissue ischemia were significantly improved and plasma markers of organ injury ameliorated by all substances. Of the plasma cytokine concentrations increased by LPS, some were differently decreased or even further increased by the substances. None of them reduced the elevated plasma nitrite and nitrate concentration. Glycine diminished the increases in tissue cytokine levels organ specifically, pyruvate decreased some cytokine concentrations in all organs, and nitrite significantly affected only a few cytokine concentrations in some organs, whereas the levels of many cytokines were raised by resveratrol. All substances except resveratrol decreased granulocyte infiltrates in the liver. ConclusionsThe present results demonstrate that glycine, pyruvate, resveratrol, and nitrite protect against LPS-induced shock and tissue injury (cell death) in rats and suggest that inhibition of the proinflammatory cytokine response is not mandatory for their protective actions.

Effect of dietary nitrate on blood pressure, endothelial function, and insulin sensitivity in type 2 diabetes

Diets rich in green, leafy vegetables have been shown to lower blood pressure (BP) and reduce the risk of cardiovascular disease. Green, leafy vegetables and beetroot are particularly rich in inorganic nitrate. Dietary nitrate supplementation, via sequential reduction to nitrite and NO, has previously been shown to lower BP and improve endothelial function in healthy humans. We sought to determine if supplementing dietary nitrate with beetroot juice, a rich source of nitrate, will lower BP and improve endothelial function and insulin sensitivity in individuals with type 2 diabetes (T2DM). Twenty-seven patients, age 67.2±4.9 years (18 male), were recruited for a double-blind, randomized, placebo-controlled crossover trial. Participants were randomized to begin, in either order, a 2-week period of supplementation with 250ml beetroot juice daily (active) or 250ml nitrate-depleted beetroot juice (placebo). At the conclusion of each intervention period 24-h ambulatory blood pressure monitoring, tests of macro- and microvascular endothelial function, and a hyperinsulinemic isoglycemic clamp were performed. After 2 weeks administration of beetroot juice mean ambulatory systolic BP was unchanged: 134.6±8.4mmHg versus 135.1±7.8mmHg (mean±SD), placebo vs active—mean difference of −0.5mmHg (placebo–active), p=0.737 (95% CI −3.9 to 2.8). There were no changes in macrovascular or microvascular endothelial function or insulin sensitivity. Supplementation of the diet with 7.5mmol of nitrate per day for 2 weeks caused an increase in plasma nitrite and nitrate concentration, but did not lower BP, improve endothelial function, or improve insulin sensitivity in individuals with T2DM.

The impact of elevated water nitrate concentration on physiology, growth and feed intake of African catfishClarias gariepinus(Burchell 1822)

The nitrate threshold concentration in rearing water of African catfish (Clarias gariepinus) was assessed. Female African catfish with an initial mean (SD) weight of 154.3 (7.5) g were exposed to 0.4 (Control), 1.5, 4.2, 9.7 and 27.0 mM nitrate for 42 days. Mean (SD) plasma concentrations of nitrate increased from 71 (29) to 6623 (921) μM at the highest ambient nitrate level. Mean (SD) plasma nitrite concentration ranged from 1.2 (0.5) to 7.9 (9.0) μM. Haematocrit, plasma concentrations of non-esterified fatty acids (NEFA), cortisol, glucose, lactate, osmolality, gill morphology and branchial Na+/K+-ATPase activity were not affected. Feed intake and specific growth rate were significantly reduced at the highest nitrate concentration. We advise not to exceed a water nitrate concentration of 10 mM (140 mg L−1 NO3-N) to prevent the risk of reduced growth and feed intake in African catfish aquaculture.

Dietary Nitrate Supplementation Improves Rowing Performance in Well-Trained Rowers

Increased plasma nitrate concentrations from dietary sources of nitrate have proven to benefit exercise performance. Beetroot (BR) contains relatively high levels of nitrate (NO₃⁻), which increases nitric oxide stores. This study investigated whether dietary nitrate supplementation, in the form of a BR beverage, would improve rowing performance during ergometer repetitions. In a randomized crossover design, 14 well-trained junior male rowers consumed 500 ml of either BR or placebo (PL) daily for 6 d. After supplementation, rowers completed 6 maximal 500-m ergometer repetitions and times were recorded. A 7-d washout period separated the 2 trials. Blood pressure, oxygen saturation, maximum heart rate, urine (specific gravity, pH, and nitrites), and lactates were collected for analysis at baseline and pre- and postperformance. Changes in the mean with 95% confidence limits were calculated. There was a likely benefit to average repetition time in the BR condition, compared with PL (0.4%, 95% confidence limits, ± 1.0%). In particular, Repetitions 4-6 showed an almost certain benefit in rowing time on BR (1.7%, 95% CL, ± 1.0%). The underlying mechanism for the observed results remains unknown, as differences observed in rowers' physiological measures between the 2 conditions were unclear. Conclusively, nitrate supplementation in the form of BR juice resulted in improved maximal rowing-ergometer repetitions, particularly in the later stages of exercise.

Simultaneous determination of nitrite and nitrate in human plasma by on‐capillary preconcentration with field‐amplified sample stacking

A simple method for the determination of nitrite and nitrate in human plasma has been developed using CZE with minimal sample preparation. Field-amplified sample stacking (FASS) was used to achieve submicromolar detection by dilution of the plasma sample with deionized water. In CZE, the separation of nitrite and nitrate was achieved within 10 min without adding EOF modifier. The optimal condition was achieved with 50 mM phosphate buffer at pH 9.3. The ninefold diluted plasma samples were injected hydrodynamically for 40 s into a 60 cm×75 μm id uncoated fused-silica capillary. The separation voltage was 20 kV (negative potential) and UV detection was performed at 214 nm. The linearity curves for nitrite and nitrate were obtained by the standard addition method. The estimated LODs for nitrite and nitrate in ninefold diluted plasma sample were 0.05 and 0.07 μM, respectively. The LODs for nitrite and nitrate in original plasma samples were 0.45 and 0.63 μM. The intra- and inter-day precisions for both analytes were <2.6% and the recovery ranged between 92.3 and 113.3%. It was found that nitrite was more stable than nitrate in the plasma after the sample preparation. This proposed method was applied to a number of human plasma samples and the measured nitrite and nitrate concentrations in human plasma were consistent with the literature ranges.

SVO2-Guided Resuscitation for Experimental Septic Shock

The pathogenetic mechanisms associated to the beneficial effects of mixed venous oxygen saturation (SvO(2))-guided resuscitation during sepsis are unclear. Our purpose was to evaluate the effects of an algorithm of SvO(2)-driven resuscitation including fluids, norepinephrine and dobutamine on hemodynamics, inflammatory response, and cardiovascular oxidative stress during a clinically resembling experimental model of septic shock. Eighteen anesthetized and catheterized pigs (35-45 kg) were submitted to peritonitis by fecal inoculation (0.75 g/kg). After hypotension, antibiotics were administered, and the animals were randomized to two groups: control (n = 9), with hemodynamic support aiming central venous pressure 8 to 12 mmHg, urinary output 0.5 mL/kg per hour, and mean arterial pressure greater than 65 mmHg; and SvO(2) (n = 9), with the goals above, plus SvO(2) greater than 65%. The interventions lasted 12 h, and lactated Ringer's and norepinephrine (both groups) and dobutamine (SvO(2) group) were administered. Inflammatory response was evaluated by plasma concentration of cytokines, neutrophil CD14 expression, oxidant generation, and apoptosis. Oxidative stress was evaluated by plasma and myocardial nitrate concentrations, myocardial and vascular NADP(H) oxidase activity, myocardial glutathione content, and nitrotyrosine expression. Mixed venous oxygen saturation-driven resuscitation was associated with improved systolic index, oxygen delivery, and diuresis. Sepsis induced in both groups a significant increase on IL-6 concentrations and plasma nitrate concentrations and a persistent decrease in neutrophil CD14 expression. Apoptosis rate and neutrophil oxidant generation were not different between groups. Treatment strategies did not significantly modify oxidative stress parameters. Thus, an approach aiming SvO(2) during sepsis improves hemodynamics, without any significant effect on inflammatory response and oxidative stress. The beneficial effects associated with this strategy may be related to other mechanisms.

Over-Expression of the Mas Receptor Decreases Arterial Thrombosis Risk in B2R KO Mice by Elevating NO and Prostacyclin and Reducing GPVI Activation

Abstract 700 Background:Bradykinin B2 receptor KO mice (B2R KO) were recognized to have delayed rose bengal and ferric chloride carotid artery thrombosis times and long tail bleeding times. In B2R KO, elevated serum angiotensin II (AngII) binds to an over-expressed angiotensin receptor 2 (AT2R) to increase plasma NO and prostacyclin (Blood 108:192, 2006). It was proposed that the combined presence of elevated AngII and AT2R are essential for the thrombosis protection phenotype. However, after losartan treatment, an angiotensin receptor 1 antagonist, thrombosis remains delayed in B2R KO despite reduction in AngII levels from 258±64 to 40±15 pg/ml. An additional pathway for thrombosis protection in B2R KO mice was sought. Methods and Results:Losartan treatment lowers ACE mRNA in mice along with lowering AngII levels. However, even in the presence of low AngII levels, plasma Ang1-7 levels, the breakdown product of AngII, in losartan-treated B2R KO are increased [21±2.2 pg/ml vs 14.4±0.7 pg/ml in wild type mice (WT)]. Upon examination, B2R KO also have elevated Mas mRNA, the receptor for angiotensin1-7, with over-expressed renal Mas protein. Treatment of B2R KO with A-779, a Mas antagonist, shortens carotid artery thrombosis time (58±4 to 38±4 min) and tail bleeding time (170±13 to 88±8 sec) and lowers plasma concentrations of nitrate (from 21.5±3.6 micromolar in untreated B2R KO to 15±5 micromolar in A-779-treated mice) and 6-keto-PGF1alpha (259±103 pg/ml in untreated B2R KO to 132±58 pg/ml). ADP- or thrombin-induced platelet (plt) activation are not abnormal in B2R KO, either by aggregation studies or by flow cytometric studies examining for fibrinogen binding or JON/A integrin epitope binding and P-Selectin expression, respectively. Although static and flow adherence to collagen was normal, B2R KO plts have defective spreading on collagen (20±0.6 microns in B2R KO versus 27±0.4 microns in WT, p<0.0001) under static conditions. Resting B2R KO plts constitutively also have increased DAF-FM fluorescence (649±41 AFU versus 405±36 AFU, p=0.0012), suggesting increased plt NO without stimulation. GSNO, an NO donor, or carbaprostacyclin, a stable derivative of prostacyclin, inhibits murine plt spreading on collagen in a concentration-dependent manner. B2R KO plts have reduced GPVI activation using CRP or convulxin even though GPVI expression on B2R KO and WT plts are similar. Further, B2R KO plts have reduced Syk phosphorylation after convulxin stimulation and reduced phosphorylation of Src family kinases when spreading on collagen. Treatment of B2R KO plts in vivo with A-779 corrects the spreading and Src family kinase phosphorylation defects upon collagen exposure, suggesting an in vivo role of the Mas receptor in the plt function defect and thrombosis delay. Conclusion:B2R KO are protected from thrombosis by over-expression of two receptors (AT2R and Mas) from the renin-angiotensin system resulting in increased NO and prostacyclin and an acquired platelet function defect. The receptors AT2R and MAS compensate for the absence of the B2R. These studies show novel mechanism(s) whereby receptors in the renin-angiotensin system influence platelet function and arterial thrombosis risk in vivo. Disclosures:No relevant conflicts of interest to declare.

Nitric oxide production is increased in severely obese children and related to markers of oxidative stress and inflammation

ObjectiveNitric oxide (NO) is the major endothelium-derived relaxing factor. The aim of the present study was to evaluate NO synthesis and metabolism in severely obese children with different degrees of metabolic risk and to ascertain their relation with the parameters of oxidative stress and inflammation. MethodsThe study involved 60 obese children evaluated with respect to metabolic risk factors (MRFs) (32<4 MRFs and 28≥4 MRFs) and 50 normal weight children between 7 and 14 years of age. Nutritional status was assessed by clinical and anthropometric examination. MRFs (serum lipid profile, insulin resistance indexes, blood pressure) in addition to uric acid, homocysteine, leptin, and inflammatory markers were measured. Plasma nitrite, nitrate and nitrotyrosine concentrations, and urinary nitrate were determined as markers of NO production and nitrosative stress. Malondialdehyde, 8-isoprostane F2α, and advanced oxidation protein products were analyzed in plasma to assess oxidative stress. ResultsCompared with healthy controls, the obese children had significantly increased concentrations of markers of NO synthesis and nitrosative and oxidative stress that were correlated with each another. Increased NO production in obese children was associated with MRFs; plasma nitrate to waist circumference (r=0.388, p=0.003), uric acid (r=0.404, p<0.001), and tumor necrosis factor α (r=0.302, p=0.021), and plasma nitrite to triglycerides (r=0.432, p<0.001). ConclusionNO synthesis and nitrosative stress are increased in severely obese children and correlated with anthropometric parameters indicative of abdominal obesity, oxidative stress and inflammatory markers.

Effect of Acute Alloxan Diabetes on Ischemic and Reperfusion Arrhythmias in Rats with Different Activity of Nitric Oxide System

Similar degree of glycemia (28-31 mmol/liter) and similar mortality (37-42%) were revealed in August rats exhibiting enhanced activity of NO system and in Wistar rats 3 weeks after alloxan treatment. Under conditions of myocardial ischemia caused by 10-min coronary artery ligation, the intensity of arrhythmias did not differ from the control in Wistar rats with diabetes mellitus and increased in August rats. Under conditions of reperfusion, diabetes produced an antiarrhythmic effect in Wistar rats and did not affect arrhythmia in August rats. Plasma concentrations of nitrates and nitrites in Wistar and August rats increased by 82 and 143%, respectively, compared to the control. The level of hemoxygenase-1 (hsp32) in the myocardium remained unchanged in Wistar rats and decreased by 26% in August rats. Thus, the absence of antiarrhythmic effect of acute diabetes in August rats is probably related to elevated NO content and reduced antioxidant activity.

Inorganic Nitrate for Blood Pressure Lowering?

To the Editor: Kapil et al1 reported that potassium nitrate ingestion lowers blood pressure (BP) in human subjects. Peak plasma nitrate (NO3−) and nitrite (NO2−) concentrations increased up to 35- and 4-fold, respectively. Increased plasma cGMP levels suggests that the depressor action of nitrate is mediated by the NO/cGMP cascade, presumably after reduction of NO3− to NO2− and its reduction to NO. Using 15NO3−, we provided evidence for 15NO3− to 15NO2− reduction in humans.2 Similar results were obtained for 15 …

Bradykinin B2 Receptor KO Mice Are Protected From Thrombosis by A Platelet Spreading Defect

AbstractAbstract 3198 Background:Bradykinin B2 receptor KO mice (B2R KO) have delayed rose bengal and ferric chloride carotid artery thrombosis times and long tail bleeding times. In B2R KO mice, elevated serum angiotensin II (AngII) binds to an over-expressed angiotensin receptor 2 (AT2R) to increase plasma NO and PGI2 (Blood 108:192, 2006). The combined presence of elevated AngII and AT2R was essential to have the thrombosis protection phenotype. However, thrombosis remained delayed in B2R KO mice despite reduction in AngII levels from 258+64 to 40+15 pg/ml using losartan, an angiotension 1 receptor antagonist. These data suggested an additional pathway for thrombosis protection in B2R KO mice. Methods and Results:B2R KO mice have elevated bradykinin and bradykinin 1–5 levels with normal thrombin generation times. Losartan treatment lowered ACE mRNA in mice. B2R KO mice also had elevated Mas mRNA, the receptor for angiotensin1-7, with overexpressed renal AT2R and Mas antigens. Treatment of B2R KO with A-779, a Mas antagonist, shortened thrombosis time (58+4 to 38+4 min) and tail bleeding time (170+13 to 88+8 sec) and lowered plasma concentrations of nitrate (21.5+3.6 to 15+5 micromolar) and 6-keto-PGF1alpha (259+103 to 132+58 pg/ml). Immunofluorescent staining and immunoblot demonstrate AT2R and Mas antigen on murine platelets. “Threshold” ADP- and thrombin-induced platelet aggregation was identical for B2R KO and wild type platelets. Platelet activation by arachidonic acid, ionomycin, thrombin, or collagen as measured by flow cytometry (the activated α2bβ3 integrin complex and P-selectin) was similar between B2R KO and wild type platelets. Although static and flow adherence to collagen was normal, B2R KO platelets had defects in spreading on collagen (20+0.6 versus 27+0.4 microns WT, p<0.0001) under both static and flow conditions. B2R KO platelets had increased DAF-FM fluorescence (649+41 versus 405+36 AFU, p=0.0012), suggesting increased platelet NO. Increasing concentrations of GSNO, a NO donor, or carbaprostacyclin, a stable derivative of prostacyclin, inhibited mouse platelet spreading in a concentration-dependent manner. B2R KO and wild type platelets did not show a difference in cGMP [631+143 (Mean+SD) vs 594+80 fmol/108 plts] or cAMP [505+227 vs 568+151 fmol/108 plts] levels. However, B2R KO platelets had increased constitutive and GSNO-induced total protein nitrosylation on biotin-switch assay. N-acetylcysteine, a protein nitrosylation inhibitor and antioxidant, corrected the spreading defect in B2R KO platelets. Conclusion:B2R KO mice are protected from thrombosis by over-expression of two receptors from the renin-angiotensin system that results in increased NO and prostacyclin and an acquired platelet spreading defect. These data suggest that a platelet spreading defect alone protects from thrombosis and indicate that the receptors AT2R and MAS compensate the absence of the B2R. Last, these studies show a novel mechanism(s) by which receptors in the renin-angiotensin system influence platelet function and arterial thrombosis risk in vivo. Disclosures:No relevant conflicts of interest to declare.

Redox modulation of the fetal cardiovascular defence to hypoxaemia

Episodes of hypoxia in utero present a potentially serious challenge to the fetus, but are counteracted by defence responses including marked redistribution of blood flow from peripheral circulations to the brain. Here, we report the novel observation that the oxidant tone is an important modulator of this cardiovascular defence. Using pregnant Welsh Mountain sheep surgically prepared for long-term recording, we investigated in vivo the effects on the fetal cardiovascular defence to acute hypoxaemia of fetal treatment with the antioxidant vitamin C. The mechanisms via which vitamin C may affect the vascular oxidant tone were investigated by monitoring fetal plasma concentrations of nitrates and nitrites, by determining changes in the activity of superoxide dismutase (SOD) in fetal plasma, and by investigating the effect of vitamin C treatment on the fetal cardiovascular defence to hypoxaemia following nitric oxide (NO) synthase blockade. Fetal treatment with vitamin C markedly depressed the normal femoral constrictor response to acute hypoxaemia in the fetus (5.2 ± 1.0 vs. 1.1 ± 0.3 mmHg (ml min(-1))(-1), mean ± s.e.m.; P < 0.05) an effect which was completely restored following NO synthase blockade (6.2 ± 1.3 mmHg (ml min(-1))(-1)). Compared to saline infusion, fetal treatment with vitamin C during acute hypoxaemia also significantly increased fetal plasma SOD activity from normoxic baseline (-8.9 ± 6.5 vs. 15.0 ± 6.6% inhibition, P < 0.05) and decreased the plasma concentration ratio of nitrate:nitrite from normoxic baseline (ΔNO3(-):NO2(-); 0.15 ± 0.30 vs. -0.29 ± 0.11, P < 0.05). The data provide in vivo evidence of redox modulation of redistribution of blood flow in the fetus, part of the fetal brain sparing during acute hypoxaemic stress.

Effects of dietary glycine supplementation and fish meal on inflammatory responses in broiler chicks

1. The effect of dietary protein source and glycine (Gly) supplementation on inflammatory responses was investigated using broiler chicks. Birds (7 d of age) were fed on a maize–soybean meal based (CS) diet with or without 20 g fish meal (FM)/kg and/or 10 g Gly/kg for 14 d. 2. Inflammatory responses were assessed by determining changes in the plasma concentrations of nitrate plus nitrite (NO x ), caeruloplasmin (Cer) and α-1 acid glycoprotein (AGP), and changes in the expression of mRNA encoding substances related to the inflammatory response, such as interleukin (IL)-1 β and −6, tumour necrosis factor like ligand (TL)1A, inducible nitrite synthase (iNOS), interferon(IFN)-γ, following lipopolysaccharide (LPS) injection. 3. IL-1 β and TL1A mRNA expression in chicks fed on the FM diet was higher than in chicks fed on the CS diet. 4. Supplementing the CS diet with Gly resulted in smaller increases in the plasma concentrations of NO x , Cer and AGP after LPS injection than were observed for chicks fed on the CS diet alone. In addition, 2 h after LPS challenge the expression of encoding inflammatory response-related substances was lower in the spleens of those chicks fed on the Gly-supplemented diet than in those fed the CS diet. 5. Supplementation of the FM diet with Gly reduced the plasma AGP concentration and IL-1 β and TL1A expression. 6. These results suggest that modulation of inflammatory responses by dietary Gly supplementation is affected by dietary composition; Gly supplementation has different effects on the cytokine responses dependent on the diet.

The correlation between the plasma nitric oxide levels and QT/QTc interval in conscious rabbits

The current study was undertaken to investigate the effects of NOx (plasma concentrations of nitrate plus nitrite which are plasma nitric oxide (NO) metabolites) over QT interval and to determine the level of correlation between them in conscious rabbits. For this purpose, twenty-one New Zealand rabbits (5-7 months old) were used and randomly assigned into the following three groups: control (CG; n = 7, 1 ml isotonic NaCl solution per animal/day), L-arginine (ARG-G; n = 7, L-arginine solution 200 mg/kg/day) and L-NAME (NAME-G; n = 7, L-NAME solution 100 mg/kg/day). Injections were performed intraperitoneally at 9:00 a.m. for 9 days. Blood samples were collected 2 h after the injections on day 1, 5 and 9 and the concentration of plasma NOx was determined using a colorimetric method. ECG was also recorded 2 h after the injection on 1st, 5th and 9th days. The heart rate, QT intervals, corrected QT intervals (QTc) and QT dispersion (QTd), QTc dispersion (QTcd) values were calculated from the ECG recordings. Statistically significant differences were observed between HR, QT and QTc values in all groups for all days (p < 0.001). QTd and QTcd values were found statistically significant different in NAME-G compared to CG and ARG-G (p < 0.001). It was also determined that there was a statistically significant correlation between the NOx and HR and QT and QTc in all days. It is concluded from this study that NO is an important molecule for the electrical activation of heart and has effects on the duration of QT/QTc interval, which should be taken into consideration by the physicians. In addition, application of the L-arginine should be further studied.

Is There Any Correlation between Insulin Resistance and Nitrate Plasma Concentration in White Coat Hypertensive Patients?

We evaluated a relationship between nitric oxide plasma correlation and insulin resistance in white coat hypertensive patients. Patients were screened for white coat hypertension using an ambulatory blood pressure monitoring. The homeostasis model assessment insulin resistance index (HOMA-IR) was used to assess insulin resistance, and plasma nitrate concentrations were determined by chemiluminescence. The HOMA-IR was significantly higher in hypertensive (3.84 ± 0.62) when compared to white coat (2.11 ± 0.36) and normotensive (1.40 ± 0.21). We found no correlation between HOMA-IR and plasma nitrate levels in all three groups. We suggest that the more important issue for these patients is to focus on the metabolic abnormalities where lifestyle interventions such as weight control and exercise have proven effective.

Variability in tumor necrosis factor-α, nitric oxide, and xanthine oxidase responses to endotoxin challenge in heifers: Effect of estrous cycle stage

The severity of host response to some disease agents differs between sexes and this dimorphism has been attributed to the immunomodulating effects of steroid hormones. Our objective was to determine in heifers whether the phase of estrous cycle affected immune response mediators after endotoxin challenge (LPS, 2.5 μg/kg BW, i.v.). Sixteen beef heifers (426 ± 9 kg) were reproductively synchronized with the two-injection protocol of dinoprost tromethamine (Lutalyse ®, Pfizer) to establish diestrus and estrus stages of the estrous cycle. Heifers were challenged with LPS on day 3 (E, estrus; n = 8) or day 10 (D, diestrus, n = 8) after the last i.m. injection of Lutalyse ®. In all heifers, plasma concentrations of tumor necrosis factor-α (TNF-α) peaked 2 h after LPS treatment ( P < 0.01) and returned to basal level by 7 h. However, the integrated TNF-α response (area under the time × concentration curve, AUC) was greater in E than in D ( P < 0.05). Plasma concentrations of nitrate + nitrite (NO x , an estimate of NO production) increased ( P < 0.01) in all heifers at 7 and 24 h after LPS; plasma NO x AUC after LPS was greater in E than D ( P < 0.01). Plasma xanthine oxidase activity (XO, a mediator of superoxide production) responses were also greater in E than D ( P < 0.05). A companion LPS challenge study in steers validated that the protocol for and use of Lutalyse ® did not affect any of the immune parameters studied in heifers in response to LPS. Results indicate that the underlying physiological attributes of the estrus and diestrus phases of the estrous cycle constitute a major source of variability in the magnitude of proinflammatory response to bacterial toxins like LPS.