Urinary Nitrate Excretion Research Articles

Increased urinary nitrate excretion associated with hepatic allograft rejection in experimental rat models and clinical cases

Abstract In this study, we investigated the relationship between excretion of urinary nitrate as a stable end-product of nitric oxide (NO) metabolism and hepatic allograft rejection. In experimental rat models, hepatic allograft rejection was associated with increased nitrate excretion with a peak on postoperative day 5. The severity of the hepatic allograft rejection was dependent on the increased urinary nitrate excretion. No significant increase in urinary nitrate excretion was observed in cases in which effective immuno-suppression was achieved. Inducible nitric oxide synthase mRNA expression was upregulated parallel to interferon-7gamma; gene expression in the graft-infiltrating mononuclear cells and spleen cells from the recipients. In clinical cases, urinary nitrate excretion increased parallel to increased serum cytosolic enzymes that accompanied rejection. These results suggest that urinary nitrate excretion is a useful indicator for the surveillance of graft rejection and the monitoring of therapeutic effects of antirejection treatments.

Induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats.

Several recent studies have shown that certain forms of genetic or acquired hypertension are associated with oxidative stress and that animals with those types of hypertension respond favorably to antioxidant therapy. We hypothesize that oxidative stress may cause hypertension via (among other mechanisms) enhanced oxidation and inactivation of nitric oxide (NO). To test this hypothesis, Sprague-Dawley rats were subjected to oxidative stress by glutathione (GSH) depletion by means of the GSH synthase inhibitor buthionine sulfoximine (BSO, 30 mmol/L in drinking water) for 2 weeks. The control group was given drug-free drinking water. In parallel experiments, subgroups of animals were provided vitamin E-fortified chow and vitamin C-supplemented drinking water. The BSO-treated group showed a 3-fold decrease in tissue GSH content, a marked elevation in blood pressure, and a significant reduction in the urinary excretion of the NO metabolite nitrate plus nitrite, which suggests depressed NO availability. These characteristics were associated with a significant accumulation in various tissues of nitrotyrosine, which is the footprint of NO inactivation by reactive oxygen species. Administration of vitamin E plus vitamin C ameliorated hypertension, improved urinary nitrate-plus-nitrite excretion, and mitigated nitrotyrosine accumulation (despite GSH depletion) in the BSO-treated animals but had no effect in the control group. In conclusion, GSH depletion resulted in perturbation of the NO system and severe hypertension in normal animals. The effects of BSO were mitigated by concomitant antioxidant therapy despite GSH depletion, which supports the notion that oxidative stress was involved in the pathogenesis of hypertension in this model.

Correlation between increased nitric oxide production and markers of endothelial activation in systemic sclerosis: findings with the soluble adhesion molecules E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1.

To determine the relationship between vascular function and the inflammatory response in systemic sclerosis (SSc), and to investigate whether production of endothelial-derived nitric oxide (NO) is disturbed in this disease. We measured plasma nitrate, urinary excretion of both nitrate and cGMP, and soluble adhesion molecules of endothelial origin in patients with SSc and in age- and sex-matched controls and compared these levels between groups. Additionally, we performed correlation analysis to determine how these variables were related to one another. Plasma nitrate and 24-hour-urinary excretion of nitrate in patients and controls were measured after a 72-hour nitrate-free-diet, using a gas chromatography/mass spectrometric method. Soluble adhesion molecules intercellular adhesion molecule 1 (sICAM-1), vascular cell adhesion molecule 1 (sVCAM-1), and E-selectin and cytokines were measured by enzyme-linked immunosorbent assay. The expression of E-selectin was further investigated in skin biopsy specimens by immunoperoxidase staining, and the presence of inducible NO synthase by immunoblotting. Plasma nitrate and 24-hour-urinary-excretion of cGMP were significantly elevated in patients compared with controls, while 24-hour-urinary-excretion of nitrate tended to be elevated in SSc patients. Levels of sICAM-1, sVCAM-1, and sE-selectin were significantly elevated in the patients. Levels of plasma nitrate in the patients correlated significantly with levels of sVCAM-1 (P = 0.020) and sE-selectin (P = 0.018) and approached a significant correlation with sICAM-1 (P = 0.055), suggesting that activated endothelial cells may produce plasma nitrate. NO synthesis is elevated in SSc patients, and the activated endothelial cell is a likely site of its production.

Sources of nitrate exposure in residents of rural areas in Quebec, Canada

Nitrate exposure was investigated in a group of 187 people using well water and living in four areas of rural Quebec (Canada) with intensive agricultural activities. Nitrate intake was evaluated using a 24-h dietary recall and a food frequency questionnaire, in conjunction with a validated food database and measurements of nitrate concentrations in private wells. The total internal dose was estimated by means of the 24-h urinary nitrate excretion, while taking into account risk factors for endogenous nitrate formation. Mean (geometric) 24-h urinary nitrate excretion was 16.9 mg N for the 100 people with low groundwater contamination (mean nitrate concentration=0.18 mg N/l) and 23.3 mg N in the 87 individuals with moderate groundwater contamination (mean nitrate concentration=7.1 mg N/l). A multivariate analysis revealed that dietary nitrate intake during the last 24 h was the principal source of exposure, followed by water intake during the last 24 h. The Quetelet index was also a significant predictor of urinary excretion. The total predictive model explained only 29% of the variability in urinary nitrate excretion (R2=0.286). Neither the inflammatory status as indicated by elevated C reactive protein, the presence of Helicobacter pylori antibodies nor the occurrence of diarrhea during the last 24 h prior to urine collection were associated with urinary nitrate excretion. In conclusion, food and to a lesser extent water contribute to nitrate exposure in this rural setting with moderate water contamination. Better predictors of endogenous nitrate production are needed to improve our ability to model nitrate body burden and estimate associated health risks.

Antihypertensive effect of 0.1-Hz blood pressure oscillations to the kidney.

Physiological blood pressure (BP) fluctuations with frequencies >0.1 Hz can override renal blood flow autoregulation. The influence of such immediate changes in renal perfusion pressure (RPP) on daily BP regulation, eg, via shear stress-stimulated liberation of renal endothelial NO, however, is unknown. Thus, we studied the effects of such RPP oscillations on renal function and on systemic BP during the onset of renal hypertension. Seven beagles (randomly assigned to each of the following protocols) were chronically instrumented for the measurement of systemic BP, RPP, and renal excretory function. An inflatable cuff was used to reduce and to oscillate RPP over 24 hours in the freely moving dog. Reducing RPP to 87+/-2 mm Hg diminished excretion of sodium and water and doubled plasma renin activity (PRA, n=7, P<0. 01) but had no significant effect on urinary nitrate excretion (n=6), a marker of NO generation. Superimposing 0.1-Hz oscillations (+/-10 mm Hg) onto the reduced RPP blunted hypertension, returned fluid excretion almost to control levels, and doubled renal sodium elimination. Nitrate excretion peaked at 8 hours, only to return to control values shortly thereafter. PRA, conversely, was significantly reduced during the last third of the experimental protocols. BP fluctuations transiently stimulate NO liberation and induce a reduction in PRA, which enhances 24-hour sodium and water excretion and markedly attenuates the acute development of renovascular hypertension.

Urinary nitrate excretion in cholesterol-fed rabbits: effect of a chronic treatment by N-iminoethyl- l-lysine, a selective inhibitor of inducible nitric oxide synthase

To evaluate the influence of atherosclerosis on the global production of NO, we studied the effect of a 0.3% cholesterol-enriched diet on urinary nitrate excretion in rabbits during 69 weeks. To examine whether the inducible nitric oxide synthase (iNOS) present in atherosclerotic lesions could participate in NO excretion, hypercholesterolemic rabbits were treated chronically with the selective iNOS inhibitor, N-iminoethyl- l-lysine ( l-NIL; 5 mg/kg/day). Urinary nitrate excretion was higher in hypercholesterolemic than in control rabbits throughout the study period and decreased progressively with time in both groups; l-NIL had no significant effect on urinary nitrate excretion. These data illustrate that systemic NO production is enhanced in hypercholesterolemia and that iNOS, present within the plaque, might not participate in this enhanced NO production.

THE ROLE OF INTRARENAL NITRIC OXIDE IN THE NATRIURETIC RESPONSE TO DOPAMINE-RECEPTOR ACTIVATION

Dopamine and dopamine-1 receptor agonists produce diuresis and natriuresis by causing changes in renal hemodynamics and by the activation of dopamine-1 receptors located within the various regions of the nephron. Nitric oxide plays an important role in the maintenance of systemic and regional hemodynamics. The present study was undertaken to investigate the effect of locally generated nitric oxide on renal function and its potential influence on the renal responses to dopamine-1 receptor agonists. The intrarenal infusion of a nitric oxide synthase inhibitor, L-NAME, (50 μg/kg min for 90 min) in anesthetized rats produced significant decreases in urine volume, urinary sodium excretion, glomerular filtration rate and fractional sodium excretion. These changes in renal function were associated with a concomitant decrease in urinary nitrate excretion, an indicator of nitric oxide release. However, L-NAME at this dose did not produce any significant changes in mean arterial pressure or heart rate. Intravenous infusion of fenoldopam (1 μg/kg min for 30 min), a selective dopamine-1 receptor agonist, produced diuresis and natriuresis without causing any changes in mean arterial pressure and heart rate. These renal effects of fenoldopam were significantly attenuated in animals that received the simultaneous infusion of L-NAME (intrarenal). Similar results were obtained with dopamine in that the natriuretic and diuretic response to dopamine was also attenuated during simultaneous infusion of dopamine with L-NAME. In addition, the diuresis and natriuresis produced by fenoldopam and dopamine was associated with increases in urinary nitrate excretion. Interestingly, these increases in the nitrate levels seen with fenoldopam and dopamine were also significantly reduced in the presence of L-NAME. These results indicate that intrarenal nitric oxide plays an important role in regulating renal sodium excretion and that an intact renal nitric oxide system is required for the full expression of diuretic and natriuretic response seen during dopamine-1 receptor activation.

Increased nitric oxide synthase-3 expression in kidneys of deoxycorticosterone acetate-salt hypertensive rats.

In addition to its hemodynamic effects, nitric oxide (NO) may play a role in the renal tubular handling of sodium. Experiments were conducted to determine possible changes in renal nitric oxide synthase-3 (NOS3) expression in rats treated with deoxycorticosterone acetate (DOCA) and high salt. All rats were uninephrectomized, and either a placebo or DOCA pellet was implanted subcutaneously. Placebo-treated rats were then given tap water to drink ad libitum, and DOCA-treated rats received a 0.9% NaCl solution to drink. Once a week, rats were placed in metabolic cages so that a 24-h urine sample could be collected. After 3 wk, the animals were sacrificed and the kidneys removed and prepared for subsequent immunohistochemical or Western blot analysis. Urinary excretion of nitrate and nitrite (NOx) was measured to provide an indication of the intrarenal production of NO. DOCA-salt hypertensive rats exhibited increased urinary NOx excretion (2.43 +/- 0.48 micromol NOx/mg creatinine) compared with the placebo control animals (1.17 +/- 0.06 micromol NOx/mg creatinine). Western blot analysis revealed that NOS3 protein levels in both the cortex and medulla were greater in DOCA-salt rats compared with placebo-treated animals. Immunohistochemical analysis of kidneys revealed that NOS3 expression in placebo rats was localized in vascular endothelial cells with slight, but detectable, immunoreactivity in medullary collecting ducts. In DOCA-salt rats, a very large increase in the intensity of immunostaining was detected in tubular epithelia of the proximal tubule, thick ascending limb of Henle's loop, and cortical and medullary collecting duct; immunoreactivity in endothelial cells appeared unchanged. These data suggest that increased tubular expression of NOS3 is responsible, at least in part, for the increased renal production of NO in DOCA-salt hypertension, and are consistent with a role for NO in the renal tubular response to salt loading.

Nitric oxide synthesis in patients with infective gastroenteritis

BACKGROUNDThere is evidence that endogenous nitrate synthesis is notably increased in patients with infective gastroenteritis.AIMSTo determine whether this is due to nitric oxide (NO) production via the l-arginine/NO pathway.METHODSSeven male...

Dietary Protein or Arginine Deficiency Impairs Constitutive and Inducible Nitric Oxide Synthesis by Young Rats

Effects of dietary protein or arginine deficiency on constitutive and lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis were determined in young rats by quantifying urinary nitrate excretion. In Experiment 1, 30-d-old rats (n = 16) were divided randomly into two groups (n = 8/group) and pair-fed on the basis of body weight semipurified isocaloric diets containing 20 or 5% casein. In Experiment 2, 30-d-old rats (n = 24) were divided randomly into three groups (n = 8) and pair-fed on the basis of body weight purified isonitrogenous and isocaloric diets (composed of amino acids) containing 0.0, 0.3 or 1.0% L-arginine. In both experiments, daily collection of urine was initiated 10 d after the start of pair-feeding. On d 17 after the pair-feeding was initiated, LPS (1 mg/kg body wt) was injected intraperitoneally into rats, and urine was collected daily for an additional 7 d. In Experiments 3 and 4, activities of constitutive and inducible NO synthases were measured in macrophages and various tissues from protein- or arginine-deficient rats (n = 6). Body weight was lower in rats fed the 5% casein diet or the 0.0 and 0.3% arginine diets than in those fed 20% casein or 1% arginine, respectively. Dietary protein or arginine deficiency decreased serum concentrations of arginine and urinary nitrate excretion before and after LPS treatment, indicating impaired constitutive and inducible NO synthesis. Protein malnutrition reduced constitutive and inducible NO synthase activities in brain, heart, jejunum, lung, skeletal muscle and spleen, and inducible NO synthase activity in macrophages. Because NO is a mediator of the immune response and is the endothelium-dependent relaxing factor, impaired NO synthesis may help explain immunodeficiency and cardiovascular dysfunction in protein- or arginine-deficient subjects.

Effects of acute euglycemic hyperinsulinemia on urinary nitrite/nitrate excretion and plasma endothelin-1 levels in men with essential hypertension and normotensive controls

Insulin stimulates the production of endothelin-1 (ET-1) and nitric oxide (NO) by isolated endothelial cells. Additionally, insulin-dependent glucose transport and insulin-mediated NO production partially share common elements in signal transduction. There are discordant data on plasma ET-1 levels during acute euglycemic systemic hyperinsulinemia in normotensive men and men with essential hypertension (EH) (known to be insulin-resistant), as well as on the relations between insulin sensitivity and vascular function. Our aim was to assess the response of approximate measures of whole-body generation of NO and ET-1 to acute euglycemic hyperinsulinemia in EH patients and controls. We studied 17 newly diagnosed untreated men with uncomplicated EH and 10 normotensive controls. Plasma ET-1 and urinary excretion of nitrite plus nitrate, stable NO metabolites (Uno x), were measured before and during a 3-hour hyperinsulinemic-euglycemic clamp. Both in hypertensives and normotensives, plasma ET-1 levels were reduced after 2 hours of the clamp (EH: baseline, 3.1 ± 1.9 pg/mL; 2 hours, 1.9 ± 1.2 pg/mL, P = .04 v baseline; controls: baseline, 4.2 ± 2.6 pg/mL; 2 hours, 2.8 ± 1.4 pg/mL, P = .04 v baseline). No significant changes in Uno x during the clamp were observed. Changes in Uno x during the clamp (ΔUno x) and differences in plasma ET-1 measured before the end and before the beginning of the clamp (ΔET-1) were correlated in the controls ( r = .75, P = .01) but not in EH ( r = −.01, P = .97). No parameter of glucose metabolism correlated with basal Uno x, basal plasma ET-1, ΔUno x, and ΔET-1, whether absolute or percent values, in either group. Thus, acute euglycemic hyperinsulinemia produces a decrease in plasma ET-1 in both EH patients and controls. The lack of correlation between ΔUno x and ΔET-1 under these conditions in EH may suggest an impairment of systems governing interactions between the NO-dependent pathway and ET-1. In addition, insulin actions on glucose metabolism and on the endothelial mediators appear dissociated.

Blood pressure, sodium intake, insulin resistance, and urinary nitrate excretion.

The objective of this study was to investigate the relationships among various humoral factors thought to be involved in the regulation of blood pressure during high NaCl intake. Nineteen healthy subjects underwent sequential 5-day periods ingesting a low-sodium (25 mmol/d) or high-sodium (200 mmol/d) diet. Insulin resistance was assessed by the steady-state plasma glucose concentration at the end of a 3-hour insulin suppression test. Insulin resistance correlated inversely with natriuresis (P=0.04) and directly with increase in weight (P=0.03). The increase in mean arterial pressure associated with the high-sodium diet correlated directly with the gain in weight (P<0.05) and inversely with the increase in urinary nitrate excretion (P<0.0001). In a multiple regression model, more than 2/3 of the variance in mean arterial pressure was accounted for by the gain in weight and change in urinary nitrate excretion. The steady-state plasma glucose concentrations obtained with the 2 diets were similar, indicating that insulin resistance was unaffected by sodium intake. During high sodium intake, plasma renin activity and aldosterone decreased and plasma atrial natriuretic peptide increased; these changes did not correlate with the change in mean arterial pressure, insulin resistance, or change in urinary nitrate excretion. To the extent that urinary nitrate excretion reflects activity of the endogenous nitric oxide system, these results suggest that the salt sensitivity of mean arterial pressure may be related to blunted generation of endogenous nitric oxide. The results also demonstrate that insulin-resistant individuals have an impaired natriuretic response to high sodium intake.

The effects of human burn injury on urinary nitrate excretion

Different studies have demonstrated both an increase and a decrease in the biosynthesis of nitric oxide (NO) during the first 2 days following experimental and human burn trauma. This study investigated changes in urinary nitrate excretion in humans following thermal injury in order to determine the temporal relationship between NO release and the initial injury. Urinary nitrate was measured in daily 24-h urine collections taken on days 1–7 following burn injury from 15 patients. The control group consisted of 11 healthy, age- and sex-matched patients who kept a nitrate-restricted diet for five days prior to collection of a single 24-h urine sample. The burns group had a mean age of 41.9±19.4 (mean±S.D.) years and a mean total burn surface area (TBSA) of 30.2±24.9% (mean±S.D.). In the burn injured patients, urinary nitrate levels peaked at day 4 and a 2-fold increase relative to day 1 was observed. Urinary nitrate levels were significantly higher in the burns group than the control group on days 4 and 5 only ( p<0.05 for both days). There was no correlation between TBSA and the measured urinary nitrate levels. This study confirms that the biosynthesis of NO is increased during the first week following burn trauma and establishes that the renal elimination of the by-products of NO metabolism is not increased during the first three days after injury. Notwithstanding the potential effects of burns on nitrate distribution, our findings may reflect a delay in the release of NO following the initial insult.

Altered vascular reactivity following partial nephrectomy in the rat: a possible mechanism of the blood-pressure-lowering effect of heparin.

This study was designed to assess whether the antihypertensive effect of heparin in rats after renal mass reduction (RMR) is related to changes in nitric oxide activity, and to study in vitro the altered behaviour of resistance-sized arteries induced by chronic administration of heparin. Male Wistar rats were assigned to one of two experimental protocols. In the first protocol, RMR rats received heparin (250 units/day s.c.) and tail systolic blood pressure (SBP) was measured weekly for 4 weeks. In a subgroup, urinary nitrate excretion (UNO3) and in vitro vascular reactivity of isolated perfused mesenteric arterial beds were measured 2 weeks after RMR. The second protocol assessed whether inhibition of NO synthesis with L-NAME (70 mg/l added to the drinking water) prevents the blood-pressure-lowering effect of heparin. In untreated RMR rats SBP increased from 111+/-3 mmHg to 127+/-5 mmHg at 2 weeks and 139+/-5 mmHg at 4 weeks. In contrast, in RMR rats treated with heparin, SBP was 114 +/-3 mmHg at 2 weeks and 115+/-4 mmHg at 4 weeks (P<0.05 for both). Treatment with L-NAME increased SBP both in untreated and heparin-treated RMR groups. Two weeks after nephrectomy daily urinary nitrate increased significantly more in RMR rats treated with heparin than in untreated RMR rats (22+/-2 vs 14.2+/-2.3 micromol/day, P<0.05). In vitro studies performed at 2 weeks showed that vessels of untreated RMR rats had a blunted vasodilator response to acetylcholine that was restored to levels similar to that of controls in the heparin-treated group. These results suggest that, in rats after renal ablation, heparin may exert its antihypertensive effect, at least in part, by affecting the altered behaviour of resistance vessels during the development phase of hypertension. Increased NO production may contribute to this effect.

Dietary Salt Loading Increases Nitric Oxide Synthesis in Transgenic Mice Overexpressing Sodium-Proton Exchanger

We studied the role of nitric oxide (NO) synthesis in amelioration of blood pressure elevation during dietary salt loading in transgenic mice overexpressing sodium proton exchanger. Systolic blood pressure rose after starting salt loading only in the high-salt group of transgenic mice. However, this elevation of blood pressure was not continued. Urinary excretion of inorganic nitrite and nitrate in the high-salt group of transgenic mice was significantly higher than in the high-salt group of control mice. These results suggest that increased NO synthesis in response to salt loading is one of the anti-hypertensive mechanisms in transgenic mice overexpressing sodium proton exchanger.

Potassium Supplementation Increases Sodium Excretion and Nitric Oxide Production in Hypertensive Dahl Rats

The present study was designed to investigate whether antihypertensive and natriuretic effects of K were achieved by elevation of nitric oxide (NO) production in Dahl salt-sensitive (DS) rats. The rats were placed in individual metabolic cage and fed a high sodium diet with or without K supplementation for 4 weeks. K supplementation counteracted the blood-pressure raising effect of NaCl. K supplementation significantly enhanced sodium excretion and reduced sodium retention, increased the urinary nitrite plus nitrate excretion and kidney constitutive NO synthase activity in salt-loaded DS rats. These effect did not occur in the rats fed a low sodium diet with K supplementation. These results suggest that K supplementation attenuates development of hypertension with reduction of sodium retension in salt-loaded DS rats, which is mediated by the recovery of salt-induced NO production mechanism.

Decreased excretion of nitrate and nitrite in essential hypertensives with renal vasoconstriction

Most hypertensive patients exhibit increased renal vascular resistance (RVR). This study was designed to investigate whether there exists any relationship between RVR and the production of nitric oxide (NO) in patients with essential hypertension. The study was performed in 49 non-treated patients with mild-to-moderate essential hypertension, and 20 age- and sex-matched normotensive subjects on a controlled sodium diet. Renal hemodynamics was measured in terms of the clearance of para-aminohippuric acid and inulin. Urinary excretion of nitrate and nitrite (NO3- plus NO2-) was determined as an index of NO production. As compared with normotensives, hypertensive patients exhibited higher (P < 0.001) RVR and lower (P < 0.05) urinary excretion of NO3- plus NO2-. With the 100% confidence (upper) limit of the normotensive population as a cut-off point, a subgroup of 30 hypertensives had an abnormally high RVR. The excretion of NO3- plus NO2- was lower (P < 0.005) in hypertensives with high RVR than in normotensives and the remaining hypertensives. No differences were found in the urinary excretion of NO3- plus NO2- between normotensives and hypertensives with normal RVR. Statistically significant associations were seen between diastolic blood pressure and RVR (r = 0.341, P < 0.05) and urinary excretion of NO3- plus NO2- (r = -0.387, P < 0.01) in all hypertensives. These results indicate that there is a subgroup (61%) of hypertensive patients with diminished urine levels of NO3- plus NO2- in which RVR is abnormally increased. Thus, it is suggested that in essential hypertension a diminished renal ability to produce NO by the endothelium may be involved in exaggerated renal vasoconstriction.

The contribution of nitric oxide to renal vascular wall thickening in rats with L-NAME-induced hypertension.

We investigated the mechanisms of renal vascular wall thickening in a rat model of N-nitro L-arginine methyl ester (L-NAME)-induced hypertension. To separate the effects of L-NAME-induced hypertension from other effects of nitric oxide (NO) inhibition, we created two models of L-NAME-induced hypertension: both had the same blood pressure level but NO inhibition was moderate in one group (group M) and severe in the other (group S). Urinary excretion of nitrates and nitrites was lower in group S than in group M. Wall thickening and lipid deposition in renal vessels were significantly greater in group S than in groups M. Simple and multiple regression analyses indicated that renal vascular wall thickening was more strongly correlated with lipid deposition than with blood pressure. The number of vessels positive for staining with Sudan black B was negatively correlated with urinary NO excretion. Expression of fibronectin and transforming growth factor-beta was greater in the Sudan black B-positive than in the Sudan black B-negative vessels, suggesting that extracellular matrix production was increased in vessels with lipid deposition. Lipid deposition and increased production of extracellular matrix may contribute to renal vascular wall thickening in L-NAME-induced hypertension. Some mechanisms independent of hypertension play important roles in vascular wall thickening induced by NO inhibition.

Dietary l-arginine and α-tocopherol reduce vascular oxidative stress and preserve endothelial function in hypercholesterolemic rabbits via different mechanisms

Vascular oxidative stress brought about by superoxide radicals and oxidized low-density lipoproteins (oxLDL) is a major factor contributing to decreased NO-dependent vasodilator function in hypercholesterolemia and atherosclerosis. We investigated whether chronic administration of l-arginine (2% in drinking water) or of α-tocopherol (300 mg/day) improves endothelium-dependent vasodilator function and systemic NO production, reduces vascular oxidative stress, and reduces the progression of atherosclerosis in cholesterol-fed rabbits with pre-existing hypercholesterolemia. Systemic NO production was assessed as urinary nitrate excretion; oxidative stress was measured by urinary 8-iso-PGF 2 α excretion in vivo, by lucigenin-enhanced chemiluminescence of isolated aortic rings ex vivo, and by copper-mediated LDL oxidation in vitro. Endothelium-dependent relaxation was almost completely abrogated in cholesterol-fed rabbits. Urinary nitrate excretion was reduced by 46±10%, and 8-iso-PGF 2 α excretion was increased by 61±18% as compared to controls (each P<0.05). Vascular superoxide radical release stimulated by PMA ex vivo was increased by 273±93% in this group, and the lag time of LDL oxidation was reduced by 35±6% (each P<0.05). Treatment with l-arginine and α-tocopherol reduced intimal lesion formation (by 68±6 and 41±11%, respectively; P<0.05) and improved endothelium-dependent relaxation. Both treatments also normalized urinary 8-iso-PGF 2 α excretion. l-Arginine increased urinary nitrate excretion by 43±13% ( P<0.05) and reduced superoxide radical release by isolated aortic rings to control levels, which was unaffected by vitamin E treatment. By contrast, vitamin E dramatically increased the resistance of isolated LDL to copper-mediated oxidation in vitro by 178±7% ( P<0.05), which was only marginally prolonged by l-arginine. Intimal thickening was reduced by both treatments. We conclude that both l-arginine and α-tocopherol reduce the progression of atherosclerotic plaques in cholesterol-fed rabbits. However, while l-arginine increases NO formation and reduces superoxide release, α-tocopherol antagonizes mainly oxLDL-related events in atherogenesis. Thus, both treatments reduce urinary isoprostane excretion and improve endothelium-dependent vasodilation via different mechanisms.

The fate of nitrogen from 15N-labeled nitrate after single intravenous administration of Na15NO3 in sheep

The metabolic fate of nitrogen from 15N-labeled sodium nitrate has been investigated in four healthy Polish Merino ewes. 15N-labeled sodium nitrate was administered intravenously at the dosage of 400 mu mol·kg-1 body weight. Blood plasma and urine concentrations of nitrate, ammonia, and urea and 15N enrichment of ammonia and urea were estimated over a 50-h period following 15N-nitrate administration. Nitrate (NO3-) was slowly eliminated from the blood plasma, and the presence of NO3- in the blood plasma above the nitrate "background" was observed for 50 h. 15N enrichment of blood plasma urea already appeared at 15 min and reached the maximum 6 h after 15N-nitrate administration. The urinary excretion of nitrate occured during 50 h after 15N-nitrate injection; the total urine excretion of NO3- was 23.63 ± 2.39% of the administered dose. The mean urinary recoveries of nitrogen as 15N-urea and 15N-ammonia were 14.76 ± 1.32% and 0.096 ± 0.015% of the administered 15N-nitrate dose, respectively. It should be pointed out that in total only 38.49% of the administered nitrate-N was excreted in urine (as nitrate, ammonia and urea nitrogen) during 50 h. The results obtained indicate that sheep are able to store nitrate nitrogen in their body. The fate of the remaining approximately 60% of the 15NO3- administered dose is unknown. The results obtained do not allow one to conclude what fraction of the unrecovered approximately 60% of the 15NO3- dose was utilized by gastrointestinal microorganisms, and (or) metabolized, or stored in sheep tissues.Key words: nitrate, ammonia, urea, metabolism, excretion, sheep.