Index Of Nitric Oxide Formation Research Articles

Sodium nitrite protects against kidney injury induced by brain death and improves post-transplant function

Renal injury induced by brain death is characterized by ischemia and inflammation and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 hours of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 hours of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain dead rats showed significant improvement in function over the first 2 to 4 days post transplantation compared to untreated brain dead animals. Gene microarray analysis after 2 hours of brain death without or with nitrite therapy showed the latter significantly altered the expression of about 400 genes. Ingenuity Pathway analysis indicated multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription and genes related to humoral immune responses. Thus, nitrite-therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.

Involvement of TRPV4-NO-cGMP-PKG pathways in the development of thermal hyperalgesia following chronic compression of the dorsal root ganglion in rats

The aim of the present study was to test the hypothesis that the TRPV4-NO-cGMP-PKG cascade is involved in the maintenance of thermal hyperalgesia following chronic compression of the dorsal root ganglion (DRG) (the procedure hereafter termed CCD) in rats. CCD rats showed thermal hyperalgesia and increased nitrite production. Intrathecal administration of ruthenium red (TRPV4 antagonist, 0.1–1nmol), TRPV4 antisense ODN (TRPV4 AS, 40μg, daily for 7 days), NG-l-nitro-arginine methyl ester (l-NAME, inhibitor of NO synthase, 30–300nmol), 1H-[1,2,4]-oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor, 50–100nmol) or 8-(4-Chlorophenylthio) guanosine 3′,5′-cyclic Monophosphothioate, Rp-Isomer sodium salt (Rp-8-pCPT-cGMPS, a PKG inhibitor, 25–50nmol) induced a significant (P<0.001) and dose-dependent increase in the paw withdrawal latency (PWL) compared with control rats, respectively. Ruthenium red (1nmol), TRPV4 AS (40μg, daily for 7 days) or l-NAME (300nmol) decreased nitrite (an index of nitric oxide formation) in the DRG of CCD rats. In addition, the phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD, TRPV4 synthetic activator, 1nmol), co-administered with l-NAME (300nmol), attenuated the suppressive effect of l-NAME on CCD-induced thermal hyperalgesia and nitrite production. Our data suggested that the TRPV4-NO-cGMP-PKG pathway could be involved in CCD-induced thermal hyperalgesia.

Arteriovenous Differences in NO2- Kinetics in Anesthetized Rabbits

Despite of the importance of plasma NO2- as an index of endothelial nitric oxide (NO) formation and a substrate for NO production, only a limited kinetic knowledge is available in vivo. To address this issue, we intravenously injected NaNO2 into anesthetized rabbits and quantified changes in arterial and venous plasma NO2- levels. Plasma NO2- levels in arterial blood (956 +/- 220 nM) were slightly, but significantly, higher than those in venous blood (889 +/- 214 nM) under control conditions. Although similar half-lives of the NO2- were observed in arterial and venous plasma, significant arteriovenous differences were observed in the volume of distribution of the central compartment (0.158 +/- 0.007 l/kg in arterial blood and 0.295 +/- 0.015 l/kg in venous blood) and the peripheral compartment (0.259 +/- 0.035 l/kg in arterial blood and 0.135 +/- 0.034 l/kg in venous blood). When NOR1 (authentic NO) was injected, increases in NO2- levels were greater in arterial plasma, and decreases in blood pressure significantly correlated with arterial NO2- levels only (r = 0.90, p < 0.01). These results indicate that changes in NO2- are larger and more easily detectable as an index of NO formation in arterial plasma.

Hypoxic conditioning suppresses nitric oxide production upon myocardial reperfusion.

Physiologically modulated concentrations of nitric oxide (NO) are generally beneficial, but excessive NO can injure myocardium by producing cytotoxic peroxynitrite. Recently we reported that intermittent, normobaric hypoxia conditioning (IHC) produced robust cardioprotection against infarction and lethal arrhythmias in a canine model of coronary occlusion-reperfusion. This study tested the hypothesis that IHC suppresses myocardial nitric oxide synthase (NOS) activity and thereby dampens explosive, excessive NO formation upon reperfusion of occluded coronary arteries. Mongrel dogs were conditioned by a 20 d program of IHC (FIO(2) 9.5-10%; 5-10 min hypoxia/cycle, 5-8 cycles/d with intervening 4 min normoxia). One day later, ventricular myocardium was sampled for NOS activity assays, and immunoblot detection of the endothelial NOS isoform (eNOS). In separate experiments, myocardial nitrite (NO(2)(-)) release, an index of NO formation, was measured at baseline and during reperfusion following 1 h occlusion of the left anterior descending coronary artery (LAD). Values in IHC dogs were compared with respective values in non-conditioned, control dogs. IHC lowered left and right ventricular NOS activities by 60%, from 100-115 to 40-45 mU/g protein (P < 0.01), and decreased eNOS content by 30% (P < 0.05). IHC dampened cumulative NO(2)(-) release during the first 5 min reperfusion from 32 +/- 7 to 14 +/- 2 mumol/g (P < 0.05), but did not alter hyperemic LAD flow (15 +/- 2 vs. 13 +/- 2 ml/g). Thus, IHC suppressed myocardial NOS activity, eNOS content, and excessive NO formation upon reperfusion without compromising reactive hyperemia. Attenuation of the NOS/NO system may contribute to IHC-induced protection of myocardium from ischemia-reperfusion injury.

Neutrophils-derived peroxynitrite contributes to acute hyperalgesia and cell influx in zymosan arthritis

We investigated the contribution of neutrophils to joint hyperalgesia and peroxynitrite formation in zymosan arthritis. Rats received 1 mg zymosan intra-articular, and joint hyperalgesia was measured using the rat knee-joint articular incapacitation test. After 6 h, joint exudates were collected by aspiration for the assessment of cell influx, myeloperoxidase activity, and nitrite (as an index of nitric oxide formation) levels. Nitrotyrosine content, used as an index of peroxynitrite formation, was measured in joint exudates, using enzyme-linked immunosorbent assay. A group of rats was rendered neutropenic through the administration of a rabbit anti-rat neutrophil antibody (2 ml kg(-1), i.p.) 30 min before injection of 1 mg zymosan intra-articular. Other groups received uric acid (100 or 250 mg kg(-1), i.p.), the peroxynitrite scavenger, 30 min before 1 mg zymosan intra-articular. Controls received the vehicle. The significant inhibition of joint hyperalgesia in neutropenic animals was associated to significantly decreased cell influx, myeloperoxidase activity, nitric oxide, and nitrotyrosine levels in the joint exudates, as compared to naive rats. Uric acid administration inhibited both hyperalgesia and cell influx, as compared to controls. Neutrophils are involved in both nitric oxide and peroxynitrite formation in zymosan arthritis, thereby contributing to acute joint hyperalgesia. Scavenging of reactive nitrogen species (e.g. peroxynitrite) inhibits neutrophil migration and joint hyperalgesia in the acute phase of zymosan arthritis in rats.

Hypoxic Conditioning Suppresses Cytotoxic Nitric Oxide Production Upon Myocardial Reperfusion

Background Physiologocal concentrations of nitric oxide (NO) are generally beneficial, but excessive NO formation can injure ischemic myocardium by producing cytotoxic peroxynitrite upon reperfusion. Recently we reported that intermittent hypoxia conditioning (IHC) produced remarkable cardioprotection against infarction and lethal arrhythmias in a canine model of coronary occlusion-reperfusion. Hypothesis IHC suppresses myocardial formation of NO by the endothelial NO synthase isoform (eNOS) upon coronary artery reperfusion. Methods Mongrel dogs were conditioned by a 20 d program of intermittent normobaric hypoxia (FIO2 9.5- 10%; 5–10 min/cycle, 5–8 cycles/d with intervening 4 min normoxia), and compared with nonhypoxic control dogs. One day later, myocardium was sampled for measurement of left ventricular NOS activity (colorimetric assay) and eNOS content (Western blot). In other anesthetized dogs, myocardial nitrite release, an index of NO formation, was measured at baseline and during reperfusion following 1 h occlusion of the left anterior descending coronary artery (LAD). Results IHC lowered left ventricular NOS activity 45%, from 98 ± 10 to 55 ± 10 mU/g protein (P<0.01), and sharply decreased eNOS content (Figure). IHC reduced cumulative NO2− release during the first 5 min reperfusion from 26.4 ± 7.5 to 11.8 ± 1.8 μmol/g (P<0.05), but did not alter hyperemic LAD flow (15.2 ± 2.1 vs. 14.4 ± 2.4 ml/g). Conclusion IHC suppressed myocardial NOS activity and eNOS content. Reperfusion NO release was decreased in IHC myocardium without compromising reactive hyperemia. (supported by NIH grants HL-64785 and HL-71684)

Antioxidants in Detoxification of Arsenic-Induced Oxidative Injury in Rabbits: Preliminary Results

To assess the oxidative injuries caused by arsenic toxicity in rabbits and evaluate the detoxifying effects of exogenous antioxidants, we administered arsenic trioxide (3–5 mg/kg/day) in rabbits through a feeding tube for seven days. These rabbits were then treated with a recipe of vitamins, zinc, selenium (VZS) or a plant polyphenol or a placebo for the next seven days. Blood samples were collected from ear vein for spectrophotometric assay of reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS), and nitrite/nitrate (NO x ; index of nitric oxide formation) before arsenic administration, seven days after arsenic administration, and seven days after antioxidant treatment. The total arsenic concentrations in hair and spot urine samples of rabbits before arsenic administration were 0.6 ± 0.21 µg/g and 34.0 ± 5.9 µg/L, respectively. Administration of arsenic trioxide significantly increased arsenic concentrations in hair and in urine to 2.8 ± 0.40 µg/g (p<0.001) and 7372 ± 1392.0 µg/L (p<0.001), respectively. Arsenic administration to rabbits significantly reduced GSH concentration (post-arsenic,17.5 ± 0.81 mg/dL vs. pre-arsenic, 32.0 ± 0.76 mg/dL, p<0.001), increased TBARS concentration (post-arsenic, 8 ± 1.1 µM vs. pre-arsenic, 5 ± 0.7 µM, p<0.05), and NO x concentration (post-arsenic, 465 ± 38.5 µM vs. pre-arsenic, 320 ± 24.7 µM, p<0.001) as compared to the pre-arsenic levels. There was a negative correlation between TBARS and GSH concentrations (r = −0.464, p<0.01) and between NO x and GSH concentrations (r = − 0.381, p<0.05) of intoxicated rabbits. The recovery of the depleted GSH was significantly greater in the polyphenols (77.0 ± 12.0%) or VZS (67.0 ± 17.0%) treatment groups compared with the placebo group (36.0 ± 7.0%). The decrease in NO x level of arsenic-treated rabbits was significantly greater in polyphenols treatment group than the placebo group (60.0 ± 9.0% vs. 17.0 ± 6.0%, p<0.001). These results indicate that arsenic induces toxicity in rabbits associated with an increase in lipid peroxidation. Arsenic toxicity increases nitric oxide production in the body. Exogenous antioxidants such as polyphenols and recipe of vitamins, zinc, and selenium are useful for arsenic detoxification.

Erythropoietin protects against brain ischemic injury by inhibition of nitric oxide formation

Erythropoietin prevents in vitro glutamate-induced neuronal death and could play a role in the central nervous system. We investigated the in vivo effects of recombinant human erythropoietin after intraperitoneal (i.p.; 25–100 U) or intracerebroventricular (i.c.v.; 0.25–25 U) administration on survival, brain malonildialdehyde (MDA) levels, brain edema, hippocampal neuronal death and brain nitric oxide (NO) synthesis after bilateral carotid occlusion (5 min), followed by reperfusion in the Mongolian gerbil. Peripheral posttreatment with recombinant human erythropoietin reduced postischemic MDA levels, brain edema and increased survival. Either peripheral or i.c.v. posttreatment with recombinant human erythropoietin significantly reduced hippocampal CA1 neuronal loss, observed 7 days after the ischemic event. Increase of nitrite and nitrate (as an index of NO formation) in the hippocampus, as observed after ischemia, was reduced in animals treated with recombinant human erythropoietin. These data suggest that in vivo recombinant human erythropoietin effects on brain ischemic injury could be due to inhibition of NO overproduction.

Plasma Nitrate as an Index of Nitric Oxide Formation in Patients with Acute Infectious Diseases

In humans, the role of nitric oxide (NO) in host defence is controversial. We prospectively studied plasma levels of nitrate, the stable end-product of NO formation, during acute infection in 43 patients controlled with regard to dietary nitrate/nitrite. During acute gastroenteritis the mean plasma nitrate level was significantly increased compared with at recovery 4-5 weeks later (118 vs. 32.5 micromol/l; p < 0.001), in contrast with the findings in patients with acute pneumonia (PN; 34.6 vs. 42.8 micromol/l) or febrile urinary tract infection (UTI; 27.7 vs. 31.3 micromol/l). In a second group of 20 retrospectively studied patients with severe PN or UTI, of whom 70% were bacteraemic, no significantly increased nitrate levels could be demonstrated during the acute stage of infection. These findings indicate that increased NO production, as measured by plasma nitrate, is not a general finding in patients with acute infectious diseases, but may rather be associated with certain pathogens or sites of infection.

Plasma nitrate as an index of nitric oxide formation in man: analyses of kinetics and confounding factors.

Nitric oxide (NO) is metabolized to nitrate in humans. Accordingly, plasma nitrate has been proposed as an index of the in vivo formation of NO. Such an application requires knowledge about the possible influence of nitrate from sources other than endogenous NO formation, as well as of the kinetics of nitrate in plasma. In the present study, plasma nitrate increased from 32 +/- 4 to 205 +/- 27 mumol/l (mean +/- SE) following intake of nitrate-rich food. It dropped during the intake of nitrate-restricted diet and stabilized at a level of 29 +/- 1 mumol/l. The urinary excretion of nitrate during nitrate restriction was 840 +/- 146 mumol/24 h. Plasma nitrate was not affected following the intake of a gastrointestinal antibiotic drug for a period of four days. Smoking three cigarettes in succession did not affect the plasma nitrate levels significantly. The oral intake of potassium nitrate (500 mg approximately 4950 mumol) elevated plasma nitrate from 29 +/- 3 to 313 +/- 12 mumol/l within 60 min. The subsequent drop in plasma nitrate, with a t1/2 of 451 +/- 42 min, was probably a reflection of the redistribution of nitrate within the body fluids and the renal excretion of nitrate. The plasma clearance of nitrate was 30 +/- 2 ml/min/1.73 m2 BSA. The distribution volume for nitrate was 28 +/- 1% of the bodyweight (BW). We conclude that plasma nitrate can be used as an index of the endogenous formation of NO, provided that the oral intake of nitrate is restricted for at least 48 h. Due to the large distribution volume and the low clearance of the ion wide-spread, marked, and chronic changes in NO formation are required to significantly affect the levels of nitrate in samples of mixed blood.

Pharmacodynamics of plasma nitrate/nitrite as an indication of nitric oxide formation in conscious dogs.

The present investigation was undertaken to better understand the production of nitric oxide (NO) in vivo as measured by alterations in plasma nitrite or nitrate in blood samples from studies in experimental animals or clinical studies in humans. Plasma samples were taken from the aorta, the coronary sinus, a peripheral vein in the leg (skeletal muscle), or the right ventricle (mixed venous) in chronically instrumented conscious dogs. Plasma nitrite was converted to NO gas in an argon environment by use of hydrochloric acid, and plasma nitrate was converted first to nitrite with nitrate reductase and then to NO gas with acid. Standard curves were constructed, and the amount of nitrite and nitrate in plasma was determined. The primary metabolite was nitrate, whereas nitrate was approximately 10% of the total and remained constant. In the resting dog, the only vascular bed with a positive arterial-venous nitrate difference, evidence for production of NO, was the heart. Nitrate infusion into quietly resting dogs resulted in increases in plasma nitrate up to 38 +/- 3.4 mmol/L, increases in systemic arterial pressure, and a marked diuresis. The plasma half-life was calculated as 3.8 hours. The volume of distribution was calculated as 0.215 L/kg, or equivalent to the extracellular volume. These studies indicate that nitrate is a reliable measure of NO metabolism in vivo but that because of the long half-life, nitrate will accumulate in plasma once it is produced. Because of the large volume of distribution (21% of body weight versus the 4% of body weight usually attributed to plasma volume, the compartment in which nitrate is measured), simple measures of plasma nitrate underestimate by a factor of 4 to 6 the actual production of nitrate or NO by the body. In disease states, such as heart failure, in which renal function and extracellular volume are altered, caution should be exercised when increases in nitrate in plasma as an index of NO formation are evaluated.

Liposomal delivery of antioxidant enzymes protects against hydrogen peroxide- but not interleukin-1β-induced inhibition of glucose metabolism in rat pancreatic islets

The aim of the present investigation was to evaluate the putative involvement of oxygen free radicals in interleukin-1 beta (IL-1 beta)-induced suppression of islet glucose oxidation. Isolated adult rat pancreatic islets were exposed for 1 h to liposomally encapsulated superoxide dismutase (SOD; 10 mg/ml), catalase (CAT; 10 mg/ml) and glutathione peroxidase (GPX; 5 mg/ml), after which IL-1 beta (25 U/ml) or hydrogen peroxide (H2O2; 0.1 mM) was added, and the incubation was continued overnight. The following day, samples were taken from the incubation media for nitrite determinations, and islet glucose oxidation rates were measured. The CAT activity increased fourfold after addition of CAT-containing liposomes. It was found that IL-1 beta induced a marked increase in islet nitrite production, as an index of nitric oxide formation, and that this was paralleled by a decrease in islet glucose oxidation rates. H2O2-treated islets exhibited a modest decrease in glucose oxidation rates and a minor increase in the release of nitrite to the media. Treatment of islets with liposomes containing the antioxidant enzymes SOD, CAT and GPX, either alone or in combination, did not decrease the effect of IL-1 beta. However, the H2O2-induced decrease in glucose oxidation rates was counteracted by the combination of the antioxidants. It was concluded that, provided the intracellular delivery of the antioxidant enzymes to the islet cells was effective, oxygen free radicals probably do not play a decisive role in IL-1 beta suppression of islet glucose metabolism.

Evidence for nitric oxide-generator cells in the brain

Nitric oxide (NO) is a free radical that has been recently recognized as a neuronal messenger molecule. In order to understand the way in which NO functions in the central nervous system (CNS), it is important to identify the NO-generator and NO-target cells in the brain. I measured firstly the distribution of NO synthase in the brain, which catalyzes L-arginine to form NO, by the measurement of citrulline formation that is also synthesized from L-arginine together with NO in equal molar bass. In the brain of adult rat, the most potent activity of NOS was apparent in the cerebellum, next in the olfactory bulb and medium in the cerebrum. Further, in the presence of NADPH and Ca2+, NOS activity was detected in the neuron cultures derived from the cerebrum of fetal rat. Astrocytes, one type of glia, prepared also from the cerebrum of fetal rat, appeared to have a small but significant NOS activity. As astrocytes possess a high amount of cytosolic guanylate cyclase that is known to be activated by NO, the changes in the intracellular cGMP levels in the astrocytes were measured as another index of NO formation. The treatment of astrocytes with NOS inhibitor caused the suppression of the intracellular cGMP levels. These results indicate that NO is definitely produced by astrocytes. In addition, in the blood vessel system of the brain, although NOS has been thought to be localized in the endothelial cells of only larger vessels, NOS activity was also observed in the microvessel endothelial cells of the cerebrum of both adult and fetal pig. These data suggest that neuronal cells may be the major site of NO generation in the brain, and that the NOS is a constitutive type. The data also suggest that astrocytes can also express constitutive NOS, although the potency is not so large. Microvessel endothelial cells of the brain are also one of the sources of NO. The NO produced by these cells increases the cGMP levels in the astrocytes and may affect some physiological and/or pathophysiological events in the brain.

Anti‐inflammatory agents and substance P depletion in experimental ileitis

To understand the interactions between substance P and gut inflammation, changes in substance P levels were evaluated in a chronic model of ileitis in response to three anti-inflammatory agents with distinct mechanisms of action. The agents were the prostaglandin E1 analogue misoprostol (30 μg/kg, s.c., b.i.d.), the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 μg/ml in drinking water) and the leumedin, N-(fluorenyl-9-methoxycarbonyl)-L-leucine (NPC 15199, 10 mg/kg, s.c.). Ileitis was induced by a transmural injection of trinitrobenzene sulphonic acid (TNBS 30 mg/kg in 50% ethanol) into the distal ileum of guinea-pigs. All anti-inflammatory therapies were introduced after TNBS administration and continued until day 7, when guinea-pigs were killed. Ileal substance P levels were measured by radioimmunoassay, and granulocyte infiltration was quantified by myeloperoxidase (MPO) activity. Protein and nitrite (an index of nitric oxide formation) levels in a luminal saline lavage were quantified in all groups. TNBS ileitis caused a marked reduction in ileal substance P content and increased MPO activity, protein and nitrite secretion. The nitric oxide synthase inhibitor, L-NAME, completely restored all parameters to baseline. Misoprostol attenuated the granulocyte infiltration and exacerbated protein leak but had no effect on substance P levels. In contrast, NPC 15199 had no effect on granulocyte infiltration but normalized substance P levels and protein leak. Only L-NAME and NPC 15199 blocked the TNBS induced increase in nitrite levels. These results suggest that the regulation of granulocyte infiltration in this model is unrelated to changes in substance P levels. Inhibition of nitric oxide synthase was the most effective therapeutic strategy in TNBS ileitis but the precise interactions between nitric oxide and the enteric nervous system during inflammatory states remain to be defined.

Role of nitric oxide in the oxidant stress during ischemia/reperfusion injury of the liver

The potential role of nitric oxide (NO) and its reaction product with superoxide, peroxynitrite, was investigated in a model of hepatic ischemiareperfusion injury in male Fischer rats in vivo. Pretreatment with the NO synthase inhibitor nitro-L-arginine (10 mg/kg) did neither affect the postischemic oxidant stress and liver injury during the initial reperfusion phase nor the subsequent infiltration of neutrophils into the liver and the later, neutrophil-induced injury phase. Furthermore, no evidence was found for a postischemic increase of the urinary excretion of nitrite, a stable oxidation metabolite of NO. In contrast, the administration of Salmonella enteritidis endotoxin (1 mg/kg) induced a significant diuresis in Fischer rats and an 800-fold enhancement of the urinary nitrite excretion. Nitro-L-arginine pretreatment inhibited the endotoxin-induced nitrite formation by 97%. Hepatic cGMP levels, as index of NO formation in the liver, were only increased significantly after endotoxin administration but not after eschemia and reperfusion. Our results provide no evidence for any enhanced generation of NO or peroxynitrite either systematically or locally during reperfusion and therefore it is unlikely that any of these metabolites are involved in the oxidant stress and liver injury during reperfusion after hepatic ischemia.