Total Nitric Oxide Production Research Articles

THE INFLUENCE OF QUERCETIN ON THE PRODUCTION OF REACTIVE OXYGEN-NITROGEN FORMS IN THE STRUCTURE OF THE INTERSTITIAL SPACE OF RAT TESTICLES WITH LONG-TERM EFFECT OF TRIPTORELIN

Quercetin is a flavonoid commonly found in fruits, vegetables, and seeds. Flavonoids can help prevent cardiovascular diseases, reduce the risk of degenerative brain processes and cancer. Studies have shown that blocking the synthesis of luteinising hormone can lead to oxidative stress in the liver, heart, and salivary glands of rats. The purpose of this study was to assess alterations in the interstitial space of rat testes, identify sources of nitric oxide production, and measure the intensity of oxidative stress in the testes during long-term experimental central blocking of luteinising hormone synthesis by tryptorelin. The study was conducted on 15 sexually mature male white rats. The animals were divided into two groups. The first group (control) received a subcutaneous injection of 0.9% sodium chloride solution. The second group (experimental) received a subcutaneous injection of tryptorelin at a dose of 0.3 mg/kg of active ingredient for 365 days, with simultaneous addition of quercetin to the diet, which was administered orally through a gastric tube three times a day. Total nitric oxide production was assessed by measuring the activity of total NO synthase (gNOS). The activity of gNOS was determined by the increase in nitrite (NO2-) after incubation in Tris-buffered saline. The nitrite concentration was determined using the Gris-Ilosvay reagent at a wavelength of 540 nm. The activity of the inducible (iNOS) and constitutive (cNOS) isoforms was also determined using a selective iNOS inhibitor, aminoguanidine hydrochloride. The production of superoxide anion radical (O2--) was measured by the increase in the formation of diformazan resulting from the reaction of O2-- with nitroblue tetrazolium after incubation in a buffered solution containing sodium hydroxide. The administration of quercetin mitigates the adverse effects of tryptorelin on the structural and functional components of the connective tissue in the rat testes. This effect is observed on day 365 and is characterised by fibrosis and impaired nitric oxide production by constitutive isoforms of NO synthase.

Quantitative evaluation of NO formation and destruction routes during methane MILD combustion using an improved calculation method

Moderate or intense low-oxygen dilution (MILD) combustion has become increasingly attractive because of its ultra-low nitric oxide (NO) emission. Although many efforts have been made to investigate the NO formation and destruction pathways of MILD combustion, their relative contribution still fails to reach an agreement when using previously-developed NO calculation methods. For this reason, these NO calculation methods are first evaluated for methane MILD combustion by the kinetic modeling of a well-stirred reactor (WSR). Then, an improved calculation method for quantifying the contribution of NO sub-routes is proposed, which involves thermal, prompt, N2O-intermediate, NNH, selective non-catalytic reduction of NO, and NO-reburning routes. Also, this improved method allows the application of a more comprehensive mechanism (Progress in Energy and Combustion Science, 2018, 67: 31–68) other than GRI-Mech 2.11. Using the improved method with the more comprehensive mechanism, the NO formation and destruction routes during methane MILD combustion are quantitatively investigated. The results show that in the WSR at 1300 K with 5% O2, the contribution of the N2O-intermediate route accounts for about 80% at an equivalent ratio (Φ) of 0.8, and it gradually decreases as Φ increases. Once Φ is >1.0, the prompt route surpasses the N2O-intermediate route to dominate the NO generation entirely. The NNH mechanism is found to be of little importance at Φ ranging from 0.5 to 1.5. Additionally, the NO-reburning reaction reduces <10% of the total NO production when Φ < 1.0, and its contribution increases abruptly at 1.0 < Φ < 1.1.

Quantitative aspects of nitric oxide production from nitrate and nitrite.

Nitric oxide (NO) is involved in many physiological and pathological processes in the human body. At least two major pathways produce NO: (1) the L-arginine-NO-oxidative pathway in which NO synthase (NOS) enzymes convert L-arginine to NO; (2) the nitrate-nitrite-NO reductive pathway in which NO is produced from the serial reduction of nitrate and nitrite. The deficiency of NO is involved in the pathophysiology of cardiometabolic disorders. Intervention with foods containing nitrate and nitrite can potentially prevent or treat some chronic diseases, including cardiovascular diseases and diabetes. A better understanding of the NO cycle would help develop effective strategies for preventing or treating the disorders in which NO homeostasis is disturbed. This review summarizes quantitative aspects of NO production, emphasizing the nitrate-nitrite-NO pathway. Available data indicates that total NO production by NOS-dependent L-arginine-NO pathway is about 1000 μmol.day-1. Of about 1700 μmol.day-1 ingested nitrate, ~25 % is extracted by the salivary glands and of which ~20 % is converted nitrite. It means that about 5 % of ingested nitrate is converted to nitrite in the oral cavity; assuming that all produced nitrite is reduced to NO in the stomach, it can be calculated that contribution of the nitrate-nitrite-NO pathway to the whole-body NO production is about 85 μmol.day-1 (1700 ×0.05=85) or approximately 100 μmol.day-1. The lower contribution of the nitrate-nitrite-NO pathway does not mean that this pathway has lower importance in the whole-body NO homeostasis. Even in the adequate L-arginine supply, NOS-dependent NO production is insufficient to meet all NO functions, and the nitrate-nitrite-NO pathway must provide the rest. In conclusion, the contribution of the nitrate-nitrite-NO pathway in the whole human body NO production is <10 %, and the nitrate-nitrite-NO pathway is complementary to the NOS-dependent NO production.

Lack of nitric oxide bioavailability in early pregnancy predisposes to dyslipidemia and surges preeclampsia and fetal growth retardation

Background: Hyperlipidemia has been reported in preeclampsia (PrE) and is linked to poor pregnancy outcome and long-term cardiovascular complications. This study aimed to elucidate the relationship between nitric oxide (NO) and blood lipids levels during normal pregnancy and in NG-nitro-l-arginine methyl ester (L-NAME) - induced preeclampsia before and after magnesium sulphate (MgSO4) therapy and its effect on the pregnancy outcome.Methods: Forty female Wistar rats were divided into four groups: non pregnant (NP) group - non pregnant healthy rats receiving no treatment, control pregnant (Con-P) group - control pregnant rats receiving no treatment, pregnant (PE) group - pregnant animals with untreated PrE, and the pregnant MgSO4 (PE-Mg) group - pregnant animals with PrE- treated with MgSO4. The nitric oxide synthase inhibitor L-NAME was used to induce experimental model of PrE in the PE and the PE-Mg groups. The changes in total NO production, total cholesterol (TC), triglycerides (TG), low density lipoproteins (LDL-C), high density lipoproteins (HDL), LDL-C/HDL-C ratio, soluble vascular endothelial growth factor receptor-1 (sVEGFR1) also known as sFlt-1, blood pressure, kidney functions, body weight, and pregnancy outcome were assessed.Results: Decreased NO production in the PE group was associated with elevated TC, TG, LDL-C/HDL-C ratio, hypertension, proteinuria, increased urea, creatinine, and sFlt-1 levels, and poor pregnancy outcome demonstrated by high pup mortality rate and low birth weight. Increased NO production in the PE-Mg group treated with MgSO4 therapy was associated with decreased signs of preeclampsia and hypolipidemia and increased pup viability and birth weight.Conclusions: NO bioavailability is crucial for the homeostasis of the lipid profile in normal pregnancy and the prevention of preeclampsia. Routine periodic assessments of the blood lipid profile and the NO production in the pregnant females may be a helpful tool in early prediction of preeclampsia.

The effect of Cinnamomum cassia extract on oxidative stress in the liver and kidney of STZ-induced diabetic rats.

Many diabetes-related complications are caused by oxidative stress. In the current study, the protective effect of Cinnamomum cassia against diabetes-induced liver and kidney oxidative stress was evaluated. The male Wistar rats (n=48) were randomly divided into six groups including; control group received 500µL normal saline orally for 42days. Diabetes groups received intraperitoneally (i.p.) streptozotocin (STZ) as single-dose (60mg/kg, i.p.). Cinnamon extract (100, 200, 400mg/kg) and metformin (300mg/kg) were orally administered to diabetic rats for 42days. After the experiment period, the animals were anesthetized and the liver and kidney tissues were quickly removed and restored for oxidative stress evaluation. The levels of malondialdehyde (MDA), total thiol content, glutathione (GSH), nitric oxide (NO) metabolites, as well as, superoxide dismutase (SOD) and catalase (CAT) activities were measured in kidney and liver tissue. The level of MDA, SOD, and CAT activities increased significantly, while the total thiol content, and NO production were significantly reduced in diabetic animals compared to the control group (from p<0.05 to p<0.001). Treatment with cinnamon extract significantly decreased the MDA level, as well as, SOD and CAT activities in the liver and kidney of diabetic rats (from p<0.05 to p<0.001). In the liver and kidney of cinnamon treated groups, GSH and total thiol contents and NO production were significantly higher than diabetic group (from p<0.05 to p<0.001). Cinnamon extract due to its potent antioxidant property could be effective in decrease of diabetes-induced oxidative stress that plays a major role in renal and hepatic complications.

Poloxamer 188 Exerts Direct Protective Effects on Mouse Brain Microvascular Endothelial Cells in an In Vitro Traumatic Brain Injury Model.

Traumatic Brain Injury (TBI), the main contributor to morbidity and mortality worldwide, can disrupt the cell membrane integrity of the vascular endothelial system, endangering blood–brain barrier function and threatening cellular subsistence. Protection of the vascular endothelial system might enhance clinical outcomes after TBI. Poloxamer 188 (P188) has been shown to improve neuronal function after ischemia/reperfusion (I/R) injury as well as after TBI. We aimed to establish an in vitro compression-type TBI model, comparing mild-to-moderate and severe injury, to observe the direct effects of P188 on Mouse Brain Microvascular Endothelial Cells (MBEC). Confluent MBEC were exposed to normoxic or hypoxic conditions for either 5 or 15 h (hours). 1 h compression was added, and P188 was administered during 2 h reoxygenation. A direct effect of P188 on MBEC was tested by assessing cell number/viability, cytotoxicity/membrane damage, metabolic activity, and total nitric oxide production (tNOp). While P188 enhanced cell number/viability, metabolic activity, and tNOp, an increase in cytotoxicity/membrane damage after mild-to-moderate injury was prevented. In severely injured MBEC, P188 improved metabolic activity only. P188, present during reoxygenation, influenced MBEC function directly in simulated I/R and compression-type TBI.

Markers of oxidative-nitrosative stress induced by artesunate are followed by clastogenic and aneugenic effects and apoptosis in human lymphocytes.

Artesunate (ARS) is a semi-synthetic derivative of artemisinin, used as an outstanding antimalarial drug, which also displays antitumor, anti-inflammatory and immunosuppressive effects. In spite of the numerous reports showing the antitumor activity of ARS, the particular mechanisms associated with its cytotoxicity and genotoxicity in non-neoplastic human cells remain unclear. Here we aimed to verify the specific chromosome damages and the changes in markers of oxidative-nitrosative stress and apoptosis triggered by ARS exposure in human peripheral blood lymphocytes. Cultures were incubated in the presence of ARS and the number of binucleated cells was determined. To discriminate between micronuclei (MN) containing a whole chromosome or an acentric chromosome, the MN test was employed in combination with the fluorescence in situ hybridization assay. Alterations in the levels of superoxide anion (O2- ) and nitric oxide (NO) were measured by the nitroblue tetrazolium and Griess assay, respectively. Changes in the expression of the apoptotic markers were assessed by immunocytochemistry. We found that ARS induced a significant formation of both centromere-positive MN (C+ MN) and centromere-negative MN (C- MN). These alterations were accompanied by an increase in both cellular levels of O2- and total NO production, and a remarkable enhancement in the expression of the apoptotic markers cytochrome c and caspases 8 and 9. Together these findings reveal that ARS induces changes in the oxidative-nitrosative status of human lymphocytes, which are followed by apoptosis and clastogenic and aneugenic effects.

Role of Nox4 in Angiotensin II‐Mediated Changes in Volume Dynamics and Nitric Oxide Production in Podocytes

Nitric oxide (NO) is a powerful vasodilator that serves important functions in the kidney such as regulation of renal hemodynamics, pressure natriuresis, and diuresis. Significant reduction in NO production has been found to play a role in the pathogenesis of hypertension. Our recent studies revealed that Angiotensin II (Ang II), in addition to causing a release of calcium, stimulates NO production in the glomerular podocytes. This signaling pathway can be also triggered by extracellular application of hydrogen peroxide (H2O2) and is significantly reduced with the development of salt‐sensitive hypertension and kidney injury in Dahl salt‐sensitive (SS) rats (Palygin et al., Nitric Oxide, 2018). Recently, using a knock out of NADPH oxidase 4 (Nox4) in SS rats (SSNox4−/−), one of the primary source of renal reactive oxygen species (ROS) and specifically H2O2 we described the reduction in intracellular calcium activation in podocytes as well as H2O2 production in isolated glomeruli in response to Ang II (Ilatovskaya et al., J Am Soc Nephrol. 2018). Overall, SSNox4−/− rats displayed a protective phenotype with a significant reduction of albuminuria, glomeruli injury and reduction in the elevation of blood pressure under high salt challenge (Cowley et al., Hypertension, 2016). Here we used this model to characterize the effects of alterations in the balance in the relationship between ROS and NO production in podocytes and establish the relation between Nox4 and glomerular/podocyte volume dynamics. The in vivo concentration of H2O2, measured by biosensors amperometry as catalase sensitive current, was significantly reduced in both cortex and medullar parts of the SSNox4−/− rat kidney compared to wild type SS rats, indicating a direct association between Nox4 and the production of renal ROS. We found that acute application of Ang II promoted opposite changes in glomeruli and podocyte volumes, where glomeruli volume decreased, and podocyte volume increased approximately by 18% in SS rats. Overall, SSNox4−/− rats exhibited an increase in total NO production (by 16% to compare with SS), calculated using confocal microscopy as a total integral of NO fluorescent transient (DAF‐FM dye) in the population of podocytes within a freshly isolated glomerulus. In SSNox4−/− rats the elevation in NO together with declining in H2O2 levels directly correlated with an increase of podocyte volume in response to Ang II (13% vs. SS control). We anticipate a rapid podocyte volume change dependent on intracellular NO production and reflecting the ability of these cells to dynamically regulate the permeability of glomeruli filtration barrier. Thus far, we can conclude that in the absence of Nox4 the podocytes shift the ROS production in response to Ang II towards the increase of intracellular NO bioavailability and a decrease of renal H2O2 production. This mechanism may play a key role in the protection of glomeruli form oxidative stress and elevation of blood pressure during the development of salt‐sensitive hypertension.Support or Funding InformationNHLBI R35 HL135749; P01 HL116264; AHA 18PRE34030127; ADA 1‐15‐BS‐172; DK‐020595 Pilot &amp; Feasibility projectThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Arginase Activity in Red Blood Cells Is Not Altered with Sub-maximal Exercise.

Nitric oxide (NO) is an important mediator of exercise remodeling. Its role has been highlighted in the promotion of vascular growth as well as in mitochondrial function. While the endothelium has long been thought to be the dominant physiological source of NO, recent work has suggested that red blood cells (RBCs) express the enzyme endothelial nitric oxide synthase (eNOS) and may contribute to total NO production. Within RBCs, the NO production can be decreased by the activity of the enzyme arginase, which competes with eNOS for the substrate L-arginine. PURPOSE: The aim of the study was to test the hypothesis that RBC arginase activity is modulated by exercise. METHODS: Nine subjects (4 male and 5 females, age; 26±5 years, VO2peak; 54,8±8,1 mL/min/kg) performed a 60-min sub-maximal exercise bout corresponding to 65% of VO2peak. Blood samples were taken at rest (T0), 30 min in to the exercise (T1), directly after exercise (T2), 30 min post-exercise (T3) and 60 min post-exercise (T4). Arginase activity in RBCs was measured by colorimetric determination of urea formed from L-arginine substrate with α-isonitrosopropiophenone as a marker of urea content. RESULTS: Contradictory to our initial hypothesis, arginase activity was unaffected by exercise. No changes in urea production (T0; 0.52±0.08, T1; 0.50±0.08, T2; 0.51±0.09, T3; 0.51±0.09 and T4; 0.52±0.08 mM/g Hb, p>0.05) was seen between the resting blood sample and the ones taken during or after exercise. CONCLUSION: Arginase activity in RBCs is not modulated by sub-maximal exercise in young healthy subjects. Hence, the increase in eNOS activity and NO production from RBCs with exercise is most likely not explained by a reduced activity of arginase.

The asymmetric dimethylarginine-mediated inhibition of nitric oxide in the rostral ventrolateral medulla contributes to regulation of blood pressure in hypertensive rats

Nitric oxide (NO) contributes to the central control of cardiovascular activity. The rostral ventrolateral medulla (RVLM) has been recognized as a pivotal region for maintaining basal blood pressure (BP) and sympathetic tone. It is reported that asymmetric dimethylarginine (ADMA), characterized as a cardiovascular risk marker, is an endogenous inhibitor of nitric oxide synthesis. The present was designed to determine the role of ADMA in the RVLM in the central control of BP in hypertensive rats. In Sprague Dawley (SD) rats, microinjection of ADMA into the RVLM dose-dependently increased BP, heart rate (HR), and renal sympathetic never activity (RSNA), but also reduced total NO production in the RVLM. In central angiotensin II (Ang II)-induced hypertensive rats and spontaneously hypertensive rat (SHR), the level of ADMA in the RVLM was increased and total NO production was decreased significantly, compared with SD rats treated vehicle infusion and WKY rats, respectively. These hypertensive rats also showed an increased protein level of protein arginine methyltransferases1 (PRMT1, which generates ADMA) and a decreased expression level of dimethylarginine dimethylaminohydrolases 1 (DDAH1, which degrades ADMA) in the RVLM. Furthermore, increased AMDA content and PRMT1 expression, and decreased levels of total NO production and DDAH1 expression in the RVLM in SHR were blunted by intracisternal infusion of the angiotensin II type 1 receptor (AT1R) blocker losartan. The current data indicate that the ADMA-mediated NO inhibition in the RVLM plays a critical role in involving in the central regulation of BP in hypertension, which may be associated with increased Ang II.

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages.

Excess nitric oxide (NO) production occurs in several pathological states, including neurodegeneration, ischemia, and inflammation, and is generally accompanied by increased oxidative/nitrosative stress. Carnosine [β-alanine-histidine (β-Ala-His)] has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of carnosine on NO production by murine RAW 264.7 macrophages stimulated with lipopolysaccharides + interferon-γ. Intracellular NO and intracellular and extracellular nitrite were measured by microchip electrophoresis with laser-induced fluorescence and by the Griess assay, respectively. Results showed that carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity endproduct, nitrite, with no inhibition of inducible nitric oxide synthase (iNOS). ESI-MS and NMR spectroscopy in a cell-free system showed the formation of multiple adducts (at different ratios) of carnosine-NO and carnosine-nitrite, involving both constituent amino acids (β-Ala and His) of carnosine, thus providing a possible mechanism for the changes in free NO and nitrite in the presence of carnosine. In stimulated macrophages, the addition of carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of IL-6, suggesting that carnosine might alter M1/M2 macrophage ratio. These results provide evidence for previously unknown properties of carnosine that modulate the NO/nitrite ratio of stimulated macrophages. This modulation is also accompanied by changes in the release of pro-inflammatory molecules, and does not involve the inhibition of iNOS activity.

Transnitrosylation: A Factor in Nitric Oxide-Mediated Penile Erection.

Nitric oxide (NO) signaling can be mediated not only through classic 3',5'-cyclic guanosine monophosphate but also through S-nitrosylation. However, the impact of S-nitrosylation on erectile function and in NO regulation and oxidative stress in the penis remains poorly understood. To characterize the role of S-nitrosoglutathione reductase (GSNOR), a major regulator of S-nitrosylation homeostasis, on erection physiology and on endothelial NO synthase (eNOS) function and oxidative-nitrosative stress in the penis. Adult GSNOR-deficient and wild-type (WT) mice were used. Erectile function was assessed in response to electrical stimulation of the cavernous nerve. Total NO in penile homogenates was measured by Griess reaction. Protein S-nitrosylation, eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS uncoupling, and markers of oxidative stress (4-hydroxy-2-nonenal, malondialdehyde, and nitrotyrosine) in the penis were measured by western blot. Erectile function, eNOS function, and oxidative stress in the penis of GSNOR-deficient mice. Erectile function was intact in GSNOR-deficient mice. Total S-nitrosylated proteins were increased (P< .05) in the GSNOR(-/-) compared with WT mouse penis. Although eNOS phosphorylation on Ser-1177 did not differ between the GSNOR(-/-) and WT mouse penises at baseline, electrical stimulation of the cavernous nerve increased (P < .05) phosphorylated eNOS in the WT mouse penis but failed to increase phosphorylated eNOS in the GSNOR(-/-) mouse penis. Total NO production was decreased (P < .05), whereas eNOS uncoupling, 4-hydroxy-2-nonenal, malondialdehyde, and nitrotyrosine were increased (P < .05) in the GSNOR-deficient mouse penis compared with the WT mouse penis. Transnitrosylation mechanisms play an important role in regulating NO bioactivity in the penis. Deficiency of GSNOR leads to eNOS dysfunction and increased oxidative damage, suggesting that homeostatic eNOS function in the penis is governed by transnitrosylation.

Characterization of the sex-dependent myocardialS-nitrosothiol proteome

Premenopausal women exhibit endogenous cardioprotective signaling mechanisms that are thought to result from the beneficial effects of estrogen, which we have shown to increase protein S-nitrosylation in the heart. S-nitrosylation is a labile protein modification that increases with a number of different forms of cardioprotection, including ischemic preconditioning. Herein, we sought to identify a potential role for protein S-nitrosylation in sex-dependent cardioprotection. We utilized a Langendorff-perfused mouse heart model of ischemia-reperfusion injury with male and female hearts, and S-nitrosylation-resin-assisted capture with liquid chromatography tandem mass spectrometry to identify S-nitrosylated proteins and modification sites. Consistent with previous studies, female hearts exhibited resilience to injury with a significant increase in functional recovery compared with male hearts. In a separate set of hearts, we identified a total of 177 S-nitrosylated proteins in female hearts at baseline compared with 109 S-nitrosylated proteins in male hearts. Unique S-nitrosylated proteins in the female group included the F1FO-ATPase and cyclophilin D. We also utilized label-free peptide analysis to quantify levels of common S-nitrosylated identifications and noted that the S-nitrosylation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a was nearly 70% lower in male hearts compared with female, with no difference in expression. Furthermore, we found a significant increase in endothelial nitric oxide synthase expression, phosphorylation, and total nitric oxide production in female hearts compared with males, likely accounting for the enhanced S-nitrosylation protein levels in female hearts. In conclusion, we identified a number of novel S-nitrosylated proteins in female hearts that are likely to contribute to sex-dependent cardioprotection.

Isoflurane favorably modulates guanosine triphosphate cyclohydrolase-1 and endothelial nitric oxide synthase during myocardial ischemia and reperfusion injury in rats.

The authors investigated the hypothesis that isoflurane modulates nitric oxide (NO) synthesis and protection against myocardial infarction through time-dependent changes in expression of key NO regulatory proteins, guanosine triphosphate cyclohydrolase (GTPCH)-1, the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin and endothelial nitric oxide synthase (eNOS). Myocardial infarct size, NO production (ozone-mediated chemiluminescence), GTPCH-1, and eNOS expression (real-time reverse transcriptase polymerase chain reaction and western blotting) were measured in male Wistar rats with or without anesthetic preconditioning (APC; 1.0 minimum alveolar concentration isoflurane for 30 min) and in the presence or absence of an inhibitor of GTPCH-1, 2,4-diamino-6-hydroxypyrimidine. NO2 production (158 ± 16 and 150 ± 13 pmol/mg protein at baseline in control and APC groups, respectively) was significantly (P < 0.05) increased 1.5 ± 0.1 and 1.4 ± 0.1 fold by APC (n = 4) at 60 and 90 min of reperfusion, respectively, concomitantly, with increased expression of GTPCH-1 (1.3 ± 0.3 fold; n = 5) and eNOS (1.3 ± 0.2 fold; n = 5). In contrast, total NO (NO2 and NO3) was decreased after reperfusion in control experiments. Myocardial infarct size was decreased (43 ± 2% of the area at risk for infarction; n = 6) by APC compared with control experiments (57 ± 1%; n = 6). 2, 4-Diamino-6-hydroxypyrimidine decreased total NO production at baseline (221 ± 25 and 175 ± 31 pmol/mg protein at baseline in control and APC groups, respectively), abolished isoflurane-induced increases in NO at reperfusion, and prevented reductions of myocardial infarct size by APC (60 ± 2%; n = 6). APC favorably modulated a NO biosynthetic pathway by up-regulating GTPCH-1 and eNOS, and this action contributed to protection of myocardium against ischemia and reperfusion injury.

Enhancement of Inducible Nitric Oxide Synthase Activity by Low Molecular Weight Peptides Derived from Protamine: A Potential Therapy for Chronic Rhinosinusitis

Nitric oxide (NO) is a key immune defense agent that is produced from l-arginine in the airways by leukocytes and airway epithelial cells, primarily via inducible nitric oxide synthase (iNOS). Deficiencies in nasal NO levels have been associated with diseases such as primary ciliary dyskinesia and chronic rhinosinusitis. Herein, we demonstrate a proof-of-concept regarding a potential new therapeutic approach for such disorders. We show that arginine-rich low molecular weight peptides (LMWPs) derived from the FDA-approved protamine (obtained from salmon sperm) are effective at significantly raising NO production in both RAW 264.7 mouse macrophage and LA4 mouse epithelial cell lines. LMWP is produced using a stable, easily produced immobilized thermolysin gel column followed by size-exclusion purification. Monomeric l-arginine induces concentration-dependent increases in NO production in stimulated RAW 264.7 and LA4 cells, as measured by stable nitrite in the cell media. In stimulated RAW 264.7 cells, LMWP significantly increases iNOS expression and total NO production 12-24 h post-treatment compared to cells given equivalent levels of monomeric l-arginine. For stimulated LA4 cells, LMWPs are effective in significantly increasing NO production compared to equivalent l-arginine monomer concentrations over 24 h but do not substantially enhance iNOS expression. The use of the arginase inhibitor S-boronoethyl-l-cysteine in combination with LMWPs results in even higher NO production by stimulated RAW 264.7 cells and LA4 cells. Increases in NO due to LMWPs, compared to l-arginine, occur only after 4 h, which may be due to iNOS elevation rather than increased substrate availability.

Influence of cyclophilin D protein expression level on endothelial cell oxidative damage resistance.

We examined the influence of cyclophilin-D (CypD) protein expression level on endothelial cell oxidative damage resistance. A model of CypD protein expression or high expression in endothelial cells was established through gene silencing or cloning. The comparable groups were normal endothelial cells cultured in phosphate-buffered solution in liquid handling cells containing 500 mM H2O2 for 90 or 120 min, and then the medium was replaced with common nutrient solution and cultured again for 24 h. The apoptosis rate and nitric oxide (NO) levels of each group were tested. The cell apoptosis rate of the CyPD low expression group (32.51 ± 6.6 %) was significantly lower than that of the control group (52.57 ± 5.84%, P = 0.001), and total NO production was 24.06 ± 3 and 13.03 ± 3.55 μM. The apoptosis rate of the CyPD high expression group (24.24 + 3.08%) was significantly higher than that of the control group (7.7 + 0.68%, P < 0.001); total NO production was 3.55 ± 1.53 and 8.46 ± 0.77 μM, which was significantly different (P = 0.008). CypD protein could increase oxidative stress and cause endothelial cell injury and apoptosis.

Pre-exposure to mechanical ventilation and endotoxin influence bacterial growth and immune response during experimental ventilator-associated pneumonia

Overproduction of nitric oxide (NO) is correlated with adverse outcomes in sepsis. NO is additionally a central part of the innate immune system defense against pathogens causing ventilator-associated pneumonia (VAP), which can complicate the clinical course during mechanical ventilation (MV). We hypothesized that pre-exposure to MV and systemic inflammation from endotoxin each would influence bacterial growth in lung tissue, based on an altered immune response in experimental pneumonia. We used a porcine Pseudomonas aeruginosa VAP model with ventilatory and inflammatory pre-exposures before inoculation to evaluate bacterial growth, development of lung damage, total NO production and inflammatory cytokine response.

To resist or to endure: exercise mode matters in arterial structure and function

To resist or to endure: exercise mode matters in arterial structure and function

A human study model for nitric oxide research in sinonasal disease

Sinus nitric oxide (NO) measurements present a novel and promising approach to help overcome difficulties and confounding variables associated with nasal NO measurements such as the nasal cycle, ostial patency, and individual contribution to total NO production of each sinus. Conflicting results reported on nasal NO measurements in various sinonasal diseases are presumed to originate from the variable diffusion of sinus NO into the nose where it is measured. This study presents a novel technique and research method for direct measurement of sinus NO. The authors' original technique of individual, non-destructive catheterization of the sinuses through their natural ostia is developed and refined to allow accurate measurements of NO produced in the sinuses. Our study indicates that reproducible catheterization of the sinuses through their natural ostia can be performed in the clinical research setting under local and topical anesthesia. The model can be used to test the effects of various conditions on nasal and sinus NO production in a variety of disease models and the variables affecting sinonasal gas exchange can be differentially studied. Volunteer healthy adult human subjects without nasal allergies are used. An endoscopic nasal exam with topical anesthesia followed by in vitro allergy testing is performed to determine eligibility. Sinus computerized tomography (CT) scans are used to delineate anatomic features and to calculate paranasal sinus volumes. Continuous flow sinus air sampling and NO measurement with a chemiluminescence analyzer is obtained through polyethylene tube catheters (PEC) placed endoscopically into an aerated major paranasal sinus. Catheters are introduced through natural ostia under local and topical anesthesia. Nasal and differential sinus NO measurements are performed.

Antioxidant and anti-inflammatory activities of different solvent fractions from ethanol extract of Synurus deltoides (Aiton) Nakai leaves

Fractions of Synurus deltoides (Aiton) Nakai leaves showed excellent antioxidant and anti-inflammatory effects in a series of antioxidant and anti-inflammatory assays including total phenolic and flavonoid contents determination assay, DPPH free radical scavenging assay, reducing power assay, superoxide radical scavenging assay, nitrite scavenging assay, total antioxidant assay, DNA damage protection assay, MTT assay, nitric oxide (NO) production inhibition assay, and reverse transcription (RT)-PCR assay. The ethyl acetate fraction exhibited stronger antioxidant effects than other fractions in most of antioxidant assays such as DPPH assay, reducing power assay, superoxide radical scavenging assay, and nitrite scavenging assay. However, the dichloromethane fraction showed the strongest total antioxidant activity and NO production inhibitory activity. It was demonstrated that S. deltoides was valuable to be researched farther and application and fractions of S. deltoides had great potential to be a source of edible antioxidant and anti-inflammatory agents.