Measurement Of Nitric Oxide Production Research Articles

Laser-Based Methods for Detection of Nitric Oxide in Plants.

Nitric oxide (NO) plays an important role in plant signaling and in response to various stress conditions. Therefore, real-time measurements of NO production provide better insights into understanding plant processes and can help developing strategies to improve food production and postharvest quality. Using laser-based spectroscopic methods, sensitive, online, in planta measurements of plant-pathogen interactions are possible. This chapter introduces the basic principle of the optical detectors using different laser sources for accurate monitoring of fast dynamic changes of NO production. Several applications are also presented to demonstrate the suitability of these detectors for detection of NO in plants.

Effect of Interleukin‐10 and Laminar Shear Stress on African American Human Umbilical Vein Endothelial Cells

African Americans (AA) have a predisposition to heightened systemic inflammation and a high prevalence of hypertension. The purpose of this study was to evaluate the influence of interleukin‐10 (IL‐10) and laminar shear stress (LSS) on AA endothelial cells by measuring total endothelial nitric oxide synthase (eNOS) protein expression and its activated, phosphorylated form (p‐eNOS) at Serine 1177, as well as nitric oxide (NO) levels, in response to IL‐10 incubation and high physiological levels of LSS, used as a mimetic for aerobic exercise training (AEXT). Human umbilical vein endothelial cells (HUVEC) from an AA donor were cultured. The experimental conditions included Static, Static with IL‐10 Incubation, LSS at 20 dynes/cm2, and LSS at 20 dynes/cm2 with IL‐10 Incubation. Western blotting was used to measure eNOS and p‐eNOS protein expression in the cells. A modified Griess assay was used to measure NO production in the cell culture media. There were significant increases in p‐eNOS, eNOS, and NO in the LSS at 20 dynes/cm2 and LSS at 20 dynes/cm2 with IL‐10 Incubation experimental conditions when compared to the Static experimental condition. There were no other statistically significant differences, demonstrating that IL‐10 did not have an additive effect on eNOS activity in the present study. The significant increases in p‐eNOS, eNOS, and NO as a result of LSS in AA HUVECs suggest that AEXT may be a viable, nonpharmacologic method to improve vascular inflammation status and vasodilation, and thereby contribute to reduction of hypertension in the AA population.AHA Scientist Development Grants 12SDG12070327NIH Grant RO1 [HL085497]

Evidence for anti-inflammatory and antioxidative properties of dried plum polyphenols in macrophage RAW 264.7 cells.

This study presents the anti-inflammatory and antioxidative properties of dried plum (Prunus domestica L.) polyphenols in macrophage RAW 264.7 cells. We hypothesized that dried plum polyphenols have strong anti-inflammatory and antioxidant properties against lipopolysaccharide (LPS)-induced production of the pro-inflammatory markers, nitric oxide (NO) and cyclooxygenase-2 (COX-2), and the lipid peroxidation product, malondialdehyde, in activated macrophage RAW 264.7 cells. To test this hypothesis, macrophage RAW 264.7 cells were stimulated with either 1 μg ml(-1) (for measurement of NO production) or 1 ng ml(-1) (for measurement of COX-2 expression) of LPS to induce inflammation and were treated with different doses of dried plum polyphenols (0.0, 0.1, 1, 10, 100 and 1000 μg ml(-1)). Dried plum polyphenols at a dose of 1000 μg ml(-1) was able to significantly (P < 0.05) reduce NO production by 43%. Additionally, LPS-induced expression of COX-2 was significantly (P < 0.05) reduced by 100 and 1000 μg ml(-1) dried plum polyphenols. To investigate the antioxidant activity of dried plum polyphenols, macrophage RAW 264.7 cells were stimulated with 100 μg ml(-1) of FeSO4 + 1 mM ml(-1) of H2O2 to induce lipid peroxidation. Dried plum polyphenols at a dose of 1000 μg ml(-1) showed a 32% reduction in malondialdehyde production. These findings indicate that dried plum polyphenols are potent anti-inflammatory and antioxidative agents in vitro.

Anandamide rescues retinal barrier properties in Müller glia through nitric oxide regulation

The blood retinal barrier (BRB) can mitigate deleterious immune response. Dysfunction at the BRB can affect disease progression. Under inflammatory conditions Müller glia produce increased pro-inflammatory factors, like nitric oxide (NO). In this study we describe molecular events at the Müller glia during inflammation which could affect inner BRB properties. Griess assay and 4,5-diaminofluorescein diacetate (DAF-2DA) time-lapse fluorescence were used to measure NO production. Western blot was used to analyze the expression of inducible nitric oxide synthase (iNOS) and mitogen-activated protein kinases (MAPK) components. Lucifer Yellow was used to measure permeability. Griess assay and DAF-2DA time-lapse fluorescence images revealed that lipopolysaccharide (LPS) induced inflammation and increased NO production. In parallel, changes were observed in tight junction proteins, zona occludens 1 (ZO-1), connexin 43 (Cx43), and permeability. This was mediated through activation of iNOS and mitogen-activated protein kinase phosphatase-1 (MKP-1), implicated in immune response. Endocannabinoids can exert a protective and anti-inflammatory effect. Exogenous arachidonoyl ethanolamide (AEA) inhibited NO generation and also abolished LPS-induced increase in permeability. Our work suggests that subtle changes in Müller glia function, which act as part of the BRB, could contribute to retinal health. AEA which can reduce inflammatory cytotoxicity has potential as treatment in several ocular manifestations where the integrity of the BRB is crucial.

Immunopathological and antimicrobial effect of black pepper, ginger and thyme extracts on experimental model of acute hematogenous pyelonephritis in albino rats.

Recent studies showed prominent antimicrobial activity of various plant extracts on certain pathogenic microorganisms, therefore we prepared crude aqueous extracts of black pepper, ginger and thyme and carried out an in vitro study by measuring antimicrobial activity of these extracts using the agar well diffusion method. An in vivo study was carried out on 50 adult healthy male albino rats which were divided into 5 groups, 10 rats each. Group 1: negative control group which received saline solution intragastrically daily; Group 2: Positive control group, injected with mixed bacterial suspension of S.aureus and E.coli as a model of pyelonephritis, then received saline solution intragastrically daily; Group 3: injected with the same dose of mixed bacterial suspension, then received 100 mg/kg/day black pepper extract intragastrically; Group 4: injected with mixed bacterial suspension then received 500 mg/kg/day ginger extract intragastrically. Group 5: injected with mixed bacterial suspension then received 500 mg/kg/day thyme extract intragastrically. All groups were sacrificed after either 1 or 4 weeks. Serum and blood samples were collected for lysozyme activity estimation using agarose lysoplate, measurement of nitric oxide production, and lymphocyte transformation test as well as for counting both total and differential leukocytes and erythrocytes. Kidney samples were tested histopathologically. Both in vivo and in vitro results confirm the efficacy of these extracts as natural antimicrobials and suggest the possibility of using them in treatment procedures.

Characterization of lipoteichoic acid structures from three probiotic Bacillus strains: involvement of D-alanine in their biological activity.

Probiotics represent a potential strategy to influence the host’s immune system thereby modulating immune response. Lipoteichoic Acid (LTA) is a major immune-stimulating component of Gram-positive cell envelopes. This amphiphilic polymer, anchored in the cytoplasmic membrane by means of its glycolipid component, typically consists of a poly (glycerol-phosphate) chain with d-alanine and/or glycosyl substitutions. LTA is known to stimulate macrophages in vitro, leading to secretion of inflammatory mediators such as Nitric Oxide (NO). This study investigates the structure–activity relationship of purified LTA from three probiotic Bacillus strains (Bacillus cereus CH, Bacillus subtilis CU1 and Bacillus clausii O/C). LTAs were extracted from bacterial cultures and purified. Chemical modification by means of hydrolysis at pH 8.5 was performed to remove d-alanine. The molecular structure of native and modified LTAs was determined by 1H NMR and GC–MS, and their inflammatory potential investigated by measuring NO production by RAW 264.7 macrophages. Structural analysis revealed several differences between the newly characterized LTAs, mainly relating to their d-alanylation rates and poly (glycerol-phosphate) chain length. We observed induction of NO production by LTAs from B. subtilis and B. clausii, whereas weaker NO production was observed with B. cereus. LTA dealanylation abrogated NO production independently of the glycolipid component, suggesting that immunomodulatory potential depends on d-alanine substitutions. d-alanine may control the spatial configuration of LTAs and their recognition by cell receptors. Knowledge of molecular mechanisms behind the immunomodulatory abilities of probiotics is essential to optimize their use.

TRPV4 activation mediates flow-induced nitric oxide production in the rat thick ascending limb

Nitric oxide (NO) regulates renal function. Luminal flow stimulates NO production in the thick ascending limb (TAL). Transient receptor potential vanilloid 4 (TRPV4) is a mechano-sensitive channel activated by luminal flow in different types of cells. We hypothesized that TRPV4 mediates flow-induced NO production in the rat TAL. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. Increasing luminal flow from 0 to 20 nl/min stimulated NO from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min (n = 5; P < 0.05). The TRPV4 antagonists, ruthenium red (15 μmol/l) and RN 1734 (10 μmol/l), blocked flow-induced NO production. Also, luminal flow did not increase NO production in the absence of extracellular calcium. We also studied the effect of luminal flow on NO production in TALs transduced with a TRPV4shRNA. In nontransduced TALs luminal flow increased NO production by 47 ± 17 AU/min (P < 0.05; n = 5). Similar to nontransduced TALs, luminal flow increased NO production by 39 ± 11 AU/min (P < 0.03; n = 5) in TALs transduced with a control negative sequence-shRNA while in TRPV4shRNA-transduced TALs, luminal flow did not increase NO production (Δ10 ± 15 AU/min; n = 5). We then tested the effect of two different TRPV4 agonists on NO production in the absence of luminal flow. 4α-Phorbol 12,13-didecanoate (1 μmol/l) enhanced NO production by 60 ± 11 AU/min (P < 0.002; n = 7) and GSK1016790A (10 ηmol/l) increased NO production by 52 ± 15 AU/min (P < 0.03; n = 5). GSK1016790A (10 ηmol/l) did not stimulate NO production in TRPV4shRNA-transduced TALs. We conclude that activation of TRPV4 channels mediates flow-induced NO production in the rat TAL.

Anti-oxidant and Anti-inflammatory Effects of &lt;i&gt;Acanthopanacia Cortex&lt;/i&gt; Hot Aqueous Extract on Lipopolysaccharide(LPS) Simulated Macrophages

Objectives : This study is to investigate the effects of Acanthopanacis Cortex hot aqueous extract on nitric oxide(NO), prostaglandin E2(PGE2) production and DPPH(1,1-diphenyl-2-picryl hydrazyl) radical scavenging activity in macrophages. Methods : Acanthopanacis Cortex(200 g) was heated at 100 °C with distilled water(2 L) for 4hrs. The extract was filtered and concentrated to 100 ml using a rotary evaporator and was frozen at -80 °C, then was freeze-dried. The RAW 264.7 macrophages were subcultured. In order to evaluate cytotoxicity, MTT assay was performed. Experimental groups were divided into five(control, AC 25, 50, 100 and 200 μg/ml) and we measured cytotoxicity. The concentrations of NO were preprocessed by Griess assay. The RAW 264.7 macrophages was pretreated by 10 μg/ml LPS and experimental groups were divided into five and we measured NO production. The concentrations of PGE2 were measured by enzyme immunoassay. The RAW 264.7 macrophages was pretreated by 10 μg/ml LPS. Experimental groups were divided into five and we measured PGE2 production. Antioxidant activity was measured by the DPPH method. experimental groups were divided into four(AC 25, 50, 100 and 200 μg/ml) and we measured DPPH radical scavenging activity. Results : 1. Viability of RAW 264.7 macrophages did not significantly decrease in 25, 50 and 100 μg/ml Acanthopanacis Cortex hot aqueous extract compared to control group. 2. NO production in LPS-stimulated RAW 264.7 macrophages significantly inhibited in 100, 200 μg/ml Acanthopanacis Cortex hot aqueous extract compared to control group. 3. PGE2 production in LPS-stimulated RAW 264.7 macrophages significantly inhibited in 100, 200 μg/ml Acanthopanacis Cortex hot aqueous extract compared to control group. 4. DPPH radical scavenging capability of Acanthopanacis Cortex hot aqueous extract in RAW 264.7 macrophages had the high level in 100, 200 μg/ml. Conclusion : According to the results, Acanthopanacis Cortexx hot aqueous extract has ability to suppress NO, PGE2 production and improve DPPH free radical scavenging activity. So Acanthopanacis Cortex hot aqueous extract may have an anti-inflammation effect and antioxidant activity.

Effect of short-term polyphenol treatment on endothelial dysfunction and thromboxane A2 levels in streptozotocin-induced diabetic mice.

Diabetes is characterized by the development of endothelial dysfunction, which affects both nitric oxide (NO)-mediated relaxation and endothelium-derived contracting factors, associated with vascular oxidative stress. There is a growing body of evidence suggesting that polyphenols have several beneficial effects, such as antioxidant and anti-inflammatory activities. This study investigated whether short-term treatment with polyphenols (chlorogenic acid (CA), morin (MO), resveratrol (RV)) can improve endothelial dysfunction related to diabetes. Aorta reactivity was determined in organ chambers, and we measured NO production and thromboxane B2 (TXB2; a metabolite of TXA2) from aortas in response to acetylcholine (ACh). Streptozotocin (STZ)-induced diabetic mice (16 weeks) were injected with solvent (ethanol, 10% v/v; intraperitoneally (i.p.)), CA (0.03 mmol/kg/d), MO (0.03 mmol/kg/d), and RV (0.03 mmol/kg/d) for 5 d. The ACh-induced endothelium-dependent relaxation was markedly reduced in rings of STZ-induced diabetic mice compared to controls. The treatment with polyphenols (significantly: MO, tendency: CA and RV) for only 5 d improved the NO components of relaxation, but did not normalize ACh-stimulated NO production. However, polyphenol treatment suppressed the ACh-stimulated level of TXB2 in aortas from STZ-induced diabetic mice. Thus, treatment with polyphenols caused basal NO production and a prompt improvement of the endothelial function in diabetic mice, and this may involve the normalization of TXA2 levels, not NO production, under ACh stimulation.

The anti-inflammatory effect of Indonesian Areca catechu leaf extract in vitro and in vivo.

BACKGROUND/OBJECTIVESOverproduction of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) enzyme can cause inflammation. Cyclooxygenase-2 (COX-2) is also involved in the inflammatory response through regulation of nuclear factor-kappa B (NF-κB). Areca catechu is one of the known fruit plants of the Palmaceae family. It has been used for a long time as a source of herbal medicine in Indonesia. In this study, we explored the effect of Indonesian Areca catechu leaf ethanol extract (ACE) in lipopolysaccharide (LPS)-induced inflammation and carrageenan-induced paw edema models. Recently, this natural extract has been in the spotlight because of its efficacy and limited or no toxic side effects. However, the mechanism underlying its anti-inflammatory effect remains to be elucidated.MATERIALS/METHODSWe measured NO production by using the Griess reagent, and determined the expression levels of inflammation-related proteins, such as iNOS, COX2, and NF-κB, by western blot. To confirm the effect of ACE in vivo, we used the carrageenan-induced paw edema model.RESULTSCompared to untreated cells, LPS-stimulated RAW 264.7 cells treated with ACE showed reduced NO generation and reduced iNOS and COX-2 expression. We found that the acute inflammatory response was significantly reduced by ACE in the carrageenan-induced paw edema model.CONCLUSIONTaken together, these results suggest that ACE can inhibit inflammation and modulate NO generation via downregulation of iNOS levels and NF-κB signaling in vitro and in vivo. ACE may have a potential medical benefit as an anti-inflammation agent.

Effect of Exercise Training on Enos Expression, NO Production and Oxygen Metabolism in Human Placenta

ObjectiveTo determine the effects of combined aerobic and resistance exercise training during the second half of pregnancy on endothelial NOS expression (eNOS), nitric oxide (NO) production and oxygen metabolism in human placenta.MethodsThe study included 20 nulliparous in gestational week 16–20, attending prenatal care at three tertiary hospitals in Colombia who were randomly assigned into one of two groups: The exercise group (n = 10) took part in an exercise session three times a week for 12 weeks which consisted of: aerobic exercise at an intensity of 55–75% of their maximum heart rate for 60 min and 25 mins. Resistance exercise included 5 exercise groups circuit training (50 repetitions of each) using barbells (1–3 kg/exercise) and low-to-medium resistance bands. The control group (n = 10) undertook their usual physical activity. Mitochondrial and cytosol fractions were isolated from human placental tissue by differential centrifugation. A spectrophotometric assay was used to measure NO production in cytosolic samples from placental tissue and Western Blot technique to determine eNOS expression. Mitochondrial superoxide levels and hydrogen peroxide were measured to determine oxygen metabolism.ResultsCombined aerobic and resistance exercise training during pregnancy leads to a 2-fold increase in eNOS expression and 4-fold increase in NO production in placental cytosol (p = 0.05). Mitochondrial superoxide levels and hydrogen peroxide production rate were decreased by 8% and 37% respectively in the placental mitochondria of exercising women (p = 0.05).ConclusionRegular exercise training during the second half of pregnancy increases eNOS expression and NO production and decreases reactive oxygen species generation in human placenta. Collectively, these data demonstrate that chronic exercise increases eNOS/NO production, presumably by increasing endothelial shear stress. This adaptation may contribute to the beneficial effects of exercise on the vascular and antioxidant system and in turn reduce the risk of preeclampsia, diabetes or hypertension during pregnancy.

NO homeostasis is a key regulator of early nitrate perception and root elongation in maize.

Crop plant development is strongly dependent on nitrogen availability in the soil and on the efficiency of its recruitment by roots. For this reason, the understanding of the molecular events underlying root adaptation to nitrogen fluctuations is a primary goal to develop biotechnological tools for sustainable agriculture. However, knowledge about molecular responses to nitrogen availability is derived mainly from the study of model species. Nitric oxide (NO) has been recently proposed to be implicated in plant responses to environmental stresses, but its exact role in the response of plants to nutritional stress is still under evaluation. In this work, the role of NO production by maize roots after nitrate perception was investigated by focusing on the regulation of transcription of genes involved in NO homeostasis and by measuring NO production in roots. Moreover, its involvement in the root growth response to nitrate was also investigated. The results provide evidence that NO is produced by nitrate reductase as an early response to nitrate supply and that the coordinated induction of non-symbiotic haemoglobins (nsHbs) could finely regulate the NO steady state. This mechanism seems to be implicated on the modulation of the root elongation in response to nitrate perception. Moreover, an improved agar-plate system for growing maize seedlings was developed. This system, which allows localized treatments to be performed on specific root portions, gave the opportunity to discern between localized and systemic effects of nitrate supply to roots.

Thiamine deficiency leads to reduced nitric oxide production and vascular dysfunction in rats

Background and aimsThiamine deficiency is a condition that is known to cause damage to the nervous and cardiovascular systems because it interferes with cellular metabolism. It is well known that the control of vascular function is highly dependent on the production of nitric oxide (NO) by NO synthases. Studies exploring the physiological relevance of NO signaling under conditions of thiamine deficiency are scarce. The present study sought to investigate whether chronic metabolic changes would cause alterations in vascular responsiveness. Methods and resultsBy removing thiamine from the diet, we observed a reduced acetylcholine-mediated relaxation and an increased phenylephrine-mediated vasoconstriction in the aortas containing functional endothelium. Removal of the endothelium or the pre-treatment of vessels with l-NAME restored the contractile responses to the level of controls. Conversely, indomethacin did not modify phenylephrine-mediated contractions. We also used carbon microsensors to continually measure NO production in situ while simultaneously measuring the vascular tone. The results revealed a significant decrease in NO production. Western blot analysis showed a decreased expression of the total eNOS in the thiamine-deficient aorta compared to the control. Concentration–response curves for phenylephrine indicated no difference between the control and deficient groups in the presence and absence of SOD or Tyron. The NO donor DEA-NONOate produced a concentration-dependent relaxation response in the endothelium-denuded vessels that did not differ between the control and thiamine-deficient rats. ConclusionThiamine deficiency modulates eNOS-dependent NO production, leading to a decreased vasorelaxation and an increased contractile response in the rat aorta.

Poly(vinyl alcohol) Physical Hydrogel Nanoparticles, Not Polymer Solutions, Exert Inhibition of Nitric Oxide Synthesis in Cultured Macrophages

Hydrogel nanoparticles (HNP) are an emerging tool of biomedicine with unique materials characteristics, scope, and utility. These hydrated, soft colloidal carriers can penetrate through voids with dimensions narrower than the size of the particle, provide stabilization for fragile biological cargo and allow diffusion and exchange of solutes with external phase. However, techniques to assemble HNP are few; solitary examples exist of biocompatible polymers being formulated into HNP; and knowledge on the biomedical properties of HNP remains rather cursory. In this work, we investigate assembly of HNP based on a polymer with decades of prominence in the biomedical field, poly(vinyl alcohol), PVA. We develop a novel method for production of PVA HNP through nanoprecipitation-based assembly of polymer nanoparticles and subsequent physical hydrogelation of the polymer. Polymer nanoparticles and HNP were visualized using scanning electron microscopy and fluorescence imaging, and characterized using dynamic light scattering and zeta potential measurements. Interaction of PVA HNP with mammalian cells was investigated using flow cytometry, viability screening, and measurements of nitric oxide production by cultured macrophages. The latter analyses revealed that PVA administered as a polymer solution or in the form of HNP resulted in no measurable increase in production of the inflammation marker. Unexpectedly, PVA HNP exerted a pronounced inhibition of NO synthesis by stimulated macrophages, that is, had an anti-inflammatory activity. This effect was accomplished with a negligible change in the cell viability and was not observed when PVA was administered as a polymer solution. To the best of our knowledge, this is the first observation of inhibition of NO synthesis in macrophages by administered nanoparticles and specifically hydrogel nanoparticles. Taken together, our results present PVA HNP as promising colloidal hydrogel nanocarriers for biomedical applications, specifically drug delivery and assembly of intracellular biosensors.

Nitro-Oxidative Stress after Neuronal Ischemia Induces Protein Nitrotyrosination and Cell Death

Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO) to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y), a murine glial (BV2), a human endothelial cell line (HUVEC), and in primary cultures of human cerebral myocytes (HC-VSMCs) after experimental ischemia in vitro. Neuronal, endothelial, and inducible NO synthase (NOS) expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells with H2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia.

Uric acid modulates vascular endothelial function through the down regulation of nitric oxide production

Endothelial dysfunction characterized by decreased nitric oxide (NO) bioavailability is the first stage of coronary artery disease. It is known that one of the factors associated with an increased risk of coronary artery disease is a high plasma level of uric acid. However, causative associations between hyperuricaemia and cardiovascular risk have not been definitely proved. In this work, we tested the effect of uric acid on endothelial NO bioavailability. Electrochemical measurement of NO production in acetylcholine-stimulated human umbilical endothelial cells (HUVECs) revealed that uric acid markedly decreases NO release. This finding was confirmed by organ bath experiments on mouse aortic segments. Uric acid dose-dependently reduced endothelium-dependent vasorelaxation. To reveal the mechanism of decreasing NO bioavailability we tested the effect of uric acid on reactive oxygen species production by HUVECs, on arginase activity, and on acetylcholine-induced endothelial NO synthase phosphorylation. It was found that uric acid increases arginase activity and reduces endothelial NO synthase phosphorylation. Interestingly, uric acid significantly increased intracellular superoxide formation. In conclusion, uric acid decreases NO bioavailability by means of multiple mechanisms. This finding supports the idea of a causal association between hyperuricaemia and cardiovascular risk.

INTRACELLULAR NO AND CALCIUM IONS CHANGE CAUSED BY EXENDIN-4 IN THE PIG ILIAC ENDOTHELIUM

ObjectivesTo investigate the effects of Exendin-4 (Ex-4) on nitric oxide (NO) produced bypig iliac endothelium cells (PIEC) and the relationship with intracellular calcium ions change.MethodsGLP-1receptor (GLP-1R) in the PIEC membrane...

Immunomodulatory effects of heat‐killed Enterococcus faecalis TH10 on murine macrophage cells

The objective of this study was to investigate the immunomodulatory effects of heat-killed Enterococcus faecalis TH10 (hk-TH10) and its signal transduction on murine macrophage RAW264 cells. RAW264 cells produced nitric oxide (NO) following hk-TH10 treatment. In order to investigate the mechanisms underlying hk-TH10-stimulated NO production, we further measured NO production in RAW264 cells treated with Toll-like receptor (TLR) 4 inhibitor peptide, NF-κB inhibitor, TLR1-siRNA, TLR2-siRNA, and TLR-6 siRNA. Furthermore, the activation of TLR2-TLR1/6 pathway molecules was analyzed by Western blotting. The result of this study showed that hk-TH10 stimulates NO in RAW264 cells through the activation of the TLR2-TLR1/6 pathway. From our findings, we can conclude that hk-TH10 isolated from a traditional side-dish fermented food (tempeh) may facilitate host immunomodulation.

Attenuated glomerular arginine transport prevents hyperfiltration and induces HIF-1α in the pregnant uremic rat

Pregnancy worsens renal function in females with chronic renal failure (CRF) through an unknown mechanism. Reduced nitric oxide (NO) generation induces renal injury. Arginine transport by cationic amino acid transporter-1 (CAT-1), which governs endothelial NO generation, is reduced in both renal failure and pregnancy. We hypothesize that attenuated maternal glomerular arginine transport promotes renal damage in CRF pregnant rats. In uremic rats, pregnancy induced a significant decrease in glomerular arginine transport and cGMP generation (a measure of NO production) compared with CRF or pregnancy alone and these effects were prevented by l-arginine. While CAT-1 abundance was unchanged in all experimental groups, protein kinase C (PKC)-α, phosphorylated PKC-α (CAT-1 inhibitor), and phosphorylated CAT-1 were significantly augmented in CRF, pregnant, and pregnant CRF animals; phenomena that were prevented by coadministrating l-arginine. α-Tocopherol (PKC inhibitor) significantly increased arginine transport in both pregnant and CRF pregnant rats, effects that were attenuated by ex vivo incubation of glomeruli with PMA (a PKC stimulant). Renal histology revealed no differences between all experimental groups. Inulin and p-aminohippurate clearances failed to augment and renal cortical expression of hypoxia inducible factor-1α (HIF-1α) significantly increased in CRF pregnant rat, findings that were prevented by arginine. These studies suggest that in CRF rats, pregnancy induces a profound decrease in glomerular arginine transport, through posttranslational regulation of CAT-1 by PKC-α, resulting in attenuated NO generation. These events provoke renal damage manifested by upregulation of renal HIF-1α and loss of the ability to increase glomerular filtration rate during gestation.

ATP mediates flow‐induced nitric oxide production in thick ascending limbs by activating luminal and basolateral P2 receptors

In the thick ascending limb (TAL) ATP mediates flow‐induced nitric oxide (NO) production via purinergic type 2 receptors (P2r) activation. Both P2r subtypes, P2X and P2Y, are differentially expressed in the luminal (LL) and basolateral (BL) sides of the TAL. We hypothesized that ATP mediates flow‐induced NO production in the TAL via activation of both LL and BL P2r. We measured NO production in isolated and perfused rat TALs using the fluorescent dye DAF FM. Increasing luminal flow from 0 to 20 nL/min stimulated NO production from 17±15 to 130±34 arbitrary units (AU)/min (p &lt; 0.04; n=7). The P2r antagonist suramin (300 μmol/L) reduced flow‐induced NO production by 83 ± 25 % (p &lt; 0.03; n = 5) when added to both LL and BL sides. Luminal hexokinase plus glucose (HK + GL) decreased flow‐induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min (p &lt; 0.02; n=7). Basolateral HK + GL reduced flow‐induced NO production from 186.4 ± 45.7 to 30.9 ± 38.4; p &lt;0.005; n=6). The P2Xr selective antagonist NF023 (200 umol/L) prevented flow‐induced NO production when added to the BL side (from 286.1 ± 8.1 to −23.5 ± 17.3; p &lt;0.01; n=6). Luminal NF023 did not affect flow‐induced NO production. We conclude that ATP mediates flow‐induced NO production in the TAL likely via activation of P2Yr at the LL and P2Xr at the BL side.