Nitric Oxide Assay Kit Research Articles

Organochlorine Pesticide-Mediated Induction of NADPH Oxidase and Nitric-Oxide Synthase in Endothelial Cell.

Organochlorine Pesticides (OCPs) are detected ubiquitously in human and have been shown to be associated with Cardiovascular Disease (CVD) and atherosclerosis. To find out the effect of organochlorine pesticides in endothelial cell with regard to oxidative stress and associated expression of enzymes producing superoxide and Nitric Oxide (NO). Human Umbilical Vein Endothelial Cells (HUVEC) were cultured and treated with four OCPs which were found in human blood at a concentration of 0.1μM. The cells were tested for Reactive Oxygen Species (ROS) generation, NO production and mRNA expression of NAPDH oxidase (p47phox) and endothelial Nitric Oxide Synthase (eNOS). ROS generation was measured by using 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) method. NO was analysed by Bioxytech nitric oxide assay kit method and mRNA of NADPH oxidase and eNOS was quantified by real time PCR. Data were expressed as the mean±SEM. Comparison between the groups were made by student's t-test (2-tailed) or one-way ANOVA with Tukey's post-hoc analysis depending on number of groups. For all statistical tests, p< 0.05 was considered to be significant. All the four pesticides generated ROS accompanied by enhanced expression of NADPH oxidase. Maximum effect was observed with β-endosulfan. Level of NO was found to be decreased significantly in endothelial cells treated with these pesticides accompanied by enhanced expression of eNOS. The antioxidant N-acetylcysteine (NAC) reduced ROS generation and enhanced NO formation. Pesticide-mediated ROS generation possibly reacts with NO forming peroxinitrite and thereby reducing the bioavailability of NO although eNOS expression is increased. OCPs induce endothelial dysfunction through increased ROS generation via NADPH oxidase expression and reduced bioavailability of nitric oxide.

BDKRB2 +9/−9 bp polymorphisms influence BDKRB2 expression levels and NO production in knee osteoarthritis

The bradykinin B2 receptor (BDKRB2) plays a key role in the inflammation process of osteoarthritis. Nitric oxide has also long been considered to be a catabolic factor that contributes to inflammatory response and the osteoarthritis disease pathology. Several studies have reported that the BDKRB2 +9/-9 bp polymorphisms are associated with transcription of the receptor. However, the roles of BDKRB2 polymorphisms in inflammation in osteoarthritis remain unclear. This study enrolled 156 subjects with primary knee osteoarthritis and 58 healthy volunteers. BDKRB2 polymorphisms were genotyped, and the mRNA and protein levels of BDKRB2 in synovial tissues from osteoarthritis patients were measured by quantitative real-time polymerase chain reaction and western blot analysis, respectively. Nitric oxide production in serum from patients with osteoarthritis was measured using a nitric oxide assay kit. We found that the mean BDKRB2 mRNA levels were significantly higher in Kallgren-Lawrence grade-4 osteoarthritis patients than patients with lower grade osteoarthritis. The +9/-9 bp polymorphisms significantly affected the BDKRB2 mRNA and protein expression levels in synovial tissues from osteoarthritis subjects. Osteoarthritis patients with +9/-9 and -9/-9 genotypes had higher BDKRB2 expression levels in synovial tissue and nitric oxide production in serum. Moreover, positive correlation was found between the BDKRB2 levels in synovial tissue and nitric oxide production. Compared with health controls, significant increases of nitric oxide production in osteoarthritis were detected which were associated with increasing severity of osteoarthritis. Multiple linear regression analysis (adjusted for gender and age) showed serum nitric oxide level was positively associated with BDKRB2 polymorphism and Kallgren-Lawrence grade and was inversely associated with obesity. Our findings showed that the BDKRB2 +9/-9 bp polymorphisms affected the gene expression and nitric oxide production, which were associated with radiographic severity of osteoarthritis, suggesting that the BDKRB2 +9/ -9 bp polymorphisms may act as a genetic modulator of osteoarthritis, and play an essential role in inflammatory process in osteoarthritis.

Effects of desloratadine citrate disodium injection on rat models of ovalbumin-induced allergic rhinitis: involvement of T-cell responses modulation.

Allergic rhinitis (AR) is a very common worldwide problem; patients display a number of symptoms, such as sneezing, nasal itching, and rhinorrhea, and their lifestyle is affected. Desloratadine is a novel, long-acting inhibitor of histamine. However, very little is known about the effect of desloratadine citrate disodium injection (DLC injection) on AR, and the underlying mechanisms are yet unexplored. Herein, we sought to explore the effects and mechanisms of actions of DLC injection in ovalbumin (OVA)-induced immune responses in a rat model of AR. Sixty rats were subjected to immunization with OVA (intraperitoneal [i.p.]), followed by a nasal challenge with OVA. Drugs or saline were given daily for treatment. Nasal symptoms and histology of the nasal mucosa were examined. Cytokines such as interleukin (IL)-4, IL-12, interferon (IFN)-γ, adhesion molecules such as soluble vascular cell adhesion molecule 1 (sVCAM-1), and inducible nitric oxide synthase (iNOS) expression were assessed by enzyme-linked immunosorbent assay (ELISA) kit. Nitric oxide (NO) concentration was also measured by NO assay kit. DLC treatment (intravenous [i.v.]) significantly decreased the frequency of sneezing and nasal scratching and alleviated nasal inflammation by increasing the serum levels of IFN-γ and IL-12, while lowering the expression of IL-4. Thus, DLC (i.v.) treatment led to a marked elevation in T-helper 1/T-helper 2 (Th1/Th2) ratio when administered in the AR rats. The expression of sVCAM-1, iNOS, and NO were also reversed. DLC (i.v.), given after an allergen challenge, improved Th1 cytokines level and restrained Th2 responses alleviating the symptoms of AR. Our results indicate that DLC injection may exhibit such effects through the modulation of T-cell responses.

Pterostilbene, an Active Constituent of Blueberries, Stimulates Nitric Oxide Production via Activation of Endothelial Nitric Oxide Synthase in Human Umbilical Vein Endothelial Cells.

Endothelial dysfunction, a key process in development of cardiovascular diseases, is largely due to reduced nitric oxide (NO) derived from endothelial NO synthase (eNOS). Resveratrol has been reported to stimulate NO production via estrogen receptor α (ERα) activation in endothelial cells. Here, we investigated whether two natural methylated analogs of resveratrol, pterostilbene (Pts) and trans-3,5,4'-trimethoxystilbene (TMS), similarly to resveratrol, could influence endothelial NO release in human umbilical vein endothelial cells (HUVECs). In HUVECs exposed to Pts or TMS, NO production and phosphorylation of eNOS, protein kinase B (Akt), and ERα were measured by using a fluorimetric NO assay kit and Western blot analysis, respectively. Dimethylated Pts, but not trimethylated TMS, stimulated dose-dependent NO production via eNOS phosphorylation. Pts also stimulated dose-dependent phosphorylation of Akt, but not of ERα. NO production and eNOS phosphorylation in response to Pts were significantly abolished by the phosphoinositide 3-kinase (PI3K)/Akt inhibitor LY294002, but not by the ERα antagonist ICI182780. Our results suggest that Pts, but not TMS, is capable of inducing eNOS phosphorylation and the subsequent NO release, presumably, by activating PI3K/Akt pathway. The potential efficacy of Pts, an active constituent of blueberries, may aid in the prevention of cardiovascular diseases characterized by endothelial dysfunction.

Abstract 354: Nitrate Improves Postresuscitation Myocardial Dysfunction by Preserving Myocardial Nitric Oxide and Phospholamban Protein

Objectives: Sarcoplasmic reticulum (SR) Ca 2+ handling proteins have been shown to play an important role in myocardial dysfunction after acute ischemic/reperfusion injury. We assessed the hypothesis that nitrite would improve postresuscitation myocardial dysfunction by increasing NO generation, and the mechanism is related to the modulation of SR Ca 2+ handling proteins. Methods: We performed a randomized prospective animal study using male Sprague-Dawley rats. Cardiac arrest was induced by intravenous bolus of potassium chloride (40 μg/g). Nitrite (1.2nmol/g) or placebo was administered when chest compression was started. No cardiac arrest was induced in sham group. Hemodynamic parameters were monitored invasively for one hour after ROSC. Echocardiogram was performed to evaluate cardiac function. Myocardial sample was harvested 5 minutes and 1 hour after ROSC respectively. The expression of sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a), phospholamban (PLB) and rynodine receptors (RyRs) were analyzed by Western blot analysis. Myocardial NO five minutes after ROSC were measured using Nitric Oxide Assay Kit. Results: Myocardial function measured as the maximal rate of LV pressure increase (dP/dtmax), the maximal rate of LV pressure decline (-dP/dtmax), ejection fraction, fractional shortening was significantly impaired in nitrite and placebo groups after resuscitation, and cardiac function in nitrite group was significantly greater than placebo group. Nitrite administration increased the level of nitric oxide in myocardium 5 min after resuscitation when compared to the other two groups. The levels of phosphorylated phospholamban were decreased after resuscitation, and nitrite preserved the phosphorylation of phospholamban in myocardium after resuscitation when compared to placebo group. No significant differences were found in the expressions of myocardial sarcoplasmic reticulum Ca 2+ ATPase and rynodine receptors after resuscitation. Conclusions: Nitrite administered during resuscitation improves postresusctation myocardial dysfunction as an exogenous donor of nitric oxide, and the modulation of phosphorylated phospholamban, an important SR Ca 2+ uptake protein, is involved in the mechanism.

Inhibitory effect of TGF-β1 on NO production in peritoneal macrophages from collagen-induced arthritis rats involving the LPS-TLR4 pathway

Transforming growth factor-β1 (TGF-β1) is critical in controlling inflammatory responses and the prevention of autoimmune diseases. Although the effect of TGF-β1 on macrophages from normal mice or rats has been established, little attention has been paid to its effect on disease conditions. In the present study, we investigated the regulatory effect, and possible mechanism, of TGF-β1 exposure on the secretion of nitric oxide (NO) in peritoneal macrophages (PMΦ) obtained from collagen-induced arthritis (CIA) rats. The CIA model was established by immunizing the emulsion of collagen type II and incomplete Freund's adjuvant (IFA) in Wistar rats. PMΦ were incubated with TGF-β1 (5 ng/ml) for 36 h and the supernatant, and cell mRNA and protein were collected. NO concentration was determined using an NO assay kit. The mRNA expression of inducible nitric oxide synthase (iNOS) and Toll-like receptor 4 (TLR4) was determined using reverse transcription-polymerase chain reaction (RT-PCR). The protein expression of iNOS was tested with western blot analysis. The expression of membrane TLR4 was determined by flow cytometry. We discovered that the secretion of NO from the PMΦ of CIA rats increased compared with normal rats. TGF-β1 significantly inhibited the production of NO in the PMΦ from CIA rats. iNOS mRNA and protein expression in the PMΦ from CIA rats may be suppressed by TGF-β1. TLR4 mRNA and protein expression in PMΦ from CIA rats were upregulated with LPS stimulation and treatment with TGF-β1 inhibited their expression. These results demonstrated that TGF-β1 inhibited lipopolysaccharide (LPS)-induced NO production in the PMΦ from CIA rats, which may be due to the inhibition of the LPS-TLR4 signaling pathway.

Effects of L-arginine and Nω-nitro-L-arginine methylester on learning and memory and α7 nAChR expression in the prefrontal cortex and hippocampus of rats

Nitric oxide (NO) is a novel type of neurotransmitter that is closely associated with synaptic plasticity, learning and memory. In the present study, we assessed the effects of L-arginine and N(ω)-nitro-L-arginine methylester (L-NAME, a nitric oxide synthase inhibitor) on learning and memory. Rats were assigned to three groups receiving intracerebroventricular injections of L-Arg (the NO precursor), L-NAME, or 0.9% NaCl (control), once daily for seven consecutive days. Twelve hours after the last injection, they underwent an electric shock-paired Y maze test. Twenty-four hours later, the rats' memory of the safe illuminated arm was tested. After that, the levels of NO and α7 nicotinic acetylcholine receptor (α7 nAChR) in the prefrontal cortex and hippocampus were assessed using an NO assay kit, and immunohistochemistry and Western blots, respectively. We found that, compared to controls, L-Arg-treated rats received fewer foot shocks and made fewer errors to reach the learning criterion, and made fewer errors during the memory-testing session. In contrast, L-NAME-treated rats received more foot shocks and made more errors than controls to reach the learning criterion, and made more errors during the memory-testing session. In parallel, NO content in the prefrontal cortex and hippocampus was higher in L-Arg-treated rats and lower in L-NAME rats, compared to controls. Similarly, α7 nAChR immunoreactivity and protein expression in the prefrontal cortex and hippocampus were higher in L-Arg-treated rats and lower in L-NAME rats, compared to controls. These results suggest that the modulation of NO content in the brain correlates with α7 nAChR distribution and expression in the prefrontal cortex and hippocampus, as well as with learning and memory performance in the Y-maze.

The involvement of nitric oxide in the physiopathology of hepatoportal sclerosis

Background and aimHepatoportal sclerosis (HPS) is a clinical syndrome of unspecified etiology depicted by enlarged spleen and portal hypertension in the lack of other chronic liver disease findings, hematological disorders or any infectious disease in the liver. Nitric oxide (NO) molecule has many important functions in human body including phagocytosis in macrophages, neural transmission and endothelial relaxation. Although there is no data in literature that depicts the role of NO in HPS pathogenesis, this study was conducted in order to evaluate the potential role of NO in patients with HPS. Patients and methodsThe study participants included 24 HPS patients and 20 healthy controls. The median age of HPS and control patients was 41.2±13.9 and 46.5±12.4years, respectively. NO was predicted as nitric oxide metabolites (NOx) by Griess reaction after transformation of nitrate to nitrite by nitrate reductase using the commercially obtainable Nitric Oxide Assay Kit. ResultsSerum NOx levels were 2.69±2.98μmol/L and 0.85±1.05μmol/L for the HPS patients and controls, respectively. Serum NO levels were significantly higher in patients with HPS compared to the control group (p<0.001). ROC curve analysis suggested that the optimum NOx cut-off point for HPS was 1.305 with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 83.3%, 90 %, 90.9 %, and 81.8% respectively. ConclusionCirculating NO concentration was notably higher in patients with HPS in comparison to the control group. Our study verified that an elevated level of NO might have a role in the pathogenesis of HPS.

Effect Of L-arginine Supplementation On Nitric Oxide And VO2 Max In Healthy Males

INTRODUCTION: Previous research has focused on the effects of IV or orally administered L-arginine (L-arg) and its subsequent enzymatic reduction by nitric oxide synthase to nitric oxide (NO) in respect to benefits for patients with various forms of heart disease. However, L-arg supplementation has not been widely investigated in terms of its possible cardiovascular effect in healthy populations. The benefits of using this supplement in cardiovascular training are related to the vasodilatory effect of NO, and the possible increase in oxygen delivery to the muscles during exercise. PURPOSE: The purpose of this study was to investigate the effects of varying doses of oral L-arg supplementation on NO concentration and maximal oxygen consumption in normal healthy male subjects. METHODS: Twenty-five healthy males 18-30 yrs of age were recruited. Subjects completed an informed consent and health history questionnaire and were measured for anthropometric measured. Testing trials occurred on three separate occasions seven days apart with dosing (HD - high dose [6 g],LD - low dose [3 g], or PLA- placebo) randomly assigned. Testing trials consisted 3 blood draws: pre-supplementation, 25 min post-supplementation, a VO2 max test, and a post-exercise test. NO concentration was measured via venous blood draw and separated with a centrifuge for plasma. Plasma was then analyzed for NO utilizing a total nitric oxide assay kit. RESULTS: No significant effect was observed following L-arg supplementation at rest; however L-arg concentrations did increase from post-treatment to post-exercise (PLA: 29.69 mol/L vs 39.18 μµmol/L; LD: 27.63 μmol/L vs 32.65 μmol/L; HD: 19.53 μmol/L vs 45.58 μmol/L). L-arg supplementation did demonstrate statistical significance within the treatments (p< 0.05) on VO2 max scores within treatments (PLA= 47.524 ml/kg/min, LD= 48.46 ml/kg/min, HD= 48.54 ml/kg/min). CONCLUSIONS: Since no significance was observed when examining NO measures between treatments, the increase in VO2 max scores might be related to other SNS effects not related to NO concentrations. Further research is needed in this particular subject matter to further explain the physiological rationale for the positive impact that was demonstrated by L-arg on cardiovascular endurance and to provide further information in this area.

Nitric oxide production and its functional link with OIPK in tobacco defense response elicited by chitooligosaccharide

Chitooligosaccharide (COS) or oligochitosan has been shown to induce tobacco defense responses which are connected with nitric oxide (NO) and OIPK (oligochitosan-induced Ser/Thr protein kinase). The aim of this study was to reveal the relationship between NO production and OIPK pathway in the defense response of tobacco elicited by COS. NO generation was investigated by epidermal strip bioassay and fluorophore microscope using fluorophore diaminofluorescein diacetate (DAF-2DA). Tobacco epidermal cells treated with COS resulted in production of NO, which was first present in chloroplast, then in nucleus, finally in the whole cell; this NO production was sensitive to NO scavenger cPTIO and the mammalian NO synthase (NOS) inhibitor L: -NAME, suggesting that NOS-like enzyme maybe involved in NO generation in tobacco epidermal cells. However, NOS and nitrate reductase (NR, EC 1.6.6.1) inhibitors reduced NO content in tobacco leaves by using NO Assay Kit, suggesting both NOS and NR were involved in NO production in tobacco leaves. Using a pharmacological approach and western blotting, we provide evidence that NO acts upstream of OIPK expression. NO scavenger, NOS inhibitor partly blocked the activation of OIPK and the activities of several defense-related enzymes induced by COS; treatment with NO donor sodium nitroprusside (SNP) induced the activation of OIPK and enhanced the defense systems. The results suggest that COS is able to induce NO generation, which results in up-regulation the activities of some defense-related enzymes through an OIPK-dependent or independent pathway.

PPARγ agonist pioglitazone inhibits microglia inflammation by blocking p38 mitogen-activated protein kinase signaling pathways

The aim of this paper was to investigate the inhibitory effect of peroxisome proliferator-activated receptor-gamma (PPARγ) agonist pioglitazone on microglia inflammation induced by lipopolysaccharide (LPS). Highly aggressively proliferating immortalized cells were used from a rat microglial cell line. Expression of PPARγ, inducible NO synthase (iNOS), the p42/44 extracellular signal-regulated kinase (ERK) MAPKs, c-Jun NH2-terminal kinases (JNKs) and p38 MAPK were determined by Western blot analysis. The protein levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were determined by enzyme-linked immunosorbent assay. The production of nitric oxide (NO) was determined by a Nitric Oxide Assay Kit. The subcellular localization of PPARγ was studied by immunofluorescence microscopy analysis and nuclear-cytosolic fractionation technology, respectively. The transcriptional activity of PPARγ was detected by PPRE-Luciferase transcription assay. Pioglitazone effectively inhibited NO, iNOS, TNF-α, IL-6, IL-1β production in LPS-stimulated microglial cells. Additionally, pioglitazone suppressed PPARγ loss; enhanced transcriptional activity of PPARγ; and inhibited nucleus-export of PPARγ in microglia induced by LPS. And p38 MAPK inhibitor SB203580 had the similarity effects with pioglitazone. Signal transduction studies indicated that pioglitazone blocked the phosphorylation of p38 MAPK challenged by LPS. The results show that pioglitazone can inhibit LPS-stimulated microglia inflammation by blocking p38 MAPK signaling pathway.

C-peptide stimulates nitrites generation via the calcium-JAK2/STAT1 pathway in murine macrophage Raw264.7 cells

Aims C-peptide is a product of pro-insulin cleavage. Numerous studies have demonstrated that C-peptide, although not influencing blood glucose control, may play a role in preventing and potentially reversing some of the chronic complications of type 1 diabetes. The aim of this paper was to present a novel function of C-peptide, focusing on its role in nitric oxide (NO) generation. Main methods Murine macrophage Raw264.7 cells and primary peritoneal macrophages were incubated under control conditions, or with C-peptide. Expression level of iNOS and phosphorylation status of JAK2/STAT1 were analyzed by Western blot. Fluorometric NO assay kit was used to assess the concentration of nitrite in culture medium. Intracellular calcium concentration was measured with calcium indicator dyes, such as Fura-2 and Fluo-3 AM. Key findings C-peptide increased the level of nitrites in murine macrophage Raw264.7 cells. The nitrites production induced by lipopolysaccharide (LPS) was further enhanced by co-treatment of C-peptide. This up-regulation of nitrites generation also correlated with the induction of inducible nitric oxide synthase (iNOS), a prominent marker of macrophage activation. In addition, C-peptide increased the intracellular concentration of calcium levels. Moreover, C-peptide-induced nitrites generation and increase in calcium was observed in freshly isolated primary peritoneal macrophages. In addition, C-peptide specifically affected the Janus activated kinase (JAK)/signal transducer and activated transcription (STAT) pathway. Finally, C-peptide-mediated nitrites generation and JAK2/STAT1 phosphorylation were not detected in the presence of the intracellular calcium chelator, BAPTA-AM. Significance These results suggest that C-peptide may elicit immune modulatory function via modulation of the calcium/JAK–STAT pathway.

The effects of PDTC on interleukin-1β-induced nitric oxide production in chondrocytes

In order to find new drugs to inhibit nitric oxide (NO) production, the effects of pyrrolidine dithiocarbamate (PDTC), a nuclear factor-kappa B (NF-kappaB) inhibitor, on recombinant human interleukin-1beta (rhIL-1beta)-induced NO production in chondrocytes were investigated. Rat chondrocytes were isolated and cultured, divided into control, P0, P1, P2, P3 and P4 groups. The chondrocytes in the P0, P1, P2, P3 and P4 groups were treated with different concentrations of PDTC (0, 3, 10, 30, and 50 micromol/L respectively) for 1 h and then incubated with 5 U/mL rhIL-1beta for 24 h. NO assay kit and RT-PCR were used to detect the NO content and the iNOS mRNA expression in the chondrocytes. The expression level of iNOS mRNA in control, P0, P1, P2, P3 and P4 groups was 0.02+/-0.01, 1.24+/-0.13, 1.21+/-0.14, 0.61+/-0.11, 0.40+/-0.09, 0.21+/-0.06, and the relative content of NO was 15.8+/-2.7, 100+/-14.8, 92.6+/-9.3, 68.3+/-14.2, 27.5+/-9.8, 19.8+/-3.6, respectively. In the P0, P1, P2, P3 and P4 groups, the expression of iNOS mRNA and NO production were significantly increased as compared with those in the control group. As compared with the P0 group, the expression of iNOS mRNA and NO content in control group were lower. In the P2, P3 and P4 groups, PDTC could significantly inhibit the expression of iNOS and NO production induced by rhIL-1beta in a concentration-dependent manner. It is suggested that PDTC can inhibit NO production and iNOS mRNA expression induced by IL-1beta, which may provide an alternative method for the treatment of osteoarthritis.

Erythropoietin attenuates hypertrophy of neonatal rat cardiac myocytes induced by angiotensin‐II in vitro

Objective: Erythropoietin (EPO) is a haematopoietic hormone that has been confirmed as a novel cardioprotective agent. In this study, we test the hypothesis that EPO inhibits angiotensin‐II (Ang‐II)‐induced hypertrophy in cultured neonatal rat cardiomyocytes. Material and methods: Cultured neonatal rat cardiomyocytes were used to evaluate the effects of EPO on Ang‐II‐induced hypertrophy in vitro. The surface area and mRNA expression of atrial natriuretic (ANF) myocytes were employed to detect cardiac hypertrophy. A phosphatidylinositol 3′‐kinase (PI3K) inhibitor LY294002 and an endothelial nitric oxide synthase (eNOS) inhibitor l‐NAME were also employed to detect the underlying mechanism of EPO. Intracellular signal molecules, such as Akt (PKB), phosphorylated Akt, eNOS and transforming growth factor‐β1 (TGF‐β1) protein expression were determined by Western blot. Nitric oxide (NO) levels in the supernatant of cultured cardiomyocytes were assayed using an NO assay kit. Results: The results indicate that EPO significantly attenuates Ang‐II‐induced hypertrophy shown as inhibition of increases in cell surface area and ANF mRNA levels. NO production was also increased proportionally in the EPO‐treated group. EPO enhanced Akt activation and eNOS protein expression, whereas LY294002 or l‐NAME partially abolished the anti‐hypertrophic effect of EPO, accompanied by a decrease in Akt activation, eNOS protein expression and/or a reduction of NO production. EPO also down‐regulated the protein expression of TGF‐β1. Conclusion: We conclude that EPO attenuates cardiac hypertrophy via activation of the PI3K‐Akt‐eNOS‐NO pathway and the down‐regulation of TGF‐β1.

TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells

IntroductionTumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer.MethodsThe present study was designed to determine the effects of the NO-releasing prodrug O2-(2,4-dinitrophenyl) 1- [(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion and the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs were determined using Matrigel invasion assays, an MMP array kit and ELISAs. The activity and expression of extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases were determined using western blot analyses.ResultsUnder conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production. JS-43-126, a non-NO-releasing analog of JS-K, had no effect on NO levels or invasion. JS-K increased (P < 0.05) TIMP-2 production, and blocking TIMP-2 activity with a neutralizing antibody significantly increased (P < 0.05) the invasive activity of JS-K-treated cells across Matrigel. JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected.ConclusionWe report the novel findings that JS-K inhibits breast cancer invasion across the Matrigel basement membrane, and NO production is vital for this activity. Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects. JS-K and other NO prodrugs may represent an innovative biological approach in the prevention and treatment of metastatic breast cancer.

Caveolin-1 is important for nitric oxide-mediated angiogenesis in fibrin gels with human umbilical vein endothelial cells

The role of caveolin-1 (Cav-1) in angiogenesis remains poorly understood. The endothelial nitric oxide (NO) synthase (eNOS), a caveolin-interacting protein, was demonstrated to play a predominant role in vascular endothelial growth factor (VEGF) -induced angiogenesis. The purpose of our study was to examine the role of Cav-1 and the eNOS complex in NO-mediated angiogenesis. Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in 3-D fibrin gels to form capillary-like tubules by VEGF stimulation. The expression of Cav-1 and eNOS was detected by semiquantitative RT-PCR. The HUVEC were treated with antisense oligonucleotides to downregulate Cav-1 expression. Both transduced and non-infected HUVEC were cultured in fibrin gels in the presence or absence of VEGF (20 ng/mL) and NG-nitro-L-arginine methyl ester (L-NAME; 5 mmol/L). NO was measured using a NO assay kit and capillary-like tubules were quantified by tubule formation index using the Image J program. RT-PCR analysis revealed that Cav-1 levels steadily increased in a time-dependent manner and reached their maximum after 5 d of incubation, but there were no obvious changes in eNOS mRNA expression in response to VEGF in the fibrin gel model. VEGF (20 ng/mL) can promote NO production and the formation of capillary-like tubules, and this promoting effect of VEGF was blocked by the addition of L-NAME (5 mmol/L). When transduced HUVEC with the antisense Cav-1 oligonucleotides were plated in the fibrin gels, the capillary-like tubules were significantly fewer than those of the non-infected cells. The capillary-like tubules formation and NO production of transduced HUVEC with the antisense Cav-1 oligonucleotides cultured in fibrin gels showed no responses to the addition of VEGF (20 ng/mL) and L-NAME (5.0 mmol/L). NO was a critical angiogenic mediator in this model. Cav-1 was essential for NO-mediated angiogenesis and may be an important target of anti-angiogenesis therapy.

The Role of Nitric Oxide in γ-Globin Induction by Hydroxyurea.

Hydroxyurea (HU) has been shown to induce fetal hemoglobin (HbF) synthesis through activation of the soluble guanosine cyclase/cyclic guanosine monophosphate signaling pathway. The release of NO from HU by heme iron is thought to be involved in this mechanism of HbF induction. Studies completed in sickle cell patients confirmed increased serum NO levels after oral HU therapy but NO generation in red blood cells and the effect on γ-gene transcription have not been extensively investigated. Therefore, we performed studies to quantify NO generated by HU in K562 cells and normal erythroid progenitors as a mechanism for γ-globin activation. NO levels were measured after drug treatments using the Nitric Oxide Assay Kit (Calbiochem) and γ-globin mRNA was measured using quantitative PCR. HU (100μM) increased NO 1.4 to 1.8-fold at 24–72 hrs in K562 cells compared to a 2.0 to 2.5-fold increase in NO produced by the known NO donor, deta-nonoate (DE; 400μM). NO levels were also measured in erythroid progenitors grown in liquid cultures; a 1.6-fold increase in NO was produced by 30μM HU after 48 hrs with comparable increases produced by 200μM DE. To understand the effects of HU on normal NO synthesis from L-arginine through the action of NO synthase (NOS), we performed studies with the NOS inhibitor, NG-Monomethyl-L-arginine (L-NMMA). Interestingly, HU increased NO levels 2.5-fold at 24 hrs when combined L-NMMA compared a 1.4-fold increase produced by HU alone; this pattern persisted up to 72 hrs. Parallel with these findings γ-globin activation by HU was augmented approximately 25% by L-NMMA; DE combined with L-NMMA did not produce the same effect. These data suggest a novel mechanism for NOS regulation by HU compared to DE. Subsequent studies were completed to determine if HbF synthesis could be augmented by combining NO donors since they have different mechanisms of action. HbF levels in K562 cells were measured by ELISA (Bethyl Laboratories) and normalized by total hemoglobin and protein. Treatment with HU or DE increased HbF 3.6-fold and 4.6-fold respectively; when HU was combined with DE an additive 7.6-fold increase in HbF was produced. These data confirm that HU treatment lead to NO generation in K562 cells and normal erythroid progenitors which plays a role in its mechanism of γ-globin activation. HU combined with DE had an additive effect on HbF synthesis. These findings are relevant to current research efforts to develop novel HbF inducers for therapy in sickle cell patients.

Plasma levels of IL-10 and nitric oxide under two different anaesthesia regimens

An alteration in production of both interleukin-10 (IL-10) and nitric oxide (NO) has been found following surgical/anaesthesia trauma. It is also suggested that IL-10 could be an important factor in regulating NO metabolism during the postoperative period. Furthermore, NO seems to play a crucial role in the anaesthetic state. The purpose of this study was to investigate plasma levels of IL-10 and NO following surgery, any possible correlation between these two variables and whether anaesthesia technique could influence NO and IL-10 circulating concentrations. Thirty-two patients scheduled to undergo elective major surgery were enrolled in the study and allocated into two groups to receive two different techniques of anaesthesia, total intravenous (i.v.) anaesthesia (Group I) and inhalational anaesthesia (Group II). Blood samples were drawn before (t0), at the end (t1) of operation and after 24 h (t2). Plasma IL-10 and NO levels were measured by using an enzyme-linked-immunosorbent assay (ELISA) and a total NO assay kit, respectively. In both patient groups there was a significant decrease of plasma NO levels at the end of surgery (30.35 +/- 2.70 mmol L(-1) at t0 to 13.76 +/- 1.51 mmol L(-1) at t1 in Group I, P < 0.0001; 28.23 +/- 2.50 mmol L(-1) at t0 to 11.38 +/- 0.95 mmol L(-1) at t1 in Group II, P < 0.0001). This reduction remained at 24 h postoperatively (14.33 +/- 1.52 mmol L(-1) in Group I, P < 0.0001; 12.52 +/- 1.11 mmol L(-1) in Group II, P < 0.0001, both vs. t0). There was an increase in IL-10 concentrations (26.35 +/- 3.42 pg mL(-1) and 75.39 +/- 8.33 pg mL(-1) at t1 and t2, respectively, vs. 4.93 +/- 0.31 pg mL(-1) at t0, P = 0.03 and P < 0.0001, respectively, in Group I; 26.18 +/- 3.22 pg mL(-1) and 69.91 +/- 7.33 pg mL(-1) at t1 and t2, respectively, vs. 5.50 +/- 0.33 pg mL(-1) at t0, P = 0.02 and P < 0.0001, respectively, in Group II). No relationship was found between circulating IL-10 and NO. During the postoperative period, IL-10 overproduction does not correlate with the decrease in systemic NO concentration.