Intracellular Nitric Oxide Levels Research Articles

Nitric oxide signaling is involved in arsenic-induced guard cell death in Vicia faba L. (Fabaceae)

Nitric oxide (NO) is a key endogenous signaling molecule in the plant response to environmental stresses. Here, we show that NO is involved in arsenic (As) cytotoxicity in Vicia faba L. guard cells. The results showed that NaAsO2 (0.1–10 mg L−1) decreased cell viability and caused cell death in a concentration-dependent manner, accompanied by significant increases in intracellular NO, reactive oxygen species (ROS), and Ca2+ levels as well as nitrate reductase (NR) activity. Arsenic-induced cell death was blocked by NO antagonists (NO scavenger c-PTIO; nitrate reductase inhibitor NaN3), ROS scavengers (AsA or CAT), and Ca2+ antagonists (Ca2+ chelator EGTA; Ca2+ channel blocker LaCl3). In addition, NaN3 and CAT led to a significant decrease in the intracellular Ca2+ level, whereas LaCl3 and EGTA barely affected the intracellular NO and ROS levels. Our results demonstrated that increased levels of NO and ROS activate Ca2+ signaling to control responses to As cytotoxicity and that NR-dependent NO generation contributes to As toxicity in V. faba guard cells.

Implication of VEGF and aquaporin 4 mediating Müller cell swelling to diabetic retinal edema.

Aquaporin 4 (AQP4), a water channel protein, is known to be expressed in retinal Müller cells. The purpose of this study was to determine the effects of VEGF and AQP4 channels on the volumetric changes in Müller cells. Retinas from diabetic rats and a cultured Müller cell line, TR-MUL5, were used in this study. Intravitreal injections of VEGF or PBS were performed on either streptozotocin (STZ)-induced diabetic or normoglycemic rats. Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-AQP4, and anti-VEGF. VEGF protein levels from collected retinas were determined by western blot analysis. Volumetric changes and nitric oxide (NO) levels in cultured Müller cells were determined using flow cytometry (FACS), in the presence or absence of VEGF and TGN-020, a selective AQP4 inhibitor. In the diabetic rat retina, VEGF immunoreactivity was concentrated in the internal retinal layers, and AQP4 immunoreactivity was higher than controls. The expressions of AQP4 were colocalized with GFAP. Protein levels of VEGF in the hyperglycemic rat retina were significantly higher than controls. FACS analyses showed that exposure to VEGF enlarged Müller cells, while exposure to TGN-020 suppressed the enlargement. Intracellular levels of NO were increased after exposure to VEGF, which was suppressed following the addition of TGN-020. The observed Müller cell swelling is mediated by VEGF and AQP4.

The Influence of Spirulina platensis Filtrates on Caco-2 Proliferative Activity and Expression of Apoptosis-Related microRNAs and mRNA.

Spirulina platensis (SP) is a blue-green microalga that has recently raised attention not only as a nutritional component, but also as a source of bioactivities that have therapeutic effects and may find application in medicine, including cancer treatment. In the present study we determined the cytotoxic effect of S. platensis filtrates (SPF) on human colon cancer cell line Caco-2. Three concentrations of SPF were tested—1.25%, 2.5%, and 5% (v/v). We have found that the highest concentration of SPF exerts the strongest anti-proliferative and pro-apoptotic effect on Caco-2 cultures. The SPF negatively affected the morphology of Caco-2 causing colony shrinking and significant inhibition of metabolic and proliferative activity of cells. The wound-healing assay showed that the SPF impaired migratory capabilities of Caco-2. This observation was consistent with lowered mRNA levels for metalloproteinases. Furthermore, SPF decreased the transcript level of pro-survival genes (cyclin D1, surviving, and c-Myc) and reduced the autocrine secretion of Wnt-10b. The cytotoxic effect of SPF involved the modulation of the Bax and Bcl-2 ratio and a decrease of mitochondrial activity, and was related with increased levels of intracellular reactive oxygen species (ROS) and nitric oxide (NO). Moreover, the SPF also caused an increased number of cells in the apoptotic sub-G0 phase and up-regulated expression of mir-145, simultaneously decreasing expression of mir-17 and 146. Obtained results indicate that SPF can be considered as an agent with anti-cancer properties that may be used for colon cancer prevention and treatment.

PPARα Agonist Stimulated Angiogenesis by Improving Endothelial Precursor Cell Function Via a NLRP3 Inflammasome Pathway

Background: Impaired wound healing is a common complication of diabetes and is the leading cause of lower extremity amputation. Treatment with fenofibrate, a peroxisome proliferators–activated receptor α (PPARα) agonist, was associated with a lower risk of amputations, particularly minor amputations without known large-vessel diseases, probably through non-lipid mechanisms. The current study aimed to test our hypothesis that fenofibrate stimulates angiogenesis and restores endothelial precursor cell (EPC) function via inhibiting Nod-like receptor protein 3 (NLRP3) inflammasome in streptozotocin (STZ)-induced diabetic mice. Methods: Male C57BL/6 mice were randomly divided into three groups: control, STZ-induced diabetic mice and fenofibrate treated diabetic group. Wound closure was assessed by wound area and CD31 positive capillaries. Both the migration and tube formation capacities of EPCs were measured. Intracellular nitric oxide (NO) and superoxide (O₂^-) levels were determined. Activity of NLRP3 inflammasome in EPCs was assessed by measuring thioredoxin-interacting protein (TXNIP), NLRP3, and caspase-1 expression. Results: Compared with the untreated diabetic mice, wound closure and capillary densities were significantly increased in fenofibrate treated group. Fenofibrate treatment restored EPC function, increased NO production, and decreased O₂^- level in EPCs of diabetic mice. Furthermore, fenofibrate deregulated the activity of NLRP3 inflammasome by reducing TXNIP, NLRP3 and caspase-1 expression in EPCs of diabetic mice. In vitro, fenofibrate prevented high glucose induced EPC dysfunction, deregulated NLRP3 inflammasome activity. In addition, fenofibrate inhibited IL-1β expression caused by combination use of high glucose and lipopolysaccharide. Conclusion: Fenofibrate can accelerate wound healing in diabetic mice, which at least in part was mediated by improving the impaired EPC function via a NLRP3 inflammasome pathway, suggesting the significance of PPARα agonists in the treatment of diabetes.

Chronic Nonmodulated Microwave Radiations in Mice Produce Anxiety-like and Depression-like Behaviours and Calcium- and NO-related Biochemical Changes in the Brain.

The present study was aimed to investigate behavioural and biochemical effects of chronic exposure of amplitude modulated and non-modulated microwave radiation on laboratory mice. Chronic microwave exposures were executed with 2.45 GHz of either modulated (power density, 0.029 mW/cm2; specific absorption rate, 0.019 W/Kg with sinusoidal modulation of 400 Hz) or nonmodulated continuous sinusoidal wave (power density, 0.033 mW/cm2; specific absorption rate, 0.023 W/Kg) for 2 hrs daily for 1 month. Mice subjected to non-modulated microwave exposure had significantly increased acetylcholinesterase activity and increased intracellular calcium and nitric oxide levels in the cerebral cortex and hippocampus, and also had increased glucose and corticosterone levels in blood compared to control mice. These non-modulated microwave-exposed mice exhibited anxiety-like and depression-like behaviours. In contrast, mice exposed to modulated microwave for the same period did not show such changes in concomitant biochemical and behavioural analyses. These results suggest that chronic non-modulated microwave, but not modulated microwave, radiation may cause anxiety-like and depression-like behaviours and calcium- and NO-related biochemical changes in the brain.

Effect of N-Perfluorooctane on Hypoxia/Reoxygenation Injury in Human Umbilical Vein Endothelial Cells.

This study investigated the effect of n-perfluorooctane (PFC) on hypoxia/reoxygenation (H/R) injury in human umbilical vein endothelial cells (HUVECs). In this study, the H/R models were prepared by chemical methods (using dithionite solution). The experimental groups included the control group, the PFC group with a culture volume ratio of 10%, the H/R model group, and treatment groups with various doses of PFC + H/R (i.e., 5%, 10%, or 20% PFC by volume). The cell counting kit-8 (CCK-8) method was used to assay cell viability. Colorimetric assays were used to estimate the leakage of lactate dehydrogenase (LDH) in the medium, the levels of intracellular malondialdehyde (MDA) and nitric oxide (NO), and the activity of superoxide dismutase (SOD). Western blot was used to analyze the expression of the apoptosis-related protein cystine aspartate proteolytic enzyme 3 (caspase-3). Compared with the control group, every detected index of 10% PFC group had no statistical significance (p > 0.05). Compared with the model group, 10% and 20% PFC treatment groups could increase cell viability A, decrease the content of NO and reduce caspase-3 expression (p < 0.05); Every PFC treatment group could significantly reduce the release of LDH and the contents of MDA, and also increase the activities of SOD (p < 0.01). PFC has a significant protective effect on HUVEC H/R injury, which may be related to the inhibition of oxidative stress and inflammation and further enhance cell antioxidant and anti-apoptotic characteristics.

Signaling pathways involved in HSP32 induction by hyperbaric oxygen in rat spinal neurons

Spinal cord injury (SCI) is a debilitating disease, effective prevention measures are in desperate need. Our previous work found that hyperbaric oxygen (HBO) preconditioning significantly protected rats from SCI after stimulated diving, and in vitro study further testified that HBO protected primary cultured rat spinal neurons from oxidative insult and oxygen glucose deprivation injury via heat shock protein (HSP) 32 induction. In this study, underlying molecular mechanisms were further investigated. The results showed that a single exposure to HBO significantly increased intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO) and activated MEK1/2, ERK1/2, p38 MAPK, CREB, Bach1 and Nrf2. The induction of HSP32 by HBO was significantly reversed by pretreatment neurons with ROS scavenger N-Acetyl-L-cysteine, p38 MAPK inhibitor or Nrf2 gene knockdown, enhanced by MEK1/2 inhibitors or gene knockdown but not by ERK1/2 inhibitor. CREB knockdown did not change the expression of HSP32 induced by HBO. N-Acetyl-L-cysteine significantly inhibited the activation of MEK1/2, ERK1/2, p38 MAPK, and Nrf2. Activation of Nrf2 was significantly inhibited by p38 MAPK inhibitor and the nuclear export of Bach1 was significantly enhanced by MEK1/2 inhibitor. The results demonstrated that HBO induces HSP32 expression through a ROS/p38 MAPK/Nrf2 pathway and the MEK1/2/Bach1 pathway contributes to negative regulation in the process. More importantly, as we know, this is the first study to delineate that ERK1/2 is not the only physiological substrates of MEK1/2.

CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p<0.05), (ii) elevation in ΔΨm (p<0.05), (iii) increased OCR and ATP formation (p<0.05), (iv) increased intracellular NO levels (p<0.05), (v) increased mitochondrial ROS production (p<0.05), and (vi) increased susceptibility to rotenone (p<0.05). Treatment with isradipine was able to partially rescue these negative effects of CNTF-ACM (p<0.05). CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

Liposome-entrapped GABA modulates the expression of nNOS in NG108-15 cells

BackgroundLiposomes are concentric lipid vesicles that allow a sustained release of entrapped substances. GABA (γ-aminobutyric acid) is the most prevalent inhibitory neurotransmitter in the central nervous system. New methodUsing GABA-containing liposomes (GL) prepared by the freeze-thawing method, we determined the effect of sustained release of GABA on expression of neuronal nitric oxide synthase (nNOS) and GABAA receptor (GABAAR) in an in vitro neuronal model. ResultsNeuronal cell line NG108-15 treated with different doses of GL during 24h showed an increase in expression of GABAAR (54 and 50% with 10 and 20ng doses, respectively) and nNOS (138, 157 and 165% with 20, 50 and 100ng doses, respectively) compared with cells treated with empty liposomes (EL). Additionally, cells treated with 50ng of GL showed an increase in GABAAR (23%) after 1h followed by an increase in nNOS (55, 46 and 55%) at 8, 12 and 24h time points, respectively. Immunofluorescence experiments confirmed an increase in nNOS (134%) and basal intracellular levels of nitric oxide (84%) after GL treatment. Further, treatment of cells with GL showed a decrease in expression of a protein inhibitor of nNOS (PIN) (26, 66 and 57% with 20, 50 and 100ng doses respectively) compared with control. Comparison with existing methodsThis is first demonstration for the development of GL that allows sustained slow release of this neurotransmitter. ConclusionThese results suggest that a slow release of GABA can change the expression of nNOS possibly via alteration in PIN levels in neuronal cells.

Cloning and Characterization of Filamentous Fungal S-Nitrosoglutathione Reductase from Aspergillus nidulans.

S-Nitrosoglutathione reductase (GSNOR) metabolizes S-nitrosoglutathione (GSNO) and has been shown to play important roles in regulating cellular signaling and formulating host defense by modulating intracellular nitric oxide levels. The enzyme has been found in bacterial, yeast, mushroom, plant, and mammalian cells. However, to date, there is still no evidence of its occurrence in filamentous fungi. In this study, we cloned and investigated a GSNOR-like enzyme from the filamentous fungus Aspergillus nidulans. The enzyme occurred in native form as a homodimer and exhibited low thermal stability. GSNO was an ideal substrate for the enzyme. The apparent Km and kcat values were 0.55 mM and 34,100 min(-1), respectively. Substrate binding sites and catalytic center amino acid residues based on those from known GSNORs were conserved in this enzyme, and the corresponding roles were verified using site-directed mutagenesis. Therefore, we demonstrated the presence of GSNOR in a filamentous fungus for the first time.

New thiazolidinediones affect endothelial cell activation and angiogenesis

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-γ (PPARγ) agonists used in treating type 2 diabetes that may exhibit beneficial pleiotropic effects on endothelial cells. In this study, we characterized the effects of three new TZDs [GQ-32 (3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-thiazolidine-2,4-dione), GQ-169 (5-(4-chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione), and LYSO-7 (5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione)] on endothelial cells. The effects of the new TZDs were evaluated on the production of nitric oxide (NO) and reactive oxygen species (ROS), cell migration, tube formation and the gene expression of adhesion molecules and angiogenic mediators in human umbilical vein endothelial cells (HUVECs). PPARγ activation by new TZDs was addressed with a reporter gene assay. The three new TZDs activated PPARγ and suppressed the tumor necrosis factor α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. GQ-169 and LYSO-7 also inhibited the glucose-induced ROS production. Although NO production assessed with 4-amino-5-methylamino-2′,7′-difluorofluorescein-FM probe indicated that all tested TZDs enhanced intracellular levels of NO, only LYSO-7 treatment significantly increased the release of NO from HUVEC measured by chemiluminescence analysis of culture media. Additionally, GQ-32 and GQ-169 induced endothelial cell migration and tube formation by the up-regulation of angiogenic molecules expression, such as vascular endothelial growth factor A and interleukin 8. GQ-169 also increased the mRNA levels of basic fibroblast growth factor, and GQ-32 enhanced transforming growth factor-β expression. Together, the results of this study reveal that these new TZDs act as partial agonists of PPARγ and modulate endothelial cell activation and endothelial dysfunction besides to stimulate migration and tube formation.

Cross talk between polysulfide and nitric oxide in rat peritoneal mast cells.

The aim of this study was to define the effects of polysulfide on intracellular Ca(2+) concentration ([Ca(2+)]i) and the underlying machinery, especially from the hydrogen sulfide (H2S) and nitric oxide (NO) perspectives, in rat peritoneal mast cells. We found that a polysulfide donor, Na2S4, increased [Ca(2+)]i, which is both extracellular and intracellular Ca(2+) dependent. Intracellular Ca(2+) release induced by Na2S4 was attenuated by the addition of a ryanodine receptor blocker. A slow-releasing H2S donor, GYY4137, dose dependently increased [Ca(2+)]i that was independent from extracellular Ca(2+) influx. The GYY4137-induced [Ca(2+)]i release was partially attenuated in the presence of the ryanodine receptor blocker. Both polysulfide and H2S donors increased the intracellular NO levels in DAF-2-loaded mast cells, which were abolished by an NO scavenger, cPTIO. Inhibition of NO synthase (NOS) significantly abolished the polysulfide- or H2S-donor-induced [Ca(2+)]i elevation in the absence of extracellular Ca(2+) An NO donor, diethylamine (DEA) NONOate, increased [Ca(2+)]i in a concentration-dependent manner, in which both extracellular and intracellular Ca(2+) are associated. At higher concentrations, the DEA NONOate-induced [Ca(2+)]i increases were attenuated in the absence of extracellular Ca(2+) and by the addition of the ryanodine receptor blocker. H2S and NO dose dependently induced polysulfide production. Curiously, polysulfide, H2S, and NO donors had no effect on mast cell degranulation. Among synthases, cystathionine-γ-lyase, and neuronal NOS seemed to be the major H2S- and NO-producing synthases, respectively. These results indicate that polysulfide acts as a potential signaling molecule that regulates [Ca(2+)]i homeostasis in rat peritoneal mast cells via a cross talk with NO and H2S.

Short-term exercise training improves flow-mediated dilation and circulating angiogenic cell number in older sedentary adults.

Cardiovascular disease risk increases with age due, in part, to impaired endothelial function and decreased circulating angiogenic cell (CAC) number and function. We sought to determine if 10 days of aerobic exercise training improves endothelial function, CAC number, and intracellular redox balance in older sedentary adults. Eleven healthy subjects (4 men, 7 women), 61 ± 2 years of age participated in 60 min of aerobic exercise at 70% maximal oxygen consumption for 10 consecutive days while maintaining body weight. Before and after training, endothelial function was measured as flow-mediated dilation of the brachial artery and fasting blood was drawn to enumerate 3 CAC subtypes. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) in CD34+ CACs were measured using fluorescent probes and reinforced via real-time quantitative polymerase chain reaction. Flow-mediated dilation improved significantly following training (10% ± 1.3% before vs. 16% ± 1.4% after training; P < 0.05). Likewise, CD34+/KDR+ number increased 104% and KDR+ number increased 151% (P < 0.05 for both), although CD34+ number was not significantly altered (P > 0.05). Intracellular NO and ROS levels in CD34+ CACs were not different after training (P > 0.05 for both). Messenger RNA expression of SOD1, endothelial nitric oxide synthase, and NADPH oxidase 2 and neutrophil cytosolic factor 1 in CD34+ CACs was not significantly altered with training (P > 0.05). In conclusion, 10 consecutive days of aerobic exercise increased flow-mediated dilation and CAC number in older, previously sedentary adults, but did not affect intracellular redox balance in CD34+ CACs. Overall, these data indicate that even short-term aerobic exercise training can have a significant impact on cardiovascular disease risk factors.

Cadmium-induced cell killing in Sacharomyces cerevisiae involves increases in intracellular NO levels.

Cadmium is a widespread environmental pollutant and poses some potential risks to human health. However, the signaling events controlling cadmium toxicity are not fully understood. In this study, we examined the effect of cadmium chloride on cell viability and the intracellular nitric oxide (NO) level in yeast cells. The results showed that exposure of yeast cells to cadmium (0-100 μM) could induce cell killing with significantly increased intracellular NO levels. Morphological analysis of the nuclei with 4('),6-diamidino-2-phenylindole staining and DNA strand breaks analysis showed that cadmium at 50 μM can induce cell apoptosis in yeast cells. Treatment of yeast cells with cadmium (50 μM) and the nitric oxide scavenger c-PTIO [2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-1-oxyl-3-oxide; 0.2 mM] showed that c-PTIO attenuated the cadmium-induced cell killing. Our findings indicated that cadmium-induced yeast cell killing is mediated by a directly increased intracellular NO level.

Overexpression of uncoupling protein 2 inhibits the high glucose-induced apoptosis of human umbilical vein endothelial cells.

Ectopic apoptosis of vascular cells plays a critical role in the early stage development of diabetic retinopathy (DR). Uncoupling protein 2 (UCP2) is a mitochondrial modulator which protects against endothelial dysfunction. However, the role which UCP2 plays in endothelial apoptosis and its association with DR was unclear. In the present study, we investigated whether UCP2 functioned as an inhibitor of DR in endothelial cells. Firstly, we noted that in UCP2-knockout mice retinal cell death and damage in vivo was similar to that of db/db diabetic mice. Additionally, UCP2 knockdown induced caspase-3 activation and exaggerated high glucose (HG)-induced apoptosis of human umbilical vein endothelial cells (HUVECs). Conversely, adenovirus-mediated UCP2 overexpression inhibited the apoptosis of HUVECs and HG-induced caspase-3 activation. Furthermore, HG treatment resulted in the opening of the permeability transition pore (PTP) and liberation of cytochrome c from mitochondria to the cytosol in HUVECs. Notably, UCP2 overexpression inhibited these processes. Furthermore, adenovirus-mediated UCP2 overexpression led to a significant increase in intracellular nitric oxide (NO) levels and a decrease in reactive oxygen species (ROS) generation in HUVECs. Collectively, these data suggest that UCP2 plays an anti-apoptotic role in endothelial cells. Thus, we suggest that approaches which augment UCP2 expression in vascular endothelial cells aid in preventing the early stage development and progression of DR.

수은 독성에 대한 Schizosaccharomyces pombe 단백질2황화물이성질화효소 2의 저항성

The present work was undertaken to address the role of protein disulfide isomerase 2 (Pdi2) in the mercury-tolerance of Schizosaccharomyces pombe, using the Pdi2-overexpressing recombinant plasmid pYPDI2 and the corresponding vector plasmid pRS316. When exposed to mercuric chloride, the PDI2 overepxression cells grew significantly better than the vector control cells. They revealed the lower levels of intracellular reactive oxygen species (ROS) and nitric oxide (NO), when incubated with mercuric chloride for 6 h, than the vector control cells. The PDI2 overepxression cells contained the higher levels of total glutathione (GSH) and superoxide dismutase (SOD) activity than the vector control cells, after 6 h of incubation in mercuric chloride. However, the PDI2 overepxression cells contained similar levels of glutathione peroxidase (GPx) activities, compared to those of the vector control cells. Taken together, the S. pombe Pdi2 promotes the tolerance against mercury toxicity through up-regulating total GSH and SOD and subsequently attenuating ROS and NO elevations.

Interconnection between nitric oxide formation and hypersensitivity parameters under guinea pig model of acute asthma with multiple challenges

An immunoregulatory role of nitric oxide (NO) in the development of adaptive immune responses associated with allergic diseases is very important. The present study extended these observations by the examination of the reciprocal changes in characteristic immunologic parameters of the disease and NO level of bronchoalveolar lavage (BAL) cells under guinea pig model of acute asthma with multiple challenges. Development of guinea pig Th2 mediated asthma was accompanied by increasing the level of allergic markers: ovalbumin (OVA) specific IgG and IL-4. We demonstrated that the infiltrate of airway cells contributes to NO synthesis in the respiratory tract during allergic inflammation. The level of intracellular NO formation significantly correlated with plasma allergen specific IgG value in OVA-induced asthma. The presented data evidence that the elevated intracellular NO level in BAL fluid may reflect a nitrosative stress in respiratory tract in general, when allergic asthma exacerbation is present.

Pretreatment with β-Boswellic Acid Improves Blood Stasis Induced Endothelial Dysfunction: Role of eNOS Activation.

Vascular endothelial cells play an important role in modulating anti-thrombus and maintaining the natural function of vascular by secreting many active substances. β-boswellic acid (β-BA) is an active triterpenoid compound from the extract of boswellia serrate. In this study, it is demonstrated that β-BA ameliorates plasma coagulation parameters, protects endothelium from blood stasis induced injury and prevents blood stasis induced impairment of endothelium-dependent vasodilatation. Moreover, it is found that β-BA significantly increases nitric oxide (NO) and cyclic guanosine 3’, 5’-monophosphate (cGMP) levels in carotid aortas of blood stasis rats. To stimulate blood stasis-like conditions in vitro, human umbilical vein endothelial cells (HUVECs) were exposed to transient oxygen and glucose deprivation (OGD). Treatment of β-BA significantly increased intracellular NO level. Western blot and immunofluorescence as well as immunohistochemistry reveal that β-BA increases phosphorylation of enzyme nitric oxide synthase (eNOS) at Ser1177. In addition, β-BA mediated endothelium-dependent vasodilatation can be markedly blocked by eNOS inhibitor L-NAME in blood stasis rats. In OGD treated HUEVCs, the protective effect of β-BA is attenuated by knockdown of eNOS. In conclusion, the above findings provide convincing evidence for the protective effects of β-BA on blood stasis induced endothelial dysfunction by eNOS signaling pathway.

IL-1 provokes electrical abnormalities in rat atrial myocardium

Using a micro-electrode technique we studied the effects of interleukin 1α and interleukin 1β on bio-electric activity of rat atrial myocardium under normal conditions and after gradual stretching. Perfusion with interleukin 1α increased the duration of the action potential at the level of 90% re-polarization. Stretch induced tachy-arrhythmia in the presence of interleukin 1α is mainly regulated via stretch increased nitric oxide production, while the ionotropic effect of the interleukin-1α during stretching is not pronounced. The perfusion with interleukin 1β did not change the values of the duration of the action potentials at the levels of 25, 50 and 90% repolarization. The interleukin lβ caused an appearance of extra-systolic patterns which turned into normal rhythm, alternating with periods of normal activity. The total intracellular nitric oxide level induced by both interleukin 1β and stretching is balanced by interleukin-1β induced cation influx.

Polymorphisms In The Nitric-Oxide Synthase 2 Gene And Prostate Cancer Pathogenesis

Nitric-oxide synthase (NOS)-polymorphisms influence the cellular amount of NO, and are associated with disease-risk in many disorders. We investigated 145 SNP-polymorphisms and a (CCTTT)n-microsatellite in the NOS2-gene in 3161 prostate-cancer patients and 2149 controls from a Swedish population-based GWAS-study. To analyze possible associations between NOS2-polymorphisms, prostate cancer, and prostate cancer pathogenesis. Two groups were analyzed, those with advanced tumours (Gleason≥6), and those with tumours of mixed Gleason-statues. Affymetrix 5.0-chip (SNP-polymorphisms), DNA Fragment-analysis and Sequencing ((CCTTT)n-microsatellite) were used for genotyping. Genotypes were combined with information on tumour stage, Gleason, PSA, metastases and cancer-specific death, using clinical follow-up. We divided the (CCTTT)n-alleles into short (S, n≤10), intermediate (M, n=11-12) and long (L, n≥13). Patients homozygous for longer repeats (LL) had decreased risk of highly aggressive (Gleason ≥7;PSA>20;T3+) tumours (OR:0.40;CI:0.14-1.08;p=0.071), but, once ill they showed a threefold increased risk of dying in prostate cancer (HR:3.31;CI:0.85-12.85;p=0.084), compared to SS-homozygotes. The SNP-alleles that co-varied with the (CCTTT)l-allele also had lower risk of aggressive tumours, as well as, once ill, a 2-4 times higher risk of dying (p=0.009). Also the proportion of patients with lymph node metastases increased with length of the (CCTTT)n-alleles of the patients (SS<SM<SL<MM<ML <LL)(trend analysis; p=0.033). Nitric oxide can induce proliferation as well as apoptosis depending on cellular context. Our results suggest that NOS2 polymorphisms may influence the risk of aggressive prostate cancer and that these polymorphisms could have an impact on disease pathogenesis, possibly by affecting intracellular nitric oxide levels.