Inducible Isoform Of Nitric Oxide Synthase Research Articles

Analyzing the FMN-heme interdomain docking interactions in neuronal and inducible NOS isoforms by pulsed EPR experiments and conformational distribution modeling.

Nitric oxide synthases (NOSs), a family offlavo-hemoproteins with relatively rigid domains linked by flexible regions, require optimal FMN domain docking to the heme domain for efficient interdomain electron transfer (IET). To probe the FMN-heme interdomain docking, the magnetic dipole interactions between the FMN semiquinone radical (FMNH•) and the low-spin ferric heme centers in oxygenase/FMN (oxyFMN) constructs of neuronal and inducible NOS (nNOS and iNOS, respectively) were measured using the relaxation-induced dipolar modulation enhancement (RIDME) technique. The FMNH•RIDME data were analyzed using the mesoscale Monte Carlo calculations of conformational distributions of NOS, which were improved to account for the native degrees of freedom of the amino acid residues constituting the flexible interdomain tethers. This combined computational and experimentalanalysis allowed for the estimation of the stabilization energies and populations of the docking complexes of calmodulin (CaM) and the FMN domain with the heme domain. Moreover, combining the five-pulse and scaled four-pulse RIDME data into a single trace has significantly reduced the uncertainty in the estimated docking probabilities. The obtained FMN-heme domain docking energies for nNOS and iNOS were similar (-3.8kcal/mol), in agreement with the high degree of conservation of the FMN-heme domain docking interface between the NOS isoforms. In spite of the similar energetics, the FMN-heme domain docking probabilities in nNOS and iNOS oxyFMN were noticeably different (~ 0.19 and 0.23, respectively), likely due to differences in the lengths of the FMN-heme interdomain tethers and the docking interface topographies. The analysis based on the IET theory and RIDME experiments indicates that the variations in conformational dynamics may account for half of the difference in the FMN-heme IET rates between the two NOS isoforms.

New advances in the protective mechanisms of acidic pH after ischemia: Participation of NO

BackgroundIt has been previously demonstrated that the maintenance of ischemic acidic pH or the delay of intracellular pH recovery at the onset of reperfusion decreases ischemic-induced cardiomyocyte death. ObjectiveTo examine the role played by nitric oxide synthase (NOS)/NO-dependent pathways in the effects of acidic reperfusion in a regional ischemia model. MethodsIsolated rat hearts perfused by Langendorff technique were submitted to 40 min of left coronary artery occlusion followed by 60 min of reperfusion (IC). A group of hearts received an acid solution (pH = 6.4) during the first 2 min of reperfusion (AR) in absence or in presence of l-NAME (NOS inhibitor). Infarct size (IS) and myocardial function were determined. In cardiac homogenates, the expression of P-Akt, P-endothelial and inducible isoforms of NOS (P-eNOS and iNOS) and the level of 3-nitrotyrosine were measured. In isolated cardiomyocytes, the intracellular NO production was assessed by confocal microscopy, under control and acidic conditions. Mitochondrial swelling after Ca2+ addition and mitochondrial membrane potential (Δψ) were also determined under control and acidosis. ResultsAR decreased IS, improved postischemic myocardial function recovery, increased P-Akt and P-eNOS, and decreased iNOS and 3-nitrotyrosine. NO production increased while mitochondrial swelling and Δψ decreased in acidic conditions. l-NAME prevented the beneficial effects of AR. ConclusionsOur data strongly supports that a brief acidic reperfusion protects the myocardium against the ischemia-reperfusion injury through eNOS/NO-dependent pathways.

Role of the iNOS isoform in the cardiovascular dysfunctions of male rats with 6-OHDA-induced Parkinsonism

IntroductionAvailable studies have shown the involvement of nitric oxide (NO) in the processes that lead to neurodegeneration in Parkinson's disease (PD). Also, the use of inhibitors of the inducible isoform of NO-synthase (iNOS) promotes neuroprotection and attenuates dopamine (DA) loss in experimental models of Parkinsonism. In addition, NO also appears to be involved in cardiovascular changes in 6-hydroxydopamine (6-OHDA)-induced Parkinsonism. The current study aimed to evaluate the effects of iNOS inhibition on cardiovascular and autonomic function in animals that were subjected to Parkinsonism by the administration of 6-OHDA. Materials and methodsThe animals underwent stereotaxic surgery for bilateral microinfusion of the neurotoxin 6-OHDA (6 mg/mL in 0.2% ascorbic acid in sterile saline solution) or vehicle solution for the Sham group. From the day of stereotaxis until the day of femoral artery catheterization, the animals were treated with the iNOS inhibitor, S-methylisothiourea (SMT; 10 mg/kg; i. p.) or saline solution (0.9%; i. p.) for 7 days. The animals were divided into four groups: Sham-Saline, Sham-SMT, 6-OHDA-Saline, and 6-OHDA-SMT. Subsequent analyses were performed on these four groups. After 6 days, they underwent catheterization of the femoral artery, and 24 h later, mean arterial pressure (MAP) and heart rate (HR) were recorded. Another group of animals (the 6-OHDA and Sham groups) was assessed for aortic vascular reactivity after 7 days of bilateral infusion of 6-OHDA or vehicle, in which cumulative concentration-effect curves (CCEC) were made for phenylephrine (Phenyl), acetylcholine and sodium nitroprusside (NPS). Also, CCEC in the presence of Nw-nitro-arginine-methyl-ester (l-NAME) (10-5 M), SMT (10-6 M), and indomethacin (10-5 M) blockers were made. ResultsThe effectiveness of the 6-OHDA lesion was confirmed with the reduction of DA in 6-OHDA animals. However, treatment with SMT could not reverse the loss of DA. Concerning the baseline parameters, SBP and MAP values were lower in 6-OHDA animals compared to their Sham control, with no effect of treatment with SMT. In the analysis of SBP variability, a decrease in variance, the VLFabs component, and the LFabs component were observed in the 6-OHDA groups when compared to their controls, regardless of treatment with SMT. It was also observed that intravenous injections of SMT resulted in an increase in BP and a decrease in HR. However, the response was not different between the Sham and 6-OHDA groups. In vascular function, there was a hyporeactivity to Phenyl in the 6-OHDA group, and when investigating the mechanisms of this hyporeactivity, it was seen that the Rmax to Phenyl increased with incubation with SMT, indicating that iNOS could be involved in the vascular hyporeactivity of animals with Parkinsonism. ConclusionThus, the set of results presented in this study suggests that part of the cardiovascular dysfunction in animals subjected to 6-OHDA Parkinsonism may be peripheral and involve the participation of endothelial iNOS.

β-cell–selective inhibition of DNA damage response signaling by nitric oxide is associated with an attenuation in glucose uptake

Nitric oxide (NO) plays a dual role in regulating DNA damage response (DDR) signaling in pancreatic β-cells. As a genotoxic agent, NO activates two types of DDR signaling; however, when produced at micromolar levels by the inducible isoform of NO synthase, NO inhibits DDR signaling and DDR-induced apoptosis in a β-cell-selective manner. DDR signaling inhibition by NO correlates with mitochondrial oxidative metabolism inhibition and decreases in ATP and NAD+. Unlike most cell types, β-cells do not compensate for impaired mitochondrial oxidation by increasing glycolytic flux, and this metabolic inflexibility leads to a decrease in ATP and NAD+. Here, we used multiple analytical approaches to determine changes in intermediary metabolites in β-cells and non-β-cells treated with NO or complex I inhibitor rotenone. In addition to ATP and NAD+, glycolytic and tricarboxylic acid cycle intermediates as well as NADPH are significantly decreased in β-cells treated with NO or rotenone. Consistent with glucose-6-phosphate residing at the metabolic branchpoint for glycolysis and the pentose phosphate pathway (NADPH), we show that mitochondrial oxidation inhibitors limit glucose uptake in a β-cell-selective manner. Our findings indicate that the β-cell-selective inhibition of DDR signaling by NO is associated with a decrease in ATP to levels that fall significantly below the KM for ATP of glucokinase (glucose uptake) and suggest that this action places the β-cell in a state of suspended animation where it is metabolically inert until NO is removed, and metabolic function can be restored.

Peripheral Purinergic Modulation in Pediatric Orofacial Inflammatory Pain Affects Brainstem Nitroxidergic System: A Translational Research.

Physiology of orofacial pain pathways embraces primary afferent neurons, pathologic changes in the trigeminal ganglion, brainstem nociceptive neurons, and higher brain function regulating orofacial nociception. The goal of this study was to investigate the nitroxidergic system alteration at brainstem level (spinal trigeminal nucleus), and the role of peripheral P2 purinergic receptors in an experimental mouse model of pediatric inflammatory orofacial pain, to increase knowledge and supply information concerning orofacial pain in children and adolescents, like pediatric dentists and pathologists, as well as oro-maxillo-facial surgeons, may be asked to participate in the treatment of these patients. The experimental animals were treated subcutaneously in the perioral region with pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), a P2 receptor antagonist, 30 minutes before formalin injection. The pain-related behavior and the nitroxidergic system alterations in the spinal trigeminal nucleus using immunohistochemistry and western blotting analysis have been evaluated. The local administration of PPADS decreased the face-rubbing activity and the expression of both neuronal and inducible nitric oxide (NO) synthase isoforms in the spinal trigeminal nucleus. These results underline a relationship between orofacial inflammatory pain and nitroxidergic system in the spinal trigeminal nucleus and suggest a role of peripheral P2 receptors in trigeminal pain transmission influencing NO production at central level. In this way, orofacial pain physiology should be elucidated and applied to clinical practice in the future.

Characterization of the L-Arginine/Nitric Oxide Pathway and Oxidative Stress in Pediatric Patients with Atopic Diseases.

Introduction: L-Arginine (Arg) is a semi-essential amino acid. Constitutive and inducible nitric oxide synthase (NOS) isoforms convert Arg to nitric oxide (NO), a potent vaso- and bronchodilator with multiple biological functions. Atopic dermatitis (AD) and bronchial asthma (BA) are atopic diseases affecting many children globally. Several studies analyzed NO in airways, yet the systemic synthesis of NO in AD and BA in children with BA, AD or both is elusive. Methods: In a multicenter study, blood and urine were obtained from 130 of 302 participating children for the measurement of metabolites of the Arg/NO pathway (BA 31.5%; AD 5.4%; AD + BA 36.1%; attention deficit hyperactivity disorder (ADHD) 12.3%). In plasma and urine amino acids Arg and homoarginine (hArg), both substrates of NOS, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), both inhibitors of NOS, dimethylamine (DMA), and nitrite and nitrate, were measured by gas chromatography–mass spectrometry. Malondialdehyde (MDA) was measured in plasma and urine samples to evaluate possible effects of oxidative stress. Results: There were no differences in the Arg/NO pathway between the groups of children with different atopic diseases. In comparison to children with ADHD, children with AD, BA or AD and BA had higher plasma nitrite (p < 0.001) and nitrate (p < 0.001) concentrations, suggesting higher systemic NO synthesis in AD and BA. Urinary excretion of DMA was also higher (p = 0.028) in AD and BA compared to patients with ADHD, suggesting elevated ADMA metabolization. Discussion/Conclusion: The Arg/NO pathway is activated in atopic diseases independent of severity. Systemic NO synthesis is increased in children with an atopic disease. Plasma and urinary MDA levels did not differ between the groups, suggesting no effect of oxidative stress on the Arg/NO pathway in atopic diseases.

How do we kNOw the individual contribution of eNOS and nNOS for cerebral blood flow regulation?

How do we kNOw the individual contribution of eNOS and nNOS for cerebral blood flow regulation?

Peculiarities of influence of NO-synthase inhibitors on behavioral parameters of rats

Aim. A comparative study of the influence of nitric oxide synthase (NO-synthase) inhibitors on the parameters of anxiety, motor activity and pain sensitivity of rats.&#x0D; Materials and Methods. The work was conducted on male rats of Wistar line. The anxiety level and locomotor activity of rats were studied in the elevated plus maze (EPM) test. Pain sensitivity of the animals was tested on the hotplate apparatus. In the work, selective inhibitor of inducible isoform of NO-synthase aminoguanidine at a dose of 50 mg/kg, and non-selective inhibitor of this enzyme N-nitro-L-arginine at a dose of 50 mg/kg, were used. Rats of the control group were introduced the equivalent quantity of normal saline. &#x0D; NO-synthase inducible inhibitor aminoguadinine did not produce any influence on the anxiety level, but led to reduction of the horizontal motor activity of rats. Introduction of non-selective NO-synthase inhibitor N-nitro-L-arginine was accompanied by reduction of the anxiety and of the locomotor activity of animals in the EPM test. Both investigated NO-synthase inhibitors induced alteration of pain sensitivity of rats in the form of hypoalgesia. Here, the most pronounced nociceptive effect was observed with introduction of non-selective NO-synthase inhibitor.&#x0D; Conclusion. In the work the evidence of participation of inducible isoform of NO-synthase in realization of the motor activity and pain sensitivity processes in rats is shown. In result of the conducted experiments it was found that introduction of non-selective NO-synthase inhibitor N-nitro-L-arginine was accompanied by evident alterations of anxious behavior, locomotor activity and nociceptive sensitivity of rats. The results obtained confirm the important role of the system of regulation of nitric oxide synthesis in neurochemical mechanisms of behavioral reactions in rats.

Evaluation of the modulation of nitric oxide synthase expression in the cerebellum of diabetic albino rats and the possible protective effect of ferulic acid

IntroductionDiabetes mellitus is a multisystem disease. Oxidative stress and nitric oxide isoforms are involved in diabetic pathogenesis. Ferulic acid is a natural substance that is distributed broadly in plants with strong potent properties. The aim of the researchThis research was designed to study the possible protective role of ferulic acid on oxidative stress and different Nitric oxide synthase isoforms (NOS) in the cerebellum of streptozotocin-induced diabetic rats. Materials and methodsTwenty-four albino male rats were randomly divided into equal four groups: control group, group 2 received ferulic acid orally (10 mg/kg), group 3 diabetic group, group 4 diabetic rats received ferulic acid. After 8 weeks, the left cerebellar hemisphere was taken for tissue homogenate for oxidative markers and real-time PCR for NOS isoforms. Paraffin sections of the right cerebellar hemisphere were stained with cresyl violet, Luxol fast blue and immnunohistochemically stained for neuronal NOS, inducible NOS and endothelial NOS. ResultsDegenerative changes were seen in the cerebella of the diabetic rats with significant elevation of Malondialdehyde, Nitric Oxide, and decrease of Superoxide dismutase levels. nNOS expression decreased and iNOS expression increased significantly. The ferulic acid-treated group showed a reduction of the degenerative changes in the cerebellum with significant improvement in oxidative stress marker, an increase of nNOS expression, and a decrease of iNOS expression. ConclusionsFerulic acid improves cerebellar functional and histopathological changes induced by diabetes which can be attributed mainly to its anti-oxidative effect and its ability to modulate NOS isoforms.

Hypoxia‐inducible factor‐1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n=5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.

ROLE OF THE REACTIVE OXYGEN AND NITROGEN SPECIES IN THE DEVELOPMENT OF FIBROSIS IN THE TESTES OF RATS WITH PROLONGED CENTRAL DEPRIVATION OF TESTOSTERONE SYNTHESIS

Prostate cancer is the second most common diagnosis in oncology and ranks the fifth among the causes of mortality in men around the world. 1,276,106 cases of newly diagnosed prostate cancer were recorded in 2018. Androgens play a large role in the development of prostate cancer. Elimination of androgens and blockade of their synthesis are key pathogenetic steps in the treatment of prostate cancer. Metabolic and morphological changes in the testes under prolonged central deprivation of testosterone synthesis are currently not well understood. The aim of this study was to establish the role of reactive oxygen and nitrogen species in the development of morphological changes in the testes of rats under prolonged central deprivation of testosterone synthesis. Materials and methods. The experiments were conducted on 20 sexually mature male Wistar rats. Animals were divided into 4 groups of 5 animals in each. The first group (control) received a subcutaneous injection of 0.9% sodium chloride for 180 days. The second group received a subcutaneous injection of diphereline (triptoreline) in a dose of 0.3 mg/kg of active agent for 30 days in order to simulate the central deprivation of testosterone synthesis. In the third group, diphereline was administered in a dose of 0.3 mg/kg of the active agent for 90 days. The fourth group received a subcutaneous injection of diphereline in a dose of 0.3 mg/kg of the active agent for 180 days. Small pieces of testes were fixed and enclosed in paraffin blocks; then 4-μm thick sections were made and stained with hematoxylin and eosin. Histological preparations were studied using an optical microscope with a digital microphotographic nozzle Olympus C 3040-ADU with programs adapted for these studies. The total activity of NO synthases (gNOS), the activity of the inducible isoform of NO synthase (iNOS) and constitutive isoforms (cNOS), as well as the production of superoxide anion radical (O2•-) were determined in 10% homogenate of testes. Results. Long-term central deprivation of testosterone synthesis leads to an increase in the interstitial space volume with the development of fibrotic changes in the testes on the 180th day of the experiment. The wall of seminiferous tubules was compacted, swollen, there were a large number of parietal macrophages from the outside compared to previous periods of the experiment. In the structure of some convoluted seminiferous tubules, first there was discompletion and disorientation, and then desquamation of spermatids. The number of spermatogonia type A and B decreased. Hypochromia and pycnosis were noted in the nuclei. The activities of gNOS and iNOS increased until the 90th day and decreased by 180th when compared with 90th day, remaining higher than the activities of the control group. Activity of cNOS was reduced in all experimental groups. O2•- production increased in all experimental groups. Conclusion: an increase in the production of nitric oxide by the inducible isoform of NO synthase leads to destructive changes in the testes of rats with the subsequent development of fibrotic changes on the 180th day of central deprivation of testosterone synthesis by enhancing the production of superoxide anion radical.

The role of gas mediators of nitrogen (II) oxide and hydrogen sulfide in the development of pathochemical changes in the mucous membrane of rats at water-immobilization and adrenaline-induced stress modeling

Acute and prolonged psycho-emotional overstrain, i.e. stress, are the most frequent factors of ulcer formation in the digestive system. Therefore, the study of molecular mechanisms of stress impact is one of the most promising directions of modern experimental gastroenterology. However, the features of its molecular-biochemical action on the metabolic processes in the digestive system remain insufficiently studied. In this regard, we performed a comparative analysis of changes in indicators of systems of gas mediators of nitrogen (II) oxide and hydrogen sulfide synthesis at water-immobilization and adrenaline-induced stress modeling in experiments on white non-linear rats. Water-immobilization stress (WIS) was modelled by immobilizing animals in a plastic container, and adrenaline-induced stress (AIS) was modelled by injecting adrenaline at a dose of 2 mg/kg intraperitoneally. Modelling WIS caused formation of erosion and spot hemorrhage, located mainly along the folds of the fundus of the stomach. In this case, more noticeable changes were observed in the 5-hour WSI model. Injecting adrenaline at AIS model caused development of structural-hemorrhagic damage in the pyloric part and body of the stomach. The biochemical response to stress is complex, and the release of catecholamines is not the only case of stress. The synthesis of hormones such as glucagon, somatotropin and renin is activated. However, the most important role is played by cortisol, which level of growth in blood is measured to assess the degree of stress development. In our studies, changes in cortisol level in blood under different types of stress modeling (WIS and AIS) had their own peculiarities: at WIS conditions cortisol concentration increased sharply and remained high for 5 hours, while adrenaline did not cause the growth of this “stress hormone”. In our studies, in both models of stress-induced ulcerogenesis there were significant changes to the content of H2S and NO, that confirms the significant role of these substances in development and progression of ulcerogenesis in the digestive system. Thus, H2S concentration decreased at WIS and AIS. There is a significant increase in nitrogen oxide production in both WIS with different duration of action and AIS, which is caused by multiple activation of the inducible isoform of NO-synthase. Therefore, in experimental stress-induced ulcerogenesis, the metabolism of L-arginine in the mucous membrane of stomach is equally shifted towards the formation of NO, that under conditions of strengthening of free radical processes serves as a prerequisite for activation of oxidative-nitric processes and leads to the formation of structural-hemorrhagic damage to the surface of the mucous membrane of stomach. Our studies also show that ulcerative damage to the mucous membrane of stomach in all the types of studied stress was accompanied by an increase in myeloperoxidase activity, indicating an increase in permeability of hemocapillaries due to the development of the inflammatory process.

INFLUENCE OF NANOSIZED SILICA OXIDE SUSPENSION ON NITRIC OXIDE PRODUCTION IN RATS TESTES DURING CHRONIC NITRATE-FLUORIDE INTOXICATION

In the Poltava region of Ukraine there is a high possibility of combined excessive intake of nitrates and fluorides, since the waters of the Buchaksʹkii aquifer are rich in fluorine ions, moreover, the Poltava region is a region with a developed agriculture.&#x0D; The purpose of this work is to determine the effect of nanosized silicon oxide suspension on the changes in the activity of nitric oxide cycle components, components, and, namely the total activity of NO-synthases (NOS), the activity of constitutive forms of NOS (cNOS), inducible form of NOS (iNOS), nitrate reductase, nitrite reductase, nitrite reductase, nitrite reductase in the testes of rats under the conditions of chronic combined nitrate-fluorodide intoxication.&#x0D; Materials and methods. The study was conducted on 15 mature male Wistar rats weighing 200-253 g. The animals were randomly divided into 3 groups of 5 animals in each. The first group received daily saline solution through a gastric tube. The second group (chronic combined intoxication group) received sodium nitrate and fluoride in a dosage of 500 mg / kg of sodium nitrate and 10 mg / kg of sodium fluoride. The third group against the background of chronic combined intoxication modelling additionally received a suspension of nanosized silicon oxide is a dosage of 100 mg / kg of active substance.&#x0D; Results. Chronic combined intoxication increases total NOS activity by 65.9% compared to the control group. The increase in NO production by NOS is the result of a 4-fold increase in iNOS activity, since the activity of cNOS under these conditions does not change significantly. Nitrite reductase-dependent NO production increases by 66.7%, nitrate reductase activity increases by 77.8%. Nitrite content in the testes homogenate grows in 2.17 times. Hydrolytic cleavage of L-arginine by arginase increases in 2.88 times. The introduction of nanosized silicon oxide suspension under the conditions of chronic combined intoxication did not statistically significantly change the total NOS activity and cNOS activity compared to the group exposed to chronic intoxication. However, iNOS activity reduces in 3.69 times. Nitrate reductase activity decreases in 1.6 times, while nitrite reductase activity decreases in 45%. The activity of arginases lowered in 2.38 times. The nitrite content of the testes cut down by 23.6%.&#x0D; Conclusions. Chronic combined nitrate-fluoride intoxication leads to the development of hyperproduction of nitric oxide in the testes of rats by activating the inducible isoform of NO synthase and nitrite reductases. The use of nanosized silica suspension during chronic combined intoxication is an effective method to correct nitric oxide overproduction in the testes of rats.

Pregnant mice submitted to Surgical Embryo Euthanasia (SEE) induce loss of expression of inducible nitric oxide synthase isoform and NO concentration in the maternal-fetal interface

The well-succeeded pregnancy in humans and rodents is the consequence of close interaction between maternal and fetal cells with intervening of cytokines and chemical mediators. In this process a pregnant uterus subset NK cells - uterine Natural Killer cells (uNK cells) play a pivotal modulatory role under the influence of local physiological hypoxia and other alterations. The aim of the present work was to evaluate the expression and commitment of induced form of nitric oxide synthase (iNOS) and NO concentration in the homeostasis of pregnant uterus. It was used normal pregnant mice on gd 10th and those submitted to surgical intervention to induce mechanical lesion in the embryos (SEE). Uterine samples were collected at 0.5, 1, 2 and 6 h after embryo lesion and processed for paraffin embedding and tissue homogenate. The samples destinate for paraffin embedding was performed the Dolichos biflorus (DBA) lectin cytochemistry and anti-iNOS immunocytochemistry. The samples destinate to tissue homogenates were processed for SDS-PAGE and Western-blot using anti-iNOS and evaluate of NO concentration. The embryo-injured uterine segments showed hyperemia and hemorrhage at mesometrial region in which the DBA lectin reaction showed altered uNK cells suggesting the degranulation. Positive reaction with anti-iNOS was seen on uNK cells, trophoblast giant cells, endometrial stromal and decidual cells and smooth muscle cells in the normal pregnant uterus, but 1 and 2 h after embryo lesion, the iNOS labeling decreased or was absent only in uNK cells. The same results was obtained with NO concentration. These results confirm the unique constitutive expression of iNOS in the pregnant mice uterus, being the uNK cells the only one responsive against stress of embryo failure, besides showing that excessive NO produced by quick activation of uNK-iNOS should affect the local vascular permeability.

Esculentoside B inhibits inflammatory response through JNK and downstream NF-κB signaling pathway in LPS-triggered murine macrophage RAW 264.7 cells

Natural compound esculentoside B (EsB), (2S,4aR,6aR,6aS,6bR,8aR,9R,10R,11S,12aR,14bS)-11-hydroxy-9-(hydroxymethyl)-2 methoxycarbonyl-2,6a,6b,9,12a-pentamethyl-10-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid with molecular weight of 664.833, isolated from roots of Phytolacca acinosa Roxb has been widely used as a constituent of traditional Chinese medicine (TCM). However, the anti-inflammatory capacity of EsB has not been reported yet. Therefore, the objective of this study was to investigate anti-inflammatory activities of EsB in LPS-treated macrophage RAW 264.7 cells. EsB could inhibit nitric oxide (NO) production. EsB also suppressed gene and protein expression levels of inducible isoform of NO synthase (NOS) and cyclooxygenase-2 in a dose-dependent manner. In addition, EsB decreased gene expression and protein secretion levels of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6. EsB remarkably suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) from cytosolic space. Phosphorylation of IκB was also inhibited by EsB. Moreover, EsB specifically down-regulated phospho-c-Jun N-terminal kinase (p-JNK), but not p-p38 or phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2). Taken together, these results suggest that EsB has inhibitory effect on inflammatory response by inactivating NF-κB and p-JNK. It could be used as a new modulatory drug for effective treatment of inflammation-related diseases.

Involvement of inducible nitric oxide synthase (iNOS) in immune-functioning of Paphia malabarica (Chemnitz, 1782)

In recent years, the role of inducible nitric oxide synthase (iNOS) isoform has been widely studied because of its immunological relevance in higher organisms as well as invertebrates including bivalves. However, little is known about the immunological role of iNOS in Paphia malabarica defense mechanism. In this study, we immunodetected the presence of iNOS in P. malabarica hemocytes using antibody N9657 monoclonal anti-nitric oxide synthase. In addition, increased iNOS activity was evident in response to a higher bacterial dosage (Vibrio parahaemolyticus and V. cholerae), highlighting the dose-dependent iNOS activity induction. Also, higher bacterial survivability was observed in the presence of iNOS inhibitor, i.e., S-methylisothiourea hemisulphate (SMIS) thus, validating the bactericidal role of iNOS. These findings implicate the involvement of iNOS in immune-functioning of P. malabarica. Future work should focus on elucidating the expression and regulation of pathogenesis in P. malabarica, involving iNOS.

Alterations in Arginase-NO-synthase System of Spermatozoa in Human Subjects with Different Fertility Potential.

SummaryBackgroundInfertility is an important worldwide problem which affects 10–15% of couples globally. Altered NO production has also been implicated in the pathogenesis of the male infertility. The present study was designed to evaluate the changes in the activity of NO-synthase (NOS) and arginase in spermatozoa of patients with infertility.MethodsThe total NOS, Ca2+-dependent constitutive (cNOS) and Ca2+-independent inducible (iNOS) activity and arginase activity were assessed in sperm cells of patients with different forms of pathospermia.ResultsWe found a significant increase in iNOS activity, but significantly decreased cNOS and arginase activity in sperm cells of infertile men vs fertile, normozoospermic men (p<0.001). The arginase/NOS ratio significantly decreased compared to control group. The iNOS/cNOS ratio was drastically increased in patients with decreased fertility potential indicating predominance of iNOS. Men with leuko cytospermia were distinguished to have the most express iNOS activity.ConclusionsThese observations provide evidence for a disturbed balance between the L-arginine metabolic pathways in sperm cells of infertile men. This imbalance includes the considerable activation of the inducible isoform of NO-synthase accompanied by significant inhibition of its constitutive isoform which indicates disturbances in NO production. In patients with decreased fertility potential the arginase/NOS was shifted towards predominance of iNOS-derived NO production.

A role for endothelial nitric oxide synthase in intestinal stem cell proliferation and mesenchymal colorectal cancer

BackgroundNitric oxide (NO) has been highlighted as an important agent in cancer-related events. Although the inducible nitric oxide synthase (iNOS) isoform has received most attention, recent studies in the literature indicate that the endothelial isoenzyme (eNOS) can also modulate different tumor processes including resistance, angiogenesis, invasion, and metastasis. However, the role of eNOS in cancer stem cell (CSC) biology and mesenchymal tumors is unknown.ResultsHere, we show that eNOS was significantly upregulated in VilCreERT2Apcfl/+ and VilCreERT2Apcfl/fl mouse intestinal tissue, with intense immunostaining in hyperproliferative crypts. Similarly, the more invasive VilCreERT2Apcfl/+Ptenfl/+ mouse model showed an overexpression of eNOS in intestinal tumors whereas this isoform was not expressed in normal tissue. However, none of the three models showed iNOS expression. Notably, when 40 human colorectal tumors were classified into different clinically relevant molecular subtypes, high eNOS expression was found in the poor relapse-free and overall survival mesenchymal subtype, whereas iNOS was absent. Furthermore, Apcfl/fl organoids overexpressed eNOS compared with wild-type organoids and NO depletion with the scavenger carboxy-PTIO (c-PTIO) decreased the proliferation and the expression of stem-cell markers, such as Lgr5, Troy, Vav3, and Slc14a1, in these intestinal organoids. Moreover, specific NO depletion also decreased the expression of CSC-related proteins in human colorectal cancer cells such as β-catenin and Bmi1, impairing the CSC phenotype. To rule out the contribution of iNOS in this effect, we established an iNOS-knockdown colorectal cancer cell line. NO-depleted cells showed a decreased capacity to form tumors and c-PTIO treatment in vivo showed an antitumoral effect in a xenograft mouse model.ConclusionOur data support that eNOS upregulation occurs after Apc loss, emerging as an unexpected potential new target in poor-prognosis mesenchymal colorectal tumors, where NO scavenging could represent an interesting therapeutic alternative to targeting the CSC subpopulation.

Anti-inflammatory effects of olive-derived hydroxytyrosol on lipopolysaccharide-induced inflammation in RAW264.7 cells.

The control of inflammation, which arises from complex biological responses to harmful stimuli, is an important determinant of both clinical outcomes and patient comfort. However, the side effects of many current therapies such as non-steroidal anti-inflammatory drugs mean that new safe treatments are required. We previously reported that 12.5 µg/ml hydroxytyrosol (HT) suppressed gene expression of the inducible nitric oxide (NO) synthase (iNOS) isoform and NO production, in mouse peritoneal macrophages treated with lipopolysaccharide (LPS), where nuclear factor-κB (NF-κB) gene expression was not altered. The present study evaluated the anti-inflammatory effects of various concentrations of HT in LPS-induced RAW264.7 mouse macrophages. HT suppressed NF-κB signaling and downregulated LPS-mediated expression of iNOS, cyclooxygenase-2, tumor necrosis factor alpha, and interleukin-1β at 12.5 µg/ml, resulting in reduced production of NO and prostaglandin E2. At lower concentrations, HT seemed to act via another signaling pathway to regulate the inflammatory response. In contrast, HT did not suppress LPS-induced expression of phosphorylated p44/42 mitogen-activated protein kinase. This study showed that HT had anti-inflammatory effects on LPS-stimulated RAW264.7 cells. HT is already available as a nutritional supplement and no toxic effects have been reported. Hence, HT represents a potential novel anti-inflammatory agent.

Aster yomena extract ameliorates pro-inflammatory immune response by suppressing NF-κB activation in RAW 264.7 cells

BackgroundAster yomena, an edible vegetable, is a perennial herb found in Korea, China, Japan, and Siberia. It is used as folk medicine to treat cough, bronchial asthma, and insect bites. A. yomena was recently shown to have antioxidant and anti-asthmatic activities. Studies have not yet evaluated the anti-inflammatory effects of the various solvent fractions of A. yomena. We investigated the anti-inflammatory activity of various solvent fractions (hexane, dichloromethane, ethyl acetate, and butanol) from ethanol extract of A. yomena in activated macrophages. MethodsAnti-inflammatory effects of A. yomena were investigated to determine the inhibitory effects of A. yomena against inflammation using RAW 264.7 mouse macrophages. To measure the effects of A. yomena on inflammatory mediators and cytokines, we used the following methods: cell viability assay, flow cytometry assay, ELISA assay, real-time PCR and western blotting. ResultsThe dichloromethane fraction exhibited marked anti-inflammatory activities by inhibiting nitric oxide (NO) and prostaglandin E2 production and mRNA expression of inducible isoforms of NO synthase, cyclooxygenase-2, and cytokines (tumor necrosis factor α, interleukin (IL)-6, IL-1β) in response to lipopolysaccharide (LPS) stimulation. Moreover, dichloromethane fraction from A. yomena significantly inhibited the transactivation of nuclear factor (NF)-κB and the nuclear translocation of the NF-κB p50 and p65 subunits. ConclusionThese results suggest that A. yomena may have anti-inflammatory activity in vitro, suggesting this herb could be a source of natural anti-inflammatory agents.