Form Of Nitric Oxide Synthase Research Articles

Down-regulation of neuronal form of Nitric oxide synthase in the Nurse cell of Trichinella spiralis.

The free radical nitric oxide (NO) and Ca2+ are critical regulators of skeletal muscle exercise performance and fatigue. The major source of NO in skeletal muscle cells is the neuronal form of the enzyme Nitric oxide synthase (nNOS). One of the most peculiar characteristics of the Nurse cell of Trichinella spiralis (T. spiralis) is the complete loss of the contractile capabilities of its derivative striated muscle fiber. The aim of the present study was to clarify the expression of nNOS protein and mRNA in striated muscles during the muscle phase of T. spiralis infection in mice. Muscle tissue samples were collected from mice at days 0, 14, 24, and 35 post infection (d.p.i.). The expression of nNOS was investigated by immunohistochemistry, and the expression levels of mRNA of mouse Nitric oxide synthase 1 (Nos1) by real-time PCR. The presence of nNOS protein was still well observable in the disintegrated sarcoplasm at the early stage of infection. The cytoplasm of the developing and mature Nurse cell showed the absence of this protein. At least at the beginning of the Nurse cell development, Trichinella uses the same repairing process of skeletal muscle cell, induced after any trauma and this corroborates very well our results concerning the nNOS expression on day 14 p.i. At a later stage, however, we could suggest that the down-regulation of nNOS in the Nurse cell of T. spiralis either serves a protective function or is an outcome of the genetic identity of the Nurse cell.

2-Styrylchromones Prevent IL-1β-Induced Pro-Inflammatory Activation of Fibroblast-like Synoviocytes while Increasing COX-2 Expression

Rheumatoid arthritis (RA) is characterized by systemic immune and chronic inflammatory features, leading to the destruction of the joints. Presently, there are no effective drugs able to control synovitis and catabolism in the process of RA. 2-Styrylchromones (2-SC) are a small group of compounds characterized by the attachment of a styryl group to the chromone core that have already been associated to a wide range of biological activities, including antioxidant and anti-inflammatory activities. The present study investigated the effect of a set of six 2-SC on the interleukin-1β (IL-1β)-induced increase of nitric oxide (•NO), inducible form of nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-3 (MMP-3) expression levels in human fibroblast-like synoviocytes (HFLS), pointing to the role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation in the process. From a set of six 2-SC, presenting hydroxy and methoxy substituents, the one presenting two methoxy substituents at C-5 and C-7 of A ring and a catechol group on B ring, significantly reduced •NO production and the expression of its inducible synthase (iNOS). It also significantly reduced the catabolic MMP-3 protein expression. This 2-SC inhibited the NF-κB pathway by reversing the IL-1β - induced levels of cytoplasmatic NF-kB inhibitor alpha (IκBα), and decreasing the p65 nuclear levels, suggesting the involvement of these pathways in the observed effects. The same 2-SC significantly increased the COX-2 expression, which may indicate a negative feedback loop mechanism of action. The properties of 2-SC may be of great value in the development of new therapies with improved efficacy and selectivity towards RA, and thus deserve further exploitation and evaluation to disclose the full potential of 2-SC.

Administration of Different Doses of Acrylamide Changed the Chemical Coding of Enteric Neurons in the Jejunum in Gilts.

Excessive consumption of highly processed foods, such as chips, crisps, biscuits and coffee, exposes the human to different doses of acrylamide. This chemical compound has a multidirectional, adverse effect on human and animal health, including the central and peripheral nervous systems. In this study, we examined the effect of different doses of acrylamide on the enteric nervous system (ENS) of the porcine jejunum. Namely, we took into account the quantitative changes of neurons located in the jejunum wall expressing substance P (SP), galanin (GAL), a neuronal form of nitric oxide synthase (nNOS), the vesicular acetylcholine transporter (VAChT) and cocaine- and amphetamine-regulated transcript (CART). The obtained results indicate that acrylamide causes a statistically significant increase in the number of neurons immunoreactive to SP, GAL, VAChT and CART in all types of examined enteric plexuses and a significant drop in the population of nNOS-positive enteric neurons. Changes were significantly greater in the case of a high dose of acrylamide intoxication. Our results indicate that acrylamide is not indifferent to ENS neurons. A 28-day intoxication with this substance caused marked changes in the chemical coding of ENS neurons in the porcine jejunum.

Особенности механизмов NO-опосредованной дилатации пиальных артерий на воздействие ацетилхолина у стареющих крыс

The article reports the results of the study focusing on age-related changes in the contribution of NO, synthesized by various forms of nitric oxide synthases (NOS) and exogenous L-arginine in acetylcholine-induced dilation of cerebral vessels in rats. A comparative assessment of the responses of pial arteries of various diameters to acetylcholine chloride (ACh, 10–7 M, 8 min) was carried in the absence and in the presence of NOS blockade and exogenous L-arginine (0.25 mM, 30 min) in Sprague-Dawley rats aged 4 and 18 months. NOS blockade was performed by using a non-selective NOS inhibitor (L-NAME, 10–3 M, 12 min) and an inducible NOS inhibitor (aminoguanidine, 1 mM, 10 min). The change in the number and degree of arterial dilation was assessed by measuring the width of the erythrocyte flow in three separate groups of arteries: small (<20 μm in diameter), medium (20–40 μm), and large (>40 μm). It was found that in 18-month-old rats, the contribution of NO, synthesized by constitutive forms of NOS, decreases in the implementation of ACh-mediated endothelium-dependent dilatatory reactions in small and medium pial arteries, while an increase was observed for pial. At the same time, the contribution of inducible NOS to dilation of all types of arteries is on the increase. These processes are accompanied by a decrease in the bioavailability of exogenous L-arginine for NOS.

Kisspeptin signaling and nNOS neurons in the VMHvl modulate lordosis behavior but not mate preference in female mice

It was recently shown that kisspeptin neurons in the anteroventral periventricular area (AVPV) orchestrate female sexual behavior, including lordosis behavior and mate preference. A potential target of AVPV kisspeptin signaling could be neurons expressing the neuronal form of nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Therefore, in the present study, we further refined the role of the VHMvl in female sexual behavior. Adult female mice received a bilateral cannula aimed at the VMHvl. A single injection with kisspeptin (Kp-10) or SNAP/BAY, a nitric oxide donor, significantly increased lordosis, whereas the nNOS inhibitor l-NAME decreased it. None of these drugs affected mate preference. Interestingly, administration of GnRH into the VMHvl had no effect on lordosis or mate preference. To determine whether the stimulatory effect of Kp-10 on lordosis was specific to the VMHvl, an additional group of females received Kp-10 directly into the paraventricular nucleus (PVN). No effect was found on lordosis and mate preference. These results suggest that kisspeptin most likely modulates lordosis behavior through nNOS neurons in the VMHvl whereas mate preference is modulated by kisspeptin through a separate neuronal circuit not including the VMHvl.

Distribution of nitric oxide in the rock cavy (Kerodon rupestris) brain II: The brainstem

In a recent paper, we described the distribution of Nitric oxide (NO) in the diencephalon of the rock cavy (Kerodon rupestris). This present paper follows this work, showing the distribution of NO synthesizing neurons in the rock cavy's brainstem. For this, we used immunohistochemistry against the neuronal form of nitric oxide synthase (NOS) and NADPH diaphorase histochemistry. In contrast to the diencephalon in the rock cavy, where the NOS neurons were seen to be limited to some nuclei in the thalamus and hypothalamus, the distribution of NOS in the brainstem is widespread. Neurons immunoreactive to NOS (NOS-ir) were seen as rostral as the precommissural nuclei and as caudal as the caudal and gelatinous parts of the spinal trigeminal nucleus. Places such as the raphe nuclei, trigeminal complex, superior and inferior colliculus, oculomotor complex, periaqueductal grey matter, solitary tract nucleus, laterodorsal tegmental nucleus, pedunculopontine tegmental, and other nuclei of the reticular formation are among the locations with the most NOS-ir neurons. This distribution is similar, but with some differences, to those described for other rodents, indicating that NO also has an important role in rock cavy's physiology.

Citrulline Recycling Supports Nitric Oxide Biosynthesis in Mouse PAM212 Keratinocytes

Nitric oxide synthases generate nitric oxide and citrulline from arginine using NADPH and molecular oxygen as co-substrates. In most mammalian cells, arginine is an essential amino acid and limiting extracellular supplies of this amino acid can reduce nitric oxide biosynthesis. This can occur during inflammation and injury where a variety of factors can reduce available arginine including high levels of tissue arginase. In cells and tissue containing urea cycle enzymes, arginine can be synthesized from citrulline. Thus, in a rate limiting step, argininosuccinate synthetase catalyzes the conversion of citrulline and aspartate to argininosuccinate which is then metabolized to arginine via argininosuccinate lyase. In earlier studies we reported that, in response to γ-interferon, PAM212 keratinocytes readily produce nitric oxide in an arginine-dependent manner via an inducible form of nitric oxide synthase. Citrulline is formed in the cells during this process while arginine is metabolized. In the present studies we determined if citrulline was taken up by PAM212 keratinocytes and utilized for nitric oxide biosynthesis. We found that citrulline effectively supported nitric oxide production; approximately 10-fold higher concentrations of citrulline were required to produce nitric oxide when compared to arginine. Both citrulline and arginine were taken up by PAM212 keratinocytes; at equimolar concentrations (50 µM), more arginine than citrulline was taken up by the cells. However, at higher concentrations, uptake of these amino acids was saturable; the apparent Km and Vmax for arginine uptake was 170 µM and 1.2 nmol/106 cells/min, respectively, and citrulline uptake was 850 µM and 0.7 nmol/106 cells/min, respectively. Nitric oxide production supported by citrulline was inhibited by the nitric oxide synthase inhibitors, N-monomethylarginine, N-nitroarginine and N-nitroarginine benzyl ester. Each of these compounds also inhibited citrulline uptake into keratinocytes. Citrulline is known to be taken up by the L amino acid transporter, and nitric oxide synthase inhibitors compete with this transporter for uptake into keratinocytes. When pulse-labeled with 14C-citrulline, approximately 17% to 25% of the label accumulated as 14C-arginine in cytoplasmic pools of PAM212 cells demonstrating that these cells contain arginosuccinate synthetase/lyase activity. Nitric oxide synthase inhibitors had no effect on citrulline recycling in the cells. These data indicate citrulline can be used for nitric oxide biosynthesis in PAM212 keratinocytes. Citrulline is less efficient than arginine in supporting nitric oxide production due to limited uptake and slow conversion to arginine. In PAM212 keratinocytes, nitric oxide synthase inhibitors have multiple modes of action: inhibition of uptake of citrulline and nitric oxide synthase activity.

Vorinostat exhibits anticancer effects in triple-negative breast cancer cells by preventing nitric oxide-driven histone deacetylation.

Triple-negative breast cancers (TNBC) that produce nitric oxide (NO) are more aggressive, and the expression of the inducible form of nitric oxide synthase (NOS2) is a negative prognostic indicator. In these studies, we set out to investigate potential therapeutic strategies to counter the tumor-permissive properties of NO. We found that exposure to NO increased proliferation of TNBC cells and that treatment with the histone deacetylase inhibitor Vorinostat (SAHA) prevented this proliferation. When histone acetylation was measured in response to NO and/or SAHA, NO significantly decreased acetylation on histone 3 lysine 9 (H3K9ac) and SAHA increased H3K9ac. If NO and SAHA were sequentially administered to cells (in either order), an increase in acetylation was observed in all cases. Mechanistic studies suggest that the "deacetylase" activity of NO does not involve S-nitrosothiols or soluble guanylyl cyclase activation. The observed decrease in histone acetylation by NO required the interaction of NO with cellular iron pools and may be an overriding effect of NO-mediated increases in histone methylation at the same lysine residues. Our data revealed a novel pathway interaction of Vorinostat and provides new insight in therapeutic strategy for aggressive TNBCs.

Analysis of NOS Gene Polymorphisms in Relation to Cluster Headache and Predisposing Factors in Sweden.

Cluster headache is characterized by activation of the autonomic-trigeminal reflex. Nitric oxide can trigger headaches in patients, and nitric oxide signaling is known to be affected in cluster headache. Based on the hypothesis of nitric oxide being involved in cluster headache pathophysiology we investigated nitric oxide synthases as potential candidate genes for cluster headache. We analyzed eight variants in the three forms of nitric oxide synthase (NOS) genes, inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS), and tested for association with cluster headache. Swedish cluster headache patients (n = 542) and controls (n = 581) were genotyped using TaqMan® assays on an Applied Biosystems 7500 qPCR cycler. This is the largest performed genetic study on NOS involvement in cluster headache so far. We found an association between cluster headache and one iNOS haplotype consisting of the minor alleles of rs2297518 and rs2779249 (p = 0.022). In addition, one of the analyzed nNOS variants, rs2682826, was associated with reported triptan use (p = 0.039). Our data suggest that genetic variants in NOS genes do not have a strong influence on cluster headache pathophysiology, but that certain combinations of genetic variants in NOS genes may influence the risk of developing the disorder or triptan use.

Insights into the Light Response of Skeletonema marinoi: Involvement of Ovothiol.

Diatoms are one of the most widespread groups of microalgae on Earth. They possess extraordinary metabolic capabilities, including a great ability to adapt to different light conditions. Recently, we have discovered that the diatom Skeletonema marinoi produces the natural antioxidant ovothiol B, until then identified only in clams. In this study, we investigated the light-dependent modulation of ovothiol biosynthesis in S. marinoi. Diatoms were exposed to different light conditions, ranging from prolonged darkness to low or high light, also differing in the velocity of intensity increase (sinusoidal versus square-wave distribution). The expression of the gene encoding the key ovothiol biosynthetic enzyme, ovoA, was upregulated by high sinusoidal light mimicking natural conditions. Under this situation higher levels of reactive oxygen species and nitric oxide as well as ovothiol and glutathione increase were detected. No ovoA modulation was observed under prolonged darkness nor low sinusoidal light. Unnatural conditions such as continuous square-wave light induced a very high oxidative stress leading to a drop in cell growth, without enhancing ovoA gene expression. Only one of the inducible forms of nitric oxide synthase, nos2, was upregulated by light with consequent production of NO under sinusoidal light and darkness conditions. Our data suggest that ovothiol biosynthesis is triggered by a combined light stress caused by natural distribution and increased photon flux density, with no influence from the daily light dose. These results open new perspectives for the biotechnological production of ovothiols, which are receiving a great interest for their biological activities in human model systems.

P0953NITRIC OXIDE AND LEFT VENTRICULAR HYPERTROPHY IN PATIENTS WITH CHRONIC RENAL FAILURE

Abstract Background and Aims Nitric oxide, nitric oxide, nitric oxide or NO is an endogenous molecule released by endothelial cells, macrophages, liver cells and neurons. There are three forms of nitric oxide synthase in the body. : the inducible form (iNO), the neuronal form and the endothelial form (eNO). In this work we are interested in (iNO) in patients with CKD and their impact on cardiac complications. Method A study of 202 patients with chronic renal failure at different stages (2.3.4.5 and 5D) was performed in our department. We investigated the correlation between mean (iNO) values in patients with CKD and its relationship with left ventricular hypertrophy. Results The mean age of the patients is 56.5 ± 17 years with a sex ratio of 1.43. The mean value of (iNO) is 65.82 ± 1.06 μmol / l. Its value increases significantly with the degradation of renal function (p <0.05) (stage 2 (61.75 ± 2.34), stage 3 (63.91 ± 2.21). ), stage 4 (64.08 ± 2.18), stage 5 (67 ± 2.7) and stage 5D (72.47 ± 2.18μmol / l). It is found that the mean value of nitric oxide NO is slightly higher in patients with HVG + ( 67.58 ± 1.48) versus HVG-patients (63.1 ± 1.39) (p <0.05).Discussion: iNOS secreted nitric oxide (NO) initiates cardiac remodeling to lead to left ventricular hypertrophy and cardiac fibrosis Conclusion Cardiac complications are the leading cause of death in patients with chronic renal failure. oxidative stress is a risk factor for these cardiac complications, iNOS accelerates cardiac aging.

How can nitric oxide help osteogenesis?

Nitric oxide (NO) is endogenously produced free radical that plays important biological roles, such as, the promotion of vasodilation, angiogenesis, tissue repair, wound healing process, antioxidant, antitumoral and antimicrobial actions. Although the regenerative effects of NO in soft tissues have been extensively reported, its role in bone tissue repair has not been completely addressed. Both constitutive and inducible forms of NO synthase (NOS) are expressed in bone-derived cells, and some important cytokines, such as IL-1 and TNF, are potent stimulators of NO production. The effects of NO on bone tissue are dependent on its concentration. NO has dichotomous biological effects, at low concentrations (pico-nano molar range), NO may promote proliferation, differentiation and survival of osteoblasts, whereas at high concentrations (micromolar range) NO may inhibit bone resorption and formation. Therefore, at a certain concentration range, NO can avoid osteoclast-mediated bone resorption and promote osteoblast growth. Due to the potential beneficial effects of NO in bone tissue regeneration, the exogenous administration of NO might find important biomedical applications. As NO is a free radical and a gas, the administration of NO donors/generators has been explored in tissue repair. The delivery of NO to the bone using macromolecular NO releasing scaffolds has been shown to increase osteogenesis with a relevant impact in dental and orthopedist areas. In this sense, this review presents and discusses the recent and important progresses in the effects of NO/NO donors in bone tissue, and highlights the promising approach in the design and use of NO donors allied to biomaterials in the sustained and localized NO release for bone tissue regeneration.

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.

Abnormalities in endothelial form of nitric oxide synthase is pathogenic in limited cutaneous systemic sclerosis.

Limited cutaneous systemic sclerosis is one subtype of systemic sclerosis which is characterized by a prototypic multisystem fibrotic disorder. This study aimed to further investigate the pathological mechanism of limited cutaneous systemic sclerosis (lcSSc). The dataset GSE76807 generated from 10 lcSSc patients and five healthy controls was used. After the preprocessing of the original data, differentially expressed genes (DEGs) were identified and then performed functional analysis, protein-protein interaction (PPI) network and module analysis. Additionally, the transcription factors (TFs) and miRNAs which potentially regulating DEGs were identified and the co-regulatory network was constructed. Finally, DEGs targeted by current drugs were identified. Real-time quantitative PCR analyses of some DEGs in mice with lcSSc were performed. Total 203 up-regulated and 189 down-regulated DEGs were obtained. The up-regulated genes were enriched in protein interactions at the synapses neuronal system, NCAM1 interactions, and CREB phosphorylation through the activation of CaMKII, while, cilium assembly, and endothelial form of nitric oxide synthase (eNOS) activation were enriched by down-regulated genes. SCRT2 and RABEP1 regulated by miR-218 were hub nodes in the network. DRD4 and GRIN2D were the main drug targets. RABEP1 and SSB were found lowly expressed in mice model with lcSSc. Endothelial form of NOS activation would be suppressed, and the process of neuronal migration and outgrowth would be activated to participant in the pathological mechanism of lcSSc.

The Role of Kisspeptin in Sexual Behavior.

Sexual behavior is essential for the perpetuation of a species. In female rodents, mate preference and lordosis behavior depend heavily on the integration of olfactory cues into the neuroendocrine brain, yet its underlying neural circuits are not well understood. We previously revealed that kisspeptin neurons in the anteroventral periventricular nucleus/periventricular nucleus continuum (AVPv/PeN) are activated by male olfactory cues in female mice. Here, we further reveal that male-directed mate preferences and lordosis are impaired in kisspeptin knockout mice but are rescued by a single injection with kisspeptin. Acute ablation of AVPV/PeN kisspeptin neurons in adult females impaired mate preference and lordosis behavior. Conversely, optogenetic activation of these neurons triggered lordosis behavior. Kisspeptin neurons act through classical GPR54/GnRH signaling in stimulating mate preferences, but unexpectedly, GPR54/GnRH neuronal ablation did not affect lordosis behavior. Therefore, to identify the downstream components of the neural circuit involved in lordosis behavior, we employed genetic transsynaptic tracing in combination with viral tract tracing from AVPV/PeN kisspeptin neurons. We observed that kisspeptin neurons are communicating with neurons expressing the neuronal form of nitric oxide synthase. These results suggest that hypothalamic nitric oxide signaling is an important mechanism downstream of kisspeptin neurons in the neural circuit governing lordosis behavior in female mice.

Role of nitric oxide in pathogenesis of tumor growth and its possible application in cancer treatment.

In this review, the role of nitric oxide (NO) in the pathogenesis of the tumor growth and possibilities of its application in the treatment of cancer patients are analyzed. NO is one of the most important mediators of physiological processes being involved in the regulation of practically all body functions in health and disease. The role of NO in the development of many pathological conditions has been extensively studied and debated in recent years. Today it is clear that NO in relation to malignant tumors may exhibit a dual activity- can stimulate tumor growth and cause an opposite antitumor effect. Effects of NO are mostly dependent on its concentration. At low concentrations, NO could inhibit apoptosis and cause mutations that potentially lead to the formation of malignant growth loci. However, a high concentration of NO appears to be detrimental to malignant cells, in particular under conditions of simultaneous exposure to ionizing radiation. In humans, the inducible NO synthase (iNOS, type II) is the most powerful form of NO synthases (NOS) and has the ability to synthesize large amounts of NO for a long time and exert a protective function. iNOS is expressed in macrophages, monocytes, neutrophils, fibroblasts, hepatocytes, and other cell types. In tissue of malignant tumors, the macrophagal iNOS is the main form. Experimental data provide an evidence that activated macrophages and leukocytes, which are the part of peritumorous inflammatory infiltrate, can provide radiosensitization of tumors by direct synthesis of NO and indirectly- through the secretion of cytokines stimulating iNOS activity in cancer cells. Such approach could be useful for the development of new schemes and methods of anticancer therapy based on the activation of endogenous NO biosynthesis pathways.

PUFA-Plasmalogens Attenuate the LPS-Induced Nitric Oxide Production by Inhibiting the NF-kB, p38 MAPK and JNK Pathways in Microglial Cells

The special lipids plasmalogens (Pls) were reported to be reduced in the neurodegenerative brains such as Alzheimer’s disease where a marked increase of glial activation is often observed. We previously found that a reduction of brain Pls can enhance the glial activation in murine brains. However, the detailed role of Pls in the prevention of glial activation was mostly elusive. Here we report that the Pls, extracted from scallop (sPls), significantly inhibited the inducible form of nitric oxide synthase (NOS2) and the production of NO in LPS (lipopolysaccharide)-activated microglial cells. We also observed that the polyunsaturated docosahexaenoic acid (DHA)-containing Pls but not the monounsaturated oleic acid-containing Pls attenuated the NOS2 induction. In addition, sPls blocked the activation of nuclear factor (NF)-kB and mitogen-activated protein kinases (MAPKs) e.g., JNK and p38 MAPK, thereby attenuated the nuclear translocation of NF-kB subunit, p65, and activator protein-1 (AP-1) proteins (c-Fos and c-Jun). Interestingly, LPS treatments suppressed the expression of Pls synthesizing enzymes, glycerone phosphate O-acyltransferase (GNPAT) and alkylglycerone phosphate synthase (AGPS) in the microglial cells by the p38MAPK and JNK pathways. Furthermore, the knockdown of GNPAT and AGPS genes by sh-RNAs accelerated the LPS-induced activation of p38MAPK and JNK, resulting in the increased production of NO. These findings suggested that a decrease of brain Pls can activate the NF-kB, p38MAPK and JNK pathways to induce a prolonged microglial activation which may downplay the neuroprotective events in the brains of neurodegenerative diseases.

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen‐induced retinopathy: beneficial effect of the absence of AQP4

Hypoxia‐dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin‐4 (AQP4) is involved in the hypoxia‐dependent VEGF upregulation in the retina of a mouse model of oxygen‐induced retinopathy (OIR). The expression levels of VEGF, the hypoxia‐inducible factor‐1α (HIF‐1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF‐1 binding site (HBS) in the VEGF gene promoter, the binding of HIF‐1α to the HBS, the retinal vascularization and function have been determined in the retina of wild‐type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF‐1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF‐1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF‐1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF‐1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF‐induced retinal damage in response to hypoxia.

Artemin transiently increases iNOS expression in primary cultured trigeminal ganglion neurons.

Artemin, a member of the glial cell line-derived neurotrophic factor family, is an important cytokine and a critical participant in trigeminal pain disorders such as tongue pain and migraine. However, the mechanisms underlying artemin's activity are largely unknown. In the present study, we used primary cultured trigeminal ganglion neurons (TGNs) to determine the effect of artemin on the expression of the inducible form of nitric oxide synthase (iNOS), which is released in response to painful and inflammatory stimuli. Following artemin treatment, western blot analysis showed that the protein level of iNOS was transiently elevated after artemin treatment for 15min (p<0.05). Immunofluorescence revealed that both the expressions of iNOS and GFRα3 were significantly up-regulated after artemin treatment for 15min. In addition, iNOS expression induced by artemin was co-localized with GFRα3 and TUJ-1 in primary cultured TGNs, respectively. Our results indicate a previously unknown role of artemin in regulating iNOS expression in primary cultured TGNs, and regulation of iNOS might be involved in the mechanism through which artemin participates in the trigeminal pain pathway.

Kininase 1 As a Preclinical Therapeutic Target for Kinin B1 Receptor in Insulin Resistance.

Kinin B1 receptor (B1R) contributes to insulin resistance, an early event in type 2 diabetes, through the upregulation and activation of the inducible form of nitric oxide synthase (iNOS), pro-inflammatory cytokines and the oxidative stress. This study addresses the hypothesis that inhibition of kininase 1 (carboxypeptidase M, CPM), the key enzyme involved in the biosynthesis of B1R agonists, could exert the same beneficial effects to B1R antagonism in insulin resistance. Male Sprague-Dawley rats were made insulin resistant with a drinking solution containing 10% D-glucose for a period of 9 weeks. Control rats received tap water. During the last week, kininase 1 was blocked with Mergetpa (1 mg kg−1 twice daily, s.c.) and the impact was determined on insulin resistance (HOMA index), metabolic hormone levels, oxidative stress and the expression of several markers of inflammation by western blot and qRT-PCR. Glucose-fed rats displayed hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin resistance, hypertension, positive body weight gain, and enhanced expression of B1R, CPM, iNOS, and IL-1β in renal cortex, aorta and liver. Markers of oxidative stress (superoxide anion and nitrotyrosine expression) were also enhanced in aorta and renal cortex. Mergetpa reversed and normalized most of those alterations, but failed to affect leptin levels and hypertension. Pharmacological blockade of kininase 1 (CPM) exerted similar beneficial effects to a 1-week treatment with a B1R antagonist (SSR240612) or an iNOS inhibitor (1,400 W). These data reinforce the detrimental role of B1R in insulin resistance and recommend CPM as a new therapeutic target.