Changes In Nitric Oxide Synthase Research Articles

Asymmetric and symmetric dimethylarginine gingival crevicular fluid levels in periodontitis.

This study aimed to evaluate the level of ADMA (asymmetric dimethylarginine), SDMA (symmetric dimethylarginine), and IL-1β (Interleukin-1β) in gingival crevicular fluid (GCF) from periodontitis patients and control subjects. ADMA and SDMA are potentially hazardous non-proteinogenic amino acids that limit nitric oxide (NO) synthesis and have many functions in various human disorders. ADMA causes a structural change in nitric oxide synthase, while SDMA blocks arginine cell uptake. Increased plasma ADMA has been widely recognized as a "trigger" initiating impaired NO bioavailability and vascular dysfunction, which ultimately leads to oxidative stress. Twenty-five patients with periodontitis (P) (Stage III, Grade C, n=25) and 20 control (C) subjects were included in the study. The IL-1β level of GCF was measured by enzyme immunoassay (ELISA) and ADMA and SDMA by liquid chromatography-mass spectrometry (LC-MS/MS). Periodontitis patients had higher clinical parameters than controls (p < .001). Levels of IL-1β, ADMA and SDMA GCF were statistically significantly higher in group P than in group C (respectively; p=.003, p < .0001, p < .0001). There was no difference in the ADMA/SDMA ratio (p=.312) between the groups. There were significant positive correlations between clinical periodontal parameters and IL-1β, ADMA, and SDMA levels (p < .05). ADMA and SDMA levels were significantly correlated with IL-1β (p < .05). These findings suggest that ADMA and SDMA may be involved in the pathogenesis of the periodontal disease.

Quantitative Assessment of the Choroidal Vessel Diameter during the Recovery of Form-Deprivation Myopia in Guinea Pigs

Purpose The development and recovery (REC) of myopia is associated with changing of choroidal thickness (CT) in the model of guinea pigs. Nitric oxide synthase (NOS) is an enzyme which can affect choroidal vasodilatation. This study wants to investigate the changes of choroidal vessel diameter (CVD) and NOS during the REC of form-deprivation (FD) myopia in guinea pigs. Methods Forty-eight guinea pigs were randomly assigned to the normal control (NC) group, FD group (FD for 21 d), and four REC groups: REC1/2 group (removal the deprivation and re-exposure to the normal environment for 1/2 d), REC1 group (1 d), REC 2 group (2 d), and REC7 group (7 d). CT was measured by optical coherence tomography (OCT), and CVD of foveal choroid was quantitatively assessed on OCT angiography images using MATLAB software at each time point. NOS in choroid was measured using enzyme-linked immunosorbent assays. Measurements were compared between groups and correlations between CT, CVD, and NOS were assessed using regression analyses. Results CVD and CT in FD group were significantly smaller than in NC group (both p < .05), while the NOS significantly larger (p < .001). When deprivation was removed, CVD and NOS were significantly larger and reached a peak in the REC1 group, while CT reached the peak in the REC2 group, then all gradually decreased, and no significant differences were observed in NC and REC7 group (all p > .05). In the REC and NC groups, there was a significant positive correlation between CVD and NOS (p < .001), CVD and CT (p = .0092), but no correlation was found between NOS and CT (p > .05). Conclusions This study indicated that the CVD in guinea pigs could be significantly dilated following myopia REC, and this change coincides with changes in NOS and CT.

In vitro antitumor activity of nano-pulse stimulation on human anaplastic thyroid cancer cells through nitric oxide-dependent mechanisms

Although most thyroid cancer patients have good clinical outcomes, there are still no effective therapies for patients with anaplastic thyroid cancers (ATC). Nano-pulse stimulation (NPS) is increasingly recognized as a non-thermal ablation technology that selectively targeting cancer cells. The aim of this study was to investigate the in vitro antitumor mechanism of NPS on human ATC cells. The cell line 8305C were treated with NPS and the CCK-8 assay, clonogenic assay, apoptosis assay and nitric oxide synthase (NOS) activity assay were used to test cell proliferation, apoptosis and NOS changes respectively. Our results showed that NPS can significantly (P < 0.05) inhibit ATC cell proliferation and induce apoptosis in a field strength-dependent manner. Moreover, the NOS activity in NPS treated groups obviously increased (P < 0.05). Further analysis indicated that NPS induced an inducible nitric oxide synthase (iNOS)-dependent killing in ATC cells. Finally, our research reveals for the first time that NPS is an effective non-thermal candidate for the treatment of ATC and supports a new possible calcium independent mechanism involvement in NPS treatment.

Omega-3 fatty acids differentially alter the expression of detoxification enzymes and nitric oxide bioavailability in endothelial cells during IL-6 exposure

Abstract Background Atherosclerotic plaques can elaborate reactive oxygen species (ROS) that reduce nitric oxide (NO) bioavailability. Cellular detoxification enzymes including various peroxiredoxin (PRDX) and superoxide dismutase (SOD) isoforms can inactivate ROS. The omega-3 fatty acid (n3-FA) eicosapentaenoic acid (EPA) reduced cardiovascular (CV) events in high-risk patients (REDUCE-IT), a benefit not observed with mixed n3-FAs containing docosahexaenoic acid (DHA). Purpose The purpose of this study was to compare the effects of EPA and DHA on NO bioavailability and expression of detoxification enzymes in the vascular endothelium in vitro. Methods Human umbilical vein endothelial cells (HUVECs) were pretreated with EPA or DHA at equimolar levels (10 μM) for 2 h, then challenged with IL-6 at 12 ng/ml for 24 h. Proteomic analysis was performed using LC/MS to measure relative protein expression. Only significant (p&amp;lt;0.05) changes between treatment groups &amp;gt;1-fold were analyzed. Cells were stimulated with calcium ionophore to measure NO and peroxynitrite (ONOO-) release using a porphyrinic nanosensor. Results EPA, but not DHA, augmented PRDX-2 and SOD1 expression in HUVECs relative to IL-6 alone (1.2-fold and 1.6-fold, respectively, p=0.03). EPA also significantly lowered other isoforms unlike DHA. Either EPA or DHA increased thioredoxin expression by 1.5-fold (p=0.001) and 1.3-fold (p=0.02), respectively and decreased SOD2 expression by 1.5-fold (p=8.75E-11) and 1.6-fold (p=6.03E-9), respectively. IL-6 alone only increased expression of 6 detoxification enzymes by at least 1.2-fold, relative to vehicle. Unlike DHA, EPA also increased the NO to ONOO- release ratio by 36% (p&amp;lt;0.05) relative to IL-6 alone, without changes in NO synthase (eNOS) expression. Conclusions n3-FAs differentially influenced NO bioavailability and expression of ROS detoxification proteins, including peroxiredoxin and SOD isoforms. The net benefits of EPA on eNOS function and ROS detoxification may contribute to reduced atherothrombotic risk compared to DHA. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amarin Pharma Inc., Elucida Research

Aldose reductase inhibitor Epalrestat alleviates high glucose-induced cardiomyocyte apoptosis via ROS.

To clarify the role of aldose reductase inhibitor (ARI) in the high glucose-induced cardiomyocyte apoptosis and its mechanism. In this study, H9c2 cardiomyocytes were employed as objects, high-glucose medium as stimulus, and ARI Epalrestat as a therapeutic drug. The cell apoptosis and activity changes of nitric oxide synthase (NOS), NO, and reactive oxygen species (ROS) were evaluated via Hoechst staining, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and Western blotting. In addition, the mitochondrial membrane potential was measured via fluorescence counting. Epalrestat inhibited the activity of AR to improve high glucose-induced oxidative stress in cardiomyocytes, weaken ROS activity, relieve the inhibition on NO activity, alleviate mitochondrial membrane potential damage, reduce the level of high glucose-induced cardiomyocyte apoptosis, and suppress the expression and activity of Caspase-3, thereby preventing high glucose-induced cardiomyocyte apoptosis. ARI protects against high glucose-induced cardiomyocyte apoptosis.

Effects of aerobic exercise on the expressions and activities of nitric oxide synthases in the blood vessel endothelium in prediabetes mellitus.

Previous studies by our group have indicated that exercise intervention can ameliorate endothelial dysfunction, which is an early pathophysiological change of prediabetes mellitus. The present study aimed to test the hypothesis that nitric oxide synthases (NOSs), which are expressed in blood vessel endothelium, contribute to the mitigation of vascular endothelium-dependent dysfunction by aerobic exercise in prediabetes mellitus. A prediabetic rat model was established by feeding the rats an additional high-energy diet, and was confirmed by testing blood glucose levels, the area-under-the-curve for the blood glucose tests (P<0.05) and the changes to the histological morphology of the thoracic aorta. Further examination identified that NOS expression changed significantly between the control and prediabetes groups, indicating endothelial dysfunction in the prediabetic rats. Following aerobic exercise, a significant increase in NOS, endothelial (eNOS) mRNA and protein expression (P<0.05), and a significant decrease in NOS, inducible (iNOS) mRNA and protein expression (P<0.05) was identified in the prediabetic rats compared with the control group. No significant change in nitric oxide synthase, brain expression was observed in the prediabetic rat group compared with the control group. Notably, there was also a significant increase and decrease in eNOS and iNOS activity, respectively, in the prediabetes group compared with the control group (P<0.05). Furthermore, nitric oxide (NO) concentration in the vascular endothelium was detected, which revealed a significant increase in NO concentration in the prediabetic rats following aerobic exercise compared with the control (P<0.05). The present study provided results that demonstrated that aerobic exercise ameliorated the vascular endothelium-dependent dysfunction through the NOS/NO signaling pathway, which is primarily regulated by NOS expression and activity, in prediabetes mellitus. The current study provided the theoretical basis for the use of exercise intervention to prevent diabetes mellitus during the early stage.

Flow Mediates Collecting Duct Histone Deacetylase 1 Nuclear Export

Urinary flow within the renal tubules, especially the inner‐medullary collecting duct (IMCD), increases after high sodium consumption. We previously reported that this natriuretic response is due, in part, to elevated nitric oxide (NO) production without associated changes in NO synthase (NOS) expression. This suggests that high flow increases NO production via increased NOS activity. Histone deacetylase 1 (HDAC1) has recently been cited as a flow‐responsive protein and may post‐translationally deacetylate lysines on non‐histone proteins. Therefore, we hypothesized that flow‐mediated increases in NO production occur via HDAC1. Mouse IMCD cells were grown to confluency on glass slides, then placed under static conditions or 10 dynes/cm2 fluid‐flow for 1 hour at 37°C in the presence of vehicle (0.1% DMSO) or MS‐275 (300 nM), HDAC1‐selective inhibitor. Nitrite production, analyzed by HPLC, increased in response to flow &gt;5‐fold (static: 231.4±31.1, flow: 1291.49.2±183.8 pmol/mg pr/hr; n=7/group, p&lt;0.0001). MS‐275 significantly decreased NO production in response to flow (MS‐275: 342.8±44.0 pmol/mg pr/hr; n=9/group, p&lt;0.0001). HDAC1 expression (static: 1.00±0.2, flow: 0.98±0.1 RDU/β‐actin; n=6/group, p&gt;0.05) and activity (static: 1.00±0.06, flow: 0.85±0.02 RDU/β‐actin; n=3/group, p&gt;0.05) was unchanged with flow in whole cell lysate. However, HDAC1 immunofluorescent staining of post‐flow IMCD cells suggested the presence of both nuclear and cytosolic HDAC1 and nuclear extraction of these flowed samples revealed a significant decrease in nuclear HDAC1 expression (static: 1.00±0.1, flow: 0.68±0.1 RDU; n=6/group, p&lt;0.05), indicating nuclear export. Calcium, a key player in classical NOS activation, has also been reported to activate HDAC nuclear export. Thus, we further hypothesized that flow activation of NO production as well as flow‐mediated export of HDAC1 is calcium‐dependent. Flow‐induced NO production was abolished in the absence of calcium (flow+vehicle: 2038.1±402.0; flow+BAPTA: 377.7±75.3 pmol/mg pr/hr; flow+calcium‐free media: 474.8±133.2 pmol/mg pr/hr; n= 5–11/group, p&lt;0.001). However, flow mediated nuclear HDAC1 export was similar in the presence and absence of BAPTA treatment (flow: 0.75±0.04, flow+BAPTA: 0.74±0.03 RDU; n=6/group, p&gt;0.05). In conclusion, IMCD NO production in response to flow is both calcium‐ and HDAC1‐dependent, but extracellular calcium appears not to facilitate the flow‐mediated nuclear export of HDAC1. Further studies will elucidate the disparate mechanism(s) of flow‐mediated HDAC1‐dependent NO production from flow‐mediated HDAC1 nuclear export.Support or Funding InformationThis work was supported by funding from the National Institutes of Health ‐ P01 HL136267 to Dr. Jennifer Pollock and F31 DK111067 to Randee Sedaka.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Peptidylarginine Deiminase Inhibitor Cl-Amidine Suppresses Inducible Nitric Oxide Synthase Expression in Dendritic Cells.

The conversion of peptidylarginine into peptidylcitrulline by calcium-dependent peptidylarginine deiminases (PADs) has been implicated in the pathogenesis of a number of diseases, identifying PADs as therapeutic targets for various diseases. The PAD inhibitor Cl-amidine ameliorates the disease course, severity, and clinical manifestation in multiple disease models, and it also modulates dendritic cell (DC) functions such as cytokine production, antigen presentation, and T cell proliferation. The beneficial effects of Cl-amidine make it an attractive compound for PAD-targeting therapeutic strategies in inflammatory diseases. Here, we found that Cl-amidine inhibited nitric oxide (NO) generation in a time- and dose-dependent manner in maturing DCs activated by lipopolysaccharide (LPS). This suppression of NO generation was independent of changes in NO synthase (NOS) enzyme activity levels but was instead dependent on changes in inducible NO synthase (iNOS) transcription and expression levels. Several upstream signaling pathways for iNOS expression, including the mitogen-activated protein kinase, nuclear factor-κB p65 (NF-κB p65), and hypoxia-inducible factor 1 pathways, were not affected by Cl-amidine. By contrast, the LPS-induced signal transducer and the activator of transcription (STAT) phosphorylation and activator protein-1 (AP-1) transcriptional activities (c-Fos, JunD, and phosphorylated c-Jun) were decreased in Cl-amidine-treated DCs. Inhibition of Janus kinase/STAT signaling dramatically suppressed iNOS expression and NO production, whereas AP-1 inhibition had no effect. These results indicate that Cl-amidine-inhibited STAT activation may suppress iNOS expression. Additionally, we found mildly reduced cyclooxygenase-2 expression and prostaglandin E2 production in Cl-amidine-treated DCs. Our findings indicate that Cl-amidine acts as a novel suppressor of iNOS expression, suggesting that Cl-amidine has the potential to ameliorate the effects of excessive iNOS/NO-linked immune responses.

Diabetic Microvascular Disease and Pulmonary Fibrosis: The Contribution of Platelets and Systemic Inflammation.

Diabetes is strongly associated with systemic inflammation and oxidative stress, but its effect on pulmonary vascular disease and lung function has often been disregarded. Several studies identified restrictive lung disease and fibrotic changes in diabetic patients and in animal models of diabetes. While microvascular dysfunction is a well-known complication of diabetes, the mechanisms leading to diabetes-induced lung injury have largely been disregarded. We described the potential involvement of diabetes-induced platelet-endothelial interactions in perpetuating vascular inflammation and oxidative injury leading to fibrotic changes in the lung. Changes in nitric oxide synthase (NOS) activation and decreased NO bioavailability in the diabetic lung increase platelet activation and vascular injury and may account for platelet hyperreactivity reported in diabetic patients. Additionally, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway has been reported to mediate pancreatic islet damage, and is implicated in the onset of diabetes, inflammation and vascular injury. Many growth factors and diabetes-induced agonists act via the JAK/STAT pathway. Other studies reported the contribution of the JAK/STAT pathway to the regulation of the pulmonary fibrotic process but the role of this pathway in the development of diabetic lung fibrosis has not been considered. These observations may open new therapeutic perspectives for modulating multiple pathways to mitigate diabetes onset or its pulmonary consequences.

Structural Studies of a Complex Between Endothelial Nitric Oxide Synthase and Calmodulin at Physiological Calcium Concentration.

The small acidic protein calmodulin (CaM) serves as a Ca2+ sensor and control element for many enzymes including nitric oxide synthase (NOS) enzymes that play major roles in key physiological and pathological processes. CaM binding causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. In this report, NMR spectroscopy was used to determine the solution structure of the endothelial NOS (eNOS) peptide in complex with CaM at the lowest Ca2+ concentration (225 nM) required for CaM to bind to eNOS and corresponds to a physiological elevated Ca2+ level found in mammalian cells. Under these conditions, the CaM-eNOS complex has a Ca2+-replete C-terminal lobe bound to the eNOS peptide and a Ca2+ free N-terminal lobe loosely associated with the eNOS peptide. With increasing Ca2+ concentration, the binding of Ca2+ by the N-lobe of CaM results in a stronger interaction with the C-terminal region of the eNOS peptide and increased α-helical structure of the peptide that may be part of the mechanism resulting in electron transfer from the FMN to the heme in the oxygenase domain of the enzyme. Surface plasmon resonance studies performed under the same conditions show Ca2+ concentration-dependent binding kinetics were consistent with the NMR structural results. This investigation shows that structural studies performed under more physiological relevant conditions provide information on subtle changes in structure that may not be apparent when experiments are performed in excess Ca2+ concentrations.

Change of nitric oxide synthase in peripheral blood of cataract patients

Background Studies showed that the content of nitric oxide synthase (NOS) in aqueous and lenses elevates in cataract eyes. But it is difficult to measure the NOS content in eye tissue. So to determine whether the overepression of NOS is associated with cataractogenesis is significant for the prevention and treatment of cataract. Objective This study was to detect the expression of NOS in the peripheral blood of cataract patients and evaluate the association of blood NOS with cataractogenesis. Methods Twenty-five patients with age-related cataract and 12 patients with diabetic cataract were collected from January to June 2014 in Affiliated First Hospital of Soochow University and Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, and 20 matched healthy volunteers were enrolled in the Health Check Centre in the same duration. The 2.0 ml peripheral blood was collected from each subject under the informed consent. The blood specimens were centrifuged to obtain cell pellets. The contents of NOS mRNA and inducible nitric oxide synthase (iNOS) mRNA and their proteins in leukocytes were measured by reverse transcription PCR (RT-PCR) and flow cytometry. Results The relative expression levels of total NOS mRNA were 1.46±0.31, 1.59±0.28 and 1.22±0.23 in the age-related cataract group, the diabetic cataract group and the healthy control group, respectively, with a significant difference among the three groups (F=7.572, P=0.001). The relative expression level of total NOS mRNA was significantly higher in the age-related cataract group and the diabetic cataract group than that of the healthy control group (q=4.095, 5.170, both at P<0.01). The relative expression levels of iNOS in blood were 1.17±0.21, 1.08±0.15 and 0.89±0.17 in the age-related cataract group, the diabetic cataract group and the healthy control group, respectively, showing a significant difference among the three groups (F=12.801, P=0.000), and the level of iNOS in blood was significantly increased in the age-related cataract group and the diabetic cataract group compared with the healthy control group (q=7.112, 3.932, both at P<0.01). The expressions of total NOS protein in the age-related cataract group, the diabetic cataract group and the healthy control group were 15.82±2.11, 17.19±3.37 and 10.63±2.28, with a significant difference among them (F=34.895, P=0.000), and the expressions of total NOS protein were significantly elevated in the age-related cataract group and the diabetic cataract group in comparison with the healthy control group (q=9.885, 10.266, both at P<0.01). Conclusions NOS content in periphery blood elevates in age-related and diabetic cataract patients. These results indicate that blood NOS may be associated with cataractogenesis. The measurement of blood NOS may be a useful indicator in monitoring and preventing cataract formation and development. Key words: Nitric oxide synthase; Nitric oxide; Cataract; Peripheral blood; Reverse transcription PCR; Flow cytometry

Modulating central gain in tinnitus: changes in nitric oxide synthase in the ventral cochlear nucleus.

A significant challenge in tinnitus research lies in explaining how acoustic insult leads to tinnitus in some individuals, but not others. One possibility is genetic variability in the expression and function of neuromodulators – components of neural signaling that alter the balance of excitation and inhibition in neural circuits. An example is nitric oxide (NO) – a free radical and potent neuromodulator in the mammalian brain – that regulates plasticity via both pre-synaptic and postsynaptic mechanisms. Changes in NO have previously been implicated in tinnitus generation, specifically in the ventral cochlear nucleus (VCN). Here, we examined nitric oxide synthase (NOS) – the enzyme responsible for NO production – in the guinea pig VCN following acoustic trauma. NOS was present in most cell types – including spherical and globular bushy cells, small, medium, and large multipolar cells, and octopus cells – spanning the entire extent of the VCN. The staining pattern was symmetrical in control animals. Unilateral acoustic over-exposure (AOE) resulted in marked asymmetries between ipsilateral and contralateral sides of the VCN in terms of the distribution of NOS across the cochlear nuclei in animals with behavioral evidence of tinnitus: fewer NOS-positive cells and a reduced level of NOS staining was present across the whole extent of the contralateral VCN, relative to the ipsilateral VCN. The asymmetric pattern of NOS-containing cells was observed as early as 1 day after AOE and was also present in some animals at 3, 7, and 21 days after AOE. However, it was not until 8 weeks after AOE, when tinnitus had developed, that asymmetries were significant overall, compared with control animals. Asymmetrical NOS expression was not correlated with shifts in the threshold hearing levels. Variability in NOS expression between animals may represent one underlying difference that can be linked to whether or not tinnitus develops after noise exposure.

Chemical shift perturbations induced by residue specific mutations of CaM interacting with NOS peptides.

The regulation of nitric oxide synthase (NOS) by calmodulin (CaM) plays a major role in a number of key physiological and pathological processes. A detailed molecular level picture of how this regulation is achieved is critical for drug development and for our understanding of protein regulation in general. CaM is a small acidic calcium binding protein and is required to fully activate NOS. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. The interaction of CaM with NOS is modified by a number of post-translation modifications including phosphorylation. Here we present backbone and sidechain (1)H, (15)N NMR assignments of modified CaM interacting with NOS peptides which provides the basis for a detailed study of CaM-NOS interaction dynamics using (15)N relaxation methods.

Activity and expression of nitric oxide synthase in pork skeletal muscles

The objective of this study was to investigate the biochemical changes of nitric oxide synthase (NOS) in pork skeletal muscles during postmortem storage. Longissimus thoracis (LT), psoas major (PM) and semimembranosus (SM) muscles of pork were removed immediately after slaughter and stored under vacuum condition at 4°C for 0, 1 and 3d. Results showed that all three muscles exhibited NOS activity until 1d while SM muscle retained NOS activity after 3d of storage. The content of nNOS in SM muscle was stable across 3d of storage while decreased intensity of nNOS was detected at 1 and 3d of aging in PM and LT muscles due to the degradation of calpain. Immunostaining showed that nNOS was located at not only sarcolemma but also cytoplasm at 0 and 1d of storage. Our data suggest that postmortem muscles possess NOS activity and nNOS expression depends on muscle type.

Endothelial Nitric Oxide Synthase and Superoxide Mediate Hemodynamic Initiation of Intracranial Aneurysms

BackgroundHemodynamic insults at arterial bifurcations are believed to play a critical role in initiating intracranial aneurysms. Recent studies in a rabbit model indicate that aneurysmal damage initiates under specific wall shear stress conditions when smooth muscle cells (SMCs) become pro-inflammatory and produce matrix metalloproteinases (MMPs). The mechanisms leading to SMC activation and MMP production during hemodynamic aneurysm initiation are unknown. The goal is to determine if nitric oxide and/or superoxide induce SMC changes, MMP production and aneurysmal remodeling following hemodynamic insult.MethodsBilateral common carotid artery ligation was performed on rabbits (n = 19, plus 5 sham operations) to induce aneurysmal damage at the basilar terminus. Ligated animals were treated with the nitric oxide synthase (NOS) inhibitor LNAME (n = 7) or the superoxide scavenger TEMPOL (n = 5) and compared to untreated animals (n = 7). Aneurysm development was assessed histologically 5 days after ligation. Changes in NOS isoforms, peroxynitrite, reactive oxygen species (ROS), MMP-2, MMP-9, and smooth muscle α-actin were analyzed by immunohistochemistry.ResultsLNAME attenuated ligation-induced IEL loss, media thinning and bulge formation. In untreated animals, immunofluorescence showed increased endothelial NOS (eNOS) after ligation, but no change in inducible or neuronal NOS. Furthermore, during aneurysm initiation ROS increased in the media, but not the intima, and there was no change in peroxynitrite. In LNAME-treated animals, ROS production did not change. Together, this suggests that eNOS is important for aneurysm initiation but not by producing superoxide. TEMPOL treatment reduced aneurysm development, indicating that the increased medial superoxide is also necessary for aneurysm initiation. LNAME and TEMPOL treatment in ligated animals restored α-actin and decreased MMPs, suggesting that eNOS and superoxide both lead to SMC de-differentiation and MMP production.ConclusionAneurysm-inducing hemodynamics lead to increased eNOS and superoxide, which both affect SMC phenotype, increasing MMP production and aneurysmal damage.

Effect of acupotomy on nitric oxide synthase and beta-endorphin in third lumbar vertebrae transverse process syndrome model rats

ObjectiveTo explore the long-term effects and pain relief mechanism of acupotomy by observing changes in nitric oxide synthase (NOS) and beta-endorphin (β-EP) in the hypothalamus, spinal cord, and peripheral blood of rats with third lumbar vertebrae (L3) transverse process syndrome. MethodsTwenty-eight SD rats were randomly assigned to normal, model, electroacupuncture (EA), and acupotomy group. The last three groups were put through an operation to emulate L3 transverse process syndrome. Fourteen days after the simulation operation, EA and acupotomy treatments were applied to the respective groups. Fifty-six days after the simulation operation, biochemistry tests and enzyme-linked immunosorbent assay were used to measure NOS and β-EP in the hypothalamus, spinal cord, and peripheral blood. ResultsRats with the simulation operation showed significantly higher levels of NOS and β-EP in the hypothalamus, spinal cord, and peripheral blood than those in the normal group. The EA and acupotomy groups had significantly lower levels of NOS and β-EP than those in the model group. There was no statistical difference between the EA and acupotomy groups. ConclusionEA and acupotomy treatments significantly lowered NOS and β-EP levels in the hypothalamus, spinal cord, and peripheral blood and alleviated L3 transverse process syndrome.

Regulation of endothelial nitric oxide synthase phosphorylated at Thr495 by interaction with calmodulin (769.8)

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes and it is important to understand how this enzyme is regulated. Calmodulin (CaM), a small acidic calcium binding protein, is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains, through a process that is thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. We have determined the solution structure of CaM bound to a peptide of eNOS that contains a phosphorylated threonine corresponding to Thr495 in order to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an α‐helix. The calcium affinity of the CaM‐target peptide complex is reduced due to phosphorylation and this leads to weaker binding under low physiological calcium concentrations. This study provides an explanation for the reduced NO production by eNOS carrying a phosphorylated Thr495 residue.Grant Funding Source: National Science and Engineering Research Council (NSERC)

Changes in nitric oxide synthase and nitrite and nitrate serum levels in patients with or without MDR-TB undergoing the intensive phase of anti-tuberculosis therapy

BackgroundThere is a paucity of published data on the effect of TB chemotherapy on nitric oxide (NO) synthesis and metabolism in newly diagnosed and relapsed patients with or without multi-drug resistant tuberculosis (MDRTB). MethodsThe pattern of NO response in 140 patients with pulmonary TB, including 74 with MDR-TB and 66 without MDR-TB has been studied and compared to the NO status of 30 healthy donors. Patients comprised those with newly diagnosed TB and recurrent or relapsed TB. The NO status was assessed by measuring inducible NO synthase (iNOS) and nitrites and nitrates levels. This was measured prior to treatment initiation and two months after the prescribed chemotherapy. ResultsIncreased levels of NO indices were found in patients with tuberculosis when compared to healthy controls. After two months of chemotherapy a significant decrease in NO indicators was observed in the patients with TB, particularly in those without MDR-TB and newly diagnosed TB. The decline in NO activity was less prominent in patients with recurrent TB and MDR-TB, which suggests lower level of immunologic and reparative processes in such patients. ConclusionChanges in serum levels of nitrites and nitrates as well as iNOS activity in neutrophils may serve as diagnostic criteria to differentiate various clinical forms of TB and help as prognostic tool to predict treatment outcome.

Solution Structure of Calmodulin Bound to the Target Peptide of Endothelial Nitric Oxide Synthase Phosphorylated at Thr495

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes, and it is important to understand how this enzyme is regulated. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. We have determined the solution structure of CaM bound to a peptide that contains a phosphorylated threonine corresponding to Thr495 in full size endothelial NOS (eNOS) to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an α-helix. The calcium affinity of the CaM-target peptide complex is reduced because of phosphorylation, and this leads to weaker binding at low physiological calcium concentrations. This study provides an explanation for the reduced level of NO production by eNOS carrying a phosphorylated Thr495 residue.

Preconditioning with thyroid hormone (3,5,3-triiodothyronine) prevents renal ischemia-reperfusion injury in mice

3,5,3-triiodothyronine (T3) was found to decrease ischemia-reperfusion (I/R) injury of liver and myocardium in animal models when preconditioned 48 hours in advance of the I/R injury. The purpose of this study was to evaluate the effects of T3 preconditioning on renal I/R injury with different time intervals and to determine the changes in antioxidants, apoptosis, and nitric oxide synthase (NOS) in each condition. In male C57BL/6 mice, renal I/R injury was induced by temporary ligation of the bilateral renal pedicles for 45 minutes followed by a reperfusion period for 24 hours. Preconditioning with intraperitoneal injection of T3 was performed 24 or 6 hours before or at the time of I/R injury. From the histologic examination, tubular injury was decreased in mice preconditioned with T3 6 hours before I/R injury. The levels of proinflammatory cytokines were decreased with T3 preconditioning, either 6 hours or at the time of I/R injury. The levels of glutathione were increased in all treatment groups. Expressions of neuronal NOS were increased when preconditioned 6 hours before or at the time of I/R injury. The number of apoptotic tubular epithelial cell evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was decreased when preconditioned immediately before I/R injury. Preconditioning with T3 6 hours or immediately before I/R injury had a protective effect on renal I/R injury. The changes of NOS and antiapoptosis, other than well-known antioxidative properties, may play a role in the effect of short-term preconditioning.