Reduction In Nitric Oxide Levels Research Articles

Manool, a diterpene from Salvia officinalis, exerts preventive effects on chromosomal damage and preneoplastic lesions

The present study aimed to evaluate the effect of the manool diterpene on genomic integrity. For this purpose, we evaluated the influence of manool on genotoxicity induced by mutagens with different mechanisms of action, as well as on colon carcinogenesis. The results showed that manool (0.5 and 1.0 µg/ml) significantly reduced the frequency of micronuclei induced by doxorubicin (DXR) and hydrogen peroxide in V79 cells but did not influence genotoxicity induced by etoposide. Mice receiving manool (1.25 mg/kg) exhibited a significant reduction (79.5%) in DXR-induced chromosomal damage. The higher doses of manool (5.0 and 20 mg/kg) did not influence the genotoxicity induced by DXR. The anticarcinogenic effect of manool (0.3125, 1.25 and 5.0 mg/kg) was also observed against preneoplastic lesions chemically induced in rat colon. A gradual increase in manool doses did not cause a proportional reduction of preneoplastic lesions, thus demonstrating the absence of a dose-response relationship. The analysis of serum biochemical indicators revealed the absence of hepatotoxicity and nephrotoxicity of treatments. To explore the chemopreventive mechanisms of manool via anti-inflammatory pathways, we evaluated its effect on nitric oxide (NO) production and on the expression of the NF-kB gene. At the highest concentration tested (4 μg/ml), manool significantly increased NO production when compared to the negative control. On the other hand, in the prophylactic treatment model, manool (0.5 and 1.0 μg/ml) was able to significantly reduce NO levels produced by macrophages stimulated with lipopolysaccharide. Analysis of NF-kB in hepatic and renal tissues of mice treated with manool and DXR revealed that the mutagen was unable to stimulate expression of the gene. In conclusion, manool possesses antigenotoxic and anticarcinogenic effects and its anti-inflammatory potential might be related, at least in part, to its chemopreventive activity.

Palm Kernel Cake Oligosaccharides Acute Toxicity and Effects on Nitric Oxide Levels Using a Zebrafish Larvae Model.

One of the beneficial effects of non-digestible oligosaccharides (NDOs) is their anti-inflammatory effects on host animals. While conventional animal studies require that analysis be done after samples have been taken from the host, zebrafish larvae are optically transparent upon hatching and this provides an opportunity for observations to be made within the living zebrafish larvae. This study aimed to take advantage of the optical transparency of zebrafish larvae to study the nitric oxide (NO) reducing effects of NDOs through the use of lipopolysaccharide (LPS) from Salmonella enterica serovar (ser.) Enteritidis (S. Enteritidis) to induce cardiac NO production. Prior to running the above experiment, an acute toxicity assay was conducted in order to determine the appropriate concentration of oligosaccharides to be used. The oligosaccharides tested consisted of oligosaccharides which were extracted from palm kernel cake with a degree of polymerization (DP) equal to or less than six (OligoPKC), commercial mannanoligosaccharide (MOS) and commercial fructooligosaccharide (FOS). Acute toxicity test results revealed that the OligoPKC has a LC50 of 488.1 μg/ml while both MOS and FOS were non-toxic up to 1,000 μg/ml. Results of the in vivo NO measurements revealed that all three NDOs were capable of significantly reducing NO levels in LPS stimulated zebrafish embryos. In summary, at 250 μg/ml, OligoPKC was comparable to MOS and better than FOS at lowering NO in LPS induced zebrafish larvae. However, at higher doses, OligoPKC appears toxic to zebrafish larvae. This implies that the therapeutic potential of OligoPKC is limited by its toxicity.

Sumatriptan reduces severity of status epilepticus induced by lithium-pilocarpine through nitrergic transmission and 5-HT1B/D receptors in rats: A pharmacological-based evidence.

Status epilepticus (SE) is a life-threatening neurologic disorder that can be as both cause and consequence of neuroinflammation. In addition to previous reports on anti-inflammatory property of the anti-migraine medication sumatriptan, we have recently shown its anticonvulsive effects on pentylenetetrazole-induced seizure in mice. In the present study, we investigated further (i) the effects of sumatriptan in the lithium-pilocarpine SE model in rats, and (ii) the possible involvement of nitric oxide (NO), 5-hydroxytryptamin 1B/1D (5-HT1B/1D ) receptor, and inflammatory pathways in such effects of sumatriptan. Status epilepticus was induced by lithium chloride (127mg/kg, i.p) and pilocarpine (60mg/kg, i.p.) in Wistar rats. While SE induction increased SE scores and mortality rate, sumatriptan (0.001-1mg/kg, i.p.) improved it (P<0.001). Administration of the selective 5-HT1B/1D antagonist GR-127935 (0.01mg/kg, i.p.) reversed the anticonvulsive effects of sumatriptan (0.01mg/kg, i.p.). Although both tumor necrosis factor-α (TNF-α) and NO levels were markedly elevated in the rats' brain tissues post-SE induction, pre-treatment with sumatriptan significantly reduced both TNF-α (P<0.05) and NO (P<0.001) levels. Combined GR-127935 and sumatriptan treatment inhibited these anti-inflammatory effects of sumatriptan, whereas combined non-specific NOS (L-NAME) or selective neuronal NOS (7-nitroindazole) inhibitors and sumatriptan further reduced NO levels. In conclusion, sumatriptan exerted a protective effect against the clinical manifestations and mortality rate of SE in rats which is possibly through targeting 5-HT1B/1D receptors, neuroinflammation, and nitrergic transmission.

Modulation of activated murine peritoneal macrophages functions by emodin, aloe-emodin and barbaloin isolated from Aloe barbadensis

Activated murine macrophages produce high level of nitric oxide by inducible nitric oxide synthase (iNOS), which is an important mechanism in macrophage-induced inflammation. In the present study, we isolated emodin, aloe-emodin and barbaloin compounds from Aloe barbadensis and investigated their inhibitory effects on the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin (IL-12) from murine macrophages activated by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Our results showed that the 50% inhibition concentration (IC50) of emodin, aloe-emodin and barbaloin on NO, TNF-α and IL-12 from activated peritoneal macrophages were 120, 30 and 200 μM respectively. Our experimental results showed that reduction of NO level by emodin might inhibit both at the pretreated LPS/IFN-γ activation and post-LPS/IFN-γ activation pathway. Activities of aloe-emodin and barbaloin could be caused by inhibition at the pretreated LPS/IFN-γ activation event. Chemical structures of all the isolated components were identified by comparison of NMR, mass spectral data and melting points with those reported in literature.

Reduced nNOS activity is responsible for impaired fatty acid-dependent mitochondrial oxygen consumption in atrial myocardium from hypertensive rat.

Fatty acid (FA)-dependent mitochondrial activities of atrial myocardium in hypertension (HTN) and its regulation by nitric oxide (NO) remain unidentified. Here, we have studied palmitic acid (PA) regulation of cardiac mitochondrial oxygen consumption rate (OCR) in left atrial (LA) myocardium of sham and angiotensin II-induced HTN rats and their regulations by endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS). The effects were compared with those of left ventricular (LV) myocytes. Our results showed that OCR was greater in HTN-LA compared with that in sham-LA. PA increased OCR in sham-LA, sham-LV, and HTN-LV but reduced it in HTN-LA. Inhibition of nNOS (S-methyl-L-thiocitrulline, SMTC) or eNOS/nNOS (Nω-nitro-L-arginine methyl ester hydrochloride, L-NAME) reduced PA increment of OCR in sham-LA but exerted no effect on OCR in HTN-LA. SMTC reduced OCR in HTN-LV and L-NAME reduced OCR in sham-LV. nNOS was the predominant source of NO in LA and LV. nNOS-derived NO was increased in HTN-LA and HTN-LV. PA reduced eNOSSer1177, nNOSSer1417, and NO level in HTN-LA but exerted no effect in sham-LA. In contrast, PA increased NO in HTN-LV and enhanced nNOSSer1417 but reduced NO level in sham-LV without affecting eNOSSer1177, eNOSThr495, or nNOSSer1417. 2-Bromopalmitate (2BP), which blocks the S-palmitoylation of target proteins, prevented PA-dependent decrease of nNOSSer1417 and OCR in HTN-LA. In HTN-LV, 2BP prevented PA-induced OCR without affecting nNOSSer1417. Our results reveal that FA-induced mitochondrial activity in atrial myocardium is impaired in HTN which is mediated by reduced nNOS activity and NO bioavailability. Metabolic dysregulation may underlie diastolic dysfunction of atrial myocardium in HTN.

Calcarea carbonica treatment rescues lipopolysaccharide-induced inflammatory response in human mononuclear cells via downregulation of inducible cyclooxygenase pathway

ObjectiveProlonged use of nonsteroidal anti-inflammatory drugs is associated with severe side effects and toxicity. Therefore, we studied the anti-inflammatory role of Calcarea carbonica which had minimal toxicity at the low doses. MethodsTHP-1 human mononuclear cells were treated with C. carbonica to evaluate the 50% cytotoxicity concentration (CC50) and 50% effective concentration (EC50). Cell survival was evaluated in lipopolysaccharide-stimulated C. carbonica-treated cells. Nitric oxide (NO) and tumor necrosis factor-α (TNF-α) were measured to evaluate the anti-inflammatory activity of C. carbonica. Cyclooxygenase-2 (COX-2) protein expression was determined by Western blotting analysis, and the interaction of C. carbonica with the COX-2 protein was evaluated using molecular docking simulation. ResultsThe CC50 and EC50 of C. carbonica were found to be 43.26 and 11.99 µg/mL, respectively. The cell survival assay showed a 1.192-fold (P = 0.0129), 1.443-fold (P = 0.0009) and 1.605-fold (P = 0.0004) increase in cell survival at 24, 48 and 72 h after initiating C. carbonica treatment, respectively. C. carbonica-treated cells showed a reduction in NO levels by 2.355 folds (P = 0.0001), 2.181 folds (P = 0.0001) and 2.071 folds (P = 0.0001) at 24, 48 and 72 h, respectively. The treated cells also showed a reduction in TNF-α levels by 1.395 folds (P = 0.0013), 1.541 folds (P = 0.0005) and 1.550 folds (P = 0.0005) at 24, 48 and 72 h, respectively. In addition, a 1.193-fold reduction (P = 0.0126) in COX-2 protein expression was found in C. carbonica-treated cells. The molecular docking showed interaction of C. carbonica with the phenylalanine 367 residue present in active site of Cox-2. ConclusionC. carbonica exhibited anti-inflammatory properties in lipopolysaccharide-stimulated cells by significantly reducing NO production and TNF-α level through downregulation of the COX-2 protein. This effect is probably mediated through interaction of C. carbonica with the phenylalanine 367 residue present in active site of Cox-2.

Hyperglycaemia and Inflammation are Prime Factors to Persuade Endothelial Dysfunction in Patients with Type 2 Diabetes Mellitus

Introduction: Inflammation and hyperglycaemia are known factors to influence on endothelial dysfunction&#x0D; (ED) in type 2 diabetic subjects. Endothelial dysfunction is a first stage indication for the development of&#x0D; atherosclerosis. Hence, ED plays a major role to enhance pathological changes and progression of micro and&#x0D; macrovascular complications in type 2 diabetes.&#x0D; Aim and Objective: The study has been designed to assess inflammation, glycemic control and endothelial&#x0D; function in type 2 diabetic subjects, which makes us to understand the cause of endothelial dysfunction in&#x0D; T2DM.&#x0D; Materials and Method: Sixty type 2 diabetic subjects with the age of 39 – 60 years and 60 healthy controls&#x0D; with the same age group were enrolled in this study. Fasting and post-prandial blood samples were collected&#x0D; to estimate Fasting and post-prandial blood sugar, lipid profile, inflammatory markers (like hsCRP &amp;&#x0D; fibrinogen) and nitric oxide (NO) for endothelial function.&#x0D; Result: The present study was identified significant high level of fasting and post-prandial blood sugar, lipid&#x0D; profile, fibrinogen and hsCRP in type 2 diabetic subjects. The study also found reduced NO level in type&#x0D; 2 diabetic subjects than healthy control, which indicates that significant correlation of hyperglycaemia and&#x0D; inflammation with endothelial dysfunction in type 2 diabetes.&#x0D; Conclusion: We have found that significant elevation of inflammation and glucose level (hyperglycaemia)&#x0D; might play a major role to develop endothelial dysfunction in type 2 diabetic subjects, it further extends the&#x0D; process of pathogenesis and development of vascular complications.

Inhibition of Rheumatoid Arthritis Using Bark, Leaf, and Male Flower Extracts of Eucommia ulmoides.

Eucommia ulmoides Oliv., a native Chinese plant species, has been used as a traditional Chinese medicine formulation to treat rheumatoid arthritis (RA), strengthen bones and muscles, and lower blood pressure. Various parts of this plant such as the bark, leaves, and flowers have been found to have anti-inflammatory properties. E. ulmoides has potential applications as a therapeutic agent against bone disorders, which were investigated in this study. In vitro, RA joint fibroblast-like synoviocytes (RA-FLS) were treated with different concentrations (0, 25, 50, 100, 200, 400, 800, and 1000 μg/mL) of E. ulmoides bark, leaf, and male flower alcoholic extracts (EB, EL, and EF, respectively) to determine their potential cytotoxicity. Tumor necrosis factor- (TNF-) α and nitric oxide (NO) levels in RA-FLS were quantified using enzyme-linked immunosorbent assay (ELISA). Furthermore, collagen-induced arthritis (CIA) rats were treated with EB, EL, EF, Tripterygium wilfordii polyglycoside (TG) or the normal control (Nor), and then ankle joint pathology, bone morphology, and serum and spleen inflammatory cytokine levels were evaluated. The results showed that, in RA-FLS, EB, EL, and EF were not cytotoxic; EB and EF reduced TNF-α supernatant levels; and EB, EL, and EF reduced NO levels. The results of in vivo experiments showed that EB, EL, and EF alleviated ankle swelling and joint inflammation, while all extracts diminished inflammatory cell infiltration, pannus and bone destruction, and bone erosion. All tested extracts inhibited interleukin- (IL-) 6, IL-17, and TNF-α mRNA in the spleen of CIA rats, while EB most effectively reduced osteoclasts and inhibited bone erosion. EF showed the most obvious inhibition of inflammatory factors and pannus. Thus, EB, EL, and EF may alleviate bone destruction by inhibiting inflammation.

Effect of sitagliptin on depression in male wistar rats

Background: Growing evidence supports relationship between depression and inflammation. The hypothesis of involvement of inflammatory pathways in depression is supported by the findings of increased levels of proinflammatory cytokines. So, we decided to evaluate the effect of sitagliptin on depression using forced swim test (FST) and possible effects of sitagliptin on serum oxidative stress markers and cytokine gene expression in rat hippocampus.Methods: FST model was used to evaluate antidepressant effect in male wistar rats. Rats in group I (control group) were given normal saline, group II (standard group) were given fluoxetine, group III and IV (test groups) were given sitagliptin 5 mg/kg and sitagliptin 9 mg/kg respectively. All the drugs in all groups were given per orally. At the end, animals were sacrificed and blood was collected. Hippocampus of rat brain was dissected out. Serum oxidative stress markers and hippocampal pro inflammatory cytokine gene expression analysis was carried out.Results: Sitagliptin 5 mg/kg and 9 mg/kg showed reduction in depressive symptoms and hippocampal cytokine gene expression in comparison to control. In case of serum oxidative stress markers, there was statistically significant reduction in nitric oxide levels with stagliptin 9 mg/kg. Although there was a decrease in the levels of catalase and increase in the levels of glutathione with standard and test groups, the results were not statistically significant.Conclusions: The present study showed significant antidepressant effect activity of standard and test groups. Hence, further research should be carried out to substantiate above results.

Isopimarane Diterpenoids from the Rhizomes of Kaempferia marginata and Their Potential Anti-inflammatory Activities.

Six new isopimarane diterpenes, marginaols A-F (1-6), along with eight known compounds (7-14), were isolated from the rhizomes of Kaempferia marginata. The structures and absolute configurations of 1-6 were established on the basis of spectroscopic methods and the experimental and calculated ECD data as well as comparison with the literature values. Most of the isolated compounds were tested for their nitric oxide (NO) inhibitory effects in lipopolysaccharide-activated RAW264.7 cells. Among them, marginaol B (2) was found to reduce NO levels in murine macrophage cells with an IC50 value of 28.1 ± 1.7 μM.

ELF-EMF'in Beyin, Karaciğer ve Kalp Dokularında Oksidatif Parametreler Üzerine Etkilerinin Değerlendirilmesi

The aim of the present study was to investigate the prospective effects of continuous and intermittent extremely low-frequency magnetic fields (ELF-EMFs) on the levels of malondialdehyde (MDA), nitric oxide (NO) and glutathione (GSH) in brain, heart, and liver tissues. The experiments performed on 18 guinea pigs were divided into three groups (n= 6 for each group). 50 Hz of 1.5 mT a magnetic field was used for exposure. Experimental groups were exposed for 4 h/day either continuously or intermittently (2h on/ 2h off/ 2h on) EMF for a period of 7 days. MDA, NO and GSH levels were determined by spectrophotometric methods. The intermittent exposure was increased GSH levels whereas that was reduced MDA levels in the brain when compared to control and continuous exposure groups. GSH concentrations of both experimental groups were found to be elevated in the liver. In the liver and heart, NO levels were increased by the continuous exposure in both administration groups, however, intermittent exposure has reduced NO levels in the both of the experimental groups. Results of this study indicated that the responses of various tissues to magnetic field exposures differed according to intensity and exposure duration of magnetic fields.

Reductions of Circulating Nitric Oxide are Followed by Hypertension during Pregnancy and Increased Activity of Matrix Metalloproteinases-2 and -9 in Rats.

Hypertensive pregnancy has been associated with reduced nitric oxide (NO), bioavailability, and increased activity of matrix metalloproteinases (MMPs). However, it is unclear if MMPs activation is regulated by NO during pregnancy. To this end, we examined activity of MMP-2 and MMP-9 in plasma, placenta, uterus and aorta, NO bioavailability, oxidative stress, systolic blood pressure (SBP), and fetal-placental development at the early, middle, and late pregnancy stages in normotensive and Nω-Nitro-L-arginine methyl-ester (L-NAME)-induced hypertensive pregnancy in rats. Reduced MMP-2 activity in uterus, placenta, and aorta and reduced MMP-9 activity in plasma and placenta with concomitant increased NO levels were found in normotensive pregnant rats. By contrast, increased MMP-2 activity in uterus, placenta, and aorta, and increased MMP-9 activity in plasma and placenta with concomitant reduced NO levels were observed in hypertensive pregnant rats. Also, elevated oxidative stress was displayed by hypertensive pregnant rats at the middle and late stages. These findings in the L-NAME-treated pregnant rats were also followed by increases in SBP and associated with fetal growth restrictions at the middle and late pregnancy stages. We concluded that NO bioavailability may regulate MMPs activation during normal and hypertensive pregnancy.

Arginase Isoform Expression in Chronic Rhinosinusitis.

Nitric oxide (NO) has emerged as an important regulator of upper airway inflammation, mainly as part of the local naso-sinusal defense mechanisms. Increased arginase activity can reduce NO levels by decreasing the availability of its precursor, L-arginine. Chronic rhinosinusitis (CRS) has been associated with low levels of nasal nitric oxide (nNO). Thus, the present study investigates the activity of arginase I (ARG1) and II (ARG2) in CRS and its possible involvement in the pathogenesis of this disease. Under endoscopic view, tissue samples of pathologic (n = 36) and normal (n = 29) rhinosinusal mucosa were collected. Arginase I and II mRNA levels were measured using real-time PCR. Our results showed low arginase I activity in all samples. The levels of ARG2 were significantly higher in patients with chronic rhinosinusitis compared to the control group (fold regulation (FR) 2.22 ± 0.42 vs. 1.31 ± 0.21, p = 0.016). Increased ARG2 expression was found in patients with CRS without nasal polyposis (FR 3.14 ± 1.16 vs. 1.31 ± 0.21, p = 0.0175), in non-allergic CRS (FR 2.55 ± 0.52 vs. 1.31 ± 0.21, p = 0.005), and non-asthmatic CRS (FR 2.42 ± 0.57 vs. 1.31 ± 0.21, p = 0.028). These findings suggest that the upregulation of ARG2 may play a role in the pathology of a distinctive phenotype of CRS.

Excessive Fructose Intake Impairs Baroreflex Sensitivity and Led to Elevated Blood Pressure in Rats.

Hypertension development with an increased intake of added sugar, especially excessive fructose intake, was shown in the National Health and Nutrition Examination Survey (NHANES) data. However, the mechanism underlying blood pressure (BP) elevation with increased fructose intake is still unclear. First, the present study showed that in rats fed 10% fructose for one week, BP and fructose/glucose levels increased in the central and peripheral nervous system. Furthermore, increased fructose intake resulted in an upregulation of fructose concentration in the cerebrospinal fluid. Second, consumption of excess fructose increased serum triglycerides. However, the inhibition of triglyceride production did not mitigate sympathetic nerve hyperactivity, but contributed to an insignificant decrease in BP. Finally, increased fructose intake reduced nitric oxide (NO) levels in the nucleus tractus solitarii (NTS) and reduced baroreflex sensitivity within a week. Collectively, the data suggested that fructose intake reduced NO levels in the NTS and caused baroreflex dysfunction, which further stimulated sympathetic nerve activity and induced the development of high BP.

Impaired l-arginine-nitric oxide pathway contributes to the pathogenesis of resistant hypertension.

The precise mechanisms underlying resistant hypertension remain elusive. Reduced nitric oxide (NO) bioavailability is frequently documented in chronic kidney disease, obesity, diabetes and advanced age, all of which are risk factors for resistant hypertension. Sympathetic overactivity and chronic activation of the renin-angiotensin system are salient features of resistant hypertension. Interestingly, recent data indicate that renal sympathetic overactivity can reduce the expression of neuronal nitric oxide synthase in the paraventricular nucleus. Reduced NO levels in the paraventricular nucleus can increase sympathetic outflow and this can create a vicious cycle contributing to resistant hypertension. Angiotensin II can reduce l-arginine transport and hence NO production. Reduced NO levels may reduce the formation of angiotensin 1-7 dampening the cardio-protective effects of the renin-angiotensin system contributing to resistant hypertension. In addition, interleukin-6 (IL-6) is demonstrated to be independently associated with resistant hypertension, and IL-6 can reduce NO synthesis. Despite this, NO levels have not been quantified in resistant hypertension. Findings from a small proof of concept study indicate that NO donors can reduce blood pressure in patients with resistant hypertension but more studies are required to validate these preliminary findings. In the present paper, we put forward the hypothesis that reduced NO bioavailability contributes substantially to the development of resistant hypertension.

Nitric oxide regulates the firing rate of neuronal subtypes in the guinea pig ventral cochlear nucleus.

The gaseous free radical, nitric oxide (NO) acts as a ubiquitous neuromodulator, contributing to synaptic plasticity in a complex way that can involve either long term potentiation or depression. It is produced by neuronal nitric oxide synthase (nNOS) which is presynaptically expressed and also located postsynaptically in the membrane and cytoplasm of a subpopulation of each major neuronal type in the ventral cochlear nucleus (VCN). We have used iontophoresis in vivo to study the effect of the NOS inhibitor L‐NAME (L‐NG‐Nitroarginine methyl ester) and the NO donors SIN‐1 (3‐Morpholinosydnonimine hydrochloride) and SNOG (S‐Nitrosoglutathione) on VCN units under urethane anaesthesia. Collectively, both donors produced increases and decreases in driven and spontaneous firing rates of some neurones. Inhibition of endogenous NO production with L‐NAME evoked a consistent increase in driven firing rates in 18% of units without much effect on spontaneous rate. This reduction of gain produced by endogenous NO was mirrored when studying the effect of L‐NAME on NMDA(N‐Methyl‐D‐aspartic acid)‐evoked excitation, with 30% of units showing enhanced NMDA‐evoked excitation during L‐NAME application (reduced NO levels). Approximately 25% of neurones contain nNOS and the NO produced can modulate the firing rate of the main principal cells: medium stellates (choppers), large stellates (onset responses) and bushy cells (primary‐like responses). The main endogenous role of NO seems to be to partly suppress driven firing rates associated with NMDA channel activity but there is scope for it to increase neural gain if there were a pathological increase in its production following hearing loss.

2162Neurofibromin 2 (NF2) drives obesity-related endothelial dysfunction by targeting Caveolin-1: a study in mice and humans

Abstract Introduction Endothelial dysfunction (ED) is a key underpinning of cardiovascular disease in obesity, but the underlying molecular mechanisms remain elusive. Neurofibromin 2 (NF2) is a scaffold-like protein implicated in various cellular processes, namely growth, differentiation and survival. NF2 is inactivated by Akt-dependent phosphorylation at Ser518, whereas its dephosphorylation by the myosin phosphatase target subunit 1 (MYPT-1) leads to an active conformation. The role of NF2 in obesity-related alterations of endothelial phenotype remains elusive. Purpose To investigate whether NF2 participates to ED in obesity. Methods Human aortic endothelial cells (HAECs) were exposed to palmitic acid (PA, 200 uM) or vehicle for 48 hours. Gene silencing of NF2 was performed by small interfering RNA (siRNA). Protein expression was assessed by Western blot. Nitric oxide (NO) levels were measured by using a colorimetric assay. The interaction of NF2 with endothelial proteins was investigated by co-immunoprecipitation. To specifically determine NF2 role in the endothelium, we generated mice with endothelium-specific deletion of NF2 (NF2 ECKO) by crossing NF2flox/flox mice with tamoxifen-inducible endothelial-specific cre mice [Cdh5(PAC)-CreERT2]. Endothelium-dependent relaxations to acetylcholine (Ach, 10–9 to 10–5 mol/L) were assessed in aortas isolated from male NF2 ECKO and wild type littermates, fed a control (10 kcal% fat) and a high fat diet (60 kcal% fat) for 20 weeks. NF2 signalling and endothelial function were also assessed in small visceral fat arteries (VFA) isolated from 18 obese and 18 age-matched healthy subjects undergoing bariatric surgery and cholecystectomy, respectively. Results Exposure of HAECs to PA decreased NF2 phosphorylation at Ser518, thus leading to an active protein conformation. Blunted NF2 phosphorylation was explained by a reduction of Akt phosphorylation at Ser473 and a concomitant increase of MYPT-1 phosphorylation at Thr696. Pull-down experiments revealed that NF2 binds and activates Caveolin 1 (Cav-1), a pivotal repressor of endothelial NO synthase (eNOS). NF2 knockdown in PA-treated HAECs prevented eNOS–Cav-1 interaction, thus preserving eNOS activity and NO levels. In aortas from obese mice, we found that NF2-Cav-1 interaction was responsible for impaired eNOS activity, reduced NO levels and endothelial dysfunction. By contrast, Ach-dependent vasorelaxation were preserved in obese mice with endothelium-specific deletion of NF2. Moreover, we found that NF2 is activated in VFA from obese patients as compared to healthy controls, and its activity negatively correlated with Ach-dependent vasorelaxation of isolated VFA, as assessed by organ chamber experiments. Conclusions The present findings – obtained in human endothelial cells, conditional mouse models and visceral fat arteries from obese patients – suggest that targeting NF2 may represent a potential therapeutic strategy to prevent ED in patients with obesity.

Pattern of sympathetic vasomotor activity in a model of hypertension induced by nitric oxide synthase blockade.

We aimed to investigate the effects of nitric oxide (NO) synthesis inhibition by NO synthase inhibitor N‐nitro‐L‐arginine‐methyl ester (L‐NAME) treatment on the sympathetic vasomotor nerve activity (SNA) on two sympathetic vasomotor nerves, the renal and splanchnic. NO plasma level and systemic oxidative stress were assessed. Hypertension was induced by L‐NAME (20 mg/kg per day, by gavage, for seven consecutive days) in male Wistar rats. At the end of the treatment, blood pressure, heart rate, arterial baroreflex sensitivity, renal SNA (rSNA), and splanchnic SNA (sSNA) were assessed in urethane anesthetized rats. L‐NAME‐treated rats presented increased blood pressure (152 ± 2 mmHg, n = 17) compared to the control group (101 ± 2 mmHg, n = 15). Both rSNA (147 ± 10, n = 15 vs. 114 ± 5 Spikes/s, n = 9) and sSNA (137 ± 13, n = 14 vs. 74 ± 13 spikes/s, n = 9) were significantly increased in the L‐NAME‐treated compared to the control group. A differential response on baroreflex sensitivity was found, with a significant reduction for rSNA but not for sSNA arterial baroreceptor sensitivity in L‐NAME‐treated rats. The adjusted regression model revealed that the reduction of systemic NO levels partially explains the variation in sSNA and blood pressure, but not rSNA. Taken together, our data show that hypertension induced by NO synthase blockade is characterized by increased SNA to the rSNA and sSNA. In addition, we found that the rats that had the greatest reduction in NO levels in plasma by L‐NAME were those that developed higher blood pressure levels. The reduction in the NO level partially explains the variations in sSNA but not in rSNA.

Antimalarial activity of vitamin D3 (VD3) does not result from VD3-induced antimicrobial agents including nitric oxide or cathelicidin

Recent evidence suggests that 1α,25-dihydroxyvitamin D3 (VD3), the active form of vitamin D, inhibits microbial proliferation. Previously, we used in vivo murine models to investigate the antimalarial activity of VD3 and confirmed potent antimalarial activity in the acute phase. This study aimed to clarify the mechanisms underlying the antimalarial activity of VD3 in vivo, particularly extensive inhibition of parasitemia in the acute phase, focusing on nitric oxide (NO), a potent antimalarial molecule. VD3 is a good NO inducer. When most Plasmodium chabaudi AS (PcAS)-infected mice treated with VD3 survived, NO was present in blood samples obtained from VD3-treated mice at a significantly higher rate at 2 and/or 3 days post-infection than that in vehicle-treated control mice. To verify the involvement of NO in the antimalarial activity of VD3, we used aminoguanidine (AG), an inducible NO synthase (iNOS) inhibitor, to abrogate the antimalarial activity of VD3. However, despite AG-induced reductions in NO levels, parasitemia remained inhibited during the acute phase, even in the presence of AG, and the antiplasmodial faculty of VD3 was not ablated. VD3-mediated antimalarial activity irrelevant of NO compelled us to consider another candidate. In a pilot experiment, we used cathelicidin (CAMP), an antimicrobial peptide, since it is known that VD3 induces CAMP synthesis. Serum CAMP levels increased on days 4 or 5 post-infection with or without VD3 administration, but experiments using exogenous CAMP did not display curative effects in PcAS-infected mice. The present study using VD3 to target the malarial parasite thus suggests a potential novel approach to treat malarial infections.

Stably Transformed Lotus japonicus Plants Overexpressing Phytoglobin LjGlb1-1 Show Decreased Nitric Oxide Levels in Roots and Nodules as Well as Delayed Nodule Senescence.

The class 1 phytoglobin, LjGlb1-1, is expressed in various tissues of the model legume Lotus japonicus, where it may play multiple functions by interacting with nitric oxide (NO). One of such functions is the onset of a proper symbiosis with Mesorhizobium loti resulting in the formation of actively N2-fixing nodules. Stable overexpression lines (Ox1 and Ox2) of LjGlb1-1 were generated and phenotyped. Both Ox lines showed reduced NO levels in roots and enhanced nitrogenase activity in mature and senescent nodules relative to the wild-type (WT). Physiological and cytological observations indicated that overexpression of LjGlb1-1 delayed nodule senescence. The application to WT nodules of the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) or the phytohormones abscisic acid (ABA) and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) repressed nitrogenase activity, induced the expression of three senescence-associated genes and caused cytological changes evidencing nodule senescence. These effects were almost completely reverted by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Our results reveal that overexpression of LjGlb1-1 improves the activity of mature nodules and delays nodule senescence in the L.japonicus-M.loti symbiosis. These beneficial effects are probably mediated by the participation of LjGlb1-1 in controlling the concentration of NO that may be produced downstream in the phytohormone signaling pathway in nodules.