Increase In Nitric Oxide Synthase Activity Research Articles

The effect of zofenopril on the cardiovascular system of spontaneously hypertensive rats treated with the ACE2 inhibitor MLN-4760

BackgroundAngiotensin converting enzyme 2 (ACE2) plays a crucial role in the infection cycle of SARS-CoV-2 responsible for formation of COVID-19 pandemic. In the cardiovascular system, the virus enters the cells by binding to the transmembrane form of ACE2 causing detrimental effects especially in individuals with developed hypertension or heart disease. Zofenopril, a H2S-releasing angiotensin-converting enzyme inhibitor (ACEI), has been shown to be effective in the treatment of patients with essential hypertension; however, in conditions of ACE2 inhibition its potential beneficial effect has not been investigated yet. Therefore, the aim of the study was to determine the effect of zofenopril on the cardiovascular system of spontaneously hypertensive rats, an animal model of human essential hypertension and heart failure, under conditions of ACE2 inhibition induced by the administration of the specific inhibitor MLN-4760 (MLN).ResultsZofenopril reduced MLN-increased visceral fat to body weight ratio although no changes in systolic blood pressure were recorded. Zofenopril administration resulted in a favorable increase in left ventricle ejection fraction and improvement of diastolic function regardless of ACE2 inhibition, which was associated with increased H2S levels in plasma and heart tissue. Similarly, the acute hypotensive responses induced by acetylcholine, L-NAME (NOsynthase inhibitor) and captopril (ACEI) were comparable after zofenopril administration independently from ACE2 inhibition. Although simultaneous treatment with zofenopril and MLN led to increased thoracic aorta vasorelaxation, zofenopril increased the NO component equally regardless of MLN treatment, which was associated with increased NO-synthase activity in aorta and left ventricle. Moreover, unlike in control rats, the endogenous H2S participated in maintaining of aortic endothelial function in MLN-treated rats and the treatment with zofenopril had no impact on this effect.ConclusionsZofenopril treatment reduced MLN-induced adiposity and improved cardiac function regardless of ACE2 inhibition. Although the concomitant MLN and zofenopril treatment increased thoracic aorta vasorelaxation capacity, zofenopril increased the participation of H2S and NO in the maintenance of endothelial function independently from ACE2 inhibition. Our results confirmed that the beneficial effects of zofenopril were not affected by ACE2 inhibition, moreover, we assume that ACE2 inhibition itself can lead to the activation of cardiovascular compensatory mechanisms associated with Mas receptor, nitrous and sulfide signaling.

MLN-4760 Induces Oxidative Stress without Blood Pressure and Behavioural Alterations in SHRs: Roles of Nfe2l2 Gene, Nitric Oxide and Hydrogen Sulfide.

Reduced angiotensin 1-7 bioavailability due to inhibition of angiotensin-converting enzyme 2 (ACE2) may contribute to increased mortality in hypertensive individuals during COVID-19. However, effects of ACE2 inhibitor MLN-4760 in brain functions remain unknown. We investigated the selected behavioural and hemodynamic parameters in spontaneously hypertensive rats (SHRs) after a 2-week s.c. infusion of MLN-4760 (dose 1 mg/kg/day). The biochemical and molecular effects of MLN-4760 were investigated in the brainstem and blood plasma. MLN-4760 had no effects on hemodynamic and behavioural parameters. However, MLN-4760 increased plasma hydrogen sulfide (H2S) level and total nitric oxide (NO) synthase activity and conjugated dienes in the brainstem. Increased NO synthase activity correlated positively with gene expression of Nos3 while plasma H2S levels correlated positively with gene expressions of H2S-producing enzymes Mpst, Cth and Cbs. MLN-4760 administration increased gene expression of Ace2, Sod1, Sod2, Gpx4 and Hmox1, which positively correlated with expression of Nfe2l2 gene encoding the redox-sensitive transcription factor NRF2. Collectively, MLN-4760 did not exacerbate pre-existing hypertension and behavioural hyperactivity/anxiety in SHRs. However, MLN-4760-induced oxidative damage in brainstem was associated with activation of NO- and H2S-mediated compensatory mechanisms and with increased gene expression of antioxidant, NO- and H2S-producing enzymes that all correlated positively with elevated Nfe2l2 expression.

Running exercise improves spatial learning and memory ability and enhances angiogenesis in the cerebral cortex via endogenous nitric oxide

BackgroundThe molecular mechanisms by which exercise improves brain function and capillaries in the cerebral cortex are unclear. Exercise can increase the expression of nitric oxide (NO) in the brain, and endogenous NO is thought to exert beneficial effects on proangiogenic factors, antiangiogenic factors and brain function. Therefore, we hypothesized that running exercise might improve brain function and enhance angiogenesis through endogenous NO. Methods and resultsThe following three groups of rats were administered intracerebroventricular (i.c.v.) injections before running exercise each day for 4 weeks: exercise+L-NAME group (i.c.v. L-NAME, an NO synthase blocker, dose: 1 μmol/μl and 5 μl/day; treadmill exercise, 20 min/day), exercise group (i.c.v. normal saline, 5 μl/day; treadmill exercise, 20 min/day), and sham group (i.c.v. normal saline, 5 μl/day; no treadmill exercise). Subsequently, the spatial learning and memory abilities were tested using a Morris water maze, and the nitric oxide synthase (NOS) activity in the cerebral cortex in each group of rats was measured using a method involving nitric acid reductase and metabolic chemistry. The parameters of the cortical capillaries were quantitatively investigated using an immunohistochemistry technique and stereological methods. The expression levels of proangiogenic factors (VEGF and FGF-2) and an antiangiogenic inhibitor (endostatin) in the cerebral cortex were tested using a Western blot analysis. Running exercise significantly improved the rats' spatial learning and memory abilities and increased NOS activity in the cortex. Running exercise also subsequently improved the expression of proangiogenic factors (VEGF and FGF-2) and the length, volume and surface area of capillaries and reduced the expression of antiangiogenic factors (endostatin) in the cortex. In contrast, the L-NAME treatment attenuated the effects of running exercise. ConclusionsRunning exercise regulates proangiogenic factors, antiangiogenic factors and angiogenesis in the cerebral cortex via a partially NO-dependent mechanism, and influencing endogenous NO might potentially affect the exercise-related beneficial effects on cognitive ability and cortical capillaries.

Duration of Social Isolation Affects Production of Nitric Oxide in the Rat Brain.

Social isolation deprives rodents of social interactions that are critical for normal development of brain and behavior. Several studies have indicated that postweaning isolation rearing may affect nitric oxide (NO) production. The aim of this study was to compare selected behavioral and biochemical changes related to NO production in the brain of rats reared in social isolation for different duration. At the age of 21 days, male Sprague Dawley rats were randomly assigned into four groups reared in isolation or socially for 10 or 29 weeks. At the end of the rearing, open-field and prepulse inhibition (PPI) tests were carried out. Furthermore, in several brain areas we assessed NO synthase (NOS) activity, protein expression of nNOS and iNOS isoforms and the concentration of conjugated dienes (CD), a marker of oxidative damage and lipid peroxidation. Social isolation for 10 weeks resulted in a significant decrease in PPI, which was accompanied by a decrease in NOS activity in the cerebral cortex and the cerebellum, an increase in iNOS in the hippocampus and an increase in CD concentration in cortex homogenate. On the other hand, a 29 week isolation had an opposite effect on NOS activity, which increased in the cerebral cortex and the cerebellum in animals reared in social isolation, accompanied by a decrease in CD concentration. The decrease in NOS activity after 10 weeks of isolation might have been caused by chronic stress induced by social isolation, which has been documented in previous studies. The increased oxidative state might result in the depleted NO bioavailability, as NO reacts with superoxide radical creating peroxynitrite. After 29 weeks of isolation, this loss of NO might be compensated by the subsequent increase in NOS activity.

Influence of Nitric Oxide-Cyclic GMP and Oxidative STRESS on Amyloid-β Peptide Induced Decrease of Na,K-ATPase Activity in Rat Hippocampal Slices.

Amyloid-β peptide (Aβ) has been shown to cause synaptic dysfunction and can render neurons vulnerable to excitotoxicity and oxidative stress. Na,K-ATPase plays an important role to maintain cell ionic equilibrium and it can be modulated by N-methyl-D-aspartate (NMDA)-nitric oxide (NO)-cyclic GMP pathway. Disruption of NO synthase (NOS) activity and reactive oxygen species (ROS) production could lead to changes in Na,K-ATPase isoforms' activities that may be detrimental to the cells. Our aim was to evaluate the signaling pathways of Aβ in relation to NMDA-NOS-cyclic GMP versus oxidative stress on α1-/α2,3-Na,K-ATPase activities in rat hippocampal slices. Aβ1-40 induced a concentration-dependent increase of NOS activity and increased cyclic guanosine monophosphate (cGMP), TBARS (thiobarbituric acid reactive substances), and 3-Nitrotyrosine (3-NT)-modified protein levels in rat hippocampal slices. The increase in NOS activity and cyclic GMP levels induced by Aβ1-40 was completely blocked by MK-801 (inhibitor of NMDA receptor) and L-NAME (inhibitor of NOS) pre-treatment but changes in TBARS levels were only partially blocked by both compounds. The Aβ treatment also decreased Na,K-ATPase activity which was reverted by N-nitro-L-arginine methyl ester hydrochloride (L-NAME) but not by MK-801 pre-treatment. The decrease in enzyme activity induced by Aβ was isoform-specific since only α1-Na,K-ATPase was affected. These findings suggest that the activation of NMDA-NOS signaling cascade linked to α2,3-Na,K-ATPase activity may mediate an adaptive, neuroprotective response to Aβ in rat hippocampus.

Study of Immunotropic Properties of Water-Soluble Polysaccharides Isolated from Conium maculatum Grass.

Addition of water-soluble polysaccharides isolated from Conium maculatum L. to the mouse peritoneal macrophage culture induces classical activation of antigen-presenting cells due to an increase in NO synthase activity and a decrease in arginase expression.

THU0084 TOFACITINIB REVERSED ENDOTHELIAL DYSFUNCTION IN RHEUMATOID ARTHRITIS: MECHANISTIC INSIGHTS FROM THE RAT ADJUVANT-INDUCED ARTHRITIS MODEL.

Background:Tofacitinib, an inhibitor of JAK3 and JAK1, is approved for the treatment of rheumatoid arthritis (RA)1. Cardiovascular (CV) risk2and events3in RA patients treated with Tofacitinib is a matter of debate, but the vascular mechanisms involved are unknown.Objectives:The aim of this study was to investigate whether Tofacitinib improves endothelial dysfunction (ED) and if so to explore the underlying mechanisms in the model of adjuvant-induced arthritis (AIA) in rats.Methods:AIA was induced by injection ofMycobacterium butyricumin the tail of male Lewis rats. A group of rats without arthritis served as controls. At the first signs of arthritis, AIA received Tofacitinib (10 mg/kg twice daily, s.c.) or 33%DMSO/PEG300(Vehicle). Arthritis score was daily evaluated. After 21 days, preconstricted isolated aortic rings were relaxed with acetylcholine (Ach, 10-11-10-4moles/liter) in the presence or not of inhibitor of nitric oxide (NO) synthase (L-NAME), cyclooxygenase-2 (NS398), arginase (nor-NOHA), endothelium-derived hyperpolarizing factor (EDHF) (apamin/charybdotoxin) and superoxide anions production (Tempol). Endothelium-denuded rings were used to determine the vasorelaxant response to the NO-donor sodium nitroprussiate (SNP, 10-11-10-4moles/liter). Blood pressure and heart rate were measured by invasive method. A radiographic score was attributed to hind paws.Results:Compared to AIA-Vehicle, Tofacitinib dramatically reduced arthritis (-76%) and radiographic (-73%) scores (p<0.001), and improved Ach-induced vasorelaxation (p<0.05). Of note, Ach-induced vasorelaxation was not different between Tofacitinib-AIA and control rats. The response to SNP was not different between groups. The effect of Tofacitinib on ED was mediated by decreased cyclooxygenase-2 and arginase activities, decreased superoxide anions production, increased NO synthase activity and EDHF synthesis. As compared to AIA-Vehicle, Tofacitinib changed neither blood pressure nor heart rate.Conclusion:The present results demonstrated that Tofacitinib reversed arthritis-induced ED, through the correction of all the acknowledged impaired endothelial pathways in the AIA model. As ED is theprimum movensof atherogenesis, these data provide a mechanistic explanation to the potential benefits of Tofacitinib on the cardiovascular comorbidities associated to RA.

Application of N-carbamylglutamate in Rex rabbits to reduce body fat deposition and its possible mechanism.

N-Carbamylglutamate (NCG) has been shown to enhance arginine synthesis and improve growth performance in animals. However, the effect of NCG on body fat deposition remains unknown. This study examined the effects of NCG on body fat deposition and evaluated the potential mechanisms involved. Rex rabbits (3 months old) were assigned to one of four dietary groups and supplemented with NCG at the following different concentrations in a feeding trial that lasted 67 d: 0 (control), 0·04, 0·08, and 0·12 %. NCG supplementation increased serum concentrations of arginine and proline by activating intestinal carbamoylphosphate synthase-І at the posttranscriptional level. Final body weights and growth performance were not affected by dietary NCG levels. However, NCG-treated rabbits had lower perirenal and subcutaneous fat percentages, serum TAG content, and hepatic fatty acid synthase (FAS) activity and increased NO synthase activity and serum levels of NO, growth hormone (GH), and insulin-like growth factor 1 (IGF-1). There were significant positive correlations between TAG content and perirenal fat percentage, as well as FAS activity and perirenal fat percentage, but significant negative correlations between TAG and NO levels, and FAS activity and IGF-1 level in rabbits after NCG treatment. NCG supplementation did not affect hepatic health indicators, except for serum ammonia concentrations, which were decreased in NCG-treated rabbits. Our results suggest that NCG can serve as a dietary supplement to reduce unfavourable fat deposition through inhibiting hepatic lipogenesis in animals since it appears to have no negative effects on growth performance or hepatic health.

Effect of pravastatin on tetrahydrobiopterin-sensitive and -resistant NO synthase activity of preeclamptic placentas

Introduction: The treatment of preeclampsia, which occurs in 3-8% of pregnancies, is not yet resolved. In preeclampsia, NO synthesis is insufficient, which can contribute to hypertension, proteinuria and abnormal vascularization of the placenta. Decreased NO synthesis in the preeclamptic placenta may also be due to a decrease in the affinity of NO synthase for tetrahydrobiopterin (BH4), resulting in BH4 resistance. In recent years, pravastatin has been shown to prevent preeclampsia in animal models and in human studies. One of the known pleiotropic effects of pravastatin is that it increases NO synthase activity. Aim: Description of the effect of pravastatin on BH4-resistant NO synthase activity in the preeclamptic placenta. Method: NO synthase activity in the placental microsome was measured with C14 arginine substrate using healthy (n = 9) and preeclamptic (n = 9) samples. NO synthase activity was measured at 0.02 µM, physiological at 0.20 µM and pharmacological at 50 µM BH4. Results: One of the 9 preeclamptic patients was BH4-resistant; physiologic BH4 concentration did not significantly increase NO synthase activity, whereas healthy placental microsomes showed a mean increase of 60% (p<0.01), and BH4-sensitive preeclamptic specimen showed a 67% (p<0.01) increase. 10 µM pravastatin increased NO synthase activity by 32-38% at each BH4 concentration in healthy, BH4-sensitive and BH4-resistant preeclampsia samples. Conclusion: 10 µM pravastatin increased BH4-resistant placental NO synthase activity to a similar extent as placental physiological BH4 concentration (0.06-0.20 µM) to BH4-sensitive NO synthase activity. The NO synthase activity of BH4-resistant preeclamptic placenta can be increased by pravastatin to physiological level. Orv Hetil. 2020; 161(10): 389-395.

Different adaptive NO-dependent Mechanisms in Normal and Hypertensive Conditions.

Myocardial infarction (MI) remains the leading cause of death worldwide. We aimed to investigate the effect of NO deficiency on selective biochemical parameters within discreet myocardial zones after experimentally induced MI. To induce MI, the left descending coronary artery was ligated in two groups of 16-week-old WKY rats. In one group, NO production was inhibited by L-NAME (20 mg/kg/day) administration four weeks prior to ligation. Sham operations were performed on both groups as a control. Seven days after MI, we evaluated levels of nitric oxide synthase (NOS) activity, eNOS, iNOS, NFҡB/p65 and Nrf2 in ischemic, injured and non-ischemic zones of the heart. Levels of circulating TNF-α and IL-6 were evaluated in the plasma. MI led to increased NOS activity in all investigated zones of myocardium as well as circulating levels of TNF-α and IL-6. L-NAME treatment decreased NOS activity in the heart of sham operated animals. eNOS expression was increased in the injured zone and this could be a compensatory mechanism that improves the perfusion of the myocardium and cardiac dysfunction. Conversely, iNOS expression increased in the infarcted zone and may contribute to the inflammatory process and irreversible necrotic changes.

Abstract 16806: Mechanisms of Sex Differences in Exercise Capacity

The current policy for NIH grants involves rigor, which not only includes adequate statistics, but also examination of both males and females. The reason for the priority to examine sex differences is because male rodents have been used almost exclusively in pre-clinical studies, which allows for biases to arise. Accordingly, the goal of this investigation was to determine sex differences in exercise capacity in mice and the mechanisms mediating these differences. C57 female wild-type (WT) mice (5 months old) exhibited greater, p&lt;0.05, maximal exercise capacity for running distance (469 ± 24 m; n=7) than age-matched male WT mice (337 ± 14 m; n=8) as well as a 21% improvement in work to exhaustion. The first goal was to look at the effect of physical factors, such as weight and muscle mass, on exercise capacity. When matched for weight females still out-performed males in both running distance (541 ± 21 vs. 456 ± 18m, p&lt;0.05; n=10/group) and demonstrated an 18% improvement in work to exhaustion. Similar outcomes were observed when matching for hind-limb muscle mass. The second goal was to study the effects of estrogen on exercise capacity. After ovariectomy (OVX), female mice no longer demonstrated enhanced exercise compared with males. Conversely, chronic administration of estrogen to male mice improved capacity in running distance and work to exhaustion by 35% after 4 weeks of treatment. Next, we investigated nitric oxide (NO), a downstream target of estrogen, a mechanism which has not been examined previously for mediating sex differences in exercise capacity. Total NO synthase (NOS) activity was higher in female mice compared with male mice (207 ± 14 U/mg vs. 138 ± 4 U/mg, p&lt;0.05), but was no longer different after OVX. Furthermore, males chronically treated with estrogen exhibited a 47% increase in NOS activity. NO blockade with L-NAME eliminated the enhanced exercise capacity observed in both females and males treated with estrogen. Thus, as expected, estrogen is a key mechanism mediating the enhanced exercise capacity in female mice. However, this investigation also demonstrated another, novel key mechanism, increased nitric oxide activity.

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.

ROLE OF MITOCHONDRIAL NO-SYNTHASE IN THE IMPLEMENTATION OF ANTITUMOR EFFECTS OF POLYUNSATURATED FATTY ACIDS IN THE MODEL OF GUERIN’S CARCINOMA UNDER IN VIVO CONDITIONS

NO-synthase activity and NO level in the mitochondrial fraction of rats Guerin’s carcinoma under conditions of different ю-3 and ю-6 polyunsaturated fatty acids (PUFAs) supplementation has been investigated. Guerin’s carcinoma was used as a cancer model. 0.5 ml of 30% carcinoma cell suspension in normal saline was implanted subcutaneously into thigh of a hind leg. The intensity of tumor growth was evaluated based on measurements of subcutaneous tumor size and coefficient of tumor mass. It was showed that ω-3 PUFAs supplementation before and after transplantation of Guerin’s carcinoma resulted in the decreases of tumor growth and coefficient of tumor mass in an organism. Additional ю-6 PUFA supplementation can stimulate tumor progression and increase tumor mass in body in the logarithmic phases of carcinogenesis as compared to the tumor-bearing rats. The effect depends on duration of ю-3 and ю-6 PUFAs administration and on the level of preliminary provision of an organism with these PUFAs. We found increased NO-synthase activity and NO content in mitochondrial fraction of Guerin’s carcinoma in animals of the group that was administered ю-3 PUFAs both before and post-implantation of the carcinoma during logarithmic phase of carcinogenesis. At the same time, the ю-6 PUFA administration does not contribute to significant changes in the NO-synthase system components in the mitochondrial fraction of Guerin’s carcinoma in comparison with the tumor-bearing rats.

Nitrergic neurons of the dorsal raphe nucleus encode information about stress duration.

Nitrergic neurons of the dorsal raphe nucleus (DRN) may play a role in physiological stress responses. The caudal lateral wings (CLW) are unique compared to other rostral-caudal DRN sub-regions because they contain distinct nitric oxide (NO) synthase (NOS) populations that are independent of tryptophan hydroxylase (TPH). NOS neurons in the CLW are also highly activated during acute restraint stress. However, the effects of acute stress duration on NOS activation in the CLW are unclear. Here NADPH-d, an index of NOS activity, is used to show that sub-regions of the DRN have differential NOS activation in response to 6 hours of restraint stress in rats. We report increased NOS activity through 6 hours of restraint in the caudal lateral wings and ventromedial sub-regions. These data suggest that, NOS neurons may play a dynamic role in the response to stress duration.

Glucocorticoids improve endothelial function in rheumatoid arthritis: a study in rats with adjuvant-induced arthritis.

To determine the effect of glucocorticoids (GCs) on endothelial dysfunction (ED) and on traditional cardiovascular (CV) risk factors in the adjuvant-induced arthritis (AIA) rat model. At the first signs of AIA, a high dose (HD) [10 mg/kg/day, intraperitoneally (i.p.), GC-HD] or low dose (LD) (1 mg/kg/day, i.p., GC-LD) of prednisolone was administered for 3 weeks. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of nitric oxide synthase (NOS), cyclooxygenase 2 (COX-2), arginase, endothelium derived hyperpolarizing factor (EDHF) and superoxide anions ( O2-°) production. Aortic expression of endothelial NOS (eNOS), Ser1177-phospho-eNOS, COX-2, arginase-2, p22phox and p47phox was evaluated by Western blotting analysis. Arthritis scores, blood pressure, heart rate and blood levels of cytokines, triglycerides, cholesterol and glucose were measured. GC-HD but not GC-LD reduced arthritis score significantly and improved Ach-induced relaxation (P < 0·05). The positive effect of GC-HD resulted from increased NOS activity and EDHF production and decreased COX-2/arginase activities and O2-° production. These functional effects relied upon increased phospho-eNOS expression and decreased COX-2, arginase-2 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression. Despite the lack of effect of GC-LD on ED, it increased NOS and EDHF and down-regulated O2-° pathways but did not change arginase and COX-2 pathways. GC-HD increased triglycerides levels and blood pressure significantly (P < 0·05). Both doses of GCs decreased to the same extent as plasma interleukin (IL)-1β and tumour necrosis factor (TNF)-α levels (P < 0·05). Our data demonstrated that subchronic treatment with prednisolone improved endothelial function in AIA via pleiotropic effects on endothelial pathways. These effects occurred independently of the deleterious cardiometabolic effects and the impact of prednisolone on systemic inflammation.

Simulated weightlessness affects the expression and activity of neuronal nitric oxide synthase in the rat brain.

Spaceflight induces pathophysiological alterations in various organs. To study pathophysiological adaptations to weightlessness on the ground, the tail suspension (TS) rat model has been used to simulate the effects of weightlessness. There is currently little information on the effect of TS on the expression and activity of nitric oxide synthase (NOS) in the brain. In this study, we examined time-dependent alterations in the expression and activity of neuronal NOS (nNOS) in the brains of TS rats. Male Sprague-Dawley rats were tail-suspended for 1 (TS1), 7 (TS7), and 14 (TS14) days or rested on the ground for 3 days after 14 days of TS. TS1 and TS7 rats exhibited no significant alterations in the expression of nNOS compared to control rats, whereas nNOS expression in TS14 rats was significantly upregulated compared to control rats. Normalized expression of nNOS mRNA and protein in TS14 rats (1.86 ± 0.48 and 1.84 ± 0.29, respectively) were significantly higher than that of control rats (P < 0.001 and P < 0.001, respectively). Consistent with these results, significant elevations in NOS activity and NO production were observed in TS14 rats. Thus, we demonstrated a significant upregulation of nNOS expression, accompanied by significant increases in NOS activity and NO production, in the brain of rats exposed to simulated weightlessness.

Modulation of Lung Function by Increased Nitric Oxide Production

Cigarette smoking reduces endogenous Nitric Oxide (NO) production by reducing Nitric Oxide Synthase (NOS) activity, which is one of the probable reason for increased rate of pulmonary diseases in smokers. Nitric oxide/oxygen blends are used in critical care to promote capillary and pulmonary dilation to treat several pulmonary vascular diseases. Among several supplements, the highest NOS activation has been proved for garlic with its unique mechanism of action. To investigate the effect of dietary supplementation of NO producing garlic on pulmonary function of smokers. The study was conducted on 40 healthy non-smoker (Group A) and 40 chronic smoker (Group B) males with matched age, height and weight. The pulmonary function tests- Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), FEV1/FVC ratio and Peak Expiratory Flow Rate (PEFR) were performed in non-smokers (Group A), smokers (Group B) and smokers after supplementation of approximately 4 gm of raw garlic (2 garlic cloves) per day for three months (Group C). Endogenous NO production was studied in smokers before and after garlic supplementation and in non-smokers without supplementation. The data obtained were compared between the groups using unpaired student's t-test. The p-value considered significant at <0.05. Our results showed that FVC, FEV1, FEV1/FVC ratio and PEFR were reduced significantly along with a significant decreased NOS activity among smokers (Group B) when compared with non-smokers (Group A). Garlic supplementation significantly improved the pulmonary function tests in Group C in comparison to Group B by increasing NOS activity. Dietary supplementation of garlic, which might be by increasing NOS activity, has significantly improved pulmonary functions in smokers.

Glucose-6-phosphate dehydrogenase contributes to the regulation of glucose uptake in skeletal muscle

ObjectiveThe development of skeletal muscle insulin resistance is an early physiological defect, yet the intracellular mechanisms accounting for this metabolic defect remained unresolved. Here, we have examined the role of glucose-6-phosphate dehydrogenase (G6PDH) activity in the pathogenesis of insulin resistance in skeletal muscle. MethodsMultiple mouse disease states exhibiting insulin resistance and glucose intolerance, as well as obese humans defined as insulin-sensitive, insulin-resistant, or pre-diabetic, were examined. ResultsWe identified increased glucose-6-phosphate dehydrogenase (G6PDH) activity as a common intracellular adaptation that occurs in parallel with the induction of insulin resistance in skeletal muscle and is present across animal and human disease states with an underlying pathology of insulin resistance and glucose intolerance. We observed an inverse association between G6PDH activity and nitric oxide synthase (NOS) activity and show that increasing NOS activity via the skeletal muscle specific neuronal (n)NOSμ partially suppresses G6PDH activity in skeletal muscle cells. Furthermore, attenuation of G6PDH activity in skeletal muscle cells via (a) increased nNOSμ/NOS activity, (b) pharmacological G6PDH inhibition, or (c) genetic G6PDH inhibition increases insulin-independent glucose uptake. ConclusionsWe have identified a novel, previously unrecognized role for G6PDH in the regulation of skeletal muscle glucose metabolism.

Antihypertensive effects of fargesin in vitro and in vivo via attenuating oxidative stress and promoting nitric oxide release

Fargesin, a bioactive neolignan isolated from magnolia plants, is widely used in the treatment of managing rhinitis, inflammation, histamine, sinusitis, and headache. To provide more biological information about fargesin, we investigated the effects of fargesin on rat aortic rings and 2-kidney, 1-clip (2K1C) hypertensive rats. In vitro, fargesin caused concentration-dependent vasorelaxation in rat isolated aortic rings induced by KCl and norepinephrine. The effect was weakened by endothelium denudation and nitric oxide (NO) synthesis inhibition. In vivo, the evolution of systolic blood pressure (SBP) was followed by weekly measurements. Angiotensin II (Ang II) and endothelin (ET) levels, NO and nitric oxide synthase (NOS), and plasma and liver oxidative stress markers were determined at the end of the experimental period. After 5 weeks of fargesin treatment, we found that fargesin treatment reduced SBP, cardiac hypertrophy, and Ang II and ET levels of hypertensive rats. Increased NOS activity and NO level were observed in fargesin-treated rats. Normalisation of plasma MDA concentrations and improvement of the antioxidant defence system in plasma and liver accompanied the antihypertensive effect of fargesin. Taken together, these results provided substantial evidences that fargesin has antihypertensive effect in 2K1C hypertensive rats via inhibiting oxidative stress and promoting NO release.

No effect of NOS inhibition on skeletal muscle glucose uptake during in situ hindlimb contraction in healthy and diabetic Sprague-Dawley rats.

Nitric oxide (NO) has been shown to be involved in skeletal muscle glucose uptake during contraction/exercise, especially in individuals with Type 2 diabetes (T2D). To examine the potential mechanisms, we examined the effect of local NO synthase (NOS) inhibition on muscle glucose uptake and muscle capillary blood flow during contraction in healthy and T2D rats. T2D was induced in Sprague-Dawley rats using a combined high-fat diet (23% fat wt/wt for 4 wk) and low-dose streptozotocin injections (35 mg/kg). Anesthetized animals had one hindlimb stimulated to contract in situ for 30 min (2 Hz, 0.1 ms, 35 V) with the contralateral hindlimb rested. After 10 min, the NOS inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME; 5 μM) or saline was continuously infused into the femoral artery of the contracting hindlimb until the end of contraction. Surprisingly, there was no increase in skeletal muscle NOS activity during contraction in either group. Local NOS inhibition had no effect on systemic blood pressure or muscle contraction force, but it did cause a significant attenuation of the increase in femoral artery blood flow in control and T2D rats. However, NOS inhibition did not attenuate the increase in muscle capillary recruitment during contraction in these rats. Muscle glucose uptake during contraction was significantly higher in T2D rats compared with controls but, unlike our previous findings in hooded Wistar rats, NOS inhibition had no effect on glucose uptake during contraction. In conclusion, NOS inhibition did not affect muscle glucose uptake during contraction in control or T2D Sprague-Dawley rats, and this may have been because there was no increase in NOS activity during contraction.