Nitric Oxide Synthase Enzyme Activity Research Articles

P1474: MOLECURAL MECHANISM OF CHEMOTHERAPEUTIC HYDROXYUREA IS MEDIATED BY NOS2

Background:: Hydroxyurea (HU) is a chemotherapeutic agent that reduces ribonucleotide reductase, stops DNA synthesis and repar, and therefore causes cell proliferation inhibition and apoptosis. Due to its cytostatic properties, HU is frequently used for treatment of myeloproliferative neoplasms, ovarian cancer, and sickle cell anemia. Nitric oxide (NO), produced by nitric oxide synthase (NOS) enzymes is a potent signaling molecule involved in blood flow regulation, neutrotransmission, and immunity. Although HU treatment increases NO levels, up to date it is not clear whether it originates from activation of NOS enzymes or HU degradation. Aims: The aim of this study was to determine the involvement of NOS2 enzyme in the cytostatic effect of HU. Methods: To examine the involvement of the NOS2 enzyme in the molecular mechanism of HU, we treated erythroleukemic HEL92.7.1 cells with pan-selective NOS inhibitor L-NAME (200µM, 1mM, and 5mM), NOS2 specific inhibitor 1400W (1, 10, and 100µM), or NOS2/NOS3 inhibitor DPI (1, 5, and 10µM), in combination with hydroxyurea (200µM), and monitored their effect on proliferation and cell cycle. Immunocytochemistry for the proliferation marker Ki67 was performed to assess proliferation, while cell distribution in cell cycle phases was determined by flow cytometry after propidium iodide staining. Colony forming assay have been performed with the bone marrow cells of Nos2 null mice after oral HU treatment to corroborate the data obtained by enzymatic inhibition. Results: In this study, we demonstrated that treatment of HEL92.7.1 cells with HU induces a dose-dependent increase in NOS2 protein levels and two products of the enzyme NOS - NO and citrulline. HU-induced citrulline levels can be reduced by treatment with the NOS inhibitor L-NAME, indicating that NO is produced de novo by the NOS enzyme rather than HU degradation. Inhibition of the NOS2 enzyme by L-NAME, 1400W, or DPI was sufficient to abolish HU-mediated inhibition of proliferation. While HU increased the number of cells in S-phase of the cycle at the expanse of the G0/G1 due to blocked DNA synthesis, combined treatment with HU and L-NAME or DPI inhibitor resulted in decreased G0/G1 phase and increased S and G2/M phases pointing to increased proliferation. These data indicate that the cytostatic properties of HU are mediated by the NOS2 enzyme. A colony formation assay showed that Nos2 deficient bone marrow cells isolated from mice treated orally with HU (200mg/kg) formed significantly more erythroid colonies (BFU-E and CFU-E) and granulocyte/macrophage progenitors (CFU-GM) compared to HU treated wild-type and untreated Nos2 null mice showing the involvement of Nos2 in the molecular mechanism of HU in vivo. Summary/Conclusion: Our results show that HU induces the enzymatic activity of the NOS2 protein which in turn is involved in the HU regulation of proliferation and cell cycle. Comprehensive knowledge of the molecular mechanism of HU might help to improve its beneficial properties and decrease adverse effects.

L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH4), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH4 were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH4 to kill E. coli, by using PMNs from NOS2−/− mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH4 and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.

Effect of Metformin Pretreatment on Serum Adropin Level in Rats with Adrenalin-Induced Acute Myocardial Infarction

AbstractBackground: Beside adropin's role in energy homeostasis and insulin sensitivity, it is also incriminated in myocardial ischemia. Level of adropin during myocardial infarction (MI) and the effect of metformin pretreatment on serum adropin level in rat models of MI is underinvestigated.Aim of Study: This study investigated the level of adropin in MI and the possible involvement of adropin and endothelial nitric oxide synthase enzyme activity (eNOS) in the cardio-protective effect of metformin during acute myocardial ischemia.Material and Methods: A total of forty adult male albino rats weighing 185-220g were divided equally into four groups Group I: Normal control group. Group II: MI group, which was induced by subcutaneous injection of adrenalin (2mg/kg) in two doses 24 hours apart. Group III: Normal rats which were treated with oral metformin via a feeding tube in a dose of 300mg/kg for 2 weeks. Group IV: Rats with adrenalin-induced MI and pretreated with oral metformin in the same dose and duration before induction of MI on the 13th and 14th days of the study. Serum adropin and eNOS were measured, in addition to cardiac troponin I, Creatine Kinase isoenzyme (CK-MB), and C-reactive protein (CRP). Sections from the heart was stained with Masson's trichrome stain and examined to quantify the degree of ischemic injury at the tissue level.Results: In MI group, both serum adropin level and the eNOS were significantly increased with significant positive correlation between adropin and troponin I, CK-MB, and eNOS in the same group (r= 0.857, 0.97 & 0.845, respectively, p<0.05). However, Metformin pretreatment in group IV, produced significant reduction in the level of adropin accom-panied by significant increase in levels of eNOS enzyme activity in comparison to rats with untreated MI. Additionally histological examination, showed reduction of the inflamma-tory cell infiltration, myocardial fibrosis and necrosis. More-over, there was a positive correlation between adropin and eNOS in the same group (r=0.682, p<0.05).Conclusion: Adropin and eNOS enzyme play a vital role in the pathophysiology of acute myocardial infarction. In addition, the cardioprotective effect of metformin pretreatment in this condition depends on enhancing the activity of eNOS enzyme independent on adropin level.

The peroxisome proliferator-activated receptor gamma agonist pioglitazone improves nitric oxide availability, renin-angiotensin system and aberrant redox regulation in the kidney of pre-hypertensive rats.

The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent nuclear receptor. It plays an important role in kidney physiology, where it might contribute to arterial blood pressure regulation and hypertension development by modulation of several signaling pathways. In our study we focused on the effect of PPARγ agonist pioglitazone on changes in the nitric oxide synthase (NOS) expression and activity, the renin-angiotensin system (RAS) cascade, and redox homeostasis signaling pathways in the renal cortex of young pre hypertensive rat models. Young (5-weeks old) spontaneously hypertensive (SHR) and borderline hypertensive (BHR) rats were treated by pioglitazone (PIO, 10 mg/kg/day) during 10 days. Blood pressure (BP) was determined by plethysmography method. Changes in lipid profile were detected in plasma with standard kits using biochemical analyser. Gene expression has been detected by qRT-PCR and protein level was determined using Western blot analysis. Superoxide dismutase (SOD) and catalase (CAT) activities were determined spectrophotometrically and the total enzyme activity of NOS was measured using a radioactive assay based on conversion of [3H] L-arginine to [3H] L- citrulline. Administration of pioglitazone decreased BP in BHR and slowed down the development of BP increase in young SHR animals. For NOS, activation by PPARγ correlated with increase in gene and protein expression of NOS isoforms and in total enzyme activity both in BHR and SHR. In the AT1R/Nox pathway, the treatment did not significantly influence mRNA expression of the p22phox subunit of NADPH oxidase (Nox) and AT1R, but up-regulated the 'pro-vasodilatatory' Mas and AT2R receptors in both BHR and SHR groups. Pioglitazone treatment affected redox regulation. Increase in gene expression of nuclear factor E2-related factor 2 (Nrf2) and SOD isoforms correlated with SOD and CAT enzyme activities. The group treatment-to-control ratios, BHR Pioglitazone to BHR control and SHR Pioglitazone to SHR control for gene expression increased by 10% to 230%. The largest effect of PPARγ has been observed in SOD1, SOD3 and the Mas receptor gene treatment-to-control ratios. The most prominent differences between BHR and SHR were observed in SOD1 and Mas receptor expressions, with large effects of opposite sign in BHR versus SHR. Our data indicate that an increase of NO release activates signaling in the renal cortex of pre-hypertensive rats after pioglitazone treatment. Improvement of NO availability, AT2R, Mas receptors and aberrant redox regulation is thought to be the major correlated mechanisms mediating the BP decrease affected by the PPARγ agonist treatment. We also observed that the most sensitive tissue responses to PPARγ-dependent activation of Nrf2 have been primarily found in the kidney of young hypertensive animals.

IL‐10 Supplementation Decreases BP in Male, But Not Female, Spontaneously Hypertensive Rats

Interleukin 10 (IL‐10) is an anti‐inflammatory cytokine that induces nitric oxide (NO) production. IL‐10 supplementation has previously been shown to lower blood pressure (BP) in male hypertensive mice, but the effect of exogenous IL‐10 in hypertensive females has not yet been studied. For the current study, we hypothesized that chronic infusion of IL‐10 in hypertensive rats would lower BP concomitant with an increase in NO synthase (NOS) activity. First, endogenous basal levels of renal IL‐10+ cells and the IL‐10 receptor were measured in a subset of male and female spontaneously hypertensive rats (SHR). In a second set of rats, baseline BP was recorded by tail cuff plethsmography, and rats were placed in metabolic cages for 24‐hr urine collection prior to initiating infusion of IL‐10. IL‐10 was infused by subcutaneous minipump (3.5 ug/kg/d) in half of the male and female SHR, with the remaining rats serving as sham controls (n=4–6 per treatment group per sex). After 2 weeks of treatment, final BP and metabolic cage timepoints were collected, and tissues were harvested. Female SHR had greater IL‐10+ renal cells than males (flow cytometry, 4.6±0.4 vs 1.9±0.4%, p=0.003, n=8) and greater expression of the IL‐10 receptor (Western blot, 0.4±0.1 vs 0.13±0.1 densitometric units, p=0.03, n=4). BP did not change in female SHR treated with IL‐10 (2‐way ANOVA, p=0.3), but BP significantly decreased following IL‐10 infusion in male rats (p=0.04, Table); BP was higher in males than females in both groups (p&lt;0.001). Urinary protein excretion (Bradford assay) did not differ between treatment and control groups in either sex (Table); but excretion was higher in males than in females (p=&lt;0.001). Contrary to our hypothesis, NOS enzymatic activity (measured by the conversion of radio‐labeled arginine to radio‐labeled citrulline) significantly decreased with IL‐10 treatment in both the inner medulla (p=0.004) and the renal cortex (p=0.003) of both sexes (Table). In conclusion, despite the finding that male SHR have less IL‐10 and IL‐10 receptor expression in the kidney compared to females, BP was selectively decreased in males. We hypothesize that the higher endogenous levels of IL‐10 in the female SHR contribute to the observed sex difference in BP and prevent further decreases in BP with exogenous IL‐10 infusion. Furthermore, our data suggests that decreases in BP in male SHR with IL‐10 is not dependent on upregulating NOS activity in the kidney, although the effect of IL‐10 on NO bioavailability was not assessed. Future studies will further explore the role of endogenous IL‐10 on BP control in female SHR. Baseline BP (mmHg) Final BP (mmHg) Baseline Proteinuria (mg/d) Final Proteinuria (mg/d) Inner Medulla NOS activity (pmol/mg) Cortex NOS activity (pmol/mg) Male CON 211±1 221±5 10±1 15±2 780±123 17287±42772 Male IL‐10 213±5 200±4* 12±1 16±2 284±29* 7316±1917* Female CON 160±8# 169±7# 2±0.4# 3±1# 1296±213# 20260±2207 Female IL‐10 163±2# 169±9# 1±0.3# 2±0# 650±65*# 8279±1215* p&lt;0.05 vs CON same sex, p&lt;0.05 vs male same treatment

Liposomal tetrahydrobiopterin preserves eNOS coupling in the post-ischemic heart conferring in vivo cardioprotection

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS), and reduced BH4 availability leads to endothelial NOS (eNOS) uncoupling and increased reactive oxygen species (ROS) generation. Questions remain regarding the functional state of eNOS and role of BH4 availability in the process of in vivo myocardial ischemia–reperfusion (I/R) injury. Rats were subjected to 60min of in vivo left coronary artery occlusion and varying periods of reperfusion with or without pre-ischemic liposomal BH4 supplementation (1mg/kg, iv). Myocardial infarction was correlated with cardiac BH4 content, eNOS protein level, NOS enzyme activity, and ROS generation. In the vehicle group, 60-min ischemia drastically reduced myocardial BH4 content in the area at risk (AAR) compared to non-ischemic (NI) area and the level remained lower during early reperfusion followed by recovery after 24-h reperfusion. Total eNOS, activated eNOS protein level (eNOS Ser1177 phosphorylation) and NOS activity were also significantly reduced during ischemia and/or early reperfusion, but recovered after 24-h reperfusion. With liposomal BH4 treatment, BH4 levels were identical in the AAR and NI area during ischemia and/or early reperfusion, and were significantly higher than with vehicle. BH4 pre-treatment preserved eNOS Ser1177 phosphorylation and NOS activity in the AAR, and significantly reduced myocardial ROS generation and infarction compared to vehicle. These findings provide direct evidence that in vivo I/R induces eNOS dysfunction secondary to BH4 depletion, and that pre-ischemic liposomal BH4 administration preserves eNOS function conferring cardioprotection with reduced oxidative stress.

Gut region-dependent alterations of nitrergic myenteric neurons after chronic alcohol consumption.

Chronic alcohol abuse damages nearly every organ in the body. The harmful effects of ethanol on the brain, the liver and the pancreas are well documented. Although chronic alcohol consumption causes serious impairments also in the gastrointestinal tract like altered motility, mucosal damage, impaired absorption of nutrients and inflammation, the effects of chronically consumed ethanol on the enteric nervous system are less detailed. While the nitrergic myenteric neurons play an essential role in the regulation of gastrointestinal peristalsis, it was hypothesised, that these neurons are the first targets of consumed ethanol or its metabolites generated in the different gastrointestinal segments. To reinforce this hypothesis the effects of ethanol on the gastrointestinal tract was investigated in different rodent models with quantitative immunohistochemistry, in vivo and in vitro motility measurements, western blot analysis, evaluation of nitric oxide synthase enzyme activity and bio-imaging of nitric oxide synthesis. These results suggest that chronic alcohol consumption did not result significant neural loss, but primarily impaired the nitrergic pathways in gut region-dependent way leading to disturbed gastrointestinal motility. The gut segment-specific differences in the effects of chronic alcohol consumption highlight the significance the ethanol-induced neuronal microenvironment involving oxidative stress and intestinal microbiota.

New Insights into Possible Role of NOS-NO-ADMA Pathway Dysfunction in the Development of Cognitive Decline and Dementia: Exploring the Vascular Features of Alzheimer’s Disease

In recent years significant overlap between cardio-metabolic risk factors and cognitive decline has been reported. Cardio-metabolic and vascular factors shown to be associated with Alzheimer’s disease (AD) and other forms of dementia include midlife diabetes and hypertension, cerebrovascular lesions, diminished vascular function, dyslipidemia, obesity, and cigarette smoking. Accordingly, it has been recently proposed that amyloid is not the cause of AD but merely a marker and a later consequence of upstream changes that lead to neuronal and synaptic losses. However, although the idea that features of vascular dysfunction and injury are present in cognitive decline and AD patients was suggested over 25 years ago, the role of cardio-metabolic and cerebrovascular mechanisms in the pathogenesis of AD is far from being fully elucidated. Based on newly proposed vascular hypothesis, there is an impaired structure and function of cerebral blood vessels and cells in patients with cognitive decline and AD which is mediated by vascular oxidative stress. Consistent with these observations, the importance of endothelial dysfunction in the development of AD has been highlighted. One of the most prominent features of endothelial dysfunction is decreased production and bioavailability of Nitric Oxide (NO). Based on versatile properties in physiological as well as in pathophysiological conditions NO is regarded as a key molecule for longevity and cardiovascular health. NO is produced by three different isoforms of the enzyme NO synthase (NOS), namely: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). Reduced NO bioavailability and altered vascular expression and activity of NOS enzymes are implicated as major molecular players in the process of vascular aging. In the process of aging, the mechanisms by which NOS enzymes promote vascular dysfunction are specific for each NOS isoform. Normal activity of eNOS is required for the balanced production/bioavailability of NO, which is the main prerequisite for optimal vascular function. Conversely, excessive amount of NO produced by iNOS contributes to vascular dysfunction. Furthermore, although the possible role of nNOS-derived NO in aging-associated vascular dysfunction is far from being fully elucidated, experimental findings indicate that impaired perivascular NO release from nNOS increases vasoconstriction in aged arterioles. Oxidative stress and inhibitors of NO synthase are regarded as most potent initiators of disturbed production and bioavailability of NO. Important endogenous inhibitor of all three isoforms of NOS is asymmetrical dimethylarginine (ADMA). It has been recently proposed that ADMA may be a possible link between vascular disease and dementia. Based on this assumption ADMA might represent a unifying pathophysiological pathway linking the presence of vascular risk factors with the onset and progression of cognitive decline and dementia. These observations were made based on good evidence from literature that higher plasma ADMA concentrations favor atherosclerosis and independently predict adverse cardiovascular and cerebrovascular outcomes in several patient groups. However, possible role of ADMA in pathophysiology of cognitive decline and AD is still not fully understood. Prospective studies are warranted aiming at further clarification of ADMA involvement in development and progression of dementia. Moreover, use of ADMA in CSF and /or plasma as prospective biomarker of AD should be explored. According to newly introduced hypothesis disturbed NOS-NO-ADMA pathway is one of the most important attributes of cerebrovascular dysfunction that plays a pathogenic role in the development of cognitive decline and dementia. Experimental data from cell cultures and animal models have demonstrated that dysfunction of the NO-NOS- ADMA pathway results in cell death, blood-brain barrier (BBB) disruption, and brain edema via different pathological mechanisms. However, exact underlining mechanisms through which disturbed NOS-NO-ADMA pathway contributes to cognitive decline and dementia remain to be elucidated. Clarification of these mechanisms may be helpful in the identification of new therapeutic targets for aging and neurodegenerative diseases. Future research should also explain whether endothelial dysfunction precede or follow neurological changes which characterize cognitive decline and dementia. Moreover, clinical and epidemiological studies have shown that healthy nutrition and physical exercise are important non-pharmacological, lifestyle-related interventions that could help in maintaining appropriate vascular tone, adequate cerebral blood flow and normal cognition during aging. It is reasonable to suggest that prevention of cardio-metabolic diseases via endorsement of healthy aging determinants such as control of blood pressure, lipids and glucose levels as well as maintenance of optimal body weight achieved by regular physical activity and nutrition with high anti-oxidant capacity might be effective strategy in the preservation of cognitive function.

Working Memory and Aphasia

In recent years significant overlap between cardio-metabolic risk factors and cognitive decline has been reported. Cardio-metabolic and vascular factors shown to be associated with Alzheimer’s disease (AD) and other forms of dementia include midlife diabetes and hypertension, cerebrovascular lesions, diminished vascular function, dyslipidemia, obesity, and cigarette smoking. Accordingly, it has been recently proposed that amyloid is not the cause of AD but merely a marker and a later consequence of upstream changes that lead to neuronal and synaptic losses. However, although the idea that features of vascular dysfunction and injury are present in cognitive decline and AD patients was suggested over 25 years ago, the role of cardio-metabolic and cerebrovascular mechanisms in the pathogenesis of AD is far from being fully elucidated. Based on newly proposed vascular hypothesis, there is an impaired structure and function of cerebral blood vessels and cells in patients with cognitive decline and AD which is mediated by vascular oxidative stress. Consistent with these observations, the importance of endothelial dysfunction in the development of AD has been highlighted. One of the most prominent features of endothelial dysfunction is decreased production and bioavailability of Nitric Oxide (NO). Based on versatile properties in physiological as well as in pathophysiological conditions NO is regarded as a key molecule for longevity and cardiovascular health. NO is produced by three different isoforms of the enzyme NO synthase (NOS), namely: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). Reduced NO bioavailability and altered vascular expression and activity of NOS enzymes are implicated as major molecular players in the process of vascular aging. In the process of aging, the mechanisms by which NOS enzymes promote vascular dysfunction are specific for each NOS isoform. Normal activity of eNOS is required for the balanced production/bioavailability of NO, which is the main prerequisite for optimal vascular function. Conversely, excessive amount of NO produced by iNOS contributes to vascular dysfunction. Furthermore, although the possible role of nNOS-derived NO in aging-associated vascular dysfunction is far from being fully elucidated, experimental findings indicate that impaired perivascular NO release from nNOS increases vasoconstriction in aged arterioles. Oxidative stress and inhibitors of NO synthase are regarded as most potent initiators of disturbed production and bioavailability of NO. Important endogenous inhibitor of all three isoforms of NOS is asymmetrical dimethylarginine (ADMA). It has been recently proposed that ADMA may be a possible link between vascular disease and dementia. Based on this assumption ADMA might represent a unifying pathophysiological pathway linking the presence of vascular risk factors with the onset and progression of cognitive decline and dementia. These observations were made based on good evidence from literature that higher plasma ADMA concentrations favor atherosclerosis and independently predict adverse cardiovascular and cerebrovascular outcomes in several patient groups. However, possible role of ADMA in pathophysiology of cognitive decline and AD is still not fully understood. Prospective studies are warranted aiming at further clarification of ADMA involvement in development and progression of dementia. Moreover, use of ADMA in CSF and /or plasma as prospective biomarker of AD should be explored. According to newly introduced hypothesis disturbed NOS-NO-ADMA pathway is one of the most important attributes of cerebrovascular dysfunction that plays a pathogenic role in the development of cognitive decline and dementia. Experimental data from cell cultures and animal models have demonstrated that dysfunction of the NO-NOS- ADMA pathway results in cell death, blood-brain barrier (BBB) disruption, and brain edema via different pathological mechanisms. However, exact underlining mechanisms through which disturbed NOS-NO-ADMA pathway contributes to cognitive decline and dementia remain to be elucidated. Clarification of these mechanisms may be helpful in the identification of new therapeutic targets for aging and neurodegenerative diseases. Future research should also explain whether endothelial dysfunction precede or follow neurological changes which characterize cognitive decline and dementia. Moreover, clinical and epidemiological studies have shown that healthy nutrition and physical exercise are important non-pharmacological, lifestyle-related interventions that could help in maintaining appropriate vascular tone, adequate cerebral blood flow and normal cognition during aging. It is reasonable to suggest that prevention of cardio-metabolic diseases via endorsement of healthy aging determinants such as control of blood pressure, lipids and glucose levels as well as maintenance of optimal body weight achieved by regular physical activity and nutrition with high anti-oxidant capacity might be effective strategy in the preservation of cognitive function.

Radiation-modifying abilities of Nigella sativa and Thymoquinone on radiation-induced nitrosative stress in the brain tissue

To investigate Nigella sativa oil (NSO) and Thymoquinone (TQ) for their antioxidant effects on the brain tissue of rats exposed to ionizing radiation.Fifty-four male albino Wistar rats, divided into six groups, were designed as group I (normal control group) did not receive NSO, TQ or irradiation; group II (control group of TQ) received dimethyl sulfoxide and sham irradiation; group III (control group of NSO) received saline and sham irradiation; group IV (irradiation plus NSO group) received both 5 Gray of gamma irradiation to total cranium and NSO; group V (irradiation plus TQ group) received both irradiation and TQ; group VI (irradiation alone group) received irradiation plus saline. Alterations in nitric oxide (NO) and peroxynitrite (ONOO−) levels, and nitric oxide synthase (NOS) enzyme activity were measured by biochemical methods in homogenized brain tissue of rats.Levels of NO and ONOO−, and enzyme activity of NOS in brain tissue of the rats treated with NSO or TQ were found to be lower than in received IR alone (p<0.002)Nigella sativa oil (NSO) and its active component, TQ, clearly protect brain tissue from radiation-induced nitrosative stress.

Sex does not impact asymmetric dimethylarginine (ADMA) or L‐arginine (L‐arg) levels in spontaneously hypertensive rats (SHR).

ADMA and L‐arg are endogenous amino acids that impact nitric oxide synthase (NOS) activity. ADMA has been implicated in nitric oxide (NO) deficiency and hypertension. We have reported that female SHR have significantly greater NO bioavailability and NOS enzymatic activity in the renal inner medulla compared to age‐matched males, yet the mechanism responsible is unknown. We hypothesized that male SHR have greater ADMA levels and less L‐arg compared to females. ADMA and L‐arg levels in plasma (N=6–7) and renal inner medullas (N=10) were measured via HPLC from 12–13 week old male and female SHR. ADMA (Male: 20±6; Female: 31±8 nmols/ml, p=0.7), L‐arg (Male: 152±7; Female: 141±11 nmols/ml, p=0.7) levels and the L‐arg/ADMA ratio (p=0.2) in plasma were comparable between sexes. Renal inner medullary ADMA (Male: 172±55 Female: 192±58 grams wet weight (gww), p=0.8), L‐arg (Male: 507±107; Female: 552±127 gww, p=0.8) levels and L‐arg/ADMA ratio (p=0.8) were also comparable between groups. We conclude that while ADMA and L‐arg may be important in maintaining renal NOS activity in SHR, they do not contribute to sex differences in renal inner medullary NOS activity. Funded by the American Heart Association.

Effect of Berberine on PPARα/NO Activation in High Glucose- and Insulin-Induced Cardiomyocyte Hypertrophy

Rhizoma coptidis, the root of Coptis chinensis Franch, has been used in China as a folk medicine in the treatment of diabetes for thousands of years. Berberine, one of the active ingredients of Rhizoma coptidis, has been reported to improve symptoms of diabetes and to treat experimental cardiac hypertrophy, respectively. The objective of this study was to evaluate the potential effect of berberine on cardiomyocyte hypertrophy in diabetes and its possible influence on peroxisome proliferator-activated receptor-α (PPARα)/nitric oxide (NO) signaling pathway. The cardiomyocyte hypertrophy induced by high glucose (25.5 mmol/L) and insulin (0.1 μmol/L) (HGI) was characterized in rat primary cardiomyocyte by measuring the cell surface area, protein content, and atrial natriuretic factor mRNA expression level. Protein and mRNA expression were measured by western blot and real-time RT-PCR, respectively. The enzymatic activity of NO synthase (NOS) was measured using a spectrophotometric assay, and NO concentration was measured using the Griess assay. HGI significantly induced cardiomyocyte hypertrophy and decreased the expression of PPARα and endothelial NOS at the mRNA and protein levels, which occurred in parallel with declining NOS activity and NO concentration. The effect of HGI was inhibited by berberine (0.1 to 100 μmol/L), fenofibrate (0.3 μmol/L), or L-arginine (100 μmol/L). MK886 (0.3 μmol/L), a selective PPARα antagonist, could abolish the effects of berberine and fenofibrate. N G-nitro-L-arginine-methyl ester (100 μmol/L), a NOS inhibitor, could block the effects of L-arginine, but only partially blocked the effects of berberine. These results suggest that berberine can blunt HGI-induced cardiomyocyte hypertrophy in vitro, through the activation of the PPARα/NO signaling pathway.

In vitro Anti-Inflammatory Effects of Beta-Carboline Alkaloids, Isolated from Picrasma quassioides, through Inhibition of the iNOS Pathway

In our previous study, fourty-eight compounds have been isolated and identified from the roots of Picrasma quassioides, which have been widely used as a traditional Chinese medicine for clearing heat and for detoxification as described in the Chinese Pharmacopoeia. β-Carboline alkaloids are commonly considered as main active constituents of P. quassioides, but the molecular mechanism remains yet unknown. In the present paper, one new β-carboline alkaloid together with two known β-carboline alkaloids have been investigated for their anti-inflammatory effect and mechanism of action in cultured macrophage RAW 264.7 cells. Griess assay was used to evaluate the inhibitory effect on the overproduction of nitric oxide. ELISA was used to determine the level of proinflammatory cytokines including TNF-α and IL-6. The inhibitory effect on the enzymatic activity of COX-2 and inducible nitric oxide synthase were tested by colorimetric and fluorimetric methods, respectively. Western blot was used to detect the expression of inducible nitric oxide synthase and COX-2. All three β-carboline alkaloids suppressed LPS-stimulated nitric oxide production and proinflammatory cytokines secretion, including TNF-α and IL-6 in a dose-dependent manner. They also strongly inhibited the expression of inducible nitric oxide synthase and inducible nitric oxide synthase enzymatic activity, whereas the expression of COX-2 and COX-2 enzymatic activity were not affected. These results indicated that potent inhibition of nitric oxide, TNF-α, and IL-6, but not COX-2 expression and COX-2 activity, might constitute the anti-inflammatory mechanism of β-carboline alkaloids. β-Carboline alkaloids suppressed the overproduction of nitric oxide through downregulation of inducible nitric oxide synthase expression and inducible nitric oxide synthase enzymatic activity in an LPS-stimulated macrophage.

Abstract 644: Tetrahydrobiopterin Supplementation Increases Renal Inner Medullary Nitric Oxide Synthase Enzymatic Activity in Male, But Not Female Spontaneously Hypertensive Rats

Tetrahydrobiopterin Supplementation Increases Renal Inner Medullary Nitric Oxide Synthase Enzymatic Activity in Male, But Not Female Spontaneously Hypertensive Rats Krystal N. Brinson and Jennifer C. Sullivan, Experimental Medicine, Georgia Health Sciences University, Augusta, GA 30912 We previously published that male spontaneously hypertensive rats (SHR) having less nitric oxide synthase (NOS) enzymatic activity in the renal inner medullary (IM) than female SHR, which cannot be explained by protein expression alone. Tetrahydrobiopterin (BH4) is an essential cofactor for NOS activity. BH4 is easily oxidized to BH2, which is not a NOS cofactor, and male SHR have greater levels of oxidative stress than females. Therefore, we hypothesized that 1) male SHR will have greater increases in NOS enzymatic activity following BH4 supplementation than females and 2) antioxidant treatment will result in greater increases in NOS enzymatic activity in male SHR regardless of BH4 supplementation. Studies measured renal IM NOS enzymatic activity in the presence and absence of BH4 from 12-13 week old control (N=5-6) or antioxidant treated (tempol, 30 mg/kg/day in drinking water) male and female SHR (N=4-5). Supplementation with BH4 increased NOS activity in male SHR (177±35 vs. 315±52 pmol/mg; p=0.04), with no effect in females (434±112 vs. 548±84 pmol/mg). Following 2 weeks of antioxidant treatment, NOS enzymatic activity in male SHR was significantly increased compared to control SHR (p=0.04), and supplementation with BH4 no longer altered NOS enzymatic activity in males (588±143 vs. 648±153 pmol/mg). Antioxidant treatment did not significantly alter renal IM NOS enzymatic activity in female SHR compared to untreated controls, and BH4 supplementation did not alter NOS activity in tempol-treated females (473±82 vs. 580±66 pmol/mg). Tempol treatment also abolished the sex difference in renal IM NOS enzymatic activity. In conclusion, our data suggest that greater levels of oxidative stress in male SHR contributes to lower levels of NOS enzymatic activity compared to females. We hypothesize that this results in greater BH4 deficiency in males contributing to decreases in NO and increases in blood pressure.

Ascorbate stimulates endothelial nitric oxide synthase enzyme activity by rapid modulation of its phosphorylation status

Long-term exposure to ascorbate is known to enhance endothelial nitric oxide synthase (eNOS) activity by stabilizing the eNOS cofactor tetrahydrobiopterin (BH4). We investigated acute effects of ascorbate on eNOS function in primary (HUVEC) and immortalized human endothelial cells (EA.hy926), aiming to provide a molecular explanation for the rapid vasodilatation seen in vivo upon administration of ascorbate. Enzymatic activity of eNOS and intracellular BH4 levels were assessed by means of an arginine–citrulline conversion assay and HPLC analysis, respectively. Over a period of 4h, ascorbate steadily increased eNOS activity, although endothelial BH4 levels remained unchanged compared to untreated control cells. Immunoblot analyses revealed that as early as 5min after treatment ascorbate dose-dependently increased phosphorylation at eNOS-Ser1177 and concomitantly decreased phosphorylation at eNOS-Thr495, a phosphorylation pattern indicative of increased eNOS activity. By employing pharmacological inhibitors, siRNA-mediated knockdown approaches, and overexpression of the catalytic subunit of protein phosphatase 2A (PP2A), we show that this effect was at least partly owing to reduction of PP2A activity and subsequent activation of AMP-activated kinase. In this report, we unravel a novel mechanism for how ascorbate rapidly activates eNOS independent of its effects on BH4 stabilization.

Oxidative stress and antioxidant activity of female rat liver tissue after sevoflurane anaesthesia: young versus old

The aim of our study was to assess the evidence of oxidative stress in the rat liver tissue by studying enzymes, such as nitric oxide synthase (NOS), superoxide dismutase (SOD), glutathione-S transferase (GST) activity, and thiobarbutiric acid-reactive substances (TBARS) levels in young versus old female rats after sevoflurane anaesthesia. The study involved 28 female Wistar Albino rats. The rats were divided into the two groups [(Group I, n=14): Young sevoflurane group (Group I-YS, n=7); Young control group (Group I-YC, n=7)], [(Group II, n=14): Old sevoflurane group (Group II-OS, n=7); Old control group (Group II-OC, n=7)]. Sevoflurane was administered at 2 % volume inspiratory concentration, 6 L.min-1 in 100 % O2 for 2 hours. The control groups were not subjected to any procedures. Accordingly, GST, SOD, and NOS enzyme activity and TBARS level, were studied in the liver tissue samples of the rats to determine the presence of oxidative stress (OS) and antioxidant activity. Following administration of sevoflurane anaesthesia; GST, SOD enzyme activity and TBARS level was significantly higher in the Group I-YS than in the Group I-YC and in the Group II-OS than in the Group II-OC. There was no difference between the groups when the mean NOS levels were compared. Although the results of our study are similar to the previous indicating that sevoflurane has the capacity to induce the oxidative stress; a new data has been recorded that sevoflurane has a similar effect on the OS level in aged and young female rat (Tab. 1, Fig. 4, Ref. 48).

Expression of nitric oxide synthase isoforms in different stages of buffalo ( Bubalus bubalis) ovarian follicles: Effect of nitric oxide on in vitro development of preantral follicle

The present study was designed to investigate the expression of nitric oxide synthase (NOS) isoforms in buffalo ovarian preantral (PFs), antral (AFs) and ovulatory (OFs) follicles (Experiment 1); effect of NO on in vitro survival and growth of PFs (Experiment 2) and NOS activity in immature oocytes by NADPH-diaphorase test (Experiment 3). In Experiment 1, NOS isoforms (neuronal, inducible and endothelial) were localized immunohistochemically; mRNA and protein expression was analyzed by semi-quantitative RT-PCR and western blot, respectively. In Experiment 2, PFs were isolated by micro-dissection method from buffalo ovaries and cultured in 0 (control), 10 −3, 10 −5, 10 −7 and 10 −9 M sodium nitroprusside (SNP). PFs were further cultured with 10 −5 M SNP + 1.0 mM N ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 μg/ml hemoglobin (Hb) to examine the reversible effect of SNP. Immunohistochemical studies demonstrated that inducible nitric oxide synthase (iNOS) immunoreactivity was predominantly localized in granulosa and theca cells whereas, neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase in the theca, granulosa and cumulus cells of PFs, AFs and OFs. The amount of mRNA as well as protein of nNOS and eNOS was found similar between different stages of follicles. In contrast, higher level of iNOS mRNA was observed in OFs and protein in the AFs. Higher doses of SNP (10 −3, 10 −5, 10 −7 M) inhibited (P < 0.05) while, lower dose of SNP (10 −9 M) stimulated (P < 0.05) the survival, growth, and antrum formation of PFs. The inhibitory effects of SNP were reversed by Hb, while L-NAME was not found effective. In conclusion, expression of NOS isoforms mRNA and protein in PFs, AFs, and OFs and NOS enzyme activity in immature follicular oocytes suggest a role for NO during ovarian folliculogenesis in buffalo. NO plays a dual role on growth and survival of PFs depending on its concentration in the culture medium.

Endogenous NO levels regulate nodule functioning

Nitric oxide is a small gaseous signaling molecule which functions in the regulation of plant development and responses to biotic and abiotic stresses. Recently, we have shown that nitric oxide is required for development of functional nodules. Here, we show that inhibition of nitric oxide synthase enzymatic activity (using Nω-nitro-L-arginine) reduces nitric oxide content in soybean root nodules and this is coupled by reduction of endogenous cyclic guanosine monophosphate content in the nodules. We postulate that the regulation of soybean nodule development by nitric oxide is transduced via cyclic guanosine monophosphate through activation of nitric oxide-responsive soluble guanylate cyclase. Furthermore, we hypothesize that this signaling cascade is mediated via modulation of the activities of antioxidant metabolic pathways.

Nitric oxide is not involved in Neisseria gonorrhoeae-induced cellular damage of human Fallopian tubes in vitro.

In the present study, we investigated whether cellular damage, as demonstrated by lactate dehydrogenase (LDH) release in the human fallopian tube (FT) infected by Neisseria gonorrhoeae (Ngo), correlated with high levels of nitric oxide synthase (NOS) mRNA and enzyme activity. Infection with Ngo induced a significant increase (~35-fold) in mRNA transcripts of the inducible isoform of NOS. Paradoxically, a reduction in NOS enzyme activity was observed in infected cultures, suggesting that gonococcal infection possibly influences translation of iNOS mRNA to the enzyme. In addition, treatment with the NOS inhibitor TRIM did not prevent gonococcal-induced cellular damage. In contrast, the addition of the inhibitor L-NAME induced a 40% reduction in LDH release, which correlated with a ~50% reduction in gonococcal numbers. Moreover, treatment of normal FT explants with an exogenous NO donor, SNAP, did not induce significant cellular damage. Taken together, our data suggest that NO does not contribute to cellular damage during infection of the human FT with Neisseria gonorrhoeae.

Nitric oxide is not involved in Neisseria gonorrhoeae-induced cellular damage of human Fallopian tubes in vitro

In the present study, we investigated whether cellular damage, as demonstrated by lactate dehydrogenase (LDH) release in the human fallopian tube (FT) infected by Neisseria gonorrhoeae (Ngo), correlated with high levels of nitric oxide synthase (NOS) mRNA and enzyme activity. Infection with Ngo induced a significant increase (~35-fold) in mRNA transcripts of the inducible isoform of NOS. Paradoxically, a reduction in NOS enzyme activity was observed in infected cultures, suggesting that gonococcal infection possibly influences translation of iNOS mRNA to the enzyme. In addition, treatment with the NOS inhibitor TRIM did not prevent gonococcal-induced cellular damage. In contrast, the addition of the inhibitor L-NAME induced a 40% reduction in LDH release, which correlated with a ~50% reduction in gonococcal numbers. Moreover, treatment of normal FT explants with an exogenous NO donor, SNAP, did not induce significant cellular damage. Taken together, our data suggest that NO does not contribute to cellular damage during infection of the human FT with Neisseria gonorrhoeae.