Enhanced Nitric Oxide Production Research Articles

Spermine oxidase is a regulator of macrophage host response to Helicobacter pylori: enhancement of antimicrobial nitric oxide generation by depletion of spermine

The gastric pathogen Helicobacter pylori causes peptic ulcer disease and gastric cancer. We have reported that in H. pylori-activated macrophages, nitric oxide (NO) derived from inducible NO synthase (iNOS) can kill the bacterium, iNOS protein expression is dependent on uptake of its substrate L-arginine (L-Arg), the polyamine spermine can inhibit iNOS translation by inhibiting L-Arg uptake, and inhibition of polyamine synthesis enhances NO-mediated bacterial killing. Because spermine oxidase (SMO), which back-converts spermine to spermidine, is induced in macrophages by H. pylori, we determined its role in iNOS-dependent host defense. SMO shRNA knockdown in RAW 264.7 murine macrophages resulted in a marked decrease in H. pylori-stimulated iNOS protein, but not mRNA expression, and a 90% reduction in NO levels; NO production was also inhibited in primary murine peritoneal macrophages with SMO knockdown. There was an increase in spermine levels after H. pylori stimulation that rapidly decreased, while SMO knockdown caused a greater increase in spermine that was sustained. With SMO knockdown, L-Arg uptake and killing of H. pylori by macrophages was prevented. The overexpression of SMO by transfection of an expression plasmid prevented the H. pylori-stimulated increase in spermine levels, and led to increased L-Arg uptake, iNOS protein expression and NO production, and H. pylori killing. In two human monocytic cell lines, U937 and THP-1, overexpression of SMO caused a significant enhancement of NO production with H. pylori stimulation. By depleting spermine, SMO can abrogate the inhibitory effect of polyamines on innate immune responses to H. pylori by enhancing antimicrobial NO production.

Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells

Hydrogen sulfide (H2S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2S, stimulated NO production whereas blockage of the activity of H2S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2S on ECs. Our results suggest that H2S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2S-induced angiogenesis.

Post-infarct treatment with [Pyr1]-apelin-13 reduces myocardial damage through reduction of oxidative injury and nitric oxide enhancement in the rat model of myocardial infarction

Apelin is a newly discovered peptide that has been recently shown to have cardioprotective effects in the animal model of myocardial infarction (MI) and ischemia/reperfusion (I/R) injuries. The aim of the present study was to investigate the long term cardioprotective effect of [Pyr1]-apelin-13 in the rat model of MI. Male Wistar rats (n=22) were randomly divided into three groups: (1) sham operated group (2) control MI group and (3) MI treated with apelin (MI-AP group). MI animals were subjected to 30min of left anterior descending coronary artery (LAD) ligation and 14 days of reperfusion. 24h after LAD ligation, apelin (10nmol/kg/day) was administered i.p. for 5 days. Blood sampling was performed at days 1, 3, 5 and 7 after MI for determination of serum changes of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), malondialdehyde (MDA) and nitric oxide (NO). Myocardial infarct size (IS) and hemodynamic function were also measured at the end of the study at day 14. We found out that post infarct treatment with apelin decreases infarct size, serum levels of LDH, CK-MB and MDA and increases heart rate and serum level of NO in the consecutive days, but there were no significant differences in blood pressure in the MI-AP group in comparison with MI. In conclusion, apelin has long term cardioprotective effects against myocardial infarction through attenuation of cardiac tissue injury and lipid peroxidation and enhancement of NO production.

In vitro immunopathogenesis of Edwardsiella tarda in catla Catla catla (Hamilton)

Immunopathogenesis of Edwardsiella tarda in Catla (Catla catla) was evaluated by in vitro interaction of catla head kidney (HK) phagocytes with the bacterium. Interactions of E. tarda with catla HK phagocytes were studied by light microscopy as well as by phagocytosis, intracellular replication, superoxide anion and nitric oxide production assays. Both opsonised and non-opsonized bacteria could adhere to, survive and replicate within phagocytes. Furthermore, there was no significant alteration in superoxide anion production by phagocytes when infected with E. tarda, indicating that they were able to avoid and/or resist reactive oxygen species mediated killing by the phagocytes. Enhancement of nitric oxide production indicated its possible involvement as inflammation mediator to facilitate pathogenesis. The results suggest the fact that E. tarda strain has the ability to resist phagocyte mediated killing, thereby proliferating within the host and causing infection.

P27: Significance of AMPK–eNOS pathway in renal protective effects of nitrite

Background Our daily meals contain significant amount of nitrate and nitrite, especially in vegetables. Recent studies have demonstrated beneficial effects of dietary nitrite in the treatment of cardiovascular related disease and hypertension. Nitrite has been believed to a waste form of nitric oxide (NO) though, nitrite could be a source of NO independently of conventional NO synthase (NOS) especially under hypoxic conditions. We have previously shown blood pressure lowering effect and glomerular protective effects of dietary nitrite administration in l -NAME, a NOS inhibitor, -treated animals, and concluded these phenomena were responsible for nitrite derived NO. NOS is still a dominant enzyme leading to NO production under normoxic conditions though, there are few reports whether nitrite affects NOS activity or not. Therefore we here revisited the function of nitrite in endotherial NOS (eNOS) action to clarify whether NOS plays a significant role in the renal protective effects of nitrite. Method and results We used human glomerular endotherial cells (HGEC) and explored eNOS-related molecules mainly by western blotting in this study. Nitrite stimulation increased phosphorylation of eNOS in HGEC in a dose-responsive manner, suggesting its activation by nitrite. Stimulation by nitrate did not cause eNOS phosphorylation. We then studied eNOS-phosphorylating molecules and found that nitrite stimulation led to phosphorylation of a catalytic subunit of 5′ AMP-activated protein kinase (AMPK), also suggesting its activation by nitrite. Phosphorylation of acetyl-CoA carboxylase, a typical substrate of AMPK, was also enhanced in the nitrite-stimulated HGEC. The phosphorylation of eNOS was curtailed by a specific inhibitor of AMPK, BML-275, while carboxy-PTIO, a specific quencher of NO, did not inhibit the AMPK phosphorylation by nitrite. Intracellular ATP levels were unchanged by nitrite stimulation. Conclusion Our data suggest that nitrite stimulation should lead to AMPK activation in normoxic endothelial cells without affecting intracellular energy levels, and it should be independent of NO. The activated AMPK should phosphorylate the downstream eNOS, leading to enhancement of NO production by the eNOS activation. In conclusion, we found that nitrite activates AMPK–eNOS pathway in HGEC. The activation of AMPK–eNOS pathway in endotherial cells is supposed to play a significant renal protective role in the nitrite action in addition to the NOS-independent NO production by nitrite. Disclosure Supported by Ministry of Education, Culture, Sports, Science and Technology.

Cigarette smoke extract causes endothelial nitric oxide synthase dysfunction through stimulation of ubiquitin proteasome system

Cigarette smoking (CS) is a major risk factor for endothelial dysfunction (ED), a prognostic predictor for the risk of future cardiovascular events. Endothelial nitric oxide synthase (eNOS) dysfunction has been linked to CS‐induced ED; however, the exact mechanism is not fully understood. This study reports the role of the ubiquitin proteasome system (UPS) in CS‐ induced eNOS dysfunction and ED. Exposure of bovine aortic endothelial cells (BAECs) to cigarette smoke extract (CSE) resulted in a concentration‐dependent decrease in nitric oxide (NO) production, accompanied by an increase in superoxide generation and formation of 4‐hydroxy‐2‐nonenal protein adducts. CSE depleted tetrahydrobiopterin (BH4). total biopterin, and decreased the expression of eNOS and guanosine triphosphate cyclohydrolase‐1 (GTPCH‐1). More importantly, exposure of BAECs to CSE led to accumulation of ubiqutinylated protein and increased the 26S proteasomal activity in a concentration‐and time‐dependent manner. Pre‐treatment of BAECs with MG132, an inhibitor of 26S proteasome, restored the levels of eNOS, BH4, GTPCH‐1, and enhanced NO production in BAECs following CS exposure. Our data reveal that CS‐induced stimulation of the UPS resulted in degradation of eNOS and GTPCH‐1 that led to eNOS dysfunction and uncoupling. Thus, controlled interference with the UPS pathway could be a novel modality in the prevention of CS‐induced ED. (NIH # HL38324 to JLZ; Egyptian government)

Calycosin and formononetin from astragalus root enhance dimethylarginine dimethylaminohydrolase 2 and nitric oxide synthase expressions in Madin Darby Canine Kidney II cells

Nitric oxide (NO) is a crucial vasodilator produced by nitric oxide synthase (NOS). Asymmetric dimethylarginine (ADMA) is an endogenous NOS inhibitor and mainly catabolized by dimethylarginine dimethylaminohydrolase (DDAH). As we reported, the antihypertensive effect of shichimotsukokato (SKT), a formula of Japanese traditional kampo medicine consisting of 7 crude drugs, in 5/6 nephrectomized rats, is mediated by the DDAH-ADMA-NO pathway. Our present study aimed to explore the effective compounds of SKT using Madin Darby Canine Kidney (MDCK) II cells. We isolated two isoflavones, calycosin and formononetin from astragalus root, one of the components of SKT, which can promote DDAH2 protein and mRNA expressions in MDCK II cells. The neuronal NOS levels were also upregulated by the treatment of calycosin and formononetin. These results suggest that calycosin and formononetin could be the active ingredients of astragalus root and SKT that cause antihypertensive effects. The increased levels of DDAH2 and NOS may enhance NO production, decrease ADMA level and improve endothelial and cardiovascular dysfunction.

Effect of six weeks of oral echinacea purpurea supplementation on nitric oxide production

Background Echinacea purpurea, a purple coneflower plant of the compositae family (Asteraceae), is native to North America and commonly used as an herbal supplement to enhance immune function. Echinacea purpurea has been shown to stimulate macrophage activity which is a known stimulator of nitric oxide (NO) production. Echinacea purpurea supplementation (8,000 mg·d) in untrained (42.5 ± 1.6 mL·kg·min) males was shown to elicit a 63% increase (p < 0.05) in serum erythropoietin (EPO) following two weeks of supplementation. This is supported in part by earlier findings which indicated that four weeks of Echinacea purpurea supplementation demonstrated a non-significant increase in maximal oxygen uptake (VO2max). It is plausible that Echinaceainduced EPO production may stimulate physiological responses independent of and/or in addition to erythropoiesis. There is also evidence suggesting EPO has vasculo-protective effects including the activation of endothelial nitric oxide synthase (eNOS). Based on these findings, a proposed non-hematological response to the Echinacea-induced increase in EPO could be enhanced NO production. The purpose of this investigation was to determine whether six weeks of oral Echinacea purpurea supplementation augmented NO production as a result of an Echinacea-induced increase in EPO and/or Echinaceainduced macrophage activity.

Bacopa monnieri and l-Deprenyl Differentially Enhance the Activities of Antioxidant Enzymes and the Expression of Tyrosine Hydroxylase and Nerve Growth Factor via ERK 1/2 and NF-κB Pathways in the Spleen of Female Wistar Rats

Aging is characterized by development of diseases and cancer due to loss of central and peripheral neuroendocrine-immune responses. Free radicals exert deleterious effects on neural-immune functions in the brain, heart, and lymphoid organs and thus, affecting the health. Bacopa monnieri (brahmi), an Ayurvedic herb, and L-deprenyl, a monoamine oxidase-B inhibitor, have been widely used in the treatment of neurodegenerative diseases. The purpose of this study was to investigate whether brahmi (10 and 40 mg/kg BW) and deprenyl (1 and 2.5 mg/kg BW) treatment of 3-month old female Wistar rats for 10 days can modulate the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)] in the brain and spleen. In addition, the effects of these compounds on the expression of tyrosine hydroxylase (TH), nerve growth factor (NGF), the intracellular signaling markers, p-ERK1/2, p-CREB, and p-NF-kB, and nitric oxide (NO) production were measured in the spleen by Western blot analysis. Both brahmi and deprenyl enhanced CAT activity, and p-TH, NGF, and p-NF-kB expression in the spleen. However, deprenyl alone was found to enhance the p-ERK1/2 and p-CREB expression in the spleen. The activities of SOD, CAT, and GPx in the thymus, mesenteric lymph nodes, heart, and brain areas (frontal cortex, medial basal hypothalamus, striatum, and hippocampus) were differentially altered by brahmi and deprenyl. Brahmi alone enhanced NO production in the spleen. Taken together, these results suggest that both brahmi and deprenyl can protect the central and peripheral neuronal systems through their unique effects on the antioxidant enzyme activities and intracellular signaling pathways.

Hydrogen Sulfide Stimulates Ischemic Vascular Remodeling Through Nitric Oxide Synthase and Nitrite Reduction Activity Regulating Hypoxia‐Inducible Factor‐1α and Vascular Endothelial Growth Factor–Dependent Angiogenesis

BackgroundHydrogen sulfide (H2S) therapy is recognized as a modulator of vascular function during tissue ischemia with the notion of potential interactions of nitric oxide (NO) metabolism. However, little is known about specific biochemical mechanisms or the importance of H2S activation of NO metabolism during ischemic tissue vascular remodeling. The goal of this study was to determine the effect of H2S on NO metabolism during chronic tissue ischemia and subsequent effects on ischemic vascular remodeling responses.Methods and ResultsThe unilateral, permanent femoral artery ligation model of hind‐limb ischemia was performed in C57BL/6J wild‐type and endothelial NO synthase–knockout mice to evaluate exogenous H2S effects on NO bioavailability and ischemic revascularization. We found that H2S selectively restored chronic ischemic tissue function and viability by enhancing NO production involving both endothelial NO synthase and nitrite reduction mechanisms. Importantly, H2S increased ischemic tissue xanthine oxidase activity, hind‐limb blood flow, and angiogenesis, which were blunted by the xanthine oxidase inhibitor febuxostat. H2S treatment increased ischemic tissue and endothelial cell hypoxia‐inducible factor‐1α expression and activity and vascular endothelial growth factor protein expression and function in a NO‐dependent manner that was required for ischemic vascular remodeling.ConclusionsThese data demonstrate that H2S differentially regulates NO metabolism during chronic tissue ischemia, highlighting novel biochemical pathways to increase NO bioavailability for ischemic vascular remodeling.

Potent vasodilation effect of amurensin G is mediated through the phosphorylation of endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) has important regulatory functions in vascular tone, and impaired endothelium-dependent vasodilatation is a key event in diabetes and atherosclerosis. Vitis amurensis grapes containing resveratrol oligomers are consumed as wine and fruit and have antioxidative and neuroprotective effects. In this study, our goal was identify the most potent eNOS-activating compound among six stilbenes and oligostilbenes found in V. amurensis and to clarify its molecular mechanism. Among the six tested compounds, amurensin G most potently relaxed endothelium-intact aortic rings and increased eNOS phosphorylation and nitric oxide (NO) production. Amurensin G increased both estrogen receptor (ER) phosphorylation and ER-dependent gene transcription, and ERα or ERβ inhibition suppressed amurensin G-mediated eNOS phosphorylation. Amurensin G enhanced the activities of phosphatidylinositol 3-kinase (PI3K) and Src and their chemical inhibitors suppressed amurensin G-stimulated eNOS phosphorylation. Moreover, amurensin G activated AMP-activated protein kinase (AMPK), and amurensin G-stimulated eNOS phosphorylation and PI3K activation were reversed by AMPK inhibition. ER inhibition reversed AMPK-dependent PI3K activation in response to amurensin G. Amurensin G-mediated endothelium-dependent relaxation was blocked by inhibition of AMPK, ER, Src, or PI3K. These results suggest that amurensin G enhances NO production via eNOS phosphorylation in endothelial cells, and ER-dependent AMPK/PI3K pathways are required. Amurensin G would be applicable to prevent atherosclerosis.

Sex Differences in Postischemic Cardiac Dysfunction and Norepinephrine Overflow in Rat Heart

The purpose of this study is to elucidate the relationship between sex difference and norepinephrine (NE) release in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury. Isolated male and female rat hearts were subjected to 40-minute global ischemia followed by 30-minute reperfusion. Compared with male hearts, I/R-induced cardiac dysfunction, such as decreased left ventricular developed pressure and dP/dtmax and increased left ventricular end diastolic pressure, was significantly attenuated in female hearts. An excessive NE overflow in the coronary effluent from the postischemic heart in females was much less than that in males. These sex differences were abolished by ovariectomy, but in vivo treatment with 17β-estradiol recovered it. This ameliorating effect of 17β-estradiol was not observed in the presence of nitric oxide (NO) synthase inhibitor N-nitro-L-arginine. When NOx (NO2/NO3) levels in the coronary effluent after onset of reperfusion were measured, reversed correlated relationships between NOx production and I/R-induced cardiac dysfunction, and NE overflow, were observed. These findings suggest that sex differences in the postischemic cardiac dysfunction are closely related to the NE overflow from the postischemic heart and that estrogen plays a key role in the cardioprotective effect against I/R injury in female rats, by suppressing NE release via the enhancement of NO production.

Correlative Analyses of Nitric Oxide Generation Rates and Nitric Oxide Synthase Levels in Individual Cells Using a Modular Cell-Retaining Device

Nitric oxide (NO) is recognized as one of the major immune system agents involved in the pathogenesis and control of various diseases that may benefit from novel drug development, by exploiting NO signaling pathways and targets. This calls for detection of both intracellular levels of NO and expression of its synthesizing enzymes (NOS) in individual, intact, living cells. Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, and inability to trace the same cells over time. The current study presents a device and methodology for correlative analysis of NO generation rates and NOS levels in the same individual cells, utilizing fluorescent imaging followed by immunohistochemistry (IHC). U937 promonocyte cell populations demonstrated significant heterogeneity in their baseline levels, in NO-generation kinetics, and in their response rates to stimuli. Individual cell analysis exposed cell subgroups which showed enhanced NO production upon stimulation, concomitantly with significant up-regulation of inducible NOS (iNOS) levels. Exogenous NO modulated the expression of iNOS in nondifferentiated cells within 1 h, in a dose-dependent manner, while treatment with lysophosphatidylcholine (LPC) enhanced the expression of iNOS, demonstrating a nondependence on NO production.

IL-27 promotes nitric oxide production induced by LPS through STAT1, NF-κB and MAPKs

Interleukin (IL)-27, a member of the IL-6/IL-12 heterodimeric cytokine family, induces pro-inflammatory responses including early T helper (Th)1 differentiation and generation of cytotoxic T lymphocytes, and also anti-inflammatory responses including the differentiation to IL-10-producing regulatory T cells, inhibition of Th2 and Th17 differentiation, and suppression of pro-inflammatory cytokine production. Nitric oxide (NO) is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli including cytokines and microbes. Recently, IL-27 was reported to play an anti-inflammatory role in microglia by blocking oncostatin M-induced iNOS expression and neuronal toxicity. In the present study, we investigated the effects of IL-27 on NO production in thioglycollate-elicited peritoneal macrophages. IL-27 together with lipopolysaccharide (LPS) induced morphological change into more spread and elongated cells and synergistically enhanced NO production. The combined stimulation also enhanced iNOS mRNA expression and the NO production was abrogated by an iNOS inhibitor, NG-monomethyl l-arginine. The synergistic NO production could be attributed to the augmented Toll-like receptor (TLR)4 mRNA expression by the combination. Signal transducer and activator of transcription (STAT)1 was indispensable for the morphological change and NO production. The combination induced nuclear factor κB (NF-κB) translocation into nuclear and phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and their inhibitors suppressed NO production. These results suggest that in contrast to the anti-proinflammatory role in microglia, IL-27 exerts a pro-inflammatory role by enhancing NO production in peritoneal macrophages stimulated with LPS through activation of STAT1, NF-κB and MAPKs.

L-Theanine promotes nitric oxide production in endothelial cells through eNOS phosphorylation

Consumption of tea (Camellia sinensis) improves vascular function and is linked to lowering the risk of cardiovascular disease. Endothelial nitric oxide is the key regulator of vascular functions in endothelium. In this study, we establish that l-theanine, a non-protein amino-acid found in tea, promotes nitric oxide (NO) production in endothelial cells. l-theanine potentiated NO production in endothelial cells was evaluated using Griess reaction, NO sensitive electrode and a NO specific fluorescent probe (4-amino-5-methylamino-2',7'-difluororescein diacetate). l-Theanine induced NO production was partially attenuated in presence of l-NAME or l-NIO and completely abolished using eNOS siRNA. eNOS activation was Ca2+ and Akt independent, as assessed by fluo-4AM and immunoblotting experiments, respectively and was associated with phosphorylation of eNOS Ser 1177. eNOS phosphorylation was inhibited in the presence of ERK1/2 inhibitor, PD-98059 and partially inhibited by PI3K inhibitor, LY-294002 and Wortmanin suggesting PI3K-ERK1/2 dependent pathway. Increased NO production was associated with vasodilation in ex ovo (chorioallantoic membrane) model. These results demonstrated that l-theanine administration in vitro activated ERK/eNOS resulting in enhanced NO production and thereby vasodilation in the artery. The results of our experiments are suggestive of l-theanine mediated vascular health benefits of tea.

Translational evidence that impaired autophagy contributes to arterial ageing

Ageing causes arterial endothelial dysfunction that increases the risk of cardiovascular diseases (CVD), but the underlying mechanisms are incompletely understood. The aim of the present study was to determine the role of autophagy, the cellular process of recycling damaged biomolecules, in endothelial dysfunction with ageing. In older humans, expression of autophagy markers in arterial endothelial cells was impaired by ∼50% (P <0.05) and was associated with an ∼30% (P <0.05) reduction in arterial endothelium-dependent dilatation (EDD). Similarly, in C57BL/6 control mice ageing was associated with an ∼40% decrease (P <0.05) in arterial markers of autophagy and an ∼25% reduction (P <0.05) in EDD. In both humans and mice, impaired EDD was mediated by reduced nitric oxide (NO) bioavailability and was associated with increased oxidative stress and inflammation (P <0.05). In old mice, treatment with the autophagy-enhancing agent trehalose restored expression of autophagy markers, rescued NO-mediated EDD by reducing oxidative stress, and normalized inflammatory cytokine expression. In cultured endothelial cells, inhibition of autophagy increased oxidative stress and reduced NO production, whereas trehalose enhanced NO production via an autophagy-dependent mechanism. These results provide the first evidence that autophagy is impaired with ageing in vascular tissues. Our findings also suggest that autophagy preserves arterial endothelial function by reducing oxidative stress and inflammation and increasing NO bioavailability. Autophagy-enhancing strategies may therefore have therapeutic efficacy for ameliorating age-associated arterial dysfunction and preventing CVD.

Cellular actions of testosterone in vascular cells: Mechanism independent of aromatization to estradiol

In this work we investigated the role of testosterone on cellular processes involved in vascular disease, and whether these effects depend on its local conversion to estradiol. Cultures of rat aortic endothelial and smooth muscle cells in vitro treated with physiological concentrations of testosterone were employed. Testosterone rapidly increased endothelial nitric oxide production. To evaluate whether this non genomic action was dependent on testosterone aromatization we used an aromatase inhibitor. Anastrozole compound did not modify the fast increase in nitric oxide production elicited by testosterone. The hormonal effect was completely blocked by an androgen receptor antagonist (flutamide); meanwhile it wasn´t modified by the presence of an estrogen receptor antagonist (ICI182780).The possibility of intracellular estradiol synthesis was ruled out when no differences were found in estradiol measurements performed in culture incubation medium from control and testosterone treated cells. The 5α-reductase inhibitor finasteride partially suppressed the enhancement in nitric oxide production, suggesting that the effect of testosterone was partially due to dihydrotestosterone conversion. Testosterone stimulated muscle cell proliferation independent of local conversion to estradiol. When cellular events that play key roles in vascular disease development were analyzed, testosterone prevented monocyte adhesion to endothelial cells induced by a proinflammatory stimulus (bacterial lipopolysaccharides), and prompted muscle cell migration in presence of a cell motility inducer. In summary, testosterone modulates vascular behavior through its direct action on vascular cells independent of aromatization to estradiol. The cellular actions exhibited by the steroid varied whether cells were under basal or inflammatory conditions.

Abstract 5456: Potential role of nitric oxide in chromium-induced lung carcinogenesis

Abstract Certain hexavalent chromium [Cr(VI)] compounds are well established human respiratory toxins and carcinogens of occupational and environmental relevance. Our recent studies have shown that intranasal (IN) exposure of Balb/c mice to particulate zinc Cr(VI) compounds leads to pronounced innate inflammation in the lung, which we postulate plays a significant role in pulmonary tumorigenesis. Nitric oxide (NO) has been shown to be induced via inflammatory response mediators, and has been implicated in the progression of different cancers, including lung. Moreover, NO-induced tumor invasiveness has been correlated with enhanced matrix metalloproteinase (MMP) expression in lung tumor cell lines. Of particular relevance to the present study is the finding that in vitro exposure of human lung cells to Cr(VI) in culture led to increased NO production. The aim of the present study was to explore the role of NO in chromium-induced lung tumorigenesis. BALB/c mice received weekly IN delivery of either saline or 10 ug particulate basic zinc chromate, which is an intermediately water soluble chromate typically encountered in mining/chromate production facilities and which is also present as an environmental atmospheric contaminant in urban areas surrounding ferrochrome production facilities. After 9 weeks, immune cells, cytokines, NO, MMPs and pathologic features of lung injury and inflammation were measured in airway lavage fluid and lung tissue, at both 24h after the final Cr(VI) dose, as well as 1 week later. As previously shown, repetitive Cr(VI) exposure induced a neutrophilic inflammatory airway response 24h after treatment. Neutrophils were subsequently replaced by increasing numbers of macrophages 1 week after treatment. Peribronchiolar inflammation was observed in chromate-exposed mice, and was accompanied by a 100-fold increase in pro-MMP9 release into the airways. Notably, airway NO was significantly increased ∼ 1.9 fold in Cr(VI)-exposed mice as compared to their saline-treated counterparts at both time points following final exposure. Moreover, immunoreactivity of induced nitric oxide synthase (iNOS) was markedly increased in airways exposed to Cr(VI), with staining localized to infiltrating immune cells. In conclusion, repetitive exposure to particulate chromate induces an inflammatory environment in the lung, accompanied by enhanced NO production, which we postulate may promote Cr(VI) carcinogenesis. Supported by NIH grants CA107972 and ES017334 to SC and ES017307 to SRP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5456. doi:1538-7445.AM2012-5456

Resveratrol augments nitric oxide generation and causes store calcium release in chromaffin cells

The cardiovascular protecting effect of the grape fruit trans-resveratrol has been explained among other factors, through augmentation of nitric oxide (NO) production in cardiovascular tissues. Another effect of low resveratrol concentration is the inhibition of single-vesicle quantal release of catecholamine from bovine adrenal chromaffin cells, that was recently suggested to be an additional factor contributing to its beneficial cardiovascular effects. We have investigated here the effects of a low concentration of trans-resveratrol (1μM) on Ca2+ and NO signaling pathways in bovine chromaffin cells, in an attempt to understand the mechanism underlying its previously reported inhibitory effects on quantal secretion. In cells loaded with fura-2 acetoxymethyl ester (fura-2), we have found that 1μM resveratrol produces a transient elevation of the cytosolic Ca2+ concentration ([Ca2+]c). This Ca2+ transient was drastically reduced when the Ca2+ store was depleted by ryanodine and dantrolene; it was also inhibited by Nω-nitro-l-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Furthermore, the Ca2+ transient was mimicked by NO donor S-nitroso-N-acetyl-penicillamine (SNAP). Resveratrol also enhanced the production of nitrites and NO, and L-NAME blocked both responses; in contrast, augmentation by SNAP of nitrites and NO was unaffected by ODQ and was only partially inhibited by L-NAME. On the basis of these results, we are proposing that resveratrol is mitigating the catecholamine surge occurring during stress, through its ability to elicit mild local [Ca2+]c transients and enhanced NO production, that blocks the last steps of exocytosis.

Eicosapentaenoic acid enhances nitric oxide production through up‐regulating SIRT1 expression in human endothelial cells

Accumulating evidence indicates that eicosapentaenoic acid (EPA), a polyunsaturated fatty acid of the n‐3 series extracted from fish oils, has anti‐atherogenic effects. However, the molecular mechanism of these beneficial effects remains unclear. Here, we show that EPA enhances nitric oxide (NO) production from human umbilical endothelial cells (HUVEC) which was determined by a fluorescent indicator DAF‐2. EPA also suppressed an increase in SA‐β‐gal positive cells induced by NG, NG‐dimethylarginine dihydrochloride (ADMA), a NO synthase inhibitor. siRNA‐mediated SIRT1 gene silencing cancelled EPA‐induced suppression of premature senescence and enhancement of NO production in these cells. These results suggest that up‐regulation of SIRT1 expression in endothelial cells may participate in the mechanism underlying the beneficial effects induced by EPA.