NO Bioactivity Research Articles

Endothelial cell-specific roles for tetrahydrobiopterin in myocardial function, cardiac hypertrophy, and response to myocardial ischemia-reperfusion injury.

The cofactor tetrahydrobiopterin (BH4) is a critical regulator of nitric oxide synthase (NOS) function and redox signaling, with reduced BH4 implicated in multiple cardiovascular disease states. In the myocardium, augmentation of BH4 levels can impact on cardiomyocyte function, preventing hypertrophy and heart failure. However, the specific role of endothelial cell BH4 biosynthesis in the coronary circulation and its role in cardiac function and the response to ischemia has yet to be elucidated. Endothelial cell-specific Gch1 knockout mice were generated by crossing Gch1fl/fl with Tie2cre mice, generating Gch1fl/flTie2cre mice and littermate controls. GTP cyclohydrolase protein and BH4 levels were reduced in heart tissues from Gch1fl/flTie2cre mice, localized to endothelial cells, with normal cardiomyocyte BH4. Deficiency in coronary endothelial cell BH4 led to NOS uncoupling, decreased NO bioactivity, and increased superoxide and hydrogen peroxide productions in the hearts of Gch1fl/flTie2cre mice. Under physiological conditions, loss of endothelial cell-specific BH4 led to mild cardiac hypertrophy in Gch1fl/flTie2cre hearts. Endothelial cell BH4 loss was also associated with increased neuronal NOS protein, loss of endothelial NOS protein, and increased phospholamban phosphorylation at serine-17 in cardiomyocytes. Loss of cardiac endothelial cell BH4 led to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia-reperfusion injury. Taken together, these studies reveal a specific role for endothelial cell Gch1/BH4 biosynthesis in cardiac function and the response to cardiac ischemia-reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction and ischemia-reperfusion injury.NEW & NOTEWORTHY We demonstrate a critical role for endothelial cell Gch1/BH4 biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH4 leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

Red blood cells from endothelial nitric oxide synthase-deficient mice induce vascular dysfunction involving oxidative stress and endothelial arginase I

Background & aimsNitric oxide bioactivity (NO) from endothelial NO synthase (eNOS) importantly contributes to the maintenance of vascular homeostasis, and reduced eNOS activity has been associated with cardiovascular disease. Emerging evidence suggests interaction(s) between red blood cells (RBCs) and the endothelium in vascular control; however, the specific role of RBC eNOS is less clear. We aimed to investigate the hypothesis that a lack of RBC eNOS induces endothelial dysfunction. Methods & ResultsRBCs from global eNOS knockout (KO) and wildtype (WT) mice were co-incubated ex vivo overnight with healthy mouse aortic rings, followed by functional and mechanistic analyses of endothelium-dependent and independent relaxations. RBCs from eNOS KO mice induced endothelial dysfunction and vascular oxidative stress, whereas WT RBC did not. No differences were observed for endothelium-independent relaxations. This eNOS KO RBC-induced endothelial dysfunctional phenotype was prevented by concomitant co-incubation with reactive oxygen species scavenger (TEMPOL), arginase inhibitor (nor-NOHA), NO donor (detaNONOate) and NADPH oxidase 4 (NOX4) inhibitor. Moreover, vessels from endothelial cell-specific arginase 1 KO mice were resistant to eNOS KO-RBC-induced endothelial dysfunction. Finally, in mice aortae co-incubated with RBCs from women with preeclampsia, we observed a significant reduction in endothelial function compared to when using RBCs from healthy pregnant women or from women with uncomplicated gestational hypertension. ConclusionsRBCs from mice lacking eNOS, and patients with preeclampsia, induce endothelial dysfunction in adjacent blood vessels. Thus, RBC-derived NO bioactivity acts to prevent induction of vascular oxidative stress occurring via RBC NOX4-derived ROS in a vascular arginase-dependent manner. Our data highlight the intrinsic protective role of RBC-derived NO bioactivity in preventing the damaging potential of RBCs. This provides novel insight into the functional relationship between RBCs and the vasculature in health and cardiovascular disease, including preeclampsia.

Endothelial Extracellular Signal-Regulated Kinase/Thromboxane A2/Prostanoid Receptor Pathway Aggravates Endothelial Dysfunction and Insulin Resistance in a Mouse Model of Metabolic Syndrome.

Background Metabolic syndrome is characterized by insulin resistance, which impairs intracellular signaling pathways and endothelial NO bioactivity, leading to cardiovascular complications. Extracellular signal-regulated kinase (ERK) is a major component of insulin signaling cascades that can be activated by many vasoactive peptides, hormones, and cytokines that are elevated in metabolic syndrome. The aim of this study was to clarify the role of endothelial ERK2 in vivo on NO bioactivity and insulin resistance in a mouse model of metabolic syndrome. Methods and Results Control and endothelial-specific ERK2 knockout mice were fed a high-fat/high-sucrose diet (HFHSD) for 24 weeks. Systolic blood pressure, endothelial function, and glucose metabolism were investigated. Systolic blood pressure was lowered with increased NO products and decreased thromboxane A2/prostanoid (TP) products in HFHSD-fed ERK2 knockout mice, and Nω-nitro-l-arginine methyl ester (L-NAME) increased it to the levels observed in HFHSD-fed controls. Acetylcholine-induced relaxation of aortic rings was increased, and aortic superoxide level was lowered in HFHSD-fed ERK2 knockout mice. S18886, an antagonist of the TP receptor, improved endothelial function and decreased superoxide level only in the rings from HFHSD-fed controls. Glucose intolerance and the impaired insulin sensitivity were blunted in HFHSD-fed ERK2 knockout mice without changes in body weight. In vivo, S18886 improved endothelial dysfunction, systolic blood pressure, fasting serum glucose and insulin levels, and suppressed nonalcoholic fatty liver disease scores only in HFHSD-fed controls. Conclusions Endothelial ERK2 increased superoxide level and decreased NO bioactivity, resulting in the deterioration of endothelial function, insulin resistance, and steatohepatitis, which were improved by a TP receptor antagonist, in a mouse model of metabolic syndrome.

Comparative study between apocynin and protocatechuic acid regarding antioxidant capacity and vascular effects

Reactive oxygen species (ROS) derived from NOX enzymes activity play an important role in the development of cardiovascular diseases. Compounds able to decrease oxidative stress damage are potential candidates as drugs and/or supplements for hypertension treatment. Here, we aimed to compare in vitro ROS scavenging potency, effective NOX inhibition and effects on vascular reactivity of apocynin to another phenolic compound, protocatechuic acid, in vascular cells from spontaneously hypertensive rat (SHR), where redox signaling is altered and contributes to the development and/or maintenance of hypertension. We evaluated the in vitro antioxidant capacity and free radical scavenging capacity of both phenolic compounds. Moreover, we investigated the effect of both compounds on lipid peroxidation, lucigenin chemiluminescence, nitric oxide (NO•) levels and ROS concentration in vascular cells of SHR or human umbilical vein endothelial cell (HUVEC). Apocynin and protocatechuic acid presented antioxidant capacity and ability as free radical scavengers, decreased thiobarbituric acid reactive substances (TBARS) in aortic cells from SHR, and increased NO• concentration in isolated HUVEC. Both compounds were able to reduce lucigenin chemiluminescence and increased the potency of acetylcholine in aorta of SHR. However, in SHR aortas, only apocynin diminished the contraction induced by phenylephrine. In conclusion, these results strongly reinforce the potential application of substances such as apocynin and protocatechuic acid that combine abilities as scavenging and/or prevention of ROS generation, establishment of NO bioactivity and modulation of vascular reactivity. Due to its phytochemical origin and low toxicity, its potential therapeutic use in vascular diseases should be considered.

Nitrite Concentration in the Striated Muscles Is Reversely Related to Myoglobin and Mitochondrial Proteins Content in Rats.

Skeletal muscles are an important reservoir of nitric oxide (NO•) stored in the form of nitrite [NO2−] and nitrate [NO3−] (NOx). Nitrite, which can be reduced to NO• under hypoxic and acidotic conditions, is considered a physiologically relevant, direct source of bioactive NO•. The aim of the present study was to determine the basal levels of NOx in striated muscles (including rat heart and locomotory muscles) with varied contents of tissue nitrite reductases, such as myoglobin and mitochondrial electron transport chain proteins (ETC-proteins). Muscle NOx was determined using a high-performance liquid chromatography-based method. Muscle proteins were evaluated using western-immunoblotting. We found that oxidative muscles with a higher content of ETC-proteins and myoglobin (such as the heart and slow-twitch locomotory muscles) have lower [NO2−] compared to fast-twitch muscles with a lower content of those proteins. The muscle type had no observed effect on the [NO3−]. Our results demonstrated that fast-twitch muscles possess greater potential to generate NO• via nitrite reduction than slow-twitch muscles and the heart. This property might be of special importance for fast skeletal muscles during strenuous exercise and/or hypoxia since it might support muscle blood flow via additional NO• provision (acidic/hypoxic vasodilation) and delay muscle fatigue.

Abstract 12114: Endothelial Extracellular Signal-Regulated Kinase 2 Contributed to the Development of Pulmonary Arterial Hypertension in Mice Through the Impairment of Endothelial Nitric Oxide Synthase Function

Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease, characterized by abnormal muscularization and endothelial dysfunction in pulmonary artery (PA). Previous study showed that endothelial extracellular signal-regulated kinase 2 (eERK2) released vasoconstrictive factors and decreased NO bioactivity resulting in obese-induced hypertension and endothelial dysfunction. However, the role of eERK2 in the pathogenesis of PAH remains to be elucidated. Hypothesis: We hypothesized eERK2 impaired eNOS function and progressed the pathogenesis of PAH. Methods and Results: We generated eERK2 knock out mice (EE2KO) crossing Tie2-Cre mice and ERK2 flox mice and induced PAH by exposing mice to chronic hypoxia (10%) for 4 weeks. The littermate ERK2 flox mice without Cre expression were used as Control (Ctr). Immuno-histochemical staining showed the deficiency of eERK2 expression in the PA of EE2KO. We assessed the medial thickness of pulmonary arteries (MTPA) and measured right ventricular systolic pressure (RVSP). Although Ctr exhibited the progression of PA muscularization with an increase in MTPA, EE2KO revealed the significantly attenuated MTPA under hypoxic condition. Moreover, we found the lower RVSPs in EE2KO than those in Ctr under hypoxia in spite of similar RVSPs in normoxia. In RT-PCR, lung endothelin1 (ET1) gene expression was significantly lower in EE2KO than Ctr under hypoxia. Interestingly, hypoxia increased eNOS protein expression both in Ctr and EE2KO with Western blot analysis. The phosphorylation of AMPKα and eNOS were up-regulated in the lungs of EE2KO relative to Ctr under hypoxia. In addition, eNOS dimer/monomer ratios with non redusing SDS-PAGE was significantly increased in EE2KO compared to the Ctr under hypoxia (1.29±0.10 vs. 0.87±0.02, P=0.02, dimer / monomer ratios), suggesting improved eNOS uncoupling. Conclusions: These findings suggest that hypoxia increased eNOS expression and function, and that eERK2 impaired eNOS function by decreasing the phosphorylation of AMPKα and eNOS, and inducing eNOS uncoupling. In addition, ET1 synthesis through eERK2 might contribute to these pathogenesis of PAH with hypoxia.

ADH5-mediated NO bioactivity maintains metabolic homeostasis in brown adipose tissue.

SUMMARYBrown adipose tissue (BAT) thermogenic activity is tightly regulated by cellular redox status, but the underlying molecular mechanisms are incompletely understood. Protein S-nitrosylation, the nitric-oxide-mediated cysteine thiol protein modification, plays important roles in cellular redox regulation. Here we show that diet-induced obesity (DIO) and acute cold exposure elevate BAT protein S-nitrosylation, including UCP1. This thermogenic-induced nitric oxide bioactivity is regulated by S-nitrosoglutathione reductase (GSNOR; alcohol dehydrogenase 5 [ADH5]), a denitrosylase that balances the intracellular nitroso-redox status. Loss of ADH5 in BAT impairs cold-induced UCP1-dependent thermogenesis and worsens obesity-associated metabolic dysfunction. Mechanistically, we demonstrate that Adh5 expression is induced by the transcription factor heat shock factor 1 (HSF1), and administration of an HSF1 activator to BAT of DIO mice increases Adh5 expression and significantly improves UCP1-mediated respiration. Together, these data indicate that ADH5 controls BAT nitroso-redox homeostasis to regulate adipose thermogenesis, which may be therapeutically targeted to improve metabolic health.

Effects of inorganic nitrate on ischaemia-reperfusion injury after coronary artery bypass surgery: a randomised controlled trial

BackgroundNitric oxide (NO) is an important signalling molecule in the cardiovascular system with protective properties in ischaemia–reperfusion injury. Inorganic nitrate, an oxidation product of endogenous NO production and a constituent in our diet, can be recycled back to bioactive NO. We investigated if preoperative administration of inorganic nitrate could reduce troponin T release and other plasma markers of injury to the heart, liver, kidney, and brain in patients undergoing cardiac surgery. MethodsThis single-centre, randomised, double-blind, placebo-controlled trial included 82 patients undergoing coronary artery bypass surgery with cardiopulmonary bypass. Oral sodium nitrate (700 mg×2) or placebo (NaCl) were administered before surgery. Biomarkers of ischaemia–reperfusion injury and plasma nitrate and nitrite were collected before and up to 72 h after surgery. Troponin T release was our predefined primary endpoint and biomarkers of renal, liver, and brain injury were secondary endpoints. ResultsPlasma concentrations of nitrate and nitrite were elevated in nitrate-treated patients compared with placebo. The 72-h release of troponin T did not differ between groups. Other plasma biomarkers of organ injury were also similar between groups. Blood loss was not a predefined outcome parameter, but perioperative bleeding was 18% less in nitrate-treated patients compared with controls. ConclusionPreoperative administration of inorganic nitrate did not influence troponin T release or other plasma biomarkers of organ injury in cardiac surgery. Clinical trial registrationNCT01348971.

Multidrug resistance proteins preferentially regulate natriuretic peptide-driven cGMP signalling in the heart and vasculature.

cGMP underpins the bioactivity of NO and natriuretic peptides and is key to cardiovascular homeostasis. cGMP-driven responses are terminated primarily by PDEs, but cellular efflux via multidrug resistance proteins (MRPs) might contribute. Herein, the effect of pharmacological blockade of MRPs on cGMP signalling in the heart and vasculature was investigated in vitro and in vivo. Proliferation of human coronary artery smooth muscle cells (hCASMCs), vasorelaxation of murine aorta and reductions in mean arterial BP (MABP) in response to NO donors or natriuretic peptides were determined in the absence and presence of the MRP inhibitor MK571. The ability of MRP inhibition to reverse morphological and contractile deficits in a murine model of pressure overload-induced heart failure was also explored. MK571 attenuated hCASMC growth and enhanced the anti-proliferative effects of NO and atrial natriuretic peptide (ANP). MRP blockade caused concentration-dependent relaxations of murine aorta and augmented responses to ANP (and to a lesser extent NO). MK571 did not decrease MABP per se but enhanced the hypotensive actions of ANP and improved structural and functional indices of disease severity in experimental heart failure. These beneficial actions of MRP inhibition were associated with a greater intracellular:extracellular cGMP ratio in vitro and in vivo. MRP blockade promotes the cardiovascular functions of natriuretic peptides in vitro and in vivo, with more modest effects on NO. MRP inhibition may have therapeutic utility in cardiovascular diseases triggered by dysfunctional cGMP signalling, particularly those associated with altered natriuretic peptide bioactivity. This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Quantitation of lymphatic transport mechanism and barrier influences on lymph node-resident leukocyte access to lymph-borne macromolecules and drug delivery systems.

Lymph nodes (LNs) are tissues of the immune system that house leukocytes, making them targets of interest for a variety of therapeutic immunomodulation applications. However, achieving accumulation of a therapeutic in the LN does not guarantee equal access to all leukocyte subsets. LNs are structured to enable sampling of lymph draining from peripheral tissues in a highly spatiotemporally regulated fashion in order to facilitate optimal adaptive immune responses. This structure results in restricted nanoscale drug delivery carrier access to specific leukocyte targets within the LN parenchyma. Herein, a framework is presented to assess the manner in which lymph-derived macromolecules and particles are sampled in the LN to reveal new insights into how therapeutic strategies or drug delivery systems may be designed to improve access to dLN-resident leukocytes. This summary analysis of previous reports from our group assesses model nanoscale fluorescent tracer association with various leukocyte populations across relevant time periods post administration, studies the effects of bioactive molecule NO on access of lymph-borne solutes to dLN leukocytes, and illustrates the benefits to leukocyte access afforded by lymphatic-targeted multistage drug delivery systems. Results reveal trends consistent with the consensus view of how lymph is sampled by LN leukocytes resulting from tissue structural barriers that regulate inter-LN transport and demonstrate how novel, engineered delivery systems may be designed to overcome these barriers to unlock the therapeutic potential of LN-resident cells as drug delivery targets.

Hydro Shot for Sports Performance and Exercise Medicine

Background. Exercise performance and recovery are impaired by excessive levels of oxidative stress and inflammation. However, both reactive oxygen species (ROS) and inflammation improve exercise performance including mitochondrial ATP production and force of muscle contraction. They also are essential mediators in providing the benefits and training adaptations that occur from exercise. Nitric oxide (NO•) is a gaseous radical that increases blood flow via dilation of the blood vessels and also improves mitochondrial function. Therefore, NO• improves exercise performance and capacity, but only when produced at the right times and in the right locations. Excessive levels of NO• contribute to nitrosative stress due to the spontaneous reaction with superoxide to form toxic peroxynitrite. This decreases the ½ life of nitric oxide resulting in less NO• benefits and in cellular damage leading to impaired exercise performance. In contrast to conventional antioxidants and anti-inflammatories, molecular hydrogen (H2) has been demonstrated to exert selective antioxidant and anti-inflammatory effects by decreasing only excessive inflammation and reducing toxic oxidants without disturbing important signaling ROS, like NO•. Moreover, H2 can regulate NO• production, increase its circulating ½ life and beneficial NO• cycling, potentiate the bioactivity of NO•, and act as a NO• mimetic by increasing cGMP levels. At the same time H2 can prevent peroxynitrite formation and reduce the harm from NO• metabolism, such as lower nitrotyrosine levels. Methods. The effects of an H2-infused, nitric oxide-producing beverage (Hydro Shot) on nitric oxide production, blood flow, aerobic and anaerobic exercise, and cognitive function were assessed. Results. Ingestion of the functional beverage significantly increased production of NO• and a concomitant increase in blood flow. It also improved aerobic performance as measured by VO2, and anaerobic performance as indicated by delayed muscle fatigue, and increased peak torque during maximal isokinetic leg extensions. Additionally, the H2/NO• combination significantly improved indices of cognitive function including, focus, speed, plasticity, etc. Conclusion. The molecular crosstalk between H2 and NO•coupled with these preliminary results indicate that Hydro Shot is uniquely qualified for sports performance and exercise medicine and warrants additional clinical and mechanistic research.

Tetrahydroxystilbene glycoside improves endothelial dysfunction and hypertension in obese rats: The role of omentin-1

RationaleHypertension in obesity has become a major threat for public health. Omentin-1, a novel adipokine, is down-regulated in obesity. Tetrahydroxystilbene glycoside (TSG) is the main ingredient extracted from Polygonum multiflorum Thunb (PMT), a traditional Chinese medicinal herb safely used for protecting cardiovascular systems over bimillennium. This study aims to examine (i) the impact of omentin-1 downregulation on obesity-related hypertension in murine models and the underlying mechanisms; (ii) whether tetrahydroxystilbene glycoside (TSG) improved endothelial dysfunction and obesity-associated hypertension via the increase of omentin-1. Methods(TSG-treated) male Zucker diabetic fatty (ZDF) rats and omentin-1 knockout (OMT-/-) mice were used. In vitro, human umbilical vein endothelial cells (HUVECs) and mature adipocytes differentiated from human visceral preadipocyte (HPA-v) were maintained in a co-culture system. ResultsTSG was the main active component of PMT reducing systolic blood pressure and improving endothelial vasodilation. Fortnight-TSG treatment (100 mg/kg/day) increased serum omentin-1 level, also activated Akt/eNOS signaling and enhanced NO bioactivity; decreased expression of NOX2 and p22phox, suppressed production of superoxide and peroxynitrite anion. OMT-/- mice showed elevated blood pressure and impaired endothelial vasorelaxation, whereas hypotensive effect of TSG was blunted. In co-culture system, TSG incubation promoted binding of peroxisome proliferator-activated receptor-γ (PPAR-γ) and Itln-1 promoter in adipocytes, activated Akt/eNOS/NO signaling and attenuated oxidative/nitrative stress in HUVECs. Suppression of Itln-1 with siRNA significantly blocked the protective effect of TSG in vitro. ConclusionsDown-regulation of omentin-1 induces endothelial dysfunction and hypertension in obesity. TSG treatment (at least partially) increases omentin-1 via promoting binding of PPAR-γ and Itln-1 promoter in adipose tissues, subsequently exerts protective effects on endothelial function via activating Akt/eNOS/NO signaling and attenuating oxidative/nitrative stress. These results suggest that TSG could be developed as a promising anti-hypertension agent that protects against endothelial dysfunction and obesity-associated cardiovascular diseases.

Nitric oxide signaling in the plant nucleus: the function of nitric oxide in chromatin modulation and transcription.

Nitric oxide (NO) is involved in a vast number of physiologically important processes in plants, such as organ development, stress resistance, and immunity. Transduction of NO bioactivity is generally achieved by post-translational modification of proteins, with S-nitrosation of cysteine residues as the predominant form. While traditionally the subcellular location of the factors involved was of lesser importance, recent studies identified the connection between NO and transcriptional activity and thereby raised the question about the route of NO into the nuclear sphere. Identification of NO-affected transcription factors and chromatin-modifying histone deacetylases implicated the important role of NO signaling in the plant nucleus as a regulator of epigenetic mechanisms and gene transcription. Here, we discuss the relationship between NO and its directly regulated protein targets in the nuclear environment, focusing on S-nitrosated chromatin modulators and transcription factors.

Cerebral oxygen sensing and the integrated regulation of hypoxic vasodilatation.

Cerebral oxygen sensing and the integrated regulation of hypoxic vasodilatation.

A role for S-nitrosylation of the SUMO-conjugating enzyme SCE1 in plant immunity

SUMOylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins, is emerging as a key modulator of eukaryotic immune function. In plants, a SUMO1/2-dependent process has been proposed to control the deployment of host defense responses. The molecular mechanism underpinning this activity remains to be determined, however. Here we show that increasing nitric oxide levels following pathogen recognition promote S-nitrosylation of the Arabidopsis SUMO E2 enzyme, SCE1, at Cys139. The SUMO-conjugating activities of both SCE1 and its human homolog, UBC9, were inhibited following this modification. Accordingly, mutation of Cys139 resulted in increased levels of SUMO1/2 conjugates, disabled immune responses, and enhanced pathogen susceptibility. Our findings imply that S-nitrosylation of SCE1 at Cys139 enables NO bioactivity to drive immune activation by relieving SUMO1/2-mediated suppression. The control of global SUMOylation is thought to occur predominantly at the level of each substrate via complex local machineries. Our findings uncover a parallel and complementary mechanism by suggesting that total SUMO conjugation may also be regulated directly by SNO formation at SCE1 Cys139. This Cys is evolutionary conserved and specifically S-nitrosylated in UBC9, implying that this immune-related regulatory process might be conserved across phylogenetic kingdoms.

Sulfur: the heart of nitric oxide-dependent redox signalling.

Nitric oxide (NO), more benign than its more reactive and damaging related molecules, reactive oxygen species (ROS), is perfectly suited for duties as a redox signalling molecule. A key route for NO bioactivity is through S-nitrosation, the addition of an NO moiety to a protein Cys thiol (-SH). This redox-based, post-translational modification (PTM) can modify protein function analogous to more well established PTMs such as phosphorylation, for example by modulating enzyme activity, localization, or protein-protein interactions. At the heart of the underpinning chemistry associated with this PTM is sulfur. The emerging evidence suggests that S-nitrosation is integral to a myriad of plant biological processes embedded in both development and environmental relations. However, a role for S-nitrosation is perhaps most well established in plant-pathogen interactions.

Volatile compounds other than CO2 emitted by different microorganisms promote distinct posttranscriptionally regulated responses in plants.

A "box-in-box" cocultivation system was used to investigate plant responses to microbial volatile compounds (VCs) and to evaluate the contributions of organic and inorganic VCs (VOCs and VICs, respectively) to these responses. Arabidopsis plants were exposed to VCs emitted by adjacent Alternaria alternata and Penicillium aurantiogriseum cultures, with and without charcoal filtration. No VOCs were detected in the headspace of growth chambers containing fungal cultures with charcoal filters. However, these growth chambers exhibited elevated CO2 and bioactive CO and NO headspace concentrations. Independently of charcoal filtration, VCs from both fungal phytopathogens promoted growth and distinct developmental changes. Plants cultured at CO2 levels observed in growth boxes containing fungal cultures were identical to those cultured at ambient CO2 . Plants exposed to charcoal-filtered fungal VCs, nonfiltered VCs, or superelevated CO2 levels exhibited transcriptional changes resembling those induced by increased irradiance. Thus, in the "box-in-box" system, (a) fungal VICs other than CO2 and/or VOCs not detected by our analytical systems strongly influence the plants' responses to fungal VCs, (b) different microorganisms release VCs with distinct action potentials, (c) transcriptional changes in VC-exposed plants are mainly due to enhanced photosynthesis signaling, and (d) regulation of some plant responses to fungal VCs is primarily posttranscriptional.

Nutraceutical Targeting of Placental Synthesis of Soluble Fms-Like Tyrosine Kinase- 1 (sFlt-1) as Strategy for Preventing and Controlling Pre-eclampsia.

The primary driving force in preeclampsia (PE) appears to be excessive secretion of fms-like tyrosine kinase-1 (sFlt-1), a truncated decoy receptor for vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) that induces systemic endotheliopathy by depriving endothelial cells of the trophic support conferred by VEGF. Factors which boost placental sFlt-1 production in PE include hypoxia - reflecting improper placentation - oxidative stress, and deficient production of hydrogen sulfide (H2S). Nutraceutical measures which may address these issues include taurine and N-acetylcysteine - which may boost placental H2S production; spirulina and phase 2 inducers of heme oxygenase-1 such as lipoic acid - which may down-regulate placental NADPH oxidase activity; and citrulline, high-dose folate, and dietary nitrate - which by supporting placental nitric oxide production may aid proper placentation and hence prevent placental hypoxia. These agents may also help to alleviate the pathogenic impact of sFlt-1 excess. If the utility of such measures can be demonstrated in rodent models of PE, functional foods incorporating these nutraceuticals can be envisioned as aids to a healthful pregnancy. Moreover, rodent studies suggest that such prenatal supplementation may reduce risk for hypertension in adult offspring of the pregnancy. And, since women who develop PE are at markedly higher risk for cardiovascular disorders in their later life, continuing use of such supplementation - promoting effective NO and H2S bioactivity while aiding control of oxidative stress - may be advisable for the mothers.

Oral nitrite restores age-dependent phenotypes in eNOS-null mice.

Alterations in the synthesis and bioavailability of NO are central to the pathogenesis of cardiovascular and metabolic disorders. Although endothelial NO synthase-derived (eNOS-derived) NO affects mitochondrial long-chain fatty acid β-oxidation, the pathophysiological significance of this regulation remains unclear. Accordingly, we determined the contributions of eNOS/NO signaling in the adaptive metabolic responses to fasting and in age-induced metabolic dysfunction. Four-month-old eNOS-/- mice are glucose intolerant and exhibit serum dyslipidemia and decreased capacity to oxidize fatty acids. However, during fasting, eNOS-/- mice redirect acetyl-CoA to ketogenesis to elevate circulating levels of β-hydroxybutyrate similar to wild-type mice. Treatment of 4-month-old eNOS-/- mice with nitrite for 10 days corrected the hypertension and serum hyperlipidemia and normalized the rate of fatty acid oxidation. Fourteen-month-old eNOS-/- mice exhibited metabolic derangements, resulting in reduced utilization of fat to generate energy, lower resting metabolic activity, and diminished physical activity. Seven-month administration of nitrite to eNOS-/- mice reversed the age-dependent metabolic derangements and restored physical activity. While the eNOS/NO signaling is not essential for the metabolic adaptation to fasting, it is critical for regulating systemic metabolic homeostasis in aging. The development of age-dependent metabolic disorder is prevented by low-dose replenishment of bioactive NO.

Abstract 365: Potential Relationship Between Nitric Oxide Bioactivity And Kidney Function Following Cardio-pulmonary Bypass

Background: Impaired kidney function is frequently observed in heart surgery patients following cardio-pulmonary bypass. Previous studies by our group have linked acute reductions in nitric oxide bioactivity, specifically declines in the main regulator of tissue blood flow S-nitrosohemoglobin (SNO-Hb), to reductions in kidney oxygenation and blood flow and subsequent renal impairment. For the current study, we hypothesized that components of the bypass procedure (viz. transfusion and red blood cell trauma) would reduce circulating SNO-Hb levels with the magnitude of this reduction correlating with markers of kidney function and patient outcome. Methods: After obtaining written informed consent, blood samples were obtained before and during bypass and on post-operative day (POD) 1. In addition to quantifying SNO-Hb samples were analyzed for change in kidney function (estimated glomerular filtration rate; eGFR), and plasma erythropoietin (EPO) concentration. Status and outcome data were secured from the patients’ medical record. Results: In the patients studied to date (n=30), there was a significant decline in SNO-Hb concentration by POD 1, the level of which inversely correlated with ICU stay (R=-0.32). In addition, there was a direct correlation between SNO-Hb and eGFR on PODs 1 & 2 (R=0.48 and 0.30, respectively), i.e. lower SNO-Hb tracked with reductions in kidney function. Consistent with a presumed reduction in kidney oxygenation, we observed a significant rise in plasma EPO levels when coming off pump that were sustained into POD 1. Furthermore, the rise in EPO was inversely correlated with both SNO-Hb (R=-0.53) and eGFR (R=-0.55). Conclusion: CPB decreases SNO-Hb and is associated with kidney dysfunction and worse patient outcome (e.g. increased ICU stay). While the mechanism for this decline remains to be determined, the results suggest that therapies directed at maintaining or increasing NO bioactivity could improve outcome in adult cardiac surgery patients.