Nitric Oxide Homeostasis Research Articles

Inhibition of nitric oxide synthesis during induced cholestasis ameliorates hepatocellular injury by facilitating S-nitrosothiol homeostasis

Cholestatic liver injury following extra- or intrahepatic bile duct obstruction causes nonparenchymal cell proliferation and matrix deposition leading to end-stage liver disease and cirrhosis. In cholestatic conditions, nitric oxide (NO) is mainly produced by a hepatocyte-inducible NO synthase (iNOS) as a result of enhanced inflow of endotoxins to the liver and also by accumulation of bile salts in hepatocytes and subsequent hepatocellular injury. This study was aimed to investigate the role of NO and S-nitrosothiol (SNO) homeostasis in the development of hepatocellular injury during cholestasis induced by bile duct ligation (BDL) in rats. Male Wistar rats (200–250 g) were divided into four groups (n=10 each), including sham-operated (SO), bile duct-ligated (BDL), tauroursodeoxycholic acid (TUDCA, 50 mg/kg) and S-methylisothiourea (SMT, 25 mg/kg) treated. After 7 days, BDL rats showed elevated serum levels of γ-glutamiltranspeptidase, aspartate aminotransferase, alanine aminotransferase, LDH, and bilirubin, bile duct proliferation and fibrosis, compared with the SO group. TUDCA treatment did not significantly alter these parameters, but the iNOS inhibitor SMT ameliorated hepatocellular injury, as shown by lower levels of circulating hepatic enzymes and bilirubin, and a decreased grade of bile duct proliferation and fibrosis. Both TUDCA and SMT treatments reversed Mrp2 canalicular pump expression to control levels. However, only SMT treatment significantly lowered the increased levels of plasma NO and S-nitrosation (S-nitrosylation) of liver proteins in BDL rats. Moreover, BDL resulted in a reduction of the S-nitrosoglutathione reductase (GSNOR/Adh5) enzymatic activity and a downregulation of the GSNOR/Adh5 mRNA expression that was reverted by SMT, but not TUDCA, treatment. A total of 25 liver proteins, including S-adenosyl methionine synthetase, betaine-homocysteine S-methyltransferase, Hsp90 and protein disulfide isomerase, were found to be S-nitrosated in BDL rats. In conclusion, the inhibition of NO production during induced cholestasis ameliorates hepatocellular injury. This effect is in part mediated by the improvement of cell proficiency in maintaining SNO homeostasis.

Pathophysiology of stroke-like episodes in MELAS: neuron–astrocyte uncoupling in neuronal hyperexcitability

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is a distinct clinical syndrome characterized by fluctuated encephalopathy, migraineous headache, seizure and stroke-like episodes. The molecular mechanism of MELAS mutations has been elucidated; however, the pathogenesis of stroke-like episodes remains largely unknown. Three main hypotheses include ischemic, metabolic and neuronal hyperexcitability hypotheses. Recently, emerging hypotheses include alterations in nitric oxide homeostasis and over-reduction/oxidative stress mechanisms. Although neuron–astrocyte communication is crucial in various physiological functions, it has not been seriously considered in the pathophysiology of stroke-like episodes. This review summarizes what is known about the molecular mechanisms of gene mutation, clinico-radiological, clinico-physiological and pathological features of stroke-like episodes, as well as its pathogenesis. We finally discuss potential mechanisms involved in the pathogenesis of stroke-like episodes based on currently available clinical data and the current understanding of the mechanisms of neuron–astrocyte communications. We propose that neuron–astrocyte uncoupling is a new target of research in mitochondrial disorders.

Dual Cross-Talk between Nitric Oxide and D-Serine in Astrocytes and Neurons in the Brain

The present review describes the role of the putative cross-talk between two neurotransmitters, nitric oxide (NO) and D-serine, in the brain. Under physiological conditions NO homeostasis guarantees the correct function of NO in a number of events in the brain such as neurotransmission and vascular tone regulation. D-serine, produced in astrocytes, acts synergistically with glutamate at NMDA receptors on postsynaptic neurons. Neuronal and endothelial NO synthase (nNOS and eNOS) in astrocytes cross-talk with serine racemase (SR) and D-amino acid oxydase (DAAO), catalyzing the synthesis and degradation of D-serine, respectively. SR is inhibited by NO which activates DAAO. D-serine inhibits nNOS but not eNOS and activates SR. Astrocytes and neurons also cross-talk through NO/D-serine system. D-serine released from astrocytes induces a rapid increase in NO contents in postsynaptic neurons. Overall, D-serine production in astrocytes is negatively regulated by NO. Under inflammatory conditions, pro-inflammatory cytokines or Abeta induce, first, a drop in NO contents and an increase in the amounts of D-serine in astrocytes. Together with enhanced glutamate release from presynaptic neurons, D-serine induces an increase in Ca(2+) up-take into presynaptic neurons. In astrocytes an initial drop in NO contents triggers NF-kappaB activation followed by inducible NOS (iNOS) expression. iNOS-derived massive amounts of NO may potentially be toxic. Under schizophrenic conditions, D-serine production is down-regulated. Together with reduced glutamate release, this situation leads to the decreased NO production in postsynaptic neurons. In astrocytes induction of iNOS expression becomes predominant. Initial drop in nNOS-derived NO is potentially toxic in this scenario.

The Effect of Short-Term Simvastatin On Markers of Vascular Dysfunction in Patients with Sickle Cell Disease (SCD).

Abstract 260Sickle cell disease (SCD) is characterized by frequent painful vaso-occlusive episodes, acute life-threatening complications, and progressive vascular injury resulting in multi-organ failure. Emerging data indicate that nitric oxide (NO) plays a central role in mediating the pathogenesis of sickle-induced vascular injury. Because antisickling agents only partially ameliorate the vasculopathy of SCD, therapeutic agents that modulate NO homeostasis are rational candidates for further exploration. Simvastatin and other HMG-CoA reductase inhibitors have been shown to exert NO-mediated vasculoprotective and anti-inflammatory effects in cardiovascular disease populations. Recent experimental and clinical studies have implicated the Rho/Rho-kinase (ROCK) signaling pathway as a principal mechanism by which statins increase NO availability. We enrolled twelve SCD patients in an ongoing FDA-approved dose-escalation study of the effect of simvastatin treatment on markers of vascular dysfunction and inflammation. Patients were treated at steady-state with low-dose oral simvastatin (20 mg/day) with serial blood samples obtained prior to treatment day 0, during treatment on days 7, 14, 21, 25 and after discontinuation of treatment on day 39. Plasma samples were assayed for nitrite/nitrate (NOx) concentration using NO chemiluminescence, and high-sensitivity C-reactive protein (hs-CRP), interleukin 6 (IL-6), tissue factor (TF), vascular endothelial growth factor (VEGF) and vascular cell adhesion molecule (VCAM1) by ELISA. The effect of simvastatin on leukocyte ROCK activity was assessed by Western blot analyses in a subset of patients. Changes in biomarker levels from baseline in response to simvastatin at 21 days were assessed using matched paired t-tests.The mean age of subjects was 31.4 years (range, 13–55); 7 males and 5 females. Four subjects experienced an uneventful vaso-occlusive pain episode during the study with an acute decrease in NO levels and these data points were removed from the analysis. Short-term simvastatin treatment resulted in a 24% increase in mean plasma NOx level (p=.02). Both hs-CRP (p=.01) and IL-6 levels (p=.05) decreased dramatically by 62% and 63%, respectively. There was no difference between baseline and day 21 levels of TF, VEGF and VCAM1. Although limited to qualitative assessment in a subset of patients, our pilot data also demonstrated a decrease in leukocyte ROCK activity after treatment with simvastatin. These preliminary data showing increased NO availability and anti-inflammatory effects of simvastatin treatment in SCD are consistent with studies in cardiovascular disease populations documenting the cholesterol-independent benefits of statins and provide a compelling rationale to further investigate the potential therapeutic efficacy of statins in SCD. Correlation of the effect of simvastatin on ROCK inhibition may provide further evidence for a possible mechanism by which statins modulate NO bioavailability in SCD. Disclosures:Off Label Use: Simvastatin (Zocor)is a cholesterol-lowering agent that has been shown to confer vascular protection beyond its lipid lowering effects. This drug is being studied in patients with sickle cell disease to obtain preliminary safety and efficacy data of its potential vasculoprotective effects.

Storage lesion in banked blood due to hemolysis-dependent disruption of nitric oxide homeostasis

Whereas blood storage is associated with an increased risk of cardiovascular events and multiorgan failure, the fundamental mechanisms underlying the 'storage lesion' in blood remain uncertain. A major abnormality in aged blood is the reduced red cell life-span after infusion, which is associated with microparticle and free hemoglobin release, and age-related loss of enzymatic functionality. However, the degree of intravascular hemolysis and microparticle formation in humans post-transfusion due to both storage and physiological shear has not been well studied. Our laboratories have discovered that even low levels of intravascular hemolysis severely disrupt nitric oxide bioavailability at the endothelium, via accelerated nitric oxide dioxygenation reactions with free plasma hemoglobin. This process contributes to endothelial dysfunction, adhesion molecule expression, platelet and hemostatic activation, and reactive oxygen species generation. Recent studies also suggest that red cells possess nitric oxide-generating functionality via nitrite reduction and red cell endothelial nitric oxide synthase activity, potentially providing novel pathways to therapeutically alleviate the 'storage lesion'. The understanding of the pathological effects of red cell hemolysis on endothelial function suggests that nitric oxide dysregulation may underlie the red cell storage lesion, driven by increased nitric oxide catabolism and loss of nitric oxide-generating functionality.

An Integrated Computational and Experimental Model of Nitric Oxide–Red Blood Cell Interactions

In blood vessels, nitric oxide homeostasis is maintained by its formation by endothelial nitric oxide synthase and its consumption in smooth muscle cells and in vascular lumen by red blood cell (RBC) encapsulated hemoglobin (Hb). Free hemoglobin has a very high reaction rate (k(Hb-NO) approximately 10(7) M(-1) s(-1)) with NO as compared to RBC-Hb. Mechanisms of reduced NO uptake by RBC-Hb has been extensively studied in recent years. A critical factor in the investigation of NO-RBC interactions is delivery of NO. Common NO delivery methods include use of NO donors and bolus saturated NO solutions, which delivers NO homogeneously and only in the vicinity of bolus, respectively. In this study, we developed a flow system that uses gaseous delivery of NO through a polymeric semi-permeable membrane to obtain precise and uniform NO concentrations for NO-RBC interactions. We conducted experiments using the flow system to study the effect of NO concentrations, hematocrit and RBC suspension flow rates on NO-RBC interactions. We developed a computational model to simulate NO transport and to estimate the reaction rate constant for NO-RBC interaction in the flow system. Our results showed that NO consumption of RBCs (i) increased linearly with an increase in available NO, and (ii) decreased with increase in RBCs suspension flow rate. We estimated the reaction rate constant for NO-RBC interactions to be 0.2 x 10(5) M(-1) s(-1) which is approximately 1250-fold lower than NO consumption by free hemoglobin and approximately 2.5-20 fold slower than reported NO-RBC reaction rate.

Nitric oxide bioactivity of traditional Chinese medicines used for cardiovascular indications

Traditional Chinese medicine (TCM) has been used for centuries to treat and prevent certain ailments and diseases. Although TCM has served as mainstream medical care throughout Asia for many generations, it is considered an alternative medical system in much of the Western world. Because many TCMs are used primarily for cardiovascular indications characterized by a nitric oxide (NO) insufficiency, we hypothesized that some, if not all, of these TCMs have a robust NO bioactivity that may act to restore NO homeostasis. We tested a group of convenience samples of TCMs obtained in the United States for endogenous nitrite, nitrate, nitroso, and nitrite reductase activity as well as their ability to relax isolated aortic rings. The results from this study reveal that all of the TCMs tested reveal NO bioactivity through their inherent nitrite and nitrate content and their ability to reduce nitrite to NO. Many of the TCM extracts contain a nitrite reductase activity greater by 1000 times that of biological tissues. Repletion of biological nitrite and nitrate by these extracts and providing a natural system for NO generation in both endothelium-dependent and -independent mechanisms may account for some of the therapeutic effects of TCMs.

Dietary sources of nitrite as a modulator of ischemia/reperfusion injury

The nitrite anion is an endogenous product of nitric oxide (NO) metabolism, a key intermediate in the nitrogen cycle in plants and bacteria, and a constituent of many foods. Research over the past 6 years has revealed a surprising biological and cytoprotective activity of this anion. Its ability to restore NO homeostasis throughout the physiological oxygen gradient in vivo has transformed this once-thought to be inert anion into a critical molecule in health and disease. Ischemia-reperfusion (I/R) injury is a major clinical problem worldwide. NO has been shown to be one of the most important molecules for the prevention of injury from I/R. Paradoxically, however, enzymatic NO formation from NO synthase (NOS) is inactive during conditions of inadequate oxygen and substrate delivery, such as in ischemia. Nitrite has emerged as a viable alternative source of NO under ischemic conditions. As nitrite is known to be derived not only from the oxidation of NO but also through diet, understanding nitrite metabolism and mechanisms of cytoprotection may offer novel and natural means to prevent disease or at least limit injury from an I/R event. Here, we review the current body of knowledge regarding dietary sources of nitrite and its modulation of cytoprotection in an I/R injury.

Regulation of macrophage nitric oxide production by the protein tyrosine phosphatase Src homology 2 domain phosphotyrosine phosphatase 1 (SHP‐1)

Nitric oxide (NO) is a potent molecule involved in the cytotoxic effects mediated by macrophages (MØ) against microorganisms. We previously reported that Src homology 2 domain phosphotyrosine phosphatase 1 (SHP-1)-deficient cells generate a greater amount of NO than wild-type cells in response to interferon-gamma (IFN-gamma). We also reported that the Leishmania-induced MØ SHP-1 activity is needed for the survival of the parasite within phagocytes through the attenuation of NO-dependent and NO-independent mechanisms. In the present study, we investigated the role of SHP-1 in regulating key signalling molecules important in MØ NO generation. Janus tyrosine kinase 2 (JAK2), mitogen-activated extracellular signal-regulated protein kinase kinase (MEK), extracellular signal-regulated kinases 1 and 2 (Erk1/Erk2) mitogen-activated protein kinases, p38 and stress-activated mitogen-activated protein kinases/c-Jun NH(2)-terminal kinase (SAPK/JNK) were examined in immortalized bone marrow-derived MØ (BMDM) from both SHP-1-deficient motheaten mice (me-3) and their respective littermates (LM-1). The results indicated that Erk1/Erk2 and SAPK/JNK are the main kinases regulated by SHP-1 because the absence of SHP-1 caused an increase in their phosphorylation. Moreover, only Apigenin, the specific inhibitor of Erk1/Erk2, was able to block IFN-gamma-induced inducible nitric oxide synthase (iNOS) transcription and translation in me-3 cells. Transcription factor analyses revealed that in the absence of SHP-1, activator protein-1 (AP-1) was activated. The activation of AP-1, and not nuclear factor-kappaB (NF-kappaB) or signal transducer and activator of transcription-1 alpha (STAT-1 alpha), may explain the enhanced NO generation in SHP-1-deficient cells. These observations emphasize the involvement of the MAPKs Erk1/Erk2 and SAPK/JNK in NO generation via AP-1 activation. Collectively, our findings suggest that SHP-1 plays a pivotal role in the negative regulation of signalling events leading to iNOS expression and NO generation. Furthermore, our observations underline the importance of SHP-1-mediated negative regulation in maintaining NO homeostasis and thus preventing the abnormal generation of NO that can be detrimental to the host.

Breathing lessons: Tor tackles the mitochondria

Breathing lessons: Tor tackles the mitochondria

Cardioprotective actions of nitrite therapy and dietary considerations

Nitrite is an intrinsic signaling molecule. Its ability to form nitric oxide (NO) under hypoxic conditions as well as its ability to form S-nitrosothiols under normoxic conditions has transformed this once inert anion into a critical molecule in maintaining NO and nitroso homeostasis throughout the entire physiological oxygen gradient in vivo in addition to serving as the endocrine mediator of NO. Steady state plasma nitrite has historically been used as a biomarker of NO availability. However, tissue specific metabolism of nitrite along with differences in nitrite and nitrate intake from diet warrant a careful assessment of plasma nitrite as an index of NO biochemistry. Metabolism and regulation of NO and nitrite are at the local cellular and tissue level dependent upon cellular redox status, redox active metal and thiol availability. Understanding nitrite metabolism and mechanisms of cytoprotection may offer novel means to prevent cardiovascular disease or limit injury from a cardiovascular event. Here the state of the art of nitrite in cytoprotection as well as maintenance of steady state blood and tissue nitrite is reviewed.

Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke

Survivors of massive inhalation of combustion smoke endure critical injuries, including lasting neurological complications. We have previously reported that acute inhalation of combustion smoke disrupts the nitric oxide homeostasis in the rat brain. In this study, we extend our findings and report that a 30-minute exposure of awake rats to ambient wood combustion smoke induces protein nitration in the rat hippocampus and that mitochondrial proteins are a sensitive nitration target in this setting. Mitochondria are central to energy metabolism and cellular signaling and are critical to proper cell function. Here, analyses of the mitochondrial proteome showed elevated protein nitration in the course of a 24-hour recovery following exposure to smoke. Mass spectrometry identification of several significantly nitrated mitochondrial proteins revealed diverse functions and involvement in central aspects of mitochondrial physiology. The nitrated proteins include the ubiquitous mitochondrial creatine kinase, F1-ATP synthase α subunit, dihydrolipoamide dehydrogenase (E3), succinate dehydrogenase Fp subunit, and voltage-dependent anion channel (VDAC1) protein. Furthermore, acute exposure to combustion smoke significantly compromised the respiratory capacity of hippocampal mitochondria. Importantly, elevated protein nitration and reduced mitochondrial respiration in the hippocampus persisted beyond the time required for restoration of normal oxygen and carboxyhemoglobin blood levels after the cessation of exposure to smoke. Thus, the time frame for intensification of the various smoke-induced effects differs between blood and brain tissues. Taken together, our findings suggest that nitration of essential mitochondrial proteins may contribute to the reduction in mitochondrial respiratory capacity and underlie, in part, the brain pathophysiology after acute inhalation of combustion smoke.

Increased nitrite reductase activity of fetal versus adult ovine hemoglobin

Growing evidence indicates that nitrite, NO2-, serves as a circulating reservoir of nitric oxide (NO) bioactivity that is activated during physiological and pathological hypoxia. One of the intravascular mechanisms for nitrite conversion to NO is a chemical nitrite reductase activity of deoxyhemoglobin. The rate of NO production from this reaction is increased when hemoglobin is in the R conformation. Because the mammalian fetus exists in a low-oxygen environment compared with the adult and is exposed to episodes of severe ischemia during the normal birthing process, and because fetal hemoglobin assumes the R conformation more readily than adult hemoglobin, we hypothesized that nitrite reduction to NO may be enhanced in the fetal circulation. We found that the reaction was faster for fetal than maternal hemoglobin or blood and that the reactions were fastest at 50-80% oxygen saturation, consistent with an R-state catalysis that is predominant for fetal hemoglobin. Nitrite concentrations were similar in blood taken from chronically instrumented normoxic ewes and their fetuses but were elevated in response to chronic hypoxia. The findings suggest an augmented nitrite reductase activity of fetal hemoglobin and that the production of nitrite may participate in the regulation of vascular NO homeostasis in the fetus.

Nitric Oxide Effects in Sickle Cell Disease.

Sickle cell disease (SCD) is a complex hemoglobinopathy characterized by microvascular occlusion and hemolytic anemia. Patients suffer from a myriad of both acute and chronic problems affecting virtually every organ system. Historically, microvascular occlusion has been the focus of scientific investigations into these manifestations and the chronic hemolysis of SCD was overlooked. More recently, however, the importance of the pathophysiology of hemolysis has been appreciated and related to a subset of the clinical manifestations of SCD, including pulmonary hypertension, priapism, skin ulcers, and possibly stroke. This subphenotype of SCD has been convincingly related to impaired nitric oxide (NO) homeostasis due to hemolysis. NO has pleiotropic effects including vaso-dilatory, antioxidative, anti-adhesion, and anti-thrombotic properties, which are all potentially important in the pathophysiology of SCD. Perturbation of NO homeostasis, therefore, could profoundly impact patients with SCD. Animal and human data support a state of “NO resistance” in SCD patients. Human studies have shown that SCD patients have a decreased response to exogenous NO donors and that is likely due to the scavenging of NO by free plasma hemoglobin that results from ongoing hemolysis. “NO resistance” is further augmented by the increased levels of reactive oxygen species (ROS) known to occur in SCD patients. High levels of ROS favor additional hemolysis through increased oxidant stress on the sickle red blood cell and reduce NO bioavailability by inactivation of circulating NO. With the substantial human and animal data to support a role for “NO resistance” in the pathophysiology of SCD, investigation with NO-based therapy have begun. Several approaches to overcoming “NO resistance” can be devised including increasing the precursors to NO, decreasing hemolysis, direct NO donors, and decreasing oxidant stress. To date, studies evaluating arginine (NO precursor), inhaled NO, and sildenafil (NO donor) have been reported. Oral arginine showed no benefit in a large clinical trial, and a preliminary trial of inhaled NO had only minimal benefit. Sildenafil may be more promising and is under further study. Lastly, although impaired NO bioavailability has been related to a subset of patients with pulmonary hypertension, skin ulcers and priapism, it will be important to determine what impact NO has on other manifestations, such as vaso-occlusive pain episodes and whether NO modulation can also be used therapeutically in this setting.

Tetrahydrobiopterin (6R-BH4): Novel Therapy for Endothelial Dysfunction in Sickle Cell Disease

Abstract Dysregulated nitric oxide (NO) homeostasis, a consequence of hemolysis, is a central feature of endothelial dysfunction (ED) in Sickle Cell disease (SCD). In addition to ED, scavenging of NO by free heme leads to increased cell adhesion and inflammation. Vascular inflammation and the production of superoxide may decrease BH4, an essential cofactor for NO production, thus creating an acquired BH4 deficiency. Restoring BH4 levels could potentially improve ED thereby favorably impacting complications of SCD. We assessed the safety and efficacy of 6R-BH4 on endothelial function in a Phase 2a, open-label, dose escalation study in SCD subjects using a non-invasive, operatorindependent technique of peripheral arterial tonometry (Endo-PAT; Itamar, Israel). Endo-PAT (PAT) scores were quantitatively determined as the ratio between the arterial pulse wave amplitude following a 5 min arterial occlusion in the forearm to the pre-occlusion value. A value of ≤1.67 represents an impaired response or endothelial dysfunction. Only patients with HbSS and HbSC disease and at least 15 years of age were enrolled. Patients were excluded if they: were on chronic hypertransfusion; had sickle cell crisis within 30 days of screening; had a history of bone marrow or stem cell transplant or were on hydroxyurea (HU) therapy during the 3 months prior to screening. Thirty-two African-American subjects, mean age 29 years (41% male) were sequentially treated for 4 weeks each with 6R-BH4 at 2.5, 5, 10, and 20 mg/kg/day at 12 US sites. Nine subjects discontinued therapy for various reasons including loss of follow up and pregnancy. Twenty-seven subjects had baseline PAT scores and the number of subjects with PAT scores varied at each treatment dose. There were no deaths and only one subject had a drug related adverse effect resulting in discontinuation. Overall, 6R-BH4 is safe and well-tolerated in subjects with SCD. The mean PAT scores for all participants at baseline was 1.58 ± 0.43 (mean ± SD). Mean PAT scores at baseline were 1.33 ± 0.17 in 18 patients with abnormal PAT scores and 2.09 ± 0.31 (p=<0.001) in 9 patients with normal PAT scores. Mean PAT score for all subjects demonstrated significant improvement at 5mg/kg/day and 10mg/kg/day (dose, N, mean change +/− SD, mean % change and p value) (5 mg/kg/day, N=24, 1.79 ± 0.64, 22.4%, p= 0.042; 10 mg/kg/day, N=24, 1.95 ± 0.46, 28.2%, p=0.003). Eighteen of the 27 (67%) subjects who had abnormal PAT scores at baseline had statistically significant dose-dependent improvements over the 16 weeks of therapy with 6R-BH4 (2.5 mg/kg, N=15, 1.63 ± 0.37, 24.7%, p=0.012; 5mg/kg, N=14, 1.69 ± 0.56, 31.2%, p=0.025; 10mg/kg, N=15, 1.84 ± 0.47, 39.9%, p<0.001; 20mg/kg, N=15, 2.01 ± 0.76, 56.6%, p=0.005). Consistent with the mechanism of action of 6R-BH4 subjects with normal Endo-PAT scores at baseline demonstrated no improvement with therapy. HbSS subjects appear to have more ED based on PAT scores compared with HbSC subjects, although the difference was not statistically significant 1.52 ± 0.45 vs 1.67 ± 0.39. More importantly, both HbSS and HbSC subjects demonstrated an improvement in mean change in endothelial function with increasing doses of 6R-BH4 with corresponding % mean changes from baseline being 48.8% and 15.5% respectively following 16 weeks of treatment. The majority of subjects in the study (17/27; 63%) were prescribed folic acid supplement by their physicians at baseline and throughout the study. Post hoc analysis demonstrated no difference in baseline PAT scores between subjects on folic acid supplementation and those not on it (1.60 ± 0.47 vs 1.55 ± 0.37). However, patients on folic acid demonstrated a better dose response to treatment with 6R-BH4 compared to patients not receiving folic acid (2.5 mg/kg: 1.72 ± 0.38 vs 1.69 ± 0.41; 5mg/kg: 1.93 ± 0.74 vs 1.56 ± 0.38; 10mg/kg: 1.89 ± 0.51 vs 2.06 ± 0.34; 20 mg/kg: 2.09 ± 0.73 vs 1.62 ± 0.34) In summary, 6R-BH4 is safe, well-tolerated and demonstrates a dose-dependent improvement in endothelial function in subjects with SCD. Best results were achieved in those with baseline endothelial dysfunction. Improvement in ED occurs regardless of genotype. Finally, patients receiving folic acid showed a better response to 6R-BH4 than those not receiving this supplement. These data provide further support for the development of 6R-BH4 as a treatment for sickle cell disease. 6R-BH4 is a potentially new effective modulator of NO for SCD patients who have ED.

A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis

Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis.

Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice

Endothelial production of nitric oxide (NO) is critical for vascular homeostasis. Nitrite and nitrate are formed endogenously by the stepwise oxidation of NO and have, for years, been regarded as inactive degradation products. As a result, both anions are routinely used as surrogate markers of NO production, with nitrite as a more sensitive marker. However, both nitrite and nitrate are derived from dietary sources. We sought to determine how exogenous nitrite affects steady-state concentrations of NO metabolites thought to originate from nitric oxide synthase (NOS)-derived NO as well as blood pressure and myocardial ischemia–reperfusion (I/R) injury. Mice deficient in endothelial nitric oxide synthase (eNOS –/–) demonstrated decreased blood and tissue nitrite, nitrate, and nitroso proteins, which were further reduced by low-nitrite (NOx) diet for 1 week. Nitrite supplementation (50 mg/L) in the drinking water for 1 week restored NO homeostasis in eNOS –/– mice and protected against I/R injury. Nitrite failed to alter heart rate or mean arterial blood pressure at the protective dose. These data demonstrate the significant influence of dietary nitrite intake on the maintenance of steady-state NO levels. Dietary nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a novel prevention/treatment modality for disease associated with NO insufficiency.

Modulation of Nitrosative Stress byS-Nitrosoglutathione Reductase Is Critical for Thermotolerance and Plant Growth inArabidopsis

Nitric oxide (NO) is a key signaling molecule in plants. This analysis of Arabidopsis thaliana HOT5 (sensitive to hot temperatures), which is required for thermotolerance, uncovers a role of NO in thermotolerance and plant development. HOT5 encodes S-nitrosoglutathione reductase (GSNOR), which metabolizes the NO adduct S-nitrosoglutathione. Two hot5 missense alleles and two T-DNA insertion, protein null alleles were characterized. The missense alleles cannot acclimate to heat as dark-grown seedlings but grow normally and can heat-acclimate in the light. The null alleles cannot heat-acclimate as light-grown plants and have other phenotypes, including failure to grow on nutrient plates, increased reproductive shoots, and reduced fertility. The fertility defect of hot5 is due to both reduced stamen elongation and male and female fertilization defects. The hot5 null alleles show increased nitrate and nitroso species levels, and the heat sensitivity of both missense and null alleles is associated with increased NO species. Heat sensitivity is enhanced in wild-type and mutant plants by NO donors, and the heat sensitivity of hot5 mutants can be rescued by an NO scavenger. An NO-overproducing mutant is also defective in thermotolerance. Together, our results expand the importance of GSNOR-regulated NO homeostasis to abiotic stress and plant development.

Differential expression of neuronal and inducible nitric oxide synthase in rat brain after subchronic administration of 3-monochloro-1,2-propanediol

The compound 3-monochloro-1,2-propanediol (3-MCPD) is a contaminant of acid-hydrolyzed vegetable protein foodstuffs. Several reports have suggested that chronic exposure to 3-MCPD can produce neurotoxicity or neurobehavioral effects in experimental animals. We sought to further explore the neurotoxic effects of 3-MCPD (10 or 30 mg/kg) administered for 13 weeks on the expression of two forms of nitric oxide synthase (NOS), neuronal NOS (nNOS), and inducible NOS (iNOS), in rat cerebral cortex and striatum. Using immunocytochemistry, the number of nNOS-expressing neurons or the optical density of iNOS staining in sections from three coronal levels (bregma 1.0, −0.4, and −2.3 mm) were compared between 3-MCPD-treated and control rats. At bregma level 1.0 mm, the number of nNOS-expressing neurons was significantly decreased in the 10 and 30 mg/kg groups. At bregma level −0.4 mm, nNOS expression was significantly decreased only in the 30 mg/kg group, in the cortex and striatum. However, at bregma level −2.3 mm, 3-MCPD administration produced no significant difference in the number of nNOS-expressing neurons in the cortex or striatum. In contrast, iNOS expression was significantly increased in the neocortex and striatum at all three rostrocaudal levels following subchronic 3-MCPD administration. These data suggest that subchronic 3-MCPD exposure may involve compensatory mechanisms acting on nNOS and iNOS expression to maintain nitric oxide homeostasis in the rostral part of the neocortex and striatum. However, in the caudal brain, increased iNOS expression did not profoundly suppress nNOS expression. Thus, the present study suggests that 3-MCPD-induced neurotoxicity is mediated, at least in part, through disturbances in the nitric oxide signaling pathway and exhibits a rostrocaudal difference, through differential expression of nNOS and iNOS in the neocortex and striatum.

Nitrite Infusion in Humans and Nonhuman Primates

The recent discovery that nitrite is an intrinsic vasodilator and signaling molecule at near-physiological concentrations has raised the possibility that nitrite contributes to hypoxic vasodilation and to the bioactivity of nitroglycerin and mediates the cardiovascular protective effects of nitrate in the Mediterranean diet. However, important questions of potency, kinetics, mechanism of action, and possible induction of tolerance remain unanswered. In the present study, we performed biochemical, physiological, and pharmacological studies using nitrite infusion protocols in 20 normal human volunteers and in nonhuman primates to answer these questions, and we specifically tested 3 proposed mechanisms of bioactivation: reduction to nitric oxide by xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin. We found that (1) nitrite is a relatively potent and fast vasodilator at near-physiological concentrations; (2) nitrite functions as an endocrine reservoir of nitric oxide, producing remote vasodilation during first-pass perfusion of the opposite limb; (3) nitrite is reduced to nitric oxide by intravascular reactions with hemoglobin and with intravascular reductants (ie, ascorbate); (4) inhibition of xanthine oxidoreductase with oxypurinol does not inhibit nitrite-dependent vasodilation but potentiates it; and (5) nitrite does not induce tolerance as observed with the organic nitrates. We propose that nitrite functions as a physiological regulator of vascular function and endocrine nitric oxide homeostasis and suggest that it is an active metabolite of the organic nitrates that can be used therapeutically to bypass enzymatic tolerance.