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Central adiposity is associated with greater sympathetic support of blood pressure. β-adrenergic receptors (β-AR) buffer sympathetically mediated vasoconstriction and β-AR-mediated vasodilation is attenuated in preclinical models of obesity. With this information, we hypothesized β-AR vasodilation would be lower in obese compared with normal weight adults. Because β-AR vasodilation in normal weight adults is limited by cyclooxygenase (COX) restraint of nitric oxide synthase (NOS), we further explored the contributions of COX and NOS to β-AR vasodilation in this cohort. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured and forearm vascular conductance (FVC) was calculated (FVC = FBF/MAP). The rise in FVC from baseline (ΔFVC) was quantified during graded brachial artery infusion of isoproterenol (Iso, 1–12 ng/100 g/min) in normal weight (n = 36) and adults with obesity (n = 22) (18–40 yr old). In a subset of participants, Iso-mediated vasodilation was examined before and during inhibition of NOS [N(G)-monomethyl-l-arginine (l-NMMA)], COX (ketorolac), and NOS + COX (l-NMMA + ketorolac). Iso-mediated increases in FVC did not differ between groups (P = 0.57). l-NMMA attenuated Iso-mediated ΔFVC in normal weight (P = 0.03) but not adults with obesity (P = 0.27). In normal weight adults, ketorolac increased Iso-mediated ΔFVC (P < 0.01) and this response was lost with concurrent l-NMMA (P = 0.67). In contrast, neither ketorolac (P = 0.81) nor ketorolac + l-NMMA (P = 0.40) altered Iso-mediated ΔFVC in adults with obesity. Despite shifts in COX and NOS, β-AR vasodilation is preserved in young adults with obesity. These data highlight the presence of a compensatory shift in microvascular control mechanisms in younger humans with obesity. NEW & NOTEWORTHY We examined β-adrenergic receptor-mediated vasodilation in skeletal muscle of humans with obesity and normal weight. Results show that despite shifts in the contribution of cyclooxygenase and nitric oxide synthase, β-adrenergic-mediated vasodilation is relatively preserved in young, otherwise healthy adults with obesity. These data highlight the presence of subclinical changes in microvascular control mechanisms early in the obesity process and suggest duration of obesity and/or the addition of primary aging may be necessary for overt dysfunction.

Objectives Thyroid disorders are important risk factor for cardiovascular diseases. Levels of methylarginines such as asymmetric dimethyl arginine (ADMA), L-monomethyl arginine (L-NMMA), symmetric dimethyl arginine (SDMA) are increase in cardiovascular diseases. Multinodular goiter (MNG) is the most common type of goiter in adults. To date, no study has been conducted to determine the levels of methylarginine in euthyroid MNG patients. Our aim in this study is to compare levels of methylarginines and related metabolites in the preoperative, postoperative MNG patients and controls. Methods Serum ADMA, SDMA, L-NMMA, homoarginine (hArg), arginine and citrulline concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results ADMA (pl-arginine (p=0.006) and citrulline (p Conclusions The levels of methylarginines and related metabolites altered in the euthyroid MNG patients compared to the control group, and more importantly, there were significant differences between the preop and postop groups. Therefore, these metabolites can be useful in the diagnosis and prognosis of thyroid disorders, even if thyroid hormone levels are normal.

Key points Preclinical models have demonstrated that nitric oxide is a key component of neurovascular coupling; this has yet to be translated to humans. We conducted two separate protocols utilizing intravenous infusion of a nitric oxide synthase inhibitor and isovolumic haemodilution to assess the influence of nitric oxide on neurovascular coupling in humans. Isovolumic haemodilution did not alter neurovascular coupling. Intravenous infusion of a nitric oxide synthase inhibitor reduced the neurovascular coupling response by ∼30%, indicating that nitric oxide is integral to neurovascular coupling in humans. Abstract Nitric oxide is a vital neurovascular signalling molecule in preclinical models, yet the mechanisms underlying neurovascular coupling (NVC) in humans have yet to be elucidated. To investigate the contribution of nitric oxide to NVC in humans, we utilized a visual stimulus paradigm to elicit an NVC response in the posterior cerebral circulation. Two distinct mechanistic interventions were conducted on young healthy males: (1) NVC was assessed during intravenous infusion of saline (placebo) and the non-selective competitive nitric oxide synthase inhibitor NG -monomethyl-l-arginine (l-NMMA, 5 mg kg-1 bolus & subsequent 50 μg kg-1 min-1 maintenance dose; n = 10). The order of infusion was randomized, counterbalanced and single blinded. A subset of participants in this study (n = 4) underwent a separate intervention with phenylephrine infusion to independently consider the influence of blood pressure changes on NVC (0.1-0.6 μg kg-1 min-1 constant infusion). (2) NVC was assessed prior to and following isovolumic haemodilution, whereby 20% of whole blood was removed and replaced with 5% human serum albumin to reduce haemoglobin concentration (n = 8). For both protocols, arterial and internal jugular venous blood samples were collected at rest and coupled with volumetric measures of cerebral blood flow (duplex ultrasound) to quantify resting cerebral metabolic parameters. l-NMMA elicited a 30% reduction in the peak (P = 0.01), but not average (P = 0.11), NVC response. Neither phenylephrine nor haemodilution influenced NVC. Nitric oxide signalling is integral to NVC in humans, providing a new direction for research into pharmacological treatment of humans with dementia.

Trichophyton rubrum (T. rubrum) is one of the most important agents of dermatophyte infection in humans. The aim of this experiment was to evaluate the effect of HaCaT cells on T. rubrum, investigate the responsible mechanism of action, and explore the role of reactive oxygen species (ROS) and nitric oxide (NO) in the inhibition of T. rubrum growth by HaCaT cells. The viability of fungi treated with HaCaT cells alone and with HaCaT cells combined with pretreatment with the NADPH oxidase inhibitor (DPI) or the nitric oxide synthase (NOS) inhibitor L-NMMA was determined by enumerating the colony-forming units. NOS, ROS, and NO levels were quantified using fluorescent probes. The levels of the NOS inhibitor asymmetric dimethylarginine (ADMA) were determined by enzyme-linked immunosorbent assay (ELISA). Micromorphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, fungal keratinase activity was assessed by measuring dye release from keratin azure. In vitro fungal viability, keratinase activity, and ADMA content decreased after HaCaT cell intervention, whereas the levels of ROS, NO, and NOS increased. The micromorphology was abnormal. Fungi pretreated with DPI and L-NMMA exhibited opposite effects. HaCaT cells inhibited the growth and pathogenicity of T. rubrum in vitro. A suggested mechanism is that ROS and NO play an important role in the inhibition of T. rubrum growth by HaCaT cells.

Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of protein disulfide isomerase (PDI) is increased in brains with sporadic Alzheimer's disease and Parkinson's disease. The aim of the present study was to clarify the role of nitric oxide (NO) in BPA-induced neurotoxicity. Since rotenone induces NO-mediated neurodegeneration through S-nitrosylation of PDI, it was used as a positive control. First, rats were treated with BPA and rotenone, and S-nitrosylation of PDI was detected in rat brain microsomes. BPA and rotenone decreased RNase oxidation activity of PDI concomitant with S-nitrosylation of PDI. Next, to clarify S-nitrosylation of PDI by BPA and rotenone in rat brains, we treated the rat pheochromocytoma cell line PC12 and primary cultured neuron cells from the rat hippocampus with BPA (5 and 10 μM) and rotenone (100 or 200 nM). BPA induced S-nitrosylation of PDI, while NG-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, exerted the opposite effects. Finally, to evaluate the toxicity of BPA in the CNS, we investigated its effects on neurite outgrowth of PC12 and primary cultured neuron cells. BPA inhibited neurite outgrowth of these cells, while L-NMMA reversed this inhibition. The involvement of PDI activity in neurite outgrowth was also examined, and bacitracin, a PDI inhibitor, is shown to decrease neurite outgrowth. Furthermore, the overexpression of PDI, but not a catalytically inactive PDI mutant, enhanced neurite outgrowth. These results suggested that S-nitrosylation of PDI induced by excessive NO caused BPA-induced neurotoxicity.

Background Maternal consumption of alcohol produces abnormalities in the developing fetus and can contribute to an increased incidence of many cardiovascular-related diseases. The first goal of this study was to determine whether in utero exposure to alcohol influences reactivity of cerebral arterioles in adult (12 to 15 weeks old) rats. The second goal of this study was to examine whether in utero exposure to alcohol increased the susceptibility of the brain to damage following an ischemic event in adult rats. Methods We fed Sprague Dawley dams a liquid diet with or without alcohol (3% ethanol) for the duration of their pregnancy (21 to 23 days). In the first series of studies, we examined reactivity of cerebral arterioles to endothelial nitric oxide synthase (eNOS)- (adenosine diphosphate [ADP]) and neuronal nitric oxide synthase (nNOS)-dependent N-methyl-D-aspartate (NMDA, and NOS-independent agonists in adult rats before and during application of l-NMMA. In another series of studies, we examined infarct volume following middle cerebral artery occlusion in adult offspring exposed to alcohol in utero. In both series of studies, we also determined the role for an increase in oxidative stress by feeding dams apocynin for the duration of their pregnancy. Results We found that in utero exposure to alcohol reduced responses of cerebral arterioles to ADP and NMDA, but not to nitroglycerin in adult rats. In addition, treatment of the dams with apocynin prevented this impairment in cerebral vascular function. We also found that in utero exposure to alcohol worsened brain damage following ischemia/reperfusion in adult rats and that treatment of dams with apocynin prevented this increase in brain damage following ischemia/reperfusion. Conclusions We suggest that our findings may have important implications for the pathogenesis of brain abnormalities associated with fetal alcohol exposure.

Bone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under various pathologic conditions, including rheumatoid arthritis. Previous studies have shown that OC formation is promoted under hypoxia. However, the precise mechanisms behind OC formation under hypoxia have not been elucidated. The present study investigated the role of inducible nitric oxide synthase (iNOS) in OC differentiation under hypoxia. Primary bone marrow cells obtained from mice were stimulated with receptor activator of NF-κB ligand and macrophage colony-stimulating factor to induce OC differentiation. The number of OCs increased in culture under hypoxia (oxygen concentration, 5%) compared with that under normoxia (oxygen concentration, 20%). iNOS gene and protein expression increased in culture under hypoxia. Addition of an iNOS inhibitor under hypoxic conditions suppressed osteoclastogenesis. Addition of a nitric oxide donor to the normoxic culture promoted osteoclastogenesis. Furthermore, insulin-like growth factor 2 expression was significantly altered in both iNOS inhibition experiments and nitric oxide donor experiments. These data might provide clues to therapies for excessive osteoclastogenesis under several hypoxic pathologic conditions, including rheumatoid arthritis.

We aimed to investigate the effects of L-NG-monomethyl-arginine on the apoptosis of articular chondrocytes. 30 rabbits were randomly divided into six groups (n=5). The pathological changes were detected by Hematoxylin and Eosin staining. The expression levels of synovial fluid and serum nitric oxide were measured. The apoptosis of chondrocytes was tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The expression levels of reconstituted B-cell lymphoma-2 and human B-cell lymphoma-2 associated X protein in cartilage tissues were detected by immunohistochemical assay. The Mankin score of the model group was significantly higher than that of the control group (p 0.05). With increasing L-NG-monomethyl-arginine treatment dose, the expression level of B-cell lymphoma-2 and B-cell lymphoma-2/B-cell lymphoma-2 associated X protein ratio were significantly elevated (p

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week–old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and N(G)-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.

Background Basal release of nitric oxide ( NO ) from the vascular endothelium regulates the tone of muscular arteries and resistance vasculature. Effects of NO on muscular arteries could be particularly important during exercise when shear stress may stimulate increased NO synthesis. Methods and Results We investigated acute effects of NO synthase inhibition on exercise hemodynamics using N G ‐monomethyl‐ l ‐arginine ( l ‐ NMMA ), a nonselective NO synthase ‐inhibitor. Healthy volunteers (n=10, 5 female, 19–33 years) participated in a 2‐phase randomized crossover study, receiving l ‐ NMMA (6 mg/kg, iv over 5 minutes) or placebo before bicycle exercise (25–150 W for 12 minutes). Blood pressure, cardiac output (measured by dilution of soluble and inert tracers) and femoral artery diameter were measured before, during, and after exercise. At rest, l ‐ NMMA reduced heart rate (by 16.2±4.3 bpm relative to placebo, P P P l ‐ NMMA and placebo, P l ‐ NMMA had no significant effect on peripheral resistance, cardiac output, or on femoral artery diameter. Conclusions These results suggest that NO plays little role in modulating muscular artery function during exercise but that it may mediate changes in muscular artery tone immediately before exercise.

Patients with chronic kidney disease have an increased cardiovascular morbidity and mortality. It has been recognized that the traditional cardiovascular risk factors could only partially explain the increased cardiovascular morbidity and mortality in patients with chronic kidney disease. Asymmetric dimethylarginine (ADMA) and N-monomethy L-arginine (L-NMMA) are endogenous inhibitors of nitric oxide synthases that attenuate nitric oxide production and enhance reactive oxidative specie generation. Increased plasma ADMA and/or L-NMMA are strong and independent risk factor for chronic kidney disease, and various cardiovascular diseases such as hypertension, coronary artery disease, atherosclerosis, diabetes, and heart failure. Both ADMA and L-NMMA are also eliminated from the body through either degradation by dimethylarginine dimethylaminohydrolase-1 (DDAH1) or urine excretion. This short review will exam the literatures of ADMA and L-NMMA degradation and urine excretion, and the role of chronic kidney diseases in ADMA and L-NMMA accumulation and the increased cardiovascular disease risk. Based on all available data, it appears that the increased cardiovascular morbidity in chronic kidney disease may relate to the dramatic increase of systemic ADMA and L-NMMA after kidney failure.

Purpose: Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC.Experimental Design: TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay.Results: In vitro, L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9.Conclusions: iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Clin Cancer Res; 24(5); 1152–62. ©2018 AACR.

Nitric oxide (NO)-mediated vasodilation contributes to the rapid rise in muscle blood flow at exercise onset. This occurs via increased cyclic guanosine monophosphate (cGMP), which is catabolized by phosphodiesterase-5 (PDE-5). Whether PDE-5 limits exercise vasodilation onset kinetics is unknown. We hypothesized the time course of exercise vasodilation would be 1) accelerated during PDE-5 inhibition (sildenafil citrate, SDF) and 2) decelerated during NO synthase inhibition ( NG-monomethyl-l-arginine, l-NMMA), and 3) the effect of SDF on vasodilation onset kinetics would be attenuated with concurrent l-NMMA. Data from 29 healthy adults were analyzed. Individuals completed 5 min of moderate-intensity forearm exercise under control conditions and during 1) oral SDF ( n = 8), 2) intra-arterial l-NMMA ( n = 15), or 3) combined SDF + l-NMMA ( n = 6). Forearm blood flow (FBF; Doppler ultrasound of the brachial artery) and mean brachial artery blood pressure (MAP) were measured continuously. Forearm vascular conductance (FVC, FBF ÷ MAP) was curve-fit with a monoexponential model, and vasodilation onset kinetics were assessed by mean response time (MRT, time to achieve 63% of steady state). SDF had no effect on MRT ( P = 0.90). NOS inhibition increased MRT ( P = 0.01). MRT during SDF+l-NMMA was not different from control exercise ( P = 0.76). PDE-5 inhibition alone has no effect on rapid-onset vasodilation. Whereas NOS inhibition decelerates vasodilator kinetics, when combined with SDF, vasodilator kinetics do not differ from control. These data suggest NO-independent activation of cGMP occurs at exercise onset; thus PDE-5 inhibition may improve vasodilation in pathologies where NO bioavailability is impaired. NEW & NOTEWORTHY We show that when NO bioavailability is reduced, PDE-5 inhibition can restore vasodilation onset kinetics of exercise-mediated vasodilation via NO-independent cGMP pathways. These data suggest PDE-5 inhibition may improve exercise vasodilation onset kinetics in pathologies where NO bioavailability is impaired.

Puerarin regulates the osteoblast differentiation of umbilical cord mesenchymal stem cells. This study, hereby, explored the effects of puerarin on the osteogenic differentiation of dental follicle cells (DFCs) for the first time. Rat DFCs (rDFCs) were isolated and identified. After the rDFCs were treated by Puerarin and cultured in osteogenic induction medium, the viability, osteogenic differentiation, and the activities of alkaline phosphatase (ALP) and nitric oxide (NO) were detected. Besides, the secretion of cyclic guanosine monophosphate (cGMP) and expressions of collagen I, osteocalcin (OC), osteopontin (OPN), runt-related transcription factor 2 (RUNX2), soluble guanylate cyclase (SGC), and protein kinase G 1 (PKG-1) were further determined or quantified. Puerarin enhanced the viability and osteogenic differentiation, and increased the activities of ALP, NO, and cGMP and the expressions of Collagen I, OC, OPN, RUNX2, SGC, and PKG-1 in rDFCs. After the co-treatment with puerarin and L-NMMA (NO synthase inhibitor), the promotive effects of Puerarin on cell viability, osteogenic differentiation, and the expressions of collagen I, OC, OPN, RUNX2, SGC, and PKG-1 in rDFCs were reversed by L-NMMA. Puerarin boosted the osteogenic differentiation of rDFCs by activating the NO pathway.

Polymorphonuclear granulocytes (PMN) are activated in inflammatory reactions. Intestinal epithelial cells are relevant for maintaining the intestinal barrier. We examined interactions of PMN and intestinal epithelial cell-like CaCo-2 cells to elucidate their regulation of inflammatory signalling and the impact of cyclooxygenase (COX), nitric oxide (NO) and platelet-activating factor (PAF). Human PMN and CaCo-2 cells, separately and in co-incubation, were stimulated with the calcium ionophore A23187 or with N-Formyl-methionyl-leucyl-phenylalanin (fMLP) that activates PMN only. Human neutrophil elastase (HNE) and respiratory Burst were measured. To evaluate the modulation of inflammatory crosstalk we applied inhibitors of COX (acetyl salicylic acid; ASA), NO-synthase (N-monomethyl-L-arginin; L-NMMA), and the PAF-receptor (WEB2086). Unstimulated, co-incubation of CaCo-2 cells and PMN led to significantly reduced Burst and elevated HNE as compared to PMN. After stimulation with A23187, co-incubation resulted in an inhibition of Burst and HNE. Using fMLP co-incubation failed to modulate Burst but increased HNE. Without stimulation, all three inhibitors abolished the effect of co-incubation on Burst but did not change HNE. ASA partly prevented modulation of Burst L-NMMA and WEB2086 did not change Burst but abolished mitigation of HNE. Without stimulation, co-incubation reduced Burst and elevated HNE. Activation of PMN and CaCo-2 cells by fMLP as compared to A23187 resulted in a completely different pattern of Burst and HNE, possibly due to single vs. dual cell activation. Anti-inflammatory effect of co-incubation might in part be due to due to COX-signalling governing Burst whereas NO- and PAF-dependent signalling seemed to control HNE release.

Syncope is sudden transient loss of consciousness and postural tone with spontaneous recovery; the most common form is vasovagal syncope (VVS). We previously demonstrated impaired post-synaptic adrenergic responsiveness in young VVS patients was reversed by blocking nitric oxide synthase (NOS). We hypothesised that nitric oxide may account for reduced orthostatic tolerance in young recurrent VVS patients.We recorded haemodynamics in supine VVS and healthy volunteers (aged 15-27 years), challenged with graded lower body negative pressure (LBNP) (-15, -30, -45 mm Hg each for 5 min, then -60 mm Hg for a maximum of 50 min) with and without NOS inhibitor NControls endured 25.9±4.0 min of LBNP during Saline+PE compared with 11.6±1.4 min for fainters (p0.001). After L-NMMA, control subjects endured 24.8±3.2 min compared with 22.6±1.6 min for fainters. Mean arterial pressure decreased more in VVS patients during LBNP with Saline+PE (p0.001) which was reversed by L-NMMA; cardiac output decreased similarly in controls and VVS patients and was unaffected by L-NMMA. Total peripheral resistance increased for controls but decreased for VVS during Saline+PE (p0.001) but was similar following L-NMMA. Splanchnic vascular resistance increased during LBNP in controls, but decreased in VVS patients following Saline+PE which L-NMMA restored.We conclude that arterial vasoconstriction is impaired in young VVS patients, which is corrected by NOS inhibition. The data suggest that both pre- and post-synaptic arterial vasoconstriction may be affected by nitric oxide.

L-NG-monomethyl-arginine (L-NMMA) is an experimental compound that suppresses nitric oxide production in animals. The compound was combined with oseltamivir to treat lethal influenza A/California/04/2009 (H1N1) pandemic virus infections in mice. Treatments were given twice a day for five days starting 4 h (oseltamivir, by oral gavage) or three days (L-NMMA, by intraperitoneal route; corresponding to the time previously reported for nitric oxide induction in the animals) after infection. Low doses of oseltamivir were used in order to demonstrate synergy or antagonism. Oseltamivir monotherapy protected 70% of mice from death at 1 mg/kg/day. L-NMMA (40 and 80 mg/kg/day) was ineffective alone in preventing mortality. Compared to oseltamivir treatment alone, L-NMMA combined with oseltamivir was synergistically effective (as evaluated by three-dimensional MacSynergy analysis), resulting in survival increases from 20 to 70% when 40 or 80 mg/kg/day of L-NMMA was combined with 0.3 mg/kg/day of oseltamivir, and from 70 to 100% survival increases when these doses were combined with 1 mg/kg/day of oseltamivir. These data demonstrate that a nitric oxide inhibitor such as L-NMMA has the potential to be beneficial when combined with oseltamivir in treating influenza virus infections.

Animal studies suggest that the inhibition of nitric oxide synthase (NOS) affects blood flow differently in different skeletal muscles according to their muscle fibre type composition (oxidative vs glycolytic). Quadriceps femoris (QF) muscle consists of four different muscle parts: vastus intermedius (VI), rectus femoris (RF), vastus medialis (VM), and vastus lateralis (VL) of which VI is located deep within the muscle group and is generally regarded to consist mostly of oxidative muscle fibres. We studied the effect of NOS inhibition on blood flow in these four different muscles by positron emission tomography in eight young healthy men at rest and during one-leg dynamic exercise, with and without combined blockade with prostaglandins. At rest blood flow in the VI (2.6 ± 1.1 ml/100 g/min) was significantly higher than in VL (1.9 ± 0.6 ml/100 g/min, p = 0.015) and RF (1.7 ± 0.6 ml/100 g/min, p = 0.0015), but comparable to VM (2.4 ± 1.1 ml/100 g/min). NOS inhibition alone or with prostaglandins reduced blood flow by almost 50% (p

L-NG-Nitro arginine methyl ester (L-NAME) has been widely applied for several decades in both basic and clinical research as an antagonist of nitric oxide synthase (NOS). Herein, we show that L-NAME slowly releases NO from its guanidino nitro group. Daily pretreatment of rats with L-NAME potentiated mesenteric vasodilation induced by nitrodilators such as nitroglycerin, but not by NO. Release of NO also occurred with the NOS-inactive enantiomer D-NAME, but not with L-arginine or another NOS inhibitor L-NMMA, consistent with the presence or absence of a nitro group in their structure and their nitrodilator-potentiating effects. Metabolic conversion of the nitro group to NO-related breakdown products was confirmed using isotopically-labeled L-NAME. Consistent with Fenton chemistry, transition metals and reactive oxygen species accelerated the release of NO from L-NAME. Both NO production from L-NAME and its nitrodilator-potentiating effects were augmented under inflammation. NO release by L-NAME can confound its intended NOS-inhibiting effects, possibly by contributing to a putative intracellular NO store in the vasculature. Keywords: L-NAME, Nitrodilator, L-arginine analogues, Fenton chemistry, Preformed intracellular NO store

The associations between arginine‐based metabolites and incident type 2 diabetes (T2D) are unknown. We employed a case‐cohort design, nested within the PREDIMED trial, to examine six plasma metabolites (arginine, citrulline, ornithine, asymmetric dimethylarginine [ADMA], symmetric dimethylarginine [SDMA] and N‐monomethyl‐l‐arginine [NMMA]) among 892 individuals (251 cases) for associations with incident T2D and insulin resistance. Weighted Cox models with robust variance were used. The 1‐year changes in arginine (adjusted hazard ratio [HR] per SD 0.68, 95% confidence interval [CI] 0.49, 0.95; Q4 vs. Q1 0.46, 95% CI 0.21, 1.04; P trend = 0.02) and arginine/ADMA ratio (adjusted HR per SD 0.73, 95% CI 0.51, 1.04; Q4 vs. Q1 0.52, 95% CI 0.22, 1.25; P trend = 0.04) were associated with a lower risk of T2D. Positive changes of citrulline and ornithine, and negative changes in SDMA and arginine/(ornithine + citrulline) were associated with concurrent 1‐year changes in homeostatic model assessment of insulin resistance. Individuals in the low‐fat‐diet group had a higher risk of T2D for 1‐year changes in NMMA than individuals in Mediterranean‐diet groups (P interaction = 0.02). We conclude that arginine bioavailability is important in T2D pathophysiology., This study was funded by the National Institutes of Health (R01DK102896, F31DK114938), the Spanish Ministry of Health (Instituto de Salud Carlos III), the Ministerio de Economía y Competitividad‐Fondo Europeo de Desarrollo Regional (Projects CNIC‐06/2007, RTIC G03/140, CIBER 06/03, PI06‐1326, PI07‐0954, PI11/02505, RTIC RD06/0045, SAF2009‐12304 and AGL2010‐22319‐C03‐03) and by the Generalitat Valenciana (ACOMP2010‐181, AP111/10, AP‐042/11, ACOM2011/145, ACOMP/2012/190, ACOMP/2013/159 and ACOMP/213/165). M.G.‐F. is supported by the American Diabetes Association (#1‐18‐PMF‐029).

Objective Our goals were to determine the influence of sex on reactivity of cerebral arterioles and whether MExT could influence sex-related differences in reactivity of cerebral arterioles. Materials and methods Responses of cerebral arterioles were measured in Sed and MExT adult male and female Sprague-Dawley rats to eNOS-dependent (ADP), nNOS-dependent (NMDA), and NOS-independent (nitroglycerin) agonists before and following L-NMMA. In addition, protein expression for eNOS and nNOS was determined. Results NOS-dependent vasodilation was enhanced in Sed and MExT female rats compared to their male counterparts. L-NMMA produced a greater decrease in baseline diameter of arterioles in females compared to males, and produced less inhibition of NOS-dependent vasodilation in females. Expression of eNOS protein was significantly increased in Sed female when compared to Sed male rats; nNOS protein was similar in Sed males and females, but increased in MExT females. Conclusions The findings from this study indicate that while NOS-dependent vascular reactivity is increased in females, MExT does not alter vasodilation in males or females. These studies provide insights into the influence of sex and MExT on the cerebral microcirculation and may have implications regarding mechanisms that protect the brain in females compared to males.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases that limits nitric oxide bioavailability and can increase production of NOS derived reactive oxidative species. Increased plasma ADMA is a one of the strongest predictors of mortality in patients who have had a myocardial infarction or suffer from chronic left heart failure, and is also an independent risk factor for several other conditions that contribute to heart failure development, including hypertension, coronary artery disease/atherosclerosis, diabetes, and renal dysfunction. The enzyme responsible for ADMA degradation is dimethylarginine dimethylaminohydrolase-1 (DDAH1). DDAH1 plays an important role in maintaining nitric oxide bioavailability and preserving cardiovascular function in the failing heart. Here, we examine mechanisms of abnormal NO production in heart failure, with particular focus on the role of ADMA and DDAH1.

We tested the hypothesis that women exhibit greater vasodilator responses to β-adrenoceptor stimulation compared with men. We further hypothesized women exhibit a greater contribution of nitric oxide synthase and cyclooxygenase to β-adrenergic-mediated vasodilation compared with men. Forearm blood flow (Doppler ultrasound) was measured in young men ( n = 29, 26 ± 1 yr) and women ( n = 33, 25 ± 1 yr) during intra-arterial infusion of isoproterenol (β-adrenergic agonist). In subset of subjects, isoproterenol responses were examined before and after local inhibition of nitric oxide synthase [ NG-monomethyl-l-arginine (l-NMMA); 6 male/10 female] and/or cyclooxygenase (ketorolac; 5 male/5 female). Vascular conductance (blood flow ÷ mean arterial pressure) was calculated to assess vasodilation. Vascular conductance increased with isoproterenol infusion ( P < 0.01), and this effect was not different between men and women ( P = 0.41). l-NMMA infusion had no effect on isoproterenol-mediated dilation in men ( P > 0.99) or women ( P = 0.21). In contrast, ketorolac infusion markedly increased isoproterenol-mediated responses in both men ( P < 0.01) and women ( P = 0.04) and this rise was lost with subsequent l-NMMA infusion (men, P < 0.01; women, P < 0.05). β-Adrenergic vasodilation is not different between men and women and sex differences in the independent contribution of nitric oxide synthase and cyclooxygenase to β-mediated vasodilation are not present. However, these data are the first to demonstrate β-adrenoceptor activation of cyclooxygenase suppresses nitric oxide synthase signaling in human forearm microcirculation and may have important implications for neurovascular control in both health and disease.

Nitric oxide (NO) is involved in skeletal muscle glucose uptake during exercise and also in the increase in insulin sensitivity after exercise. Given that neuronal nitric oxide synthase (NOS) isoform mu (nNOSμ) is a major isoform of NOS in skeletal muscle, we examined if the increase in skeletal muscle insulin-stimulated glucose uptake 3.5 h following ex vivo contraction of extensor digitorum longus (EDL) is reduced in muscles from nNOSμ+/− and nNOSμ−/− mice compared with nNOSμ+/+ mice. 3.5 h post-contraction/basal, muscles were exposed to saline or insulin (120μU/ml) with or without the presence of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) during the last 30 min and glucose uptake was determined by radioactive tracers. Skeletal muscle insulin-stimulated glucose uptake from nNOSμ+/+, nNOSμ+/−, and nNOSμ−/− mice increased approximately twofold 3.5 h following ex vivo contraction when compared to rest. L-NMMA significantly attenuated this increase in muscle insulin-stimulated glucose uptake by around 50%, irrespective of genotype. Low levels of NOS activity were detected in muscles from nNOSμ−/− mice. In conclusion, NO mediates increases in mouse skeletal muscle insulin response following ex vivo contraction independently of nNOSμ.

Renal denervation (RDN) has been shown to restore endogenous neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) and reduce sympathetic drive during chronic heart failure (CHF). The purpose of the present study was to assess the contribution of afferent renal nerves to the nNOS-mediated sympathetic outflow within the PVN in rats with CHF. CHF was induced in rats by ligation of the left coronary artery. Four weeks after surgery, selective afferent RDN (A-RDN) was performed by bilateral perivascular application of capsaicin on the renal arteries. Seven days after intervention, nNOS protein expression, nNOS immunostaining signaling, and diaphorase-positive stained cells were significantly decreased in the PVN of CHF rats, changes that were reversed by A-RDN. A-RDN reduced basal lumbar sympathetic nerve activity in rats with CHF (8.5%±0.5% versus 17.0%±1.2% of max). Microinjection of nNOS inhibitor L-NMMA (L-N G -monomethyl arginine citrate) into the PVN produced a blunted increase in lumbar sympathetic nerve activity in rats with CHF. This response was significantly improved after A-RDN (Δ lumbar sympathetic nerve activity: 25.7%±2.4% versus 11.2%±0.9%). Resting afferent renal nerves activity was substantially increased in CHF compared with sham rats (56.3%±2.4% versus 33.0%±4.7%). These results suggest that intact afferent renal nerves contribute to the reduction of nNOS in the PVN. A-RDN restores nNOS and thus attenuates the sympathoexcitation. Also, resting afferent renal nerves activity is elevated in CHF rats, which may highlight a crucial neural mechanism arising from the kidney in the maintenance of enhanced sympathetic drive in CHF.

Spinal nitric oxide is involved in the mechanisms of pain generation and transmission during inflammatory and neuropathic pain. The aim of the present study was to explore the role of spinal nitric oxide in the development of bone cancer pain. 2 x 10(5) osteosarcoma cells were implanted into the intramedullary space of right femurs of C3H/HeJ mice to induce a model of ongoing bone cancer. Polymerase chain reaction and immunohistochemical analyses were performed to assess the expression of neuronal nitric oxide synthase (nNOS) and inducible (i)NOS in the spinal cord following inoculation. The results showed that inoculation of osteosarcoma cells induced progressive bone cancer, accompanied with pain-associated behavior. The levels of nNOS mRNA in the spinal cord of tumor mice began to increase at day 10 and then decreased to the level in sham mice at day 14, while iNOS mRNA markedly increased in the tumor group at days 10 and 14. Immunohistochemical analysis showed that nNOS- and iNOS-positive neurons were mainly located in the superficial dorsal horn and around the central canal of the L3-L5 spinal cord. Intrathecal injection of 50 µg NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) attenuated cancer-evoked pain behaviors at day 14. These findings indicated that an upregulation of nNOS and iNOS in the spinal cord is associated with bone cancer pain and suggests that exogenously administered L-NMMA may have beneficial effects to alleviate bone cancer pain.

We studied the dynamics of indexes for the system of endogenous regulation of NO bioavailability. The content of NO synthase inhibitors (monomethylarginine and asymmetric dimethylarginine) in the blood of mice was measured after intraperitoneal injections of a nonionic surfactant poloxamer 407 for 2 and 14 weeks. The concentrations of both methylarginines in animals with atherosclerosis due to 14-week administration of poloxamer were much higher than in control specimens. The amount of arginine and symmetric dimethylarginine practically did not differ from the control. Poloxamer-induced model of atherosclerosis is characterized by increased content of NO synthase inhibitors. These changes contribute to the development of endothelial dysfunction and atherosclerosis.

The passive leg movement (PLM) model is a novel approach to assess vascular function. Increasing femoral perfusion pressure (FPP) by moving from the supine to the upright-seated posture augments the vasodilatory response to PLM in the young, with no effect in the old, but whether this augmented vasodilatation is nitric oxide (NO) dependent is unknown. Using an intra-arterial infusion of N(G) -monomethyl-L -arginine (L -NMMA) to inhibit nitric oxide synthase (NOS), the posture-induced increases in the PLM responses in the young were nearly ablated, with no effect of NOS inhibition in the old. Therefore, PLM in combination with alterations in posture can be used to determine changes in NO-mediated vasodilatation with age, and thus, may be a clinically useful tool for assessing NO bioavailability across the human lifespan. We sought to better understand the contribution of nitric oxide (NO) to passive leg movement (PLM)-induced vasodilatation with age, with and without a posture-induced increase in femoral perfusion pressure (FPP). PLM was performed in eight young (24 ± 1 years) and eight old (74 ± 3 years) healthy males, with and without NO synthase inhibition via intra-arterial infusion of N(G) -monomethyl-L -arginine (L -NMMA) into the common femoral artery in both the supine and upright-seated posture. Central and peripheral haemodynamic responses were determined second-by-second with finger photoplethysmography and Doppler ultrasound, respectively. PLM-induced increases in heart rate, stroke volume, cardiac output and reductions in mean arterial pressure were similar between age groups and conditions. In the young, L -NMMA attenuated the peak change in leg vascular conductance (ΔLVCpeak ) in both the supine (control: 7.4 ± 0.9; L -NMMA: 5.2 ± 1.1 ml min(-1) mmHg(-1) , P0.05) and upright-seated (control: 12.3 ± 2.0; L -NMMA: 6.4 ± 1.0 ml min(-1) mmHg(-1) , P0.05) posture, with no significant change in the old (supine control: 4.2 ± 1.3; supine L -NMMA: 3.4 ± 0.8; upright-seated control: 4.5 ± 0.8; upright-seated L -NMMA: 3.4 ± 0.8 ml min(-1) mmHg(-1) , P0.05). Increased FPP augmented the ΔLVCpeak in the young control condition only (P0.05). In the upright-seated posture, NOS inhibition attenuated the FPP-induced augmentation of rapid vasodilatation in the young (control: 1.25 ± 0.23; L -NMMA: 0.74 ± 0.11 ml min(-1) mmHg(-1) s(-1) ; P0.05), but not the old (control: 0.37 ± 0.07; L -NMMA: 0.25 ± 0.07 ml ml min(-1) mmHg(-1) s(-1) ; P0.05). These data reveal that greater FPP increases the role of NO in PLM-induced vasodilatation in the young, but not the old, due to reduced NO bioavailability with age. Therefore, PLM involving alterations in posture may be useful to determine changes in NO bioavailability with age.

High-fat diets are associated with an increased risk of cardiovascular disease. A potential underlying mechanism for the increased cardiovascular risk is endothelial dysfunction. Nitric oxide (NO)-mediated endothelium-dependent vasodilation is critical in the regulation of vascular tone and overall vascular health. The aim of this study was to determine the influence of dietary fat intake on endothelium-dependent vasodilation. Forty-four middle-aged and older sedentary, healthy adults were studied: 24 consumed a lower fat diet (LFD; 29% ± 1% calories from fat) and 20 consumed a high-fat diet (HFD; 41% ± 1% calories from fat). Four-day diet records were used to assess fat intake, and classifications were based on American Heart Association guidelines (G-monomethyl-l-arginine (L-NMMA), as well as responses to sodium nitroprusside were determined by plethysmography. The FBF response to acetylcholine was lower (∼15%; P < 0.05) in the HFD group (4.5 ± 0.2 to 12.1 ± 0.8 mL/100 mL tissue/min) than in the LFD group (4.6 ± 0.2 to 14.4 ± 0.6 mL/100 mL tissue/min). L-NMMA significantly reduced the FBF response to acetylcholine in the LFD group (∼25%) but not in the HFD group. There were no differences between groups in the vasodilator response to sodium nitroprusside. These data indicate that a high-fat diet is associated with endothelium-dependent vasodilator dysfunction due, in part, to diminished NO bioavailability. Impaired NO-mediated endothelium-dependent vasodilation may contribute to the increased cardiovascular risk with high dietary fat intake.

Objective— The insulinotropic gut–derived hormone glucagon-like peptide-1 (GLP-1) increases capillary perfusion via a nitric oxide–dependent mechanism in rodents. This improves skeletal muscle glucose use and cardiac function. In humans, the effect of clinically used GLP-1 receptor agonists (GLP-1RAs) on capillary density is unknown. We aimed to assess the effects of the GLP-1RA exenatide on capillary density as well as the involvement of nitric oxide in humans. Approach and Results— We included 10 healthy overweight men (age, 20–27 years; body mass index, 26–31 kg/m 2 ). Measurements were performed during intravenous infusion of placebo (saline 0.9%), exenatide, and a combination of exenatide and the nonselective nitric oxide–synthase inhibitor l - N G -monomethyl arginine. Capillary videomicroscopy was performed, and baseline and postocclusive (peak) capillary densities were counted. Compared with placebo, exenatide increased baseline and peak capillary density by 20.1% and 8.3%, respectively (both P =0.016). Concomitant l - N G -monomethyl arginine infusion did not alter the effects of exenatide. Vasomotion was assessed using laser Doppler fluxmetry. Exenatide nonsignificantly reduced the neurogenic domain of vasomotion measurements ( R =−5.6%; P =0.092), which was strongly and inversely associated with capillary perfusion ( R =−0.928; P =0.036). Glucose levels were reduced during exenatide infusion, whereas levels of insulin were unchanged. Conclusions— Acute exenatide infusion increases capillary perfusion via nitric oxide–independent pathways in healthy overweight men, suggesting direct actions of this GLP-1RA on microvascular perfusion or interaction with vasoactive factors.

Background and aims The mechanisms involved in endothelial dysfunction in humans during aging are largely unknown at the level of conduit arteries. We aimed to asses the role of NO and CYP450 epoxygenases-derived epoxyeicosatrienoic acids (EETs) in the regulation of endothelium-dependent flow-mediated dilatation of conduit arteries during aging. Methods Radial artery diameter and mean wall shear stress were determined by echotracking coupled with Doppler in 83 subjects (19–71 years old) during a sustained flow increase induced by hand skin heating, with the brachial infusion of saline or NO-synthase and cytochrome P450 epoxygenase inhibitors (L-NNMA and fluconazole respectively). Local blood sampling was performed for the quantification of NO metabolite nitrite and EETs. Results The magnitude of flow-mediated dilatation was independently and negatively correlated with age, baseline artery diameter and systolic blood pressure, and positively correlated with the increase in shear stress induced by heating. There was an increase in nitrite level during heating until the age of 35–40 years, which declined thereafter. However, the inhibitory effect of L-NMMA on flow-mediated dilatation progressively decreased during aging, demonstrating a decrease in functional NO availability. Moreover, aging progressively reduced the increase in EET level during heating as well as the inhibitory effect of fluconazole on flow-mediated dilatation. Conclusions These results show that aging impairs the availability of EETs and NO and epoxyeicosatrienoic acids in peripheral conduit arteries, contributing to the development of endothelial dysfunction.

ABSTACT Hydrogen sulfide (H2S), nitric oxide (NO), and renin-angiotensin system (RAS) are involved in hypertension. We examined whether early treatment with sodium hydrosulfide (NaHS), an exogenous H2S donor, can regulate H2S-generating pathway, NO pathway, and the RAS, to prevent the transition from prehypertension to hypertension in spontaneously hypertensive rats (SHRs). Four-week-old SHRs and control normotensive Wistar-Kyoto (WKY) rats were assigned into three groups: WKY, SHRs, and SHR + NaHS; SHRs were injected intraperitoneally with sodium hydrosulfide (14 μmol/kg/day) for 4 weeks. SHRs exhibited hypertension at 12 weeks of age, which was blocked by early sodium hydrosulfide administration. Concentrations of H2S were increased in the kidney in SHR + NaHS group versus WKY. Sodium hydrosulfide reduces mRNA expression of four H2S-generating enzymes and decreased 3-mercaptopyruvate sulphurtransferase protein level in SHRs. Early administration of sodium hydrosulfide decreases plasma NG monomethyl-l-arginine (l-NMMA, an inhibitor of NO synthase) level and increases plasma NO level in SHRs. Next, sodium hydrosulfide administration reduces renal mRNA expression of Ren, Atp6ap2, Agt, Ace, and Agtr1a in SHRs. We conclude that early short-term sodium hydrosulfide treatment increases renal H2S concentrations, restores NO bioavailability, and blocks the RAS in the kidney, in favor of vasodilatation to prevent the development of hypertension in adult SHRs.

Occlusion of retinal vessels leads to retinal ischaemia and hypoxia, which induces vasodilatation in adjacent retinal areas in order to normalize retinal oxygenation. Previous studies have shown that NO and COX products are involved in hypoxia-induced dilatation of retinal arterioles in vitro and in vivo, and that this response is disturbed in patients with diabetes mellitus. However, it is unknown to what extent post-hypoxic recovery of the diameter of retinal arterioles depends on NO and COX products in normal persons and in diabetic patients. The Dynamic Vessel Analyzer (DVA) was used to study the post-hypoxic diameter changes of larger retinal vessels in 20 normal persons, 20 diabetic patients without diabetic retinopathy, and in 18 patients with diabetic maculopathy before and after inhibition of the synthesis of nitric oxide and COX products. In normal persons, the arterioles had re-constricted (p > 0.99) 2 minutes after termination of hypoxia in the absence of antagonists, but not after treatment with L-NMMA and diclofenac (p < 0.01 for all comparisons). In diabetic patients without retinopathy, the arterioles showed no diameter changes after termination of hypoxia during any of the interventions. In patients with diabetic maculopathy hypoxia had not dilated retinal arterioles (p > 0.1 for all comparisons) to allow the study of re-constriction. In all groups, the dilatation of venules remained significantly increased during the post-hypoxic observation period, both in the absence and in the presence of L-NMMA and diclofenac.Post-hypoxic constriction of retinal arterioles depends on NO and COX products, and is impaired in diabetic patients before the development of retinopathy. This disturbance may contribute to the development of diabetic retinopathy, and should be the target of future interventional studies aimed at preventing and treating the disease.ClinicalTrials.gov identifier: NCT01689090.

The assessment of passive leg movement (PLM)-induced leg blood flow (LBF) and vascular conductance (LVC) is a novel approach to assess vascular function that has recently been simplified to only a single PLM (sPLM), thereby increasing the clinical utility of this technique. As the physiological mechanisms mediating the robust increase in LBF and LVC with sPLM are unknown, we tested the hypothesis that nitric oxide (NO) is a major contributor to the sPLM-induced LBF and LVC response. In nine healthy men, sPLM was performed with and without NO synthase inhibition by intra-arterial infusion of NG-monomethyl-l-arginine (l-NMMA). Doppler ultrasound and femoral arterial pressure were used to determine LBF and LVC, which were characterized by the peak change (ΔLBFpeak and ΔLVCpeak) and area under the curve (LBFAUC and LVCAUC). l-NMMA significantly attenuated ΔLBFpeak [492 ± 153 (l-NMMA) vs. 719 ± 238 (control) ml/min], LBFAUC [57 ± 34 (l NMMA) vs. 147 ± 63 (control) ml], ΔLVCpeak [4.7 ± 1.1 (l-NMMA) vs. 8.0 ± 3.0 (control) ml·min−1·mmHg−1], and LVCAUC [0.5 ± 0.3 (l-NMMA) vs. 1.6 ± 0.9 (control) ml/mmHg]. The magnitude of the NO contribution to LBF and LVC was significantly correlated with the magnitude of the control responses ( r = 0.94 for ΔLBFpeak, r = 0.85 for LBFAUC, r = 0.94 for ΔLVCpeak, and r = 0.95 for LVCAUC). These data establish that the sPLM-induced hyperemic and vasodilatory response is predominantly (~65%) NO-mediated. As such, sPLM appears to be a promising, simple, in vivo assessment of NO-mediated vascular function and NO bioavailability. NEW & NOTEWORTHY Passive leg movement (PLM), a novel assessment of vascular function, has been simplified to a single PLM (sPLM), thereby increasing the clinical utility of this technique. However, the role of nitric oxide (NO) in mediating the robust sPLM hemodynamic responses is unknown. This study revealed that sPLM induces a hyperemic and vasodilatory response that is predominantly NO-mediated and, as such, appears to be a promising simple, in vivo, clinical assessment of NO-mediated vascular function and, therefore, NO bioavailability.

Chronic kidney disease (CKD) is associated with increased risk of renal and cardiovascular events. It has been claimed that endogenous methylarginines, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), are contributing factors. However, earlier studies were partly contradictory and mainly focused on prevalent dialysis patients. Moreover, the potential contribution of degradation products, such as acetylated ADMA and SDMA (AcADMA and AcSDMA) and other methylarginines including L-NG-monomethylarginine (LNMMA) remains unknown. To better understand their potential pathophysiological contribution to renal and cardiovascular events, we aimed to provide a comprehensive analysis of methylarginines in a cohort of patients with non-dialysis CKD. Blood samples of 528 patients with CKD KDIGO G2 to G4 were obtained from the CARE FOR HOMe study. Baseline plasma levels of ADMA, SDMA, AcADMA, AcSDMA, and LNMMA were measured by liquid chromatography—tandem mass spectrometry. All patients were followed annually for CKD progression and for incident atherosclerotic cardiovascular events. During 5.1 ± 2.1 years follow-up, 80 patients displayed CKD progression and 145 patients developed incident atherosclerotic cardiovascular events. In univariate Cox regression analyses, elevated plasma levels of all five metabolites were associated with both CKD progression and atherosclerotic cardiovascular disease. However, adjustment for confounders attenuated the prognostic implications of ADMA, LNMMA, AcADMA and AcSDMA. In contrast, patients in the highest tertile of plasma SDMA remained at highest risk for CKD progression and incident atherosclerotic cardiovascular events in fully adjusted Cox regression analyses. Our results underline a potential pathophysiological role of SDMA in CKD progression and atherosclerotic cardiovascular disease among non-dialysis CKD patients. SDMA predicts CKD progression and future atherosclerotic cardiovascular events more consistently than other methylarginines. Future experimental and clinical studies should therefore focus upon SDMA rather than upon ADMA.

Rationale:\ud \ud Nitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with cycles which regulate the endogenous inhibitors ADMA and L-NMMA. The relationship between amines and the consequence of plasma changes on iNOS activity in early sepsis is not known.\ud \ud Objective:\ud \ud Our objective was to apply a metabolomics approach to determine the influence of sepsis on a full array of amines and what consequence these changes may have on predicted iNOS activity.\ud \ud Methods and measurements:\ud \ud 34 amino acids were measured using ultra purification mass spectrometry in the plasma of septic patients (n = 38) taken at the time of diagnosis and 24–72 hours post diagnosis and of healthy volunteers (n = 21). L-arginine and methylarginines were measured using liquid-chromatography mass spectrometry and ELISA. A top down approach was also taken to examine the most changed metabolic pathways by Ingenuity Pathway Analysis. The iNOS supporting capacity of plasma was determined using a mouse macrophage cell-based bioassay.\ud \ud Main results:\ud \ud Of all the amines measured 22, including L-arginine and ADMA, displayed significant differences in samples from patients with sepsis. The functional consequence of increased ADMA and decreased L-arginine in context of all cumulative metabolic changes in plasma resulted in reduced iNOS supporting activity associated with sepsis.\ud \ud Conclusions:\ud \ud In early sepsis profound changes in amine levels were defined by dominant changes in the iNOS canonical pathway resulting in functionally meaningful changes in the ability of plasma to regulate iNOS activity ex vivo.

OBJECTIVE: Cholinergic vasodilation has been thought to play little if any role in the regulation of blood pressure in humans. Autonomic denervation potentiates the vasoconstriction evoked by nitric oxide synthase inhibition in humans, but the mechanism is unclear. We hypothesized that this may be related to loss of neuronal, non-nitric-oxide-dependent vasodilation. METHODS: To test this hypothesis, we examined effects of cholinergic blockade on blood pressure, heart rate and peripheral vascular responses to systemic infusion of the nitric-oxide-dependent vasoconstrictor L-NMMA (0.5 mg/kg/min over 15 min) in eight normal subjects. RESULTS: The L-NMMA-induced increase in mean (+/-S.E.) arterial pressure was roughly three times larger (P=0.002) in the presence than in the absence of cholinergic blockade (38+/-6 vs. 13+/-2 mmHg). Similarly, the increase in systemic and calf vascular resistance was more than twofold larger during L-NMMA-atropine. This potentiation was specific for nitric-oxide-dependent vasoconstriction, because atropine did not alter the responses to phenylephrine infusion. Cholinergic blockade also altered (P=0.004) the heart rate response to nitric oxide synthase inhibition; during L-NMMA alone heart rate decreased by 10+/-2 beats/min, whereas during L-NMMA-atropine infusion it increased by 14+/-4 beats/min. CONCLUSION: Cholinergic mechanisms play an important hitherto unrecognized role in offsetting the hypertension and cardiac sympathetic activation caused by nitric oxide synthase inhibition in humans. Decreased parasympathetic activity and impaired nitric oxide synthesis characterize several cardiovascular disease states, as well as normal aging. The conjunction of these two defects could trigger sudden death and contribute to the hypertension of the elderly.

OBJECTIVE: Nitric oxide exerts its cardiovascular actions at least in part by modulation of the sympathetic vasoconstrictor tone. There is increasing evidence that nitric oxide inhibits central neural sympathetic outflow, and preliminary evidence suggests that it may also modulate peripheral sympathetic vasoconstrictor tone. METHODS: To test this latter concept, in six subjects having undergone thoracic sympathectomy for hyperhydrosis, we compared the vascular responses to systemic L-NMMA infusion (1 mg/kg/min over 10 min) in the innervated and the denervated limb. We also studied vascular responses to the infusion of the non-nitric-oxide-dependent vasoconstrictor phenylephrine. RESULTS: L-NMMA infusion evoked a roughly 3-fold larger increase in vascular resistance in the denervated forearm than in the innervated calf. In the denervated forearm, vascular resistance increased by 58 +/- 10 percent (mean +/- SE), whereas in the innervated calf it increased only by 21 +/- 6 percent (P < 0.01, forearm vs. calf). This augmented vasoconstrictor response was specific for L-NMMA, and not related to augmented non-specific vasoconstrictor responsiveness secondary to sympathectomy, because phenylephrine infusion increased vascular resistance similarly in the denervated forearm and the innervated calf (by 24 +/- 7, and 29 +/- 8 percent, respectively). The augmented vasoconstrictor response was related specifically to denervation, because in control subjects, the vasoconstrictor responses to L-NMMA were comparable in the forearm and the calf. CONCLUSIONS: These findings indicate that in the absence of sympathetic innervation, the vasoconstrictor responses to nitric oxide synthase inhibition are augmented.

Macrophages are remarkably versatile in their ability to recognize and respond to a wide range of stimuli by expressing a variety of surface and intracellular receptors and triggering multiple signal transduction pathways. The onset of microbial infection is primarily determined by the initial contacts made by the microbes with the host macrophages. Although there prevail a relationship between the chemokine receptor and Toll like receptors during disease, particularly TLR-2 and CCR-2 signaling interdependence on each other has not been yet investigated during acute staphylococcal infection. Thus, the present study was aimed to trace possible interaction between CCR-2 and TLR-2 in peritoneal macrophages during acute Staphylococcus aureus infection. We found that neutralization of CCR-2 attenuates TLR-2 expression and restricts S. aureus burden but TLR-2 neutralization augments CCR-2 expression in macrophages, along with compromised host-derived reactive oxygen species production. S. aureus infection to CCR-2 intact but TLR-2 neutralized macrophages triggered production of IL-1β, TNF-α, IL-6, IFN-γ, MCP-1 and expression of iNOS, TNFR-1 and GPx with concomitant decrease in IL-10 production. Further, study with NG-monomethyl-l-arginine (L-NMMA) [iNOS blocker] and buthionine sulfoximine (BSO) [GPx blocker] revealed that S. aureus infection enhanced TLR-2 expression in CCR-2 intact and TLR-2 neutralized macrophages possibly via iNOS and TNFR-1 up regulation and GPx down regulation. Overall, our data indicate that targeting CCR-2 with neutralizing antibody in the early phase of S. aureus infection could restrict excessive inflammation with less compromised bacterial killing. It certainly would be a therapeutic strategy in S. aureus induced inflammatory and infective diseases.

Lüneburg, Nicole, Patricia Siques, Julio Brito, Juan José De La Cruz, Fabiola León-Velarde, Juliane Hannemann, Cristian Ibanez, and Rainer Böger. Long-term intermittent exposure to high altitude elevates asymmetric dimethylarginine in first exposed young adults. High Alt Med Biol. 18:226–233, 2017.—Hypoxia-induced dysregulation of pulmonary and cerebral circulation may be related to an impaired nitric oxide (NO) pathway. We investigated the effect of chronic intermittent hypobaric hypoxia (CIH) on metabolites of the NO pathway. We measured asymmetric and symmetric dimethylarginine (ADMA and SDMA) and monomethyl-L-arginine (L-NMMA) and assessed their associations with acclimatization in male draftees (n = 72) undergoing CIH shifts at altitude (3550 m) during 3 months. Sixteen Andean natives living at altitude (3675 m) (chronic hypobaric hypoxia [CH]) were included for comparison. In CIH, ADMA and L-NMMA plasma concentrations increased from 1.14 ± 0.04 to 1.95 ± 0.09 μmol/L (mean ± SE) and from 0.22 ± 0.07 to 0.39 ± 0.03 μmol/L, respectively, (p

Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l -citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including Streptomyces coelicolor and Pseudomonas aeruginosa , the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or factors regulating DDAH expression in P. aeruginosa PAO1. First, results revealed that P. aeruginosa can utilize N G , N G -dimethyl- l -arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the ddaH gene was observed to be induced in the presence of methylarginines, including N G -monomethyl- l -arginine ( l -NMMA) and ADMA. Third, induction of the ddaH gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative sigma factor RpoN. Both PA1196 and RpoN were essential for the expression of the ddaH gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for d imethylarginine d imethyl a minohydrolase r egulator. Interestingly, DdaR and its target ddaH gene are conserved only among P. aeruginosa strains, suggesting that this particular Pseudomonas species has evolved to utilize methylarginines from its environment. IMPORTANCE Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium Pseudomonas aeruginosa PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the sigma factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among P. aeruginosa strains.

Nitric oxide (NO) contributes to the central control of cardiovascular activity. The rostral ventrolateral medulla (RVLM) has been recognized as a pivotal region for maintaining basal blood pressure (BP) and sympathetic tone. It is reported that asymmetric dimethylarginine (ADMA), characterized as a cardiovascular risk marker, is an endogenous inhibitor of nitric oxide synthesis. The present was designed to determine the role of ADMA in the RVLM in the central control of BP in hypertensive rats. In Sprague Dawley (SD) rats, microinjection of ADMA into the RVLM dose-dependently increased BP, heart rate (HR), and renal sympathetic never activity (RSNA), but also reduced total NO production in the RVLM. In central angiotensin II (Ang II)-induced hypertensive rats and spontaneously hypertensive rat (SHR), the level of ADMA in the RVLM was increased and total NO production was decreased significantly, compared with SD rats treated vehicle infusion and WKY rats, respectively. These hypertensive rats also showed an increased protein level of protein arginine methyltransferases1 (PRMT1, which generates ADMA) and a decreased expression level of dimethylarginine dimethylaminohydrolases 1 (DDAH1, which degrades ADMA) in the RVLM. Furthermore, increased AMDA content and PRMT1 expression, and decreased levels of total NO production and DDAH1 expression in the RVLM in SHR were blunted by intracisternal infusion of the angiotensin II type 1 receptor (AT1R) blocker losartan. The current data indicate that the ADMA-mediated NO inhibition in the RVLM plays a critical role in involving in the central regulation of BP in hypertension, which may be associated with increased Ang II.

CONTEXT: Arginine, its methylated metabolites, and other metabolites related to the urea cycle have been independently associated with cardiovascular risk, but the potential causal meaning of these associations (positive for some metabolites and negative for others) remains elusive due to a lack of studies measuring metabolite changes over time. OBJECTIVE: To examine the association between baseline and 1-year concentrations of urea cycle metabolites and cardiovascular disease (CVD) in a case-cohort setting. DESIGN: A case-cohort study was nested within the Prevención con Dieta Mediterránea trial. We used liquid chromatography-tandem mass spectrometry to assess metabolite levels at baseline and after 1-year follow-up. The primary CVD outcome was a composite of myocardial infarction, stroke and cardiovascular death. We used weighted Cox regression models (Barlow weights) to estimate multivariable-adjusted hazard ratios (HRs) and their 95% confidence intervals (CIs). SETTING: Multicenter randomized trial in Spain. PARTICIPANTS: Participants were 984 participants accruing 231 events over 4.7 years' median follow-up. MAIN OUTCOME MEASURE: Incident CVD. RESULTS: Baseline arginine/asymmetric dimethylarginine ratio [HR per standard deviation (SD) = 0.80; 95% CI, 0.67 to 0.96] and global arginine availability [arginine / (ornithine + citrulline)] (HR per SD = 0.83; 95% CI, 0.69 to 1.00) were significantly associated with lower risk of CVD. We observed no significant association for 1-year changes in these ratios or any effect modification by the Mediterranean diet (MD) intervention. CONCLUSIONS: A higher baseline arginine/asymmetric dimethylarginine ratio was associated with lower CVD incidence in a high cardiovascular risk population. The intervention with the MD did not change 1-year levels of these metabolites.

Aim A major feature of endothelial dysfunction is reduced endothelium-dependent vasodilation, which in ageing may be due to decreased production of endothelial prostacyclin, or nitric oxide (NO), or both. Method We tested this hypothesis in 12 younger (age 18–38 years, six women) and 12 older healthy adults (age 55–73 years, six post-menopausal women). Endothelium-dependent vasodilation was assessed by the forearm vascular conductance (FVC) response to intra-arterial acetylcholine (ACh) (0.5, 1.0, 2.0, 4.0 μg dl−1 forearm tissue min−1) before and 90 min after inhibition of the enzyme cyclo-oxygenase-2 (COX-2) with oral celecoxib (400 mg), followed by the addition of endothelial NO synthase inhibition with intra-arterial NG-monomethyl-l arginine acetate (L-NMMA). Results Ageing was associated with a significantly reduced FVC response to ACh (P = 0.009, age-by-dose interaction; highest dose FVC ± SEM in ageing: 11.2 ± 1.4 vs. younger: 17.7 ± 2.4 units, P = 0.02). Celecoxib did not reduce resting FVC or the responses to ACh in any group. L-NMMA significantly reduced resting FVC and the responses to ACh in all groups, and absolute FVC values following L-NMMA were similar between groups. Conclusion In healthy normotensive younger and older adults, there is minimal contribution of prostacyclin to ACh-mediated vasodilation, yet the NO component of vasodilation is reduced with ageing. In the clinical context, these findings suggest that acute administration of medications that inhibit prostacyclin (i.e. COX-2 inhibitors) evoke modest vascular consequences in healthy persons. Additional studies are necessary to test whether chronic use of COX-2 medications reduces endothelium dependent vasodilation in older persons with or without cardiovascular risk factors.

The hippocampus is a key structure for learning and memory in mammals, and long-term potentiation (LTP) is an important cellular mechanism responsible for learning and memory. Despite a number of studies indicating that nitric oxide (NO) is involved in the formation and maintenance of LTP as a retrograde messenger, few studies have used neurotransmitter release as a visual indicator in awake animals to explore the role of NO in learning-dependent long-term enhancement of synaptic efficiency. Therefore, in the present study, the effects of l-NMMA (a NO synthase inhibitor) and SNP (a NO donor) on extracellular glutamate (Glu) concentrations and amplitudes of field excitatory postsynaptic potential (fEPSP) were measured in the hippocampal dentate gyrus (DG) region during the acquisition and extinction of active-avoidance behavior in freely-moving conscious rats. In the control group, the extracellular concentration of Glu in the DG was significantly increased during the acquisition of active-avoidance behavior and gradually returned to baseline levels following extinction training. In the experimental group, the change in Glu concentration was significantly reduced by local microinjection of l-NMMA, as was the acquisition of the active-avoidance behavior. In contrast, the change in Glu concentration was significantly enhanced by SNP, and the acquisition of the active-avoidance behavior was significantly accelerated. Furthermore, in all groups, the changes in extracellular Glu were accompanied by corresponding changes in fEPSP amplitude and active-avoidance behavior. Our results suggest that NO in the hippocampal DG facilitates active avoidance learning via enhancements of glutamate levels and synaptic efficiency in rats.

Asymmetric dimethylarginine (ADMA) is an endogenously produced nitric oxide synthase (NOS) inhibitor. L-Arginine can be metabolised by NOS and arginase, and arginase is the first step in polyamine production necessary for cellular proliferation. We tested the hypothesis that ADMA would inhibit NOS but not arginase activity and that this pattern of inhibition would result in greater L-arginine bioavailability to arginase, thereby increasing viable cell number. Bovine arginase was used in in vitro activity assays with various concentrations of substrate (L-arginine, ADMA, N(G) -monomethyl-L-arginine (L-NMMA) and N(G) -nitro-L-arginine methyl ester (L-NAME)). Only L-arginine resulted in measurable urea production (Km = 6.9 ± 0.8 mmol/L; Vmax = 6.6 ± 0.3 μmol/mg protein per min). We then incubated bovine arginase with increasing concentrations of ADMA, L-NMMA and L-NAME in the presence of 1 mmol/L l-arginine and found no effect of any of the tested compounds on arginase activity. Using bovine pulmonary arterial endothelial cells (bPAEC) we determined the effects of ADMA on nitric oxide (NO) and urea production and found significantly lower NO production and greater urea production (P < 0.003) with ADMA, without changes in arginase protein levels. In addition, ADMA treatment resulted in an approximately 30% greater number of viable cells after 48 h than in control bPAEC. These results demonstrate that ADMA is neither a substrate nor an inhibitor of arginase activity and that in bPAEC ADMA inhibits NO production and enhances urea production, leading to more viable cells. These results may have pathophysiological implications in disorders associated with higher ADMA levels, such as pulmonary hypertension.

Summary Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O2) due to production of superoxide (O2−). In this study, we show that the PMNs also consume O2 for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with NG-monomethyl-L-arginine (L-NMMA) resulted in reduced O2 consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO3− (P < 0·04, r = 0·66, n = 10) and NO2− (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O2 for production of reactive oxygen species, the PMNs in CF sputum also consume O2 for production of NO.

The methylarginines asymmetric dimethylarginine (ADMA) and monomethylarginine (L-NMMA) are endogenously formed inhibitors of nitric oxide synthases (NOS), which have extensively been investigated as risk markers and used as pharmacological tools to study the L-arginine-nitric oxide (NO) pathway in vitro and in vivo. It is the aim of the present review to summarize the clinical and experimental data on the pharmacological properties that are of relevance when planning and conducting experiments and clinical studies involving methylarginines. Key pharmacodynamic and pharmacokinetic data including IC50 values of ADMA and L-NMMA for NOS isoforms and transport proteins, as well as metabolism by dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) and alanine-glyoxylate aminotransferase 2 (AGXT2) are discussed.

Objective— Abnormalities in nitric oxide (NO) bioavailability have been reported in blacks. Whether there are differences in endothelium-derived hyperpolarizing factor (EDHF) in addition to NO between blacks and whites and how these affect physiological vasodilation remain unknown. We hypothesized that the bioavailability of vascular NO and EDHF, at rest and with pharmacological and physiological vasodilation, varies between whites and blacks. Approach and Results— In 74 white and 86 black subjects without known cardiovascular disease risk factors, forearm blood flow was measured using plethysmography at rest and during inhibition of NO with N G -monomethyl- l -arginine and of K + Ca channels (EDHF) with tetraethylammonium. The reduction in resting forearm blood flow was greater with N G -monomethyl- l -arginine ( P =0.019) and similar with tetraethylammonium in whites compared with blacks. Vasodilation with bradykinin, acetylcholine, and sodium nitroprusside was lower in blacks compared with whites (all P G -monomethyl- l -arginine was greater in whites compared with blacks with bradykinin, acetylcholine, and exercise. Inhibition with tetraethylammonium was lower in blacks with bradykinin, but greater during exercise and with acetylcholine. Conclusions— The contribution to both resting and stimulus-mediated vasodilator tone of NO is greater in whites compared with blacks. EDHF partly compensates for the reduced NO release in exercise and acetylcholine-mediated vasodilation in blacks. Preserved EDHF but reduced NO bioavailability and sensitivity characterizes the vasculature in healthy blacks. Clinical Trial Registration— URL: http://clinicaltrials.gov/ . Unique identifier: NCT00166166.

Previous animal experiments have shown that the transport of LDL into arterial walls is shear stress dependent. However, little work has probed shear effects on LDL transport in vitro where conditions are well defined and mechanisms are more easily explored. Therefore, we measured shear induced water and LDL fluxes across cultured bovine aortic endothelial (BAEC) monolayers in vitro and developed a three-pore model to describe the transport dynamics. Cell apoptosis was quantified by TdT-mediated dUTP nick end labeling (TUNEL) assay. We also examined the role of nitric oxide (NO) in shear induced water and LDL fluxes by incubating BAEC monolayers with a NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA). Our results show that direct exposure of endothelial monolayers to 12 dyn/cm2 shear stress for 3 hours elicited a 2.37-fold increase in water flux (Jv), a 3.00-fold increase in LDL permeability (Pe), a 1.32-fold increase in LDL uptake, and a 1.68-fold increase in apoptotic rate. L-NMMA treatment of BAEC monolayers blocked shear induced Jv response, but had no significant effect on shear responses of Pe and cell apoptosis. A long time shear exposure (12 h) of endothelial monolayers reduced Pe and apoptotic rate close to the baseline. These results suggest that an acute change in shear stress from a static baseline state induces increases in water flux that are mediated by a NO dependent mechanism. On the other hand, the permeability of endothelial monolayers to LDL is enhanced by a short term-shear application and reduced nearly to the baseline level by a longer time shear exposure, positively correlated to the leaky junctions forming around apoptotic cells.