Endogenous Endothelial Nitric Oxide Research Articles

RBC eNOS Transduces Regional O2 Gradients to Orchestrate RBC O2 Delivery Phenotype during Circulatory Transit

BACKGROUND: The role of endogenous endothelial NO synthase (eNOS) in mature RBCs is poorly understood. We have previously demonstrated functionally O2-dependent eNOS migration in RBCs, mediating (via s-nitrosylation) glycolytic metabolon assembly/disassembly during circulation in support of antioxidant systems during stress. We have further explored eNOS migration and now propose the following mechanism along with its functional significance. We (and others) suggest that hemoglobin (Hb) conformational change resulting from deoxygenation leads to its docking at the cytoplasmic domain of Band 3 (cdB3). We propose that (1) cdB3 consequently undergoes a conformational change that is transduced by Piezo1 (a mechanosensitive ion channel located in the RBC membrane); (2) Piezo1 activation results in calcium entry into the RBC, which (3) aids in the disassembly of the RBC cytoskeleton, (4) facilitates eNOS migration to the membrane (where calmodulin is mainly localized - thereby bringing these two important proteins together), and (5) binds to calmodulin, activating eNOS to generate nitric oxide (NO) - which then influences RBC phenotype that facilitates O 2 delivery, specifically related to RBC deformability and regulation of Hb~O 2 release, via enhanced Bohr effect. METHODS: Heparinized human, eNOS (-/-), or wt murine (C57BL/6J) red blood cells (RBCs) were imaged (Nikon) for the analysis of cytoskeletal protein arrangement and eNOS migration. RBCs were subjected to oxygenation/deoxygenation, with/without pre-treatment with the Piezo1 agonist Yoda1, or a membrane tension buffer grammostola mechanotoxin #4 (GSMTx4; decoupling Piezo1), or carbon monoxide (CO) to maintain R-state Hb conformation upon RBC deoxygenation. RBCs were fixed (paraformaldehyde 4%/glutaraldehyde 0.08%), permeabilized (Triton X100 amount 0.1%) and incubated with primary antibodies (spectrin, actin, protein 4.1, or eNOS), then with the secondary antibody prior to visualization. Interaction between eNOS and cytoskeleton proteins, as well as calmodulin, was determined using a proximity ligation assay (Duolink). Intracellular RBC calcium levels were assessed in oxygenated/deoxygenated RBCs utilizing an environmentally controlled fluorescent plate reader, and the calcium probe Fluo 3. NO production in RBCs was assessed using DAF-FM. To assess the functional significance of this mechanism, Hb-O 2 affinity was determined from murine blood (eNOS (-/-) or wt). Oxygen dissociation curves (ODCs), were constructed at 2 fixed CO 2 levels (thin film rotating tonometer; in conjunction with a gas blender; with samples measured by arterial blood gas machine).Bohr effect was assessed by determining the area between the ODCs (ABC) at the 2 fixed CO2 levels (~40mmHg and ~70mmHg). RESULTS: We observed several significant findings related to red RBC deoxygenation. Upon RBC deoxygenation, there is an increase in calcium levels indicated by the Fluo3 signal, which is Piezo1 dependent. Cytoskeleton proteins, such as Actin, Spectrin, and protein 4.1, undergo disassembly upon RBC deoxygenation. This disassembly process is contingent on both Piezo1-mediated calcium flux and the conformational changes in hemoglobin (Hb) (and not pO 2). We found that eNOS is associated with 4.1 throughout the oxy/deoxy cycle and that eNOS exhibits the same migratory pattern as cytoskeleton proteins to the RBC membrane, where calmodulin is located, irrespective of the RBC's oxygenation state. Additionally, when eNOS localizes in the membrane, it exhibits interaction with calmodulin. The generation of nitric oxide (NO) by eNOS, as indicated by the DAF-FM signal, increases in a manner dependent on both calcium, Piezo1 and Hb conformational transition. The functional consequence of this mechanism appears to be related to O 2 delivery phenotype during circulatory transit, with cell deformability being affected in addition to an inhibition of the Bohr effect in eNOS (-/-) compared to wt mice (2006 ± 61 vs 1892 ± 69 ABC in WT (n = 3) vs eNOS (-/-) (n = 4), respectively; p = 0.0747). SUMMARY: Upon circulatory transit, RBC deoxygenation triggers the activation of eNOS. This activated eNOS is associated with Piezo1-mediated calcium flux, which is facilitated by calmodulin. These events are coordinated with the migration of eNOS within the red blood cell between the cytosol and the cell membrane. The migration of eNOS is influenced by cytoskeleton proteins.

Asymmetric Dimethylarginine as a Biomarker in Coronary Artery Disease.

As atherosclerosis remains a leading cause of morbidity and mortality worldwide despite the advances in its medical and interventional management, the identification of markers associated with its incidence and prognosis constitutes an appealing prospect. In this regard, asymmetric dimethylarginine (ADMA), a well-studied endogenous endothelial nitric oxide synthase inhibitor, represents a core mediator of endothelial dysfunction in atherosclerotic diseases. Given the pathophysiologic background of this molecule, its importance in the most frequent atherosclerotic manifestation, coronary artery disease (CAD), has been extensively studied in the past decades. The available evidence suggests elevation of ADMA in the presence of common cardiovascular risk factors, namely diabetes mellitus, arterial hypertension, and hypertriglyceridemia, being related to endothelial dysfunction and incident major adverse cardiovascular events in these groups of patients. Moreover, ADMA is associated with CAD occurrence and severity, as well as its prognosis, especially in populations with renal impairment. Interestingly, even in the absence of obstructive CAD, increased ADMA may indicate coronary endothelial dysfunction and epicardial vasomotor dysfunction, which are prognostication markers for incident cardiovascular events. In the case of acute coronary syndromes, high ADMA levels signify an augmented risk of incomplete ST-segment elevation resolution and poorer prognosis. Abnormal ADMA elevations may indicate adverse outcomes following percutaneous or surgical coronary revascularization, such as in-stent restenosis, graft patency, and hard cardiovascular endpoints. Finally, since its association with inflammation is significant, chronic inflammatory conditions may present with coronary endothelial dysfunction and subclinical coronary atherosclerosis by means of increased coronary artery calcium, with augmented ADMA acting as a biomarker.

The relationship between reduced choroidal thickness due to high plasma asymmetrical dimethylarginine level and increased severity of diabetic retinopathy.

To evaluate the relationship between subfoveal choroidal thickness and plasma asymmetrical dimethylarginine level and the severity of diabetic retinopathy in patients with type 2 diabetes mellitus. A total of 68 cases, including 15 patients without diabetic retinopathy, 17 patients with nonproliferative diabetic retinopathy, 16 patients with type 2 diabetes mellitus and proliferative diabetic retinopathy, and 20 healthy patients (control group), were enrolled in this study. Subfoveal choroidal thickness was measured manually using the enhanced depth imaging optical coherence tomography scanning program, and plasma asymmetrical dimethylarginine level was measured using a commercial micro enzyme-linked immunosorbent assay kit. The subfoveal choroidal thickness values and plasma asymmetrical dimethylarginine levels were significantly different between the four groups (p<0.001 and p<0.001). The subfoveal choroidal thickness values were significantly lower in the proliferative diabetic retinopathy group than in the other three groups (no diabetic retinopathy, nonproliferative diabetic retinopathy, and control groups; p<0.001, p=0.045, and p<0.001, respectively). The plasma asymmetrical dimethylarginine levels were significantly higher in the proliferative diabetic retinopathy group than in the other three groups (p<0.001, p<0.04, and p<0.001, respectively). In addition, a significant negative correlation was also found between plasma asymmetrical dimethylarginine level and subfoveal choroidal thickness (p<0.001, r=-0.479). Asymmetrical dimethylarginine is an important marker of endothelial dysfunction and endogenous endothelial nitric oxide synthase inhibitor. The severity of diabetic retinopathy was related to increased plasma asymmetrical dimethylarginine level and reduced subfoveal choroidal thickness in type 2 diabetic patients with diabetic retinopathy.

Endogenous Endothelial Nitric Oxide Synthase (eNOS) Modulates Oxygen (O2) Responsive Control of Red Blood Cell (RBC) Energy Metabolism and Antioxidant Systems

Endogenous Endothelial Nitric Oxide Synthase (eNOS) Modulates Oxygen (O2) Responsive Control of Red Blood Cell (RBC) Energy Metabolism and Antioxidant Systems

Recovery of hyperoxia‐induced DDAH1 suppression with FXR agonist GW4064 in human pulmonary microvascular endothelial cells

ObjectivePulmonary hypertension (PH) predicts mortality in patients with bronchopulmonary dysplasia (BPD), and chronic hyperoxia exposure contributes to BPD‐PH disease progression. BPD‐PH is characterized by aberrant pulmonary vascular wall proliferation/remodeling, which occludes the microvascular lumen, increasing pulmonary vascular resistance. Endogenous nitric oxide (NO) production by pulmonary vascular endothelial cells promotes apoptosis of pulmonary vascular endothelial and smooth muscle cells. In vitro studies of human pulmonary microvascular endothelial cells (hPMVEC) have shown that dimethylarginine dimethylaminohydrolase‐1 (DDAH1) promotes NO‐mediated apoptosis. DDAH1 metabolizes the majority of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial NO synthase. Therefore, our hypothesis is that pharmacologically enhancing DDAH1 expression in hPMVEC will restore endogenous endothelial NO production.MethodshPMVEC were grown at 37 ̊C to ~70% confluence. Farnesoid X receptor (FXR) agonist, GW4064 (1.25 µM) was used to bind FXR element (FXRE) of the DDAH1 promoter to enhance transcription. hPMVEC were pre‐treated with FXR agonist for 24 hours and then exposed to 85%O2 or 21%O2 for an additional 24 hours. DDAH1 protein expression was analyzed by Western blot. Groups were compared by 1‐way ANOVA.ResultsIn hPMVEC, hyperoxia exposure resulted in lower DDAH1 protein levels compared to hPMVEC grown in normoxia (N=6 in each group, p=0.003). Treatment with GW4064 prior to exposing to hyperoxia resulted in 4.3‐fold greater DDAH1 protein levels than in vehicle/85%O2 hPMVEC (N=6, p=0.044), and restored DDAH1 protein levels to 72% of that seen in the vehicle/21%O2exposed hPMVEC.ConclusionsChronic hyperoxia suppresses DDAH1 levels, and FXR agonist promotes DDAH1 recovery in hPMVEC. We speculate that promoting endogenous DDAH1 expression may prevent aberrant proliferation in the pulmonary vascular wall and thereby attenuate or prevent the vascular remodeling characteristic of pulmonary hypertension in BPD.

Brain-derived neurotrophic factor is a full endothelium-derived factor in rats

Most of what is known on vascular brain-derived neurotrophic factor (BDNF) derived from experiments on cultured endothelial cells. Therefore, the present study compared BDNF levels/localization in artery (aorta) vs vein (vena cava) from a same territory in rats either sedentary (SED) or exposed to treadmill exercise (EX) as a mean to stimulate endogenous endothelial nitric oxide (NO) production. In SED rats, for both artery and vein, BDNF was strongly expressed by endothelial cells, while only a faint and scattered expression was observed throughout the media. Endothelial and muscular BDNF staining as vascular BDNF protein levels were however higher in artery than in vein, while BDNF mRNA levels did not differ between vessels. Irrespective of the vessels, EX resulted in an increase (+50%) in BDNF protein levels with no change in BDNF mRNA levels, a selective endothelial BDNF overexpression (x4) and an increase in vascular levels of tropomyosin related kinase B receptors (TrkB) phosphorylated at tyrosine 816 (p-TrkBTyr816). Endothelial expressions of BDNF and p-TrkBTyr816 were positively associated when SED and EX rats were simultaneously examined. The results incite to consider endothelial BDNF as a full and NO-dependent endothelium-derived factor that exerts autocrine effects.

Enhancing kidney DDAH-1 expression by adenovirus delivery reduces ADMA and ameliorates diabetic nephropathy.

Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Low DDAH levels have been associated with cardiac and renal dysfunction, but its effects on DN are unknown. We hypothesized that enhanced renal DDAH-1 expression would improve DN by reducing ADMA and restoring NOS3 levels. DBA/2J mice injected with multiple low doses of vehicle or streptozotocin were subsequently injected intrarenally with adenovirus expressing DDAH-1 (Ad-h-DDAH-1) or vector control [Ad-green fluorescent protein (GFP)], and mice were followed for 6 wk. Diabetes was associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but had no effect on kidney DDAH-2 expression. Ad-GFP-treated diabetic mice showed significant increases in albuminuria, histological changes, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid reactive substances excretion as an indicator of oxidative stress, along with a significant reduction in kidney DDAH activity and kidney NOS3 mRNA compared with normal mice. In contrast, Ad-h-DDAH-1 treatment of diabetic mice reversed these effects. These data indicate, for the first time, that DDAH-1 mediates renal tissue protection in DN via the ADMA-NOS3-interaction. Enhanced renal DDAH-1 activity could be a novel therapeutic tool for treating patients with diabetes.

Evidence that links loss of cyclooxygenase-2 with increased asymmetric dimethylarginine: novel explanation of cardiovascular side effects associated with anti-inflammatory drugs.

Cardiovascular side effects associated with cyclooxygenase-2 inhibitor drugs dominate clinical concern. Cyclooxygenase-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclooxygenase-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified. Transcriptome analysis of wild-type and cyclooxygenase-2(-/-) mouse tissues revealed 1 gene altered in the heart and aorta, but >1000 genes altered in the renal medulla, including those regulating the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-l-arginine. Cyclo-oxygenase-2(-/-) mice had increased plasma levels of ADMA and monomethyl-l-arginine and reduced endothelial nitric oxide responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors. Wild-type mice or human volunteers taking cyclooxygenase-2 inhibitors also showed increased plasma ADMA. Endothelial nitric oxide is cardio-protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction with nonsteroidal anti-inflammatory drug usage. We identify the endogenous endothelial nitric oxide synthase inhibitor ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction.

Endothelial dysfunction in subjects with chronic cadmium exposure.

Vascular endothelium is a target of cadmium (Cd) toxicity. Cd exposure has been reported to be associated with vascular disorders. In this study, we aimed to investigate the effects of Cd exposure on markers of endothelial function in human subjects chronically exposed to Cd. Based on blood Cd levels, seventy-five women were categorized into non-exposed, Cd-exposed and severely Cd-exposed groups. Nitrite, L-arginine, asymmetric dimethylarginine (ADMA), and soluble thrombomodulin levels in blood were measured. Nitrite levels were lower in Cd-exposed subjects than non-exposed subjects. Plasma L-arginine decreased while ADMA, an endogenous endothelial nitric oxide synthase (eNOS) inhibitor, increased in Cd-exposed subjects. Soluble thrombomodulin also increased in Cd-exposed subjects. In Cd-exposed subjects, plasma malondialdehyde and protein carbonyl groups increased while the erythrocytic glutathione decreased. Multiple linear regression analysis revealed a negative association between urinary Cd and nitrite levels in erythrocytes. Our research suggests that subjects with chronic Cd exposure have endothelial dysfunction.

Exercise performance and peripheral vascular insufficiency improve with AMPK activation in high-fat diet-fed mice

Intermittent claudication is a form of exercise intolerance characterized by muscle pain during walking in patients with peripheral artery disease (PAD). Endothelial cell and muscle dysfunction are thought to be important contributors to the etiology of this disease, but a lack of preclinical models that incorporate these elements and measure exercise performance as a primary end point has slowed progress in finding new treatment options for these patients. We sought to develop an animal model of peripheral vascular insufficiency in which microvascular dysfunction and exercise intolerance were defining features. We further set out to determine if pharmacological activation of 5'-AMP-activated protein kinase (AMPK) might counteract any of these functional deficits. Mice aged on a high-fat diet demonstrate many functional and molecular characteristics of PAD, including the sequential development of peripheral vascular insufficiency, increased muscle fatigability, and progressive exercise intolerance. These changes occur gradually and are associated with alterations in nitric oxide bioavailability. Treatment of animals with an AMPK activator, R118, increased voluntary wheel running activity, decreased muscle fatigability, and prevented the progressive decrease in treadmill exercise capacity. These functional performance benefits were accompanied by improved mitochondrial function, the normalization of perfusion in exercising muscle, increased nitric oxide bioavailability, and decreased circulating levels of the endogenous endothelial nitric oxide synthase inhibitor asymmetric dimethylarginine. These data suggest that aged, obese mice represent a novel model for studying exercise intolerance associated with peripheral vascular insufficiency, and pharmacological activation of AMPK may be a suitable treatment for intermittent claudication associated with PAD.

IGF-1 and ADMA levels are inversely correlated in nondiabetic ankylosing spondylitis patients undergoing anti-TNF-alpha therapy.

Like rheumatoid arthritis, ankylosing spondylitis (AS) is also an inflammatory disease associated with accelerated atherosclerosis and the presence of metabolic syndrome (MeS) features. AS patients often display osteoporosis as well as new bone formation. Insulin-like growth factor 1 (IGF-1) is a protein involved in both inflammation and bone metabolism. In the present study we assessed whether disease activity, systemic inflammation, MeS features, adipokines, and biomarkers of endothelial activation were associated with IGF-1 and insulin-like growth factor binding protein-3 (IGFBP-3) levels in a series of 30 nondiabetic AS patients without CV disease undergoing TNF-α antagonist-infliximab therapy. All determinations were made in the fasting state, immediately before an infliximab infusion. Although no association of IGF-1 and IGFBP-3 levels with angiopoietin-2 or osteopontin was found, an inverse correlation between IGF-1 levels and asymmetric dimethylarginine (ADMA), an endogenous endothelial nitric oxide synthase inhibitor that impairs nitric oxide production and secretion promoting endothelial dysfunction, was found (r = −0.397; P = 0.04). However, no significant association was found between IGF-1 and IGFBP-3 levels and disease activity, systemic inflammation, metabolic syndrome features, or adipokines. In conclusion, in nondiabetic patients with AS undergoing periodic anti-TNF-α therapy, IGF-1 and ADMA are inversely correlated.

Dynamin2 S-nitrosylation regulates adenovirus type 5 infection of epithelial cells

Dynamin2 is a large GTPase that regulates vesicle trafficking, and the GTPase activity of dynamin2 is required for the multistep process of adenovirus infection. Activity of dynamin2 may be regulated by post-translational phosphorylation and S-nitrosylation modifications. In this study, we demonstrate a role for dynamin2 S-nitrosylation in adenovirus infection of epithelial cells. We show that adenovirus serotype 5 (Ad5) infection augments production of nitric oxide (NO) in epithelial cells and causes the S-nitrosylation of dynamin2, mainly on cysteine 86 (C86) and 607 (C607) residues. Forced overexpression of dynamin2 bearing C86A and/or C607A mutations decreases Ad5 infection. Diminishing NO synthesis by RNAi-induced knockdown of endogenous endothelial NO synthase (eNOS) expression attenuates virus infection of target cells. Ad5 infection promotes the kinetically dynamic S-nitrosylation of dynamin2 and eNOS: there is a rapid decrease in eNOS S-nitrosylation and a concomitant increase in the dynamin2 S-nitrosylation. These results support the hypothesis that dynamin2 S-nitrosylation following eNOS activation facilitates adenovirus infection of host epithelial cells.

Abstract 5269: Asymmetric Dimethylarginine Triggers Vasculo-Renal Interaction in CKD Mice

Background: Patients with chronic kidney disease (CKD) also suffer increased morbidity and mortality from cardiovascular diseases (CVD). Endothelial dysfunction is a major factor underlying the pathogenesis of CVD. Asymmetric dimethylarginine (ADMA), an endogenous endothelial nitric oxide synthase (eNOS) inhibitor, is elevated in CKD. We hypothesized that ADMA has a role in endothelial dysfunction in CKD. Methods and Results: We created a CKD model by performing 5/6 nephrectomy (Nx) in mice. Blood and 24-hour urine were collected for measurements of renal function and ADMA, and NOx levels at 4 weeks after Nx. Nx induced renal failure (serum blood urea nitrogen, controls: 26.9±1.6 mg/dL, Nx mice: 61.0±3.8 mg/dL, p &lt;0.001; serum creatinine, controls: 0.19±0.01 mg/dl, Nx mice: 0.31±0.03 mg/dL, p &lt;0.01) without changing blood pressure. The serum ADMA level was higher (p &lt;0.01) in Nx mice (1.20±0.05 nmol/mL) than in controls (1.00±0.05 nmol/mL). Urinary NOx was lower in Nx mice (controls: 2153±189 nmol/L, Nx mice: 706±335 nmol/L, p &lt;0.05). Isometric tension study using aortic rings showed that endothelium-mediated relaxation induced by acetylcholine was impaired in Nx mice. Furthermore, pretreatment with exogenously administered ADMA at a serum concentration comparable to that in Nx mice reduced acetylcholine-induced relaxation in the aorta of controls. Western blotting showed the phosphorylated eNOS level of the aorta was lower in Nx mice than in controls, whereas eNOS levels were similar. The expression level of dimethylarginine dimethylaminohydrolase-1 (DDAH-1), a degradation enzyme of ADMA, was reduced in the kidney, but not in the aorta of Nx mice. In an ex vivo experiment using cultured human umbilical vein endothelial cells, ADMA treatment with a concentration corresponding to the serum level in Nx mice reduced the eNOS phosphorylation level. Conclusions: Elevated levels of circulating ADMA, probably resulting from decreased DDAH-1 expression in the kidney, appear to impair endothelial function by inhibiting eNOS activity in CKD. ADMA may thus have a triggering role in the vasculo-renal interaction in CKD.

Asymmetric Dimethylarginine is a Paracrine Regulator of Vascular Function in CKD

Background: Patients with chronic kidney disease (CKD) suffer increased morbidity and mortality from cardiovascular diseases (CVD). Asymmetric dimethylarginine (ADMA), an endogenous endothelial nitric oxide synthase (eNOS) inhibitor, is elevated in CKD. ADMA has been suggested to increase CVD in CKD, but direct evidence is missing. We assessed the effect of ADMA in endothelial function in CKD. Methods and Results: We created a CKD model by performing 5/6 nephrectomy (Nx) in mice. Samples were collected for measurements of renal function and ADMA, and NOx levels at 4 weeks after Nx.

Asymmetric Dimethylarginine (ADMA) and other Endogenous Nitric Oxide Synthase (NOS) Inhibitors as an Important Cause of Vascular Insulin Resistance

Asymmetric dimethylarginine (ADMA) and NG-monomethyl- L-arginine ( L-NMMA) are important endogenous endothelial nitric oxide synthase (eNOS) inhibitors. Studies have shown that patients with insulin resistance have elevated plasma levels of ADMA. Moreover, ADMA levels have a prognostic value on long-term outcome of patients with coronary artery disease. Insulin resistance, a disorder associated to inadequate biological responsiveness to the actions of exogenous or endogenous insulin, is a metabolic condition, which exists in patients with cardiovascular diseases. This disorder affects the functional balance of vascular endothelium via changes of nitric oxide (NO) metabolism. Nitric oxide is produced in endothelial cells from the substrate L-arginine via eNOS. Elevated ADMA levels cause eNOS uncoupling, a mechanism which leads to decreased NO bioavailability and increased production of hydrogen peroxide. According to clinical studies, the administration of L-arginine to patients with high ADMA levels improves NO synthesis by antagonizing the deleterious effect of ADMA on eNOS function, although in specific populations such as diabetes mellitus, this might even been harmful. More studies are required in order to certify the role of NOS inhibitors in insulin resistance and endothelial dysfunction. It is still difficult to say whether increased ADMA levels in certain populations is only a reason or the result of the molecular alterations, which take place in vascular disease states.

Statins restore ischemic limb blood flow in diabetic microangiopathy via eNOS/NO upregulation but not via PDGF-BB expression

3-Hydroxy-3-methyl-glutaryl CoA reductase inhibitors, or statins, have pleiotropic effects and can protect the vasculature in a manner independent of their lipid-lowering effect. The effectiveness of statins in reducing the risk of coronary events has been shown even in patients with diabetes, and their effects on diabetic complications have been reported. Using a model of severe hindlimb ischemia in streptozotocin-induced diabetic mice (STZ-DM), we investigated the effects and mechanisms of statin therapy in diabetic angiopathy in ischemic hindlimbs. As a result, STZ-DM mice frequently lost their hindlimbs after induced ischemia, whereas non-DM mice did not. Supplementation with statins significantly prevented autoamputation. We previously showed that diabetic vascular complications are caused by impaired expression of PDGF-BB, but statin therapy did not enhance PDGF-BB expression. Statins helped enhance endogenous endothelial nitric oxide (NO) synthase (eNOS) expression. Furthermore, the inhibition of NO synthesis by the administration of N(omega)-nitro-l-arginine methyl ester impaired the ability of statins to prevent STZ-DM mouse limb autoamputation, indicating that the therapeutic effect of statins in hindlimb ischemia in STZ-DM mice occurs via the eNOS/NO pathway. A combination therapy of statins and PDGF-BB gene supplementation was more effective for diabetic angiopathy than either therapy alone. In conclusion, these findings indicate that statin therapy might be useful for preventing intractable diabetic foot disease in patients with diabetic angiopathy.

Swim training sensitizes myocardial response to insulin: Role of Akt-dependent eNOS activation

Physical activity has been well known to benefit heart function. The improved autonomic nervous activity is considered to be mainly responsible for this beneficial effect. However, the precise mechanism behind the intrinsic myocardial responsiveness to exercise is still unclear. This study was designed to examine the effect of swim training on myocardial response to insulin with a special focus on the endogenous endothelial nitric oxide synthase (eNOS)-nitric oxide (NO) cascade. Adult male Sprague-Dawley (SD) rats were subjected to a 10-week free-loading swim training (3 h/day, 5 days/week). Contractile response to insulin at the levels of cardiomyocytes and isolated perfused heart, myocardial glucose uptake and post-insulin receptor signaling cascades were evaluated. Swim training enhanced cardiac contractile response to insulin in cardiomyocytes and isolated perfused heart, respectively. The improved cardiac response was accompanied by facilitated insulin-stimulated glucose uptake, GLUT4 translocation and upregulation of Akt and eNOS expression (p<0.01). Treatment with insulin resulted in a 3.6- and 2.2-fold increase of eNOS phosphorylation (p<0.01), as well as a 3.0- and 1.9-fold increase of Akt phosphorylation in exercise and sedentary groups, respectively (p<0.01). In addition, exercise significantly facilitated insulin-induced myocardial NO production (p<0.01 vs. sedentary). Moreover, pretreatment with either LY294002, a phosphatidylinositol-3 kinase (PI-3K) inhibitor or L-NAME, a NOS inhibitor, abolished the exercise-induced sensitization of myocardial contractile response to insulin, insulin-induced NO production and phosphorylation of Akt and eNOS. These results demonstrate that swim training is capable of sensitizing myocardial contractile response to insulin via upregulation of Akt- and eNOS signaling cascades.

Inhaled nitric oxide does not reduce systemic vascular resistance in mice

Inhaled nitric oxide (NO) is a highly selective pulmonary vasodilator. It was recently reported that inhaled NO causes peripheral vasodilatation after treatment with a NO synthase (NOS) inhibitor. These findings suggested the possibility that inhibition of endogenous NOS uncovered the systemic vasodilating effect of NO or NO adducts absorbed via the lungs during NO inhalation. To learn whether inhaled NO reduces systemic vascular resistance in the absence of endothelial NOS, we studied the systemic vascular effects of NO breathing in wild-type mice treated without and with the NOS inhibitor N(omega)-nitro-l-arginine methyl ester and in NOS3-deficient (NOS3(-/-)) mice. During general anesthesia, the cardiac output, left ventricular function, and systemic vascular resistance were not altered by NO breathing at 80 parts/million in both genotypes. Breathing NO in air did not alter blood pressure and heart rate, as measured by tail-cuff and telemetric methods, in either awake wild-type mice (whether or not they were treated with N(omega)-nitro-l-arginine methyl ester), or in awake NOS3(-/-) mice. Our findings suggest that absorption of NO or adducts during NO breathing is insufficient to cause systemic vasodilation in mice, even when endogenous endothelial NO production is congenitally absent.

Function and regulation of mitochondrially produced nitric oxide in cardiomyocytes.

this series of six papers covers the presentations from the Experimental Biology 2003 Symposium entitled “ The Function and Regulation of Mitochondrially Produced Nitric Oxide in Cardiomyocytes ,” sponsored by the American Physiological Society. Broadly, the topics include views on nitric oxide

Chronic inhibition of endothelial nitric oxide synthase activity in nucleus tractus solitarii enhances baroreceptor reflex in conscious rats.

In acute experiments, we demonstrated previously that nitric oxide (NO) donors exogenously applied to the nucleus tractus solitarii (NTS) depressed the baroreceptor cardiac reflex. In this study, we determined a role for endogenous endothelial nitric oxide synthase (eNOS) activity in the NTS for chronically regulating baroreceptor reflex function in conscious rats. A recombinant adenoviral vector directing expression of a truncated form of eNOS was microinjected bilaterally into the NTS to inhibit endogenous eNOS activity. Arterial pressure was monitored continuously using radio-telemetry in freely moving animals and spontaneous baroreceptor reflex gain (sBRG) determined by a time-series method. sBRG showed a gradual increase from day 7 to 21 after gene transfer and the value at day 21 (1.68 +/- 0.20 ms mmHg(-1), n = 6) was significantly higher than that before gene transfer (1.13 +/- 0.09 ms mmHg(-1), P < 0.001). This value was also significantly higher than that in rats in which enhanced green fluorescent protein (eGFP) was expressed in the NTS (1.04 +/- 0.21 ms mmHg(-1); n = 6, P < 0.01) and saline-treated groups (1.12 +/- 0.15 ms mmHg(-1); n = 4, P < 0.05), which did not change from control levels. In addition, heart rate decreased from 336 +/- 6 to 318 +/- 8 b.p.m. (P < 0.05) 21 days after gene transfer. This value was also significantly lower than that in control groups (eGFP: 348 +/- 9 b.p.m., n = 6, P < 0.01; saline: 347 +/- 5 b.p.m., n = 4, P < 0.05). Gene transfer did not affect arterial pressure. These findings suggest that in the conscious rat eNOS is constitutively active within the NTS and is a factor regulating baroreceptor reflex gain and heart rate.