Role Of eNOS Research Articles

Ameliorative effect of lixisenatide on diabetic cardiovascular damage and its enhancement via ticagrelor co-administration in rats: possible role of eNOS and NrF₂ /HO-1 signaling.

In this study, we hypothesized that lixisenatide (LIX) and ticagrelor (TIC) could have a protective effect against type 2 diabetes mellitus (T2DM)-induced vascular damage. Furthermore, we explored the possible additional protective effect of co-administering LIX and TIC in the treatment regimen. Methods: 50 male rats were divided into five groups, each comprising 10 rats: C (control), D (T2DM rats), D + LIX (T2DM rats treated with LIX for 4 weeks), D + TIC (T2DM rats treated with TIC for 4 weeks), and D + LIX + TIC (T2DM rats treated with LIX + TIC for 4 weeks). Results: The D group showed an increase in body weight, blood glucose, hemostatic model assessment for insulin resistance (HOMA-IR), aorta reactive oxygen species (ROS), and nuclear factor kappa B (NF-κ B), along with a reduction in serum insulin, aorta superoxide dismutase (SOD), glutathione reduced (GSH), nuclear factor erythroid-2 (NrF₂), hemeoxygenase-1 (HO-1), and endothelial nitric oxide synthase (eNOS). Deterioration in the aorta histopathological condition, coupled with a noticeable impairment in vascular reactivity compared to the C group, was observed. A single administration of LIX showed a reduction in body weight, blood glucose, HOMA-IR, aorta ROS, and NF-κ B, accompanied by an increase in serum insulin, aorta SOD, GSH, NrF₂, HO-1, and eNOS. Amelioration in the aorta histopathological condition and improved vascular reactivity compared to the D group were reported. Similarly, a single administration of TIC showed a reduction in aorta ROS and NF-κ B, along with an increase in aorta SOD, GSH, NrF₂, HO-1, and eNOS. A slight amelioration was detected in the aorta histopathological condition, with improved vascular reactivity compared to the D group. The combined administration of LIX and TIC showed a reduction in aorta ROS and NF-κ B, along with an increase in aorta GSH, SOD, HO-1, and eNOS. This was combined with evident amelioration in the aorta histopathological condition and noticeable improvement in vascular reactivity compared to the single treatment with either LIX or TIC group. Conclusion: The present study introduces clear evidence that the administration of LIX and TIC can improve metabolic and vascular complications of T2DM through modulating eNOS and NrF₂ /HO-1 signaling. The combined administration of LIX and TIC produced more significant effects than a single treatment.

Photobiomodulation Inhibits Ischemia-Induced Brain Endothelial Senescence via Endothelial Nitric Oxide Synthase.

Recent research suggests that photobiomodulation therapy (PBMT) positively impacts the vascular function associated with various cerebrovascular diseases. Nevertheless, the specific mechanisms by which PBMT improves vascular function remain ambiguous. Since endothelial nitric oxide synthase (eNOS) is crucial in regulating vascular function following cerebral ischemia, we investigated whether eNOS is a key element controlling cerebrovascular function and the senescence of vascular endothelial cells following PBMT treatment. Both rat photothrombotic (PT) stroke and in vitro oxygen-glucose deprivation (OGD)-induced vascular endothelial injury models were utilized. We demonstrated that treatment with PBMT (808 nm, 350 mW/cm2, 2 min/day) for 7 days significantly reduced PT-stroke-induced vascular permeability. Additionally, PBMT inhibited the levels of endothelial senescence markers (senescence green and p21) and antiangiogenic factor (endostatin), while increasing the phospho-eNOS (Ser1177) in the peri-infarct region following PT stroke. In vitro study further indicated that OGD increased p21, endostatin, and DNA damage (γH2AX) levels in the brain endothelial cell line, but they were reversed by PBMT. Intriguingly, the beneficial effects of PBMT were attenuated by a NOS inhibitor. In summary, these findings provide novel insights into the role of eNOS in PBMT-mediated protection against cerebrovascular senescence and endothelial dysfunction following ischemia. The use of PBMT as a therapeutic is a promising strategy to improve endothelial function in cerebrovascular disease.

NNOS-derived NO modulates force production and iNO-derived NO the excitability in C2C12-derived 3D tissue engineering skeletal muscle via different NO signaling pathways.

Nitric oxide (NO) is a bioactive gas produced by one of the three NO synthases: neuronal NOS (nNOS), inducible (iNOS), and endothelial NOS (eNOS). NO has a relevant modulatory role in muscle contraction; this takes place through two major signaling pathways: (i) activation of soluble guanylate cyclase and, thus, protein kinase G or (ii) nitrosylation of sulfur groups of cysteine. Although it has been suggested that nNOS-derived NO is the responsible isoform in muscle contraction, the roles of eNOS and iNOS and their signaling pathways have not yet been clarified. To elucidate the action of each pathway, we optimized the generation of myooids, an engineered skeletal muscle tissue based on the C2C12 cell line. In comparison with diaphragm strips from wild-type mice, 180 myooids were analyzed, which expressed all relevant excitation–contraction coupling proteins and both nNOS and iNOS isoforms. Along with the biochemical results, myooids treated with NO donor (SNAP) and unspecific NOS blocker (L-NAME) revealed a comparable NO modulatory effect on force production as was observed in the diaphragm strips. Under the effects of pharmacological tools, we analyzed the myooids in response to electrical stimulation of two possible signaling pathways and NO sources. The nNOS-derived NO exerted its negative effect on force production via the sGG-PKG pathway, while iNOS-derived NO increased the excitability in response to sub-threshold electrical stimulation. These results strengthen the hypotheses of previous reports on the mechanism of action of NO during force production, showed a novel function of iNOS-derived NO, and establish the myooid as a novel and robust alternative model for pathophysiological skeletal muscle research.

Role of eNOS and TGF\u03b21 gene polymorphisms in the development of diabetic nephropathy in type 2 diabetic patients in South Indian population

BackgroundDiabetic nephropathy is known to be a leading complication of diabetes mellitus, characterized by diverse aspects such as high urinary albumin level, elevated blood pressure, and genetic susceptibility leading to end-stage renal disease. The current study was carried out to investigate the association of eNOS and TGFβ1 gene polymorphisms in the progression of diabetic nephropathy among type 2 diabetic patients in the South Indian population. The eNOS and TGFβ1 genetic variants were genotyped in 280 T2DM patients, 140 with DN, 140 without DN, and 140 controls. Genotyping was performed using ARMS PCR and the genomic variants were confirmed by the Sanger sequencing method.ResultsA significant (p < 0.05) association was observed in the genotypic frequencies of eNOS (G > T) polymorphism in the T2DM patients with diabetic nephropathy when compared to controls. The frequency of TT (heterozygous) genotype was observed to increase in patients with type 2 diabetes and DN when compared to the diabetic patients without DN and controls. This indicates that diabetic patients with TT genotype are at an increased risk to develop DN. However, TGFβ1 (G > C) polymorphism did not show any association in the allele and genotypic frequencies with DN when compared with T2DM and controls.ConclusionThe results of the study propose a strong influence of TT genotype of eNOS gene be significantly linked with diabetic nephropathy in T2DM patients. Whereas no association was examined concerning TGFβ1 gene polymorphism and DN. Nevertheless, large sample size studies are required to confirm the part of these genetic variants in the development of DN.

Endothelial nitric oxide synthase activation is required for heparin receptor effects on vascular smooth muscle cells.

Published studies indicate that TMEM184A is a heparin receptor that interacts with and transduces stimulation from heparin in vascular cells. Previous studies have indicated that heparin increases endothelial nitric oxide synthase (eNOS) activity in bovine endothelial cells. However, the precise mechanism remains unknown. In this study, we investigated the impact of heparin treatment and TMEM184A on eNOS's activation and the role of eNOS in heparin signaling in the cloned A7r5 rat vascular smooth muscle cell line and confirmed results in endothelial cells. We employed a combination of TMEM184A knockdown A7r5 cells along with transient eNOS knockdown and enzyme inhibitor strategies. The results indicate that heparin induces phosphorylation of eNOS. eNOS can be immunoprecipitated with TMEM184A and is internalized to the perinuclear region in a TMEM184A-dependent manner in response to heparin. We also examined how heparin treatment leads to phosphorylation of eNOS and confirmed that TMEM184A and Ca2+ were required to mediate heparin-elicited eNOS phosphorylation. Evidence supporting the involvement of transient receptor potential cation channel subfamily V member 4 with TMEM184A in this eNOS activation process is also presented.

Abstract 138: A Novel Cellular and Genetic Approach to Investigate the Cardioprotective Role Played by Endothelial Nitric Oxide Synthase in Myocardial Infarction

The loss of regenerative properties in adult cardiomyocytes (CMs) is directly linked to their inability to proliferate. Following an extensive ischaemic event in an aged heart, fibrotic scar formation is the only repair process and eventually heart failure develops. However, molecular and cellular cues in the neonatal heart support that cardiac regeneration is possible in presence of proliferating CMs. Based on previous studies demonstrating that endothelial nitric oxide synthase (eNOS) regulates proliferation in both endothelial cells (ECs) and CMs, we hypothesized that eNOS signaling could play a cardioprotective role. To test our hypothesis, we injected different combinations of co-cultured ECs and CMs in the LV muscle wall of MI mice (permanent LAD ligation). First, injected cells were isolated from either WT or KO eNOS neonatal mice and then co-cultured to form 3D vascularized cardiac spheroids (VCSs), which were eventually transplanted in adult MI mice on the day of the procedure. Control infarcted animals received media-only (vehicle). Other mice received a suspension of co-cultured VCSs in media as follows: i ) WT CMs and ECs; ii ) WT CMs and KO ECs; iii ) KO CMs and WT ECs. Following 28 days, injection of WT cells increased the ejection fraction (EF%) by 20% compared with control animals (61%±4% and 41%±11%, respectively). When eNOS was absent in either CMs or ECs, the EF% was 40%±5% and 46%±2%, respectively, suggesting that the eNOS-mediated protection is dependent on its presence in both cells. Histological analyses confirmed the presence of WT VCSs in MI mice, contributing to a thicker wall thickness compared to vehicle MI mice. No VCSs were observed in the LV wall when KO cells were injected. Therefore, our results strongly suggest that eNOS may play a major role via both an autocrine (CMs) and paracrine (ECs) mechanism. Current studies are focusing on further evaluating the mechanism(s) for this eNOS-mediated protective role. To our knowledge, this is the first study combining cellular and genetic approaches to evaluate the cardioprotective role of eNOS in the heart. A better understanding of this mechanisms may have significant impact for the development of improved molecular and cell therapeutics (including stem cells) for heart failure patients.

Mechanisms mediating the cardioprotective effect of carvedilol in cadmium induced cardiotoxicity. Role of eNOS and HO1/Nrf2 pathway

Cadmium (Cd) is a highly toxic heavy metal with several harmful effects including cardiotoxicity. For the first time, we aimed to evaluate the possible cardioprotective effect of carvedilol (CAR) in Cd induced cardiotoxicity and study the mechanisms involved in such protection including endothelial nitric oxide synthase (eNOS) and HO1/Nrf2 pathway. CAR (1,10 mg/kg/d) was administered orally for 4 weeks with Cd induced cardiac injury (3 mg/kg/d) orally for 4 weeks. We measured cardiac enzymes, mean arterial pressure changes, heme oxygenase-1 (HO1) and total antioxidant capacity (TAC). Moreover; cardiac tissue malondialdehyde (MDA), tumor necrosis factor alpha (TNFα), western blotting of caspase3 and eNOS levels and histopathology were evaluated. Immunoexpression of eNOS in cardiac tissue, gene expression changes of HO1, and nuclear factor erythroid 2-related factor 2 (Nrf2) using real time polymerase chain reactions (rtPCR) were detected. Our results showed that CAR could significantly decrease Cd induced cardiotoxicity.

Roles of eNOS in atherosclerosis treatment.

Atherosclerosis (AS) is the main pathogeny of coronary heart disease, cerebral infarction and peripheral vascular disease. Endothelial dysfunction is one of the important pathogenesis of AS. As an important endothelium-derived relaxation factor, nitric oxide (NO) plays a role in cardiovascular protection and anti-AS function; but in the pathological state, endothelial nitric oxide synthase (eNOS) disorder causes an abnormal production of NO, which may damage endothelial function and trigger AS. This review summarized the research progresses in the treatment strategies for AS based on correcting the disordered eNOS/ NO signaling pathway. According to the topic, select the search terms 'atherosclerosis,' 'nitric oxide,' 'eNOS,' 'treatment,' 'management,' 'medication,' 'maintenance,' 'remission'. Using these terms, a structured literature search via multiple electronic databases was performed for the most recent trial evidence in recent years. We read and analyze these literatures carefully, classified these literatures according to their content, and then summarized and outlined the common main points in these classified literatures. Finally, literature data were organized to discuss these main points logically. We found that both aberrant expression and dysfunction of eNOS are closely related to AS development, and some new treatment strategies aimed at eNOS have been proposed, including upregulation of eNOS expression and inhibition of eNOS uncoupling. The former one is mainly related to inflammatory inhibition and protection of the PKB-eNOS signaling pathway; whereas the latter one is associated with the addition of the L-arginine substrate of eNOS, arginase inhibition, and the supplement of tetrahydrobiopterin, which can elevate no level. eNOS can be an important target for prevention and treatment of AS, and eNOS drugs may be another potent class of effective therapeutic treatment for AS following traditional lipid-lowering, anti-platelet, vasodilator drugs. But applying these experimental results to clinic treatment still requires further studies and development of biotechnology.

Abstract TP320: Endothelial Nitric Oxide Synthase (eNOS) Inhibition Abolishes the Exercise-Induced Improvement in Neurovascular and Neurobehavioral Stroke Outcomes

Background: Clinically, it was shown that increased physical activity reduces the risk of stroke and severity of outcomes. However, the underlying mechanism is not clear. Endothelial nitric oxide synthase (eNOS) activation was shown to improve vascular function and reduce the risk of cardiovascular diseases. However, the role of eNOS in mediating short term exercise-induced improvement in stroke outcome was not previously studied. Accordingly, we tested the hypothesis that acute short term pre-stroke exercise improves stroke outcomes through increased eNOS activity, and that eNOS inhibition abolishes these beneficial effects. Methods: Male Wistar rats (300 g) were subjected to high intensity interval training (HIIT) through treadmill running sessions for four days (25 minutes/day), break for 2 days and then one acute bout for 30 minutes. Exercised animals were subjected to thromboembolic stroke at 1h, 6h, 24h or 72h after the last exercise session. At 24h after stroke, sedentary and exercised rats were tested for neurological outcomes, infarct size and cerebral edema. Western Blotting was used to measure the expression of active eNOS (p-S1177-eNOS) in brain and cerebral vessels. Additional control and 1h exercise groups were treated with eNOS inhibitor (L-NIO (L-N 5 -1-Iminoethyl ornithine)) right after stroke (I.P. injection, dose: 20 mg/Kg). Results: Acute exercise significantly reduced infarct size, edema and improved functional outcomes, and increased the expression of peNOS in the brain and cerebral vessels. eNOS inhibition significantly increased the infarct size, edema and worsened the neurobehavioral outcomes in exercised animals compared to non-treated group (table). Conclusion: Short term acute pre-stroke exercise significantly reduced neurovascular injury and improved neurobehavioral and functional outcomes after stroke, and eNOS inhibition abolished these beneficial effects.

P6570Ambivalent role of eNOS in murine intima formation depends on caveolin-1 expression

P6570Ambivalent role of eNOS in murine intima formation depends on caveolin-1 expression

Effects of Linoleic and Alpha‐Linolenic Ratios and Concentrations on In Vitro Endothelial Cell Responses

Although many studies have found that two essential fatty acids, linoleic acid (LA; n‐6 polyunsaturated fatty acid, PUFA) and α‐linolenic acid (ALA; n‐3 PUFA), are closely related to the occurrence and development of cardiovascular disease (CVD), the effects of LA and ALA ratios and concentrations on CVD risk are not well known. The aim of this study is to investigate the effects of various LA/ALA ratios (1:1, 5:1, 10:1, and 20:1) and their concentrations on inflammation, oxidative stress, endothelial cell functions, and the mechanism in human umbilical vein endothelial cells (EA.hy926 cell line). The results indicate that low LA/ALA ratio might provide CVD benefits at low PUFA concentration while low LA/ALA ratio (1:1) increases CVD risk at high PUFA concentration. The results show that the effects of LA and ALA on inflammation, oxidative stress, endothelial cell functions, and the related gene expression are influenced by not only LA/ALA ratios but also their concentrations, and the mechanism may be mediated through PKCα/NF‐κB inflammatory and NO/eNOS signaling pathways by regulation of the NOX4 pathway.Practical Applications: Lipid metabolism, inflammation, oxidative stress, and endothelial cell functions play an important role in the pathogenesis of CVD, which may be affected by an imbalance in the LA/ALA ratio. However, the effects of LA/ALA ratios on CVD risk are influenced by PUFA concentrations. The study provides a method to design diets beneficial for CVD prevention.The low LA/ALA ratio (1:1) could up‐regulate increase the gene expression level of PKCα and eNOS gene while NF‐κB and NOX4 gene expression levels are up‐regulated to increase oxidative stress, which could cause the oxidation of BH4 and uncoupling of eNOS under high PUFA concentration. Uncoupling eNOS generated , instead of NO, which could damage vascular endothelial cell function. This is different from the role of eNOS in the production of NO under normal physiological conditions, which shows the duality of eNOS on atherosclerosis. The results indicate both LA and ALA ratios and concentrations are important for CVD risk.

A role for endothelial nitric oxide synthase in intestinal stem cell proliferation and mesenchymal colorectal cancer

BackgroundNitric oxide (NO) has been highlighted as an important agent in cancer-related events. Although the inducible nitric oxide synthase (iNOS) isoform has received most attention, recent studies in the literature indicate that the endothelial isoenzyme (eNOS) can also modulate different tumor processes including resistance, angiogenesis, invasion, and metastasis. However, the role of eNOS in cancer stem cell (CSC) biology and mesenchymal tumors is unknown.ResultsHere, we show that eNOS was significantly upregulated in VilCreERT2Apcfl/+ and VilCreERT2Apcfl/fl mouse intestinal tissue, with intense immunostaining in hyperproliferative crypts. Similarly, the more invasive VilCreERT2Apcfl/+Ptenfl/+ mouse model showed an overexpression of eNOS in intestinal tumors whereas this isoform was not expressed in normal tissue. However, none of the three models showed iNOS expression. Notably, when 40 human colorectal tumors were classified into different clinically relevant molecular subtypes, high eNOS expression was found in the poor relapse-free and overall survival mesenchymal subtype, whereas iNOS was absent. Furthermore, Apcfl/fl organoids overexpressed eNOS compared with wild-type organoids and NO depletion with the scavenger carboxy-PTIO (c-PTIO) decreased the proliferation and the expression of stem-cell markers, such as Lgr5, Troy, Vav3, and Slc14a1, in these intestinal organoids. Moreover, specific NO depletion also decreased the expression of CSC-related proteins in human colorectal cancer cells such as β-catenin and Bmi1, impairing the CSC phenotype. To rule out the contribution of iNOS in this effect, we established an iNOS-knockdown colorectal cancer cell line. NO-depleted cells showed a decreased capacity to form tumors and c-PTIO treatment in vivo showed an antitumoral effect in a xenograft mouse model.ConclusionOur data support that eNOS upregulation occurs after Apc loss, emerging as an unexpected potential new target in poor-prognosis mesenchymal colorectal tumors, where NO scavenging could represent an interesting therapeutic alternative to targeting the CSC subpopulation.

Abstract P196: Effect of Combined Oral Intake of Ginger Extract and Rice Vinegar on Blood Pressure with 2-kidney,1-clip Renovascular Hypertensive Rats

Objective: Ginger is widely used as traditional Asian herbal medicine. Ginger has the same pungent ingredient as chili and vanillyl. We showed that administration of capsaicin to renovascular hypertension (RH) model rats increased endothelial nitric oxide (NO) synthase (eNOS) mRNA expression and NO production, and suppressed blood pressure (BP). Traditionally in Japan, ginger is pickled and eaten. Ginger and vinegar each are supposed to have an effect of suppressing an increase in BP in RH rats. The aim of this study was to investigate the effect. Method: Male Sprague-Dawley rats (6wks) were treated with sham operation (SHAM) as controls or clipping the left renal artery (2K1C) as RH model. After surgery, the rats started receiving a control diet (C) or a diet with 0.08% (w/w) of Ginger Extract (GE) for 6 weeks, and a tap water (W) or a water with 4.5% (v/v) rice vinegar (V). The systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the protocol, the mean arterial BP (MAP) was measured under anesthesia. Then, the aortas were removed for extracting mRNA. mRNA for angiotensin type 2 receptor (AT 2 ) and eNOS was evaluated by real-time RT-PCR. Results: Through the experiment period, SBP was significantly effects in time, model (SHAM vs 2K1C), diet (C vs GE) , timeхanimal ( P &lt;0.001, each) and water (W vs V) ( P &lt;0.05). At the end of the protocol, 2K1C-C+W was higher in SBP than SHAM-C+W (176 ± 6 vs 138 ± 1 mmHg, P &lt;0.05). 2K1C-GE+W showed lower SBP (150 ± 2 mmHg) than -C+W ( P &lt;0.05). SBP was not significantly different in 2K1C-GE+V (149 ± 4mmHg) from in -GE+W. The observations in MAP were similar to those in SBP. AT 2 R mRNA expression showed significant effects in model ( P &lt;0.05) : the mRNA in 2K1C-C+W (0.9 ± 0.2) was significantly greater than in SHAM-C+W (0.4 ± 0.1) ( P &lt;0.05). There were no significant differences among the 2K1Cs: -C+W, -C+V (0.9 ± 0.1), -GE+W (0.8 ± 0.1) and -GE+V (0.9 ± 0.2). eNOS mRNA expression showed significant effects only in diet (CTL vs GE, P &lt;0.05), but not in water and any interactions. Conclusion: Continuous ingestion of GE and V may suppress BP increase in 2K1C, respectively. Simultaneous ingestion of GE and V showed no enhanced effects compared to GE or V solo ingestion in 2K1C. The roles of eNOS and AT 2 R in the mechanism did not become clear in this study.

The role of eNOS on the compensatory regulation of vascular tonus by H2S in mouse carotid arteries

The gasotransmitter nitric oxide (NO) has an important role in vascular function and a decrease in its bioavailability is accepted as a main pathological mechanism for cardiovascular diseases. However, other gasotransmitters such as hydrogen sulfide (H2S) are also generated by the endothelium and can also affect vascular tone and a crosstalk may exist between H2S and NO. We therefore investigated the consequences of deficiency, replacement or overexpression of endothelial nitric oxide synthase (eNOS) on H2S-induced vascular responses in murine carotid arteries. In pre-contracted carotid arteries from wild-type (WT) mice, l-cysteine elicited relaxation that was inhibited by the H2S synthesis inhibitor amino-oxyacetic acid (AOAA). Genetic deletion of eNOS increased l-cysteine-induced relaxation compared to WT, but the replacement of eNOS by adenoviral transfection or H2S synthesis inhibition by AOAA reversed it. Furthermore, eNOS deletion did not alter NaHS-induced relaxation in carotid arteries while eNOS overexpression/replacement increased NaHS-induced relaxation responses in carotid arteries from WT or eNOS−/−. We suggest that, endogenously produced H2S can compensate for impaired vasodilatory responses in the absence of NO to maintain vascular patency; while, eNOS abundance can limit endogenous H2S-induced vascular responses in mice carotid arteries. Our result suggests that endogenous vs. exogenous H2S-induced relaxation are reciprocally regulated by NO in mice carotid arteries.

Chronic Hypoxia uncouples Ca2+ and eNOS in bradykinin‐induced relaxation of Ovine pulmonary arteries

Bradykinin‐induced activation of the pulmonary endothelium triggers a rise in intracellular Ca2+, which activates a nitric oxide (NO)‐dependent signaling pathway. This pathway leads to vasodilation, thereby regulating pulmonary blood flow and 02 uptake. This vasodilation process includes stimulation of endothelial nitric oxide synthase (eNOS) and downstream activation of large‐conductance K+ (Maxi K+) channels. Chronic hypoxia (CH) is known to increase pulmonary pressures and restrict arterial relaxation, and can contribute to the development of pulmonary hypertension. We thus examined the effects of CH on bradykinin‐induced Ca2+ signals, bradykinin‐induced vasorelaxation, and the roles of eNOS and Maxi K+ channels in this relaxation. Wire‐myography and confocal microscopy studies were performed on pulmonary arteries (PA) from nonpregnant ewes that lived in a normoxic state at low altitude or a hypoxic state at high altitude (3,801 m) for &gt;100 days. eNOS was inhibited with 100 μM NG‐nitro‐L‐arginine methyl ester (LNAME), and Maxi K+ channels were blocked with 1mM tetraethylammonium (TEA). The data show CH augmented endothelial Ca2+ signals, but restricted bradykinin relaxation. Further, CH caused bradykinin‐induced contraction. LNAME sensitivity was restricted, which suggests eNOS dysfunction is central to the uncoupling of Ca2+ signals and bradykinin relaxation. CH also abolished TEA‐sensitivity in bradykinin relaxation, suggesting there is loss of Maxi K+ function following CH. Overall, these results suggest that CH causes uncoupling of endothelial Ca2+ signaling and eNOS function and mediates major changes in the mechanisms of membrane hyperpolarization. It follows that CH‐induced Ca2+‐eNOS uncoupling and Maxi K+ channel dysfunction are important mechanisms to examine as future therapeutic avenues for pulmonary hypertension.Support or Funding InformationSupport from NIH, NSF and Loma Linda University School of Medicine

The synthetic peptide PnPP-19 induces peripheral antinociception via activation of NO/cGMP/KATP pathway: Role of eNOS and nNOS

Backgroundand purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway. Experimental approachNociceptive thresholds were measured by paw pressure test. PGE2 (2 μg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot. Key resultsPnPP-19 (5, 10 and 20 μg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of KATP (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 μg/paw). Tissue nitrite concentration increased after PnPP-19 (10 μg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect. Conclusions and implicationsThe peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.

Role of eNOS‐ and NOX‐containing microparticles in endothelial dysfunction in patients with obesity

To explore the pathophysiological profile of patients who have obesity and to investigate the potential role of circulating microparticles (MPs) in endothelial dysfunction in patients who have obesity. The inflammatory and oxidative status and the cutaneous microvascular blood flow were characterized in 69 patients with android obesity and 46 subjects with normal weight (controls) by using laser Doppler flowmetry. Circulating MP levels were measured by flow cytometry, and endothelial nitric oxide synthase (eNOS) and NADPH oxidase (NOX) expression in MPs was investigated by Western blotting. MP effect on vascular reactivity was assessed in rat aorta rings. Patients with obesity showed endothelial dysfunction, hyperglycemia, inflammation, and oxidative stress. In controls, low MP levels were positively correlated with normal microvascular function. Western blot analysis revealed reduced eNOS and increased NOX4D expression in MPs from subjects with obesity compared with controls. However, this was not correlated with endothelial dysfunction parameters and did not impair ex vivo endothelium-dependent vasodilation. These results suggest that MPs do not contribute directly to endothelial dysfunction associated with obesity. Conversely, eNOS- and NOX-containing MPs could be involved in the compensatory mechanism of vascular endothelial cells to counteract the pathologic mechanisms underlying endothelial dysfunction.

Evidence for a novel role for eNOS in the regulation of hepatocellular mitophagy

INTRODUCTIONConsistent with the well characterized role for endothelial nitric oxide synthase (eNOS) to stimulate mitochondrial biogenesis, we have previously demonstrated that eNOS knockout (eNOS−/−) mice have reduced hepatic expression of PGC1α and TFAM in association with reduced mitochondrial respiratory capacity in western diet‐induced nonalcoholic steatohepatitis (NASH). Interestingly, hepatic mitochondrial content was preserved in eNOS−/−relative to wild‐type (WT) mice. This led us to hypothesize that eNOS concurrently regulates mitophagy (mitochondrial autophagy).METHODSWT and eNOS−/−mice were fed either a control or western‐style diet (WD; 45% fat, 1% cholesterol, 17% sucrose) for 18‐weeks. Whole liver homogenate and isolated hepatic mitochondria were assessed for markers of autophagy (cleaved microtubule‐associated protein 1A/1B‐light chain 3 [LC3‐II]) and mitophagy (BCL2/Adenovirus E1B 19kDa Interacting Protein 3 [Bnip3]). In a separate cohort of chow‐fed mice, primary hepatocytes from WT and eNOS−/− were isolated and cultured for 5‐days in a collagen matrix. Mitophagic flux was assessed via LC3‐II and Bnip3 accumulation following treatment for 16‐hours with 10 μM chloroquine (CQ; an inhibitor of lysosomal acidification) and 1 μM carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) to induce mitochondrial depolarization in the presence or absence of 500 μM free fatty acids (FFA; 250 μM oleate + 250 μM palmitate).RESULTSWD increased the mitochondrial localization of LC3‐II and Bnip3 in both genotypes (p &lt; 0.05, diet main effect), suggesting that WD increased hepatic mitophagic activity. However, eNOS−/−mice had reduced LC3‐II in liver homogenate (−45%) and isolated mitochondria (−28%) and lower Bnip3 (−25%) content in liver homogenate (genotype main effect; p &lt; 0.05). eNOS−/−primary hepatocytes exhibited a blunted accumulation of LC3‐II (−31%) and Bnip3 (−22%) following 16‐hours of CQ+CCCP treatment compared to WT cells, and failed to further respond to FFA exposure (+32% in Bnip3 in WT vs. eNOS−/− hepatocytes).CONCLUSIONSCollectively these data identify a hepatocellular autonomous function of eNOS in the regulation of mitophagy in response to metabolic insult. This suggests that dysregulation of hepatocellular eNOS may contribute to the accumulation of damaged mitochondria and to the etiology of NASH.Support or Funding InformationSupport: MU Life Sciences Pre‐doctoral Fellowship (RDS), AHA 14POST20110034 (EMM), NIH R01DK088940 (JPT), NIH HL‐36088 (MHL), VHA‐CDA2 IK2BX001299 (RSR).

Targeted overexpression of endothelial nitric oxide synthase in endothelial cells improves cerebrovascular reactivity in Ins2Akita-type-1 diabetic mice

Reduced bioavailability of nitric oxide due to impaired endothelial nitric oxide synthase (eNOS) activity is a leading cause of endothelial dysfunction in diabetes. Enhancing eNOS activity in diabetes is a potential therapeutic target. This study investigated basal cerebral blood flow and cerebrovascular reactivity in wild-type mice, diabetic mice (Ins2Akita+/−), nondiabetic eNOS-overexpressing mice (TgeNOS), and the cross of two transgenic mice (TgeNOS-Ins2Akita+/−) at six months of age. The cross was aimed at improving eNOS expression in diabetic mice. The major findings were: (i) Body weights of Ins2Akita+/− and TgeNOS-Ins2Akita+/− were significantly different from wild-type and TgeNOS mice. Blood pressure of TgeNOS mice was lower than wild-type. (ii) Basal cerebral blood flow of the TgeNOS group was significantly higher than cerebral blood flow of the other three groups. (iii) The cerebrovascular reactivity in the Ins2Akita+/− mice was significantly lower compared with wild-type, whereas that in the TgeNOS-Ins2Akita+/− was significantly higher compared with the Ins2Akita+/− and TgeNOS groups. Overexpression of eNOS rescued cerebrovascular dysfunction in diabetic animals, resulting in improved cerebrovascular reactivity. These results underscore the possible role of eNOS in vascular dysfunction in the brain of diabetic mice and support the notion that enhancing eNOS activity in diabetes is a potential therapeutic target.

0275 : Key role of eNOS in exercise-induced cardioprotection is dependent from coronary endothelium

To date, it is clearly accepted that the activation of endothelial isoform of NOS (eNOS) by exercise training constitutes a key trigger of exercise-induced cardioprotection against ischemia-reperfusion (IR). However, this enzyme is expressed both in coronary endothelial cells and cardiomyocytes, but the contribution of the one or the other one to such cardioprotection has never been challenged. The aim of this study was then to investigate the role of cardiomyocytes vs. endothelial eNOS in exercise cardioprotection. To this, rats were assigned to sedentary (Sed) or chronic aerobic exercised (Ex) group. Effects of exercise on vulnerability to IR or anoxia-reoxygenation (AR) were respectively evaluated at whole heart or cardiomyocytes and coronary artery levels. On a first set of rats, isolated cardiomyocytes were submitted to AR in presence or not of L-NAME, an eNOS inhibitor. Exercise reduced cells death and improved cells contractility after AR, but no effect of L-NAME was observed. Interestingly, exercise had no effect neither on eNOS phosphorylation on its activation site (Ser1177) nor on S-nitrosylation at cardiomyocytes level, whereas at whole heart level exercise increased both of them, suggesting that exercise impacted endothelial cells rather than cardiomyocytes. Then, to evaluate the contribution of endothelial cells on exercise-induced cardioprotection, on a Langendorff apparatus we treated hearts with Triton X-100 before IR to abolish coronary endothelial cells activity. Inactivation of endothelial cells totally suppressed cardioprotective effects of exercise. Finally, coronary arteries were isolated from hearts submitted to IR and endothelial function was assessed on a wire-myograph. We observed that alteration of endothelium-dependent coronary relaxation induced by IR was reduced in Ex group. In conclusion, these results show that coronary endothelial cells rather than cardiomyocytes play a key role in eNOS-dependent cardioprotection in Ex rat hearts.