ACh-induced Nitric Oxide Research Articles

Accelerated Electron Ionization-Induced Changes in the Myenteric Plexus of the Rat Stomach.

The influence of accelerated electrons on neuronal structures is scarcely explored compared to gamma and X-rays. This study aims to investigate the effects of accelerated electron radiation on some pivotal neurotransmitter circuits (cholinergic and serotonergic) of rats' myenteric plexus. Male Wistar rats were irradiated with an electron beam (9 MeV, 5 Gy) generated by a multimodality linear accelerator. The contractile activity of isolated smooth muscle samples from the gastric corpus was measured. Furthermore, an electrical stimulation (200 μs, 20 Hz, 50 s, 60 V) was performed on the samples and an assessment of the cholinergic and serotonergic circuits was made. Five days after irradiation, the recorded mechanical responses were biphasic-contraction/relaxation in controls and contraction/contraction in irradiated samples. The nature of the contractile phase of control samples was cholinergic with serotonin involvement. The relaxation phase involved ACh-induced nitric oxide release from gastric neurons. There was a significant increase in serotonergic involvement during the first and second contractile phases of the irradiated samples, along with a diminished role of acetylcholine in the first phase. This study demonstrates an increased involvement of serotonergic neurotransmitter circuits in the gastric myenteric plexus caused by radiation with accelerated electrons.

Methylglyoxal-Modified Albumin Effects on Endothelial Arginase Enzyme and Vascular Function.

Advanced glycation end products (AGEs) contribute significantly to vascular dysfunction (VD) in diabetes. Decreased nitric oxide (NO) is a hallmark in VD. In endothelial cells, NO is produced by endothelial NO synthase (eNOS) from L-arginine. Arginase competes with NOS for L-arginine to produce urea and ornithine, limiting NO production. Arginase upregulation was reported in hyperglycemia; however, AGEs' role in arginase regulation is unknown. Here, we investigated the effects of methylglyoxal-modified albumin (MGA) on arginase activity and protein expression in mouse aortic endothelial cells (MAEC) and on vascular function in mice aortas. Exposure of MAEC to MGA increased arginase activity, which was abrogated by MEK/ERK1/2 inhibitor, p38 MAPK inhibitor, and ABH (arginase inhibitor). Immunodetection of arginase revealed MGA-induced protein expression for arginase I. In aortic rings, MGA pretreatment impaired acetylcholine (ACh)-induced vasorelaxation, which was reversed by ABH. Intracellular NO detection by DAF-2DA revealed blunted ACh-induced NO production with MGA treatment that was reversed by ABH. In conclusion, AGEs increase arginase activity probably through the ERK1/2/p38 MAPK pathway due to increased arginase I expression. Furthermore, AGEs impair vascular function that can be reversed by arginase inhibition. Therefore, AGEs may be pivotal in arginase deleterious effects in diabetic VD, providing a novel therapeutic target.

Vena cava presents endothelial dysfunction prior to thoracic aorta in heart failure: the pivotal role of nNOS uncoupling/oxidative stress.

Arterial endothelial dysfunction has been extensively studied in heart failure (HF). However, little is known about the adjustments shown by the venous system in this condition. Considering that inferior vena cava (VC) tone could influence cardiac performance and HF prognosis, the aim of the present study was to assess the VC and thoracic aorta (TA) endothelial function of HF-post-myocardial infarction (MI) rats, comparing both endothelial responses and signaling pathways developed. Vascular reactivity of TA and VC from HF post-MI and sham operated (SO) rats was assessed with a wire myograph, 4 weeks after coronary artery occlusion surgery. Nitric oxide (NO), H2O2 production and oxidative stress were evaluated in situ with fluorescent probes, while protein expression and dimer/monomer ratio was assessed by Western blot. VC from HF rats presented endothelial dysfunction, while TA exhibited higher acetylcholine (ACh)-induced vasodilation when compared with vessels from SO rats. TA exhibited increased ACh-induced NO production due to a higher coupling of endothelial and neuronal NO synthases isoforms (eNOS, nNOS), and enhanced expression of antioxidant enzymes. These adjustments, however, were absent in VC of HF post-MI rats, which exhibited uncoupled nNOS, oxidative stress and higher H2O2 bioavailability. Altogether, the present study suggests a differential regulation of endothelial function between VC and TA of HF post-MI rats, most likely due to nNOS uncoupling and compromised antioxidant defense.

Synchronous detection of vascular tension and nitric oxide release in pulmonary artery: A combined application of confocal wire myograph with confocal laser scanning microscopy.

To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy. In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100-300 μM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate Calbiochem (2.5 μM), and then precontracted with phenylephrine (Phen, 10-6 M), and subsequently dilated in acetylcholine (ACh, 10-6 M - 10-4 M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10-4 M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10-6 M) followed by ACh (10-5 M). The Ach-induced vasodilation and NO release were recorded simultaneously. ACh (10-6 M - 10-4 M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10-4 M) significantly attenuated the vasodilatation and the intracellular NO release. This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.

Arginase overexpression and NADPH oxidase stimulation underlie impaired vasodilation induced by advanced glycation end products

BackgroundAdvanced glycation endproducts (AGEs) play a major role in the development of many vascular complications that are mediated by endothelial dysfunction. The present work aimed to investigate the mechanism by which AGEs impair vasodilation. MethodsThe effect of AGEs on vasodilation induced by acetylcholine or D NONOate was examined by incubating isolated rat aortae with different AGEs concentrations. ACh-induced nitric oxide generation was assessed using the fluorescent probe diaminofluorecein (DAF-FM). The effect of AGEs on expression of mRNA for arginase 2, NADPH oxidase and endothelial nitric oxide synthase (eNOS) were determined by real-time PCR. ResultsOne-hour in vitro incubation of rat aortae with AGEs impaired endothelial-dependent vasodilation produced by ACh, while increasing D NONOate-induced vasodilation. Preincubation of aortae with l-ornithine, an arginase 2-inhibitor, prevented the impairment effect induced by AGEs on endothelial-dependent vasodilation. Superoxide scavenging by tempol or NADPH oxidase inhibition by apocynin also blocked the effect of AGEs. AGEs decreased ACh-induced NO production and this was inhibited by both l-ornithine and apocynin. Furthermore, AGEs exposure increased arginase mRNA expression but decreased mRNA expression for eNOS in isolated rat aortae. ConclusionThe present results indicate that AGEs impairs endothelial-dependent vasodilation, and this effect is mediated via arginase overexpression and NADPH oxidase stimulation.

Exercise training induces eNOS coupling and restores relaxation in coronary arteries of heart failure rats.

Exercise training (ET) has emerged as a nonpharmacological therapy for cardiovascular diseases because of its helpful milieu for improving vascular function. The aim of the present study was to assess whether ET reverses the alterations in vascular reactivity observed in heart failure (HF)-related coronary arteries and to elucidate the molecular mechanisms involved in these adjustments. Male Wistar rats were subjected to either coronary artery ligation or sham operation. Four weeks after the surgery, rats were divided into two groups: untrained HF (UHF) and exercise-trained HF (THF). ET was conducted on a treadmill for 8 wk. An untrained SO group was included in the study as a normal control. ET restored the impaired acetylcholine (ACh)- and sodium nitroprusside-induced relaxation in coronary arteries to levels of the control. Oxidative stress and reduced nitric oxide (NO) production were observed in UHF, whereas ET restored both parameters to the levels of the control. Expression levels of endothelial NO synthase (eNOS) and soluble guanylyl cyclase subunits were increased in coronary arteries of UHF rats but reduced in THF rats. Tetrahydrobiopterin restored ACh-induced NO production in the UHF group, indicating that eNOS was uncoupled. ET increased the eNOS dimer-to-monomer ratio and expression of GTP cyclohydrolase 1, thus increasing NO bioavailability. Taken together, these findings demonstrate that ET reverses the dysfunction of the NO/soluble guanylyl cyclase pathway present in coronary arteries of HF rats. These effects of ET are associated with increased GTP cyclohydrolase 1 expression, restoration of NO bioavailability, and reduced oxidative stress through eNOS coupling. NEW & NOTEWORTHY The present study provides a molecular basis for the exercise-induced improvement in coronary arteries function in heart failure. Increasing the expression of GTP cyclohydrolase 1, the rate-limiting enzyme in the de novo biosynthesis of tetrahydrobiopterin, exercise training couples endothelial nitric oxide synthase, reduces oxidative stress, and increases nitric oxide bioavailability and sensitivity in coronary arteries of heart failure rats.

Acetylcholine induces intracellular Ca2+ oscillations and nitric oxide release in mouse brain endothelial cells

Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca2+ concentration recruits a multitude of endothelial Ca2+-dependent pathways, such as Ca2+/calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca2+ signaling in Ach-induced NO production in bEND5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca2+-sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Ach induced dose-dependent Ca2+ oscillations in bEND5 cells, 300 μM being the most effective dose to generate a prolonged Ca2+ burst. Pharmacological manipulation revealed that Ach-evoked Ca2+ oscillations required metabotropic muscarinic receptor (mAchR) activation and were patterned by a complex interplay between repetitive ER Ca2+ release via inositol-1,4,5-trisphosphate receptors (InsP3Rs) and store-operated Ca2+ entry (SOCE). A comprehensive real time-polymerase chain reaction analysis demonstrated the expression of the transcripts encoding for M3-mAChRs, InsP3R1 and InsP3R3, Stim1-2 and Orai2. Next, we found that Ach-induced NO production was hindered by L-NAME, a selective NOS inhibitor, and BAPTA, a membrane permeable intracellular Ca2+ buffer. Moreover, Ach-elicited NO synthesis was blocked by the pharmacological abrogation of the accompanying Ca2+ spikes. Overall, these data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels.

Acetylcholine induces intracellular Ca2+ oscillations and nitric oxide release in mouse brain endothelial cells

Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca2 + concentration recruits a multitude of endothelial Ca2 +-dependent pathways, such as Ca2 +/calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca2 + signaling in Ach-induced NO production in bEnd5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca2+-sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Overall, our data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels.

Glucose and angiotensin II-derived endothelial extracellular vesicles regulate endothelial dysfunction via ERK1/2 activation.

In various diseases, including diabetes, extracellular vesicles (EVs) have been detected in circulation and tissues. EVs are small membrane vesicles released from various cell types under varying conditions. Recently, endothelial cell-derived EVs (EEVs) were identified as a marker of endothelial dysfunction in diabetes, but the ensuing mechanisms remain poorly understood. In this study, we dissected the ensuing pathways with respect to nitric oxide (NO) production under the condition of type 2 diabetes. Human umbilical vein endothelial cells (HUVECs) were stimulated with glucose alone and with glucose in combination with angiotensin II (Ang II) for 48h. In supernatants from glucose + Ang II-stimulated HUVECs, release of EEVs was assessed using Western blotting with an anti-CD144 antibody. EEV release was significantly increased after stimulation of HUVECs, and high glucose + Ang II-derived EEVs impaired ACh-induced vascular relaxation responses and NO production in mice aortic rings. Furthermore, high glucose + Ang II-derived EEVs induced ERK1/2 signalling and decreased endothelial NO synthase (eNOS) protein expression in mice aortas. Furthermore, in the presence of the MEK/ERK1/2 inhibitor PD98059, high glucose plus Ang II treatment stimulated EEVs in HUVECs and those EEVs prevented the impairments of ACh-induced relaxation and NO production in mice aortas. These data strongly indicate that high glucose and Ang II directly affect endothelial cells and the production of EEVs; the resultant EEVs aggravate endothelial dysfunction by regulating eNOS protein levels and ERK1/2 signalling in mice aortas.

Abstract P623: Endothelial Dysfunction and Abnormal Perivascular Adipose Tissue Signaling in Subcutaneous Vessels from HIV-infected individuals

Objective: Perivascular adipose tissue (PVAT) normally promotes vascular endothelial function, whereas is impaired by reactive oxygen species (ROS) in CVD. But PVAT effects on microvessels in HIV are unexplored. We previously reported endothelial dysfunction in living subcutaneous microvascular arterioles (SMAs) dissected from a gluteal biopsy in HIV infected individuals. We test hypothesis that HIV increases microvascular ROS and asymmetric dimethylarginine (ADMA) leading to impair microvascular function and PVAT signaling. Methods: Isolated SMAs with or without PVAT were prepared from young African American HIV-infected (n=8) and matched HIV-uninfected women (n=6) enrolled in the DC Women’s Interagency HIV Study (DC-WIHS). HIV-infected participants were virally suppressed on HAART without identified CVD risk factors (except obesity). SMA’s acetylcholine (ACh)-induced endothelium dependent relaxation (EDR), nitric oxide (NO) activity (DAF-FM), ACh-induced endothelium dependent contractions (EDC) and ROS generation (temp-9AC), plasma L-arginine, ADMA and adipose adipokines and malondialdehyde (MDA) were measured. Results: HIV-infected participants had significantly ( p <0.05) reduced plasma ratio of L-arginine : ADMA (99 ± 13 vs 182 ± 32 μmol/μmol), increased adipose MDA (15.1 ± 2.5 vs 10.9 ± 2.6 ng/mg protein) and leptin (40 ± 9 vs 28 ± 7 ng/mg protein); and reduced adiponectin in plasma (14 ± 2 vs 23 ± 2 ng/ml) and in adipose (2.1 ± 0.3 vs 4.6 ± 1.3 ng/mg protein). ACh-induced EDR (57 ± 4 vs 71± 4%) and NO (0.20 ± 0.03 vs 0.58 ± 0.07 Δfluoresce unit) were significantly attenuated (p<0.05) in vessels from HIV-infected participants, whereas EDC (22 ± 2 vs 7 ± 2%) and O 2 - (0.17 ± 0.02 vs 0.09 ± 0.02 Δfluoresce unit) were significantly increased ( p <0.05) . PVAT significantly increased ( p <0.05) EDR (87 ± 3 vs 72 ± 4%) and NO (0.84 ± 0.09 vs 0.58 ± 0.08 Δfluoresce unit) only in control vessels. Conclusion: HIV-infected individuals have reductions in NO synthase substrate: inhibitor ratio (L-arginine:ADMA) and consequent increased intrinsic vascular effects of ROS leading to disruption of the beneficial microvascular PVAT signaling pathway. Therapeutic targets for vascular dysfunction in HIV should include ROS and its extravascular actions on ADMA and PVAT.

Acetylcholine induces nitric oxide production by inducing intracellular Ca2+ oscillations in mouse brain endothelial cells

Basal forebrain neurons control intracortical arterioles by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism by which Ach induces NO synthesis in brain ECs is still unknown. An increase in intracellular Ca2+ concentration ([Ca2+]i) recruits a multitude of endothelial Ca2+-dependent pathways, such as Ca2+/Calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca2+ signaling in Ach-induced NO production in bEnd5 cells, an established model of mouse brain microvascular ECs. Ach induced dose-dependent asynchronous Ca2+ oscillations in bEnd5 cells. Ach-evoked Ca2+ oscillations did not arise in the absence of external Ca2+ but rapidly resumed on Ca2+ restitution to the bath. However, nicotine, a selective agonist of the Ca2+-permeable nicotinic receptors, did not cause any detectable increase in [Ca2+]i. Pharmacological manipulation indeed revealed that Ach-induced Ca2+ spikes in bEnd5 cells are triggered by the interaction between intracellular Ca2+ release from InsP3 receptors (InsP3Rs) SOCE. Next, we found that Ach-induced NO production was hindered by L-NAME, a selective NOS inhibitor, and BAPTA, a membrane permeable intracellular Ca2+ buffer. Moreover, Ach-elicited NO synthesis was blocked by the pharmacological abrogation of the accompanying Ca2+ spikes. Ach stimulates bEnd5 cells by inducing a burst of intracellular Ca2+ spikes which is patterned by the interplay between ER-dependent Ca2+ mobilization and SOCE. Ach-elicited Ca2+ spikes result in NO production and are, therefore, predicted to control local CBF in mouse brain. Future experiments will assess whether this signaling pathway is altered in neurodegenerative disorders, such as Alzheimer’s Disease.

Ferulic acid enhances nitric oxide production through up-regulation of argininosuccinate synthase in inflammatory human endothelial cells

AimIn this study, we investigated the protective effect of ferulic acid (FA) on nitric oxide (NO) production in tumor necrosis factor (TNF)-α-stimulated inflammatory human umbilical vein endothelial cells (HUVECs), and elucidated the mechanism(s) involved. Main methodsThe TNF-α-stimulated inflammatory HUVECs were treated with acetylcholine (ACh) and/or FA. NO productions were measured by monitoring nitrite and nitrate using a 2,3-diaminonaphthalene Kit. Expressions of mRNA and proteins were evaluated by RT-PCR and Western blotting, respectively. Key findingsFA treatment resulted in a dose-dependent (10–200μM) restoration of ACh-mediated NO production in TNF-α-treated HUVECs, whereas treatment with the FA analogues, coumaric acid, and apocynin resulted in no significant effect. FA treatment had no effect on O2− production in TNF-α-stimulated HUVECs. NG-monomethyl-l-arginine acetate (a nitric oxide synthase (NOS) inhibitor) and α-methyl-dl-aspartic acid (an argininosuccinate synthase (ASS) inhibitor) counteracted the effects of FA on the NO production. While FA treatment did not significantly affect the protein expression of p-eNOS or eNOS, the protein expression of ASS as well as mRNA expression was restored to normal levels upon exposure to FA in TNF-α-stimulated HUVECs. In nucleus, FA attenuated the increase of nuclear factor-kappa B (NF-κB) expression by TNF-α. SignificanceFA treatment rescues the defect in ACh-induced NO production resulting from TNF-α-stimulation in inflammatory HUVECs. This effect was likely due, in part, to the FA-mediated up-regulation of ASS expression via the suppression of NF-κB inflammatory signaling cascade.

Abstract 290: Endothelial Dysfunction in a Rat Model of Asphyxia-Induced Cardiac Arrest

Background: Altered vasoreactivity and elevated markers of endothelial activation are found following cardiac arrest (CA) and resuscitation. However, it is unknown whether the described altered vasoreactivity reflects influence from humoral factors, affection of the underlying smooth muscle or a change in the endothelium itself. Furthermore, it is unknown how the inflammatory markers correlate to endothelial function. The aim of the present study was to assess endothelial function and the relation to plasma markers of inflammation and adhesion, CA and resuscitation. Methods: Male Sprague Dawley rats underwent 5 min. of asphyxia-induced CA and subsequent resuscitation. After return of spontaneous circulation (ROSC) animals were euthanized after 30 min. or 2 hrs. Sham operated animals were used as controls. At the end of the experiment blood was drawn and analyzed for IL-1β, IL-6, IL-10, TNF-α, ICAM-1, VCAM-1, VEGF-α, vWF and sP-Selectin. Segments of the Left coronary artery (LCA) and mesenteric resistance arteries were mounted in microvascular myographs. Concentration-response curves for acetylcholine (ACH), NS309, and sodium nitroprusside where obtained. Results: LCA-segments exhibited an impaired vasodilation to ACH after 30 min. of ROSC as CA animals dilated to a max of 66 ± 2 vs. 94 ± 8% in the sham group (Mean ± SE; P=0.0014). After 2 hrs. of ROSC vasoreactivity was comparable to sham groups for all drugs tested. The mesenteric arteries exhibited a larger response to NS309 after 2 hrs. of ROSC compared to 2 hr. sham (logEC50 ± SE: 0.43 ± 0.05 vs. 1.85 ± 0.23 µM; P<0,001). Levels of sP-selectin, vWF and sVCAM-1 were elevated in both CA groups vs. sham groups, with no correlation to vasoreactivity. Conclusion: Endothelium-dependent ACH-induced nitric oxide (NO)-mediated vasodilation was blunted in LCA segments in the early post-resuscitation phase, NS309-induced endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation was enhanced in mesenteric resistance arteries in the intermediate post-resuscitation phase. There was no relation of vasoreactivity to plasma markers of adhesion and inflammation. The altered vascular reactivity may explain impaired coronary flow and hypotension in the restitution phase after cardiac arrest.

Rosiglitazone, a peroxisome proliferator‐activated receptor γ stimulant, abrogates diabetes‐evoked hypertension by rectifying abnormalities in vascular reactivity

In addition to insulin sensitization, rosiglitazone exhibits favourable circulatory effects. In the present study, we tested the hypothesis that rosiglitazone protects against hypertension and vascular derangements caused by diabetes. Diabetes was induced by a single bolus injection of streptozotocin (50 mg/kg, i.p.). After 2 weeks, rats were started on a treatment regimen of 5 mg/kg rosiglitazone daily for a period of 6 weeks. The control group consisted of rats treated with vehicle (distilled water) for the same period of time. After 6 weeks treatment, blood pressure (BP) was recorded and serum levels of glucose, advanced glycation end-products (AGE), triglycerides, total cholesterol and low-density lipoprotein-cholesterol (LDL-C) were determined. In in vitro experiments, concentration-response curves were constructed to phenylephrine (PE), KCl and acetylcholine (ACh) in thoracic aorta rings. In addition, ACh-induced nitric oxide (NO) generation and KCl-induced intracellular Ca accumulation were determined in the aorta. Compared with values in control rats, both diastolic and systolic BP were increased in diabetic rats. Rosiglitazone treatment of diabetic rats abolished the increase in diastolic BP and significantly reduced the increased systolic BP without affecting the development of hyperglycaemia. The possibility that changes in vascular reactivity and/or lipid profile contributed to the effects of rosiglitazone on BP in diabetic rats was investigated. In aortic rings from diabetic rats, contractile responses to KCl were increased, whereas the relaxant responses to ACh were decreased. In rings from diabetic rosiglitazone-treated rats, the exaggerated response to KCl and the impaired response to ACh were abolished. Furthermore, rosiglitazone abrogated impaired ACh-stimulated NO generation in aortas isolated from diabetic rats. Diabetes in rats was accompanied by elevated levels of triglycerides, total cholesterol, LDL-C and AGE. Rosiglitazone treatment abrogated the increased levels of triglycerides, total cholesterol and LDL-C, but only partially reduced AGE levels. Collectively, these observations indicate that rosiglitazone abrogates diabetes-evoked hypertension by ameliorating detrimental changes in vascular reactivity and lipid profiles.

Pentoxifylline alleviates vascular impairment in insulin resistance via TNF-α inhibition

Deterioration of vascular reactivity plays a pivotal role in vascular complications. Pentoxifylline (PTX) is a well-tolerated drug used to treat vascular insufficiency. We investigated the protective effect of PTX against vascular impairment in insulin resistance. Insulin resistance was induced by fructose (10%) in drinking water while PTX was concurrently administered (50mg/kg(-1)) for 8weeks. Serum levels of glucose, insulin, tumor necrosis factor alpha (TNF-α) were determined. Isolated aorta reactivity to phenylephrine (PE), potassium chloride (KCl), and acetylcholine (ACh) was studied, as was nitric oxide (NO) generation and histopathology. Insulin resistance was accompanied with a significant elevation in serum TNF-α level, marked leukocytes infiltration, and endothelial pyknosis. PTX inhibited insulin resistance and prevented TNF-α elevation, leukocyte infiltration and endothelial pyknosis. Vascular dysfunction was evident in insulin resistance as increased vascular contractility to PE and decreased relaxation to ACh, whereas PTX protected against this dysfunction. Notably, in vitro incubation with TNF-α (1ng/ml(-1)) increased contractility to PE and decreased relaxation to ACh while concomitant PTX (1mM) incubation partially restored response to ACh but not to PE. Furthermore, TNF-α reduced ACh-induced NO generation, whereeas PTX restored it. In conclusion, PTX protects from the impairment in vascular reactivity in insulin resistance, by a mechanism involving TNF-α inhibition.

Mechanisms underlying the losartan treatment-induced improvement in the endothelial dysfunction seen in mesenteric arteries from type 2 diabetic rats

It is well known that type 2 diabetes mellitus is frequently associated with vascular dysfunction and an elevated systemic blood pressure, yet the underlying mechanisms are not completely understood. We previously reported that in mesenteric arteries from established type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats, which exhibit endothelial dysfunction, there is an imbalance between endothelium-derived vasodilators [namely, nitric oxide (NO) and hyperpolarizing factor (EDHF)] and vasoconstrictors [contracting factors (EDCFs) such as cyclooxygenase (COX)-derived prostanoids]. Here, we investigated whether the angiotensin II receptor antagonist losartan might improve endothelial dysfunction in OLETF rats at the established stage of diabetes. In mesenteric arteries isolated from OLETF rats [vs. those from age-matched control Long-Evans Tokushima Otsuka (LETO) rats]: (1) the acetylcholine (ACh)-induced relaxation was impaired, (2) the NO- and EDHF-mediated relaxations were reduced, (3) the ACh-induced EDCF-mediated contraction and the production of prostanoids were increased, and (4) superoxide generation was increased. After such OLETF rats had received losartan (25 mg/kg/day p.o. for 4 weeks), their isolated mesenteric arteries exhibited: (1) improvements in ACh-induced NO- and EDHF-mediated relaxations, (2) reduced EDCF- and arachidonic acid-induced contractions, (3) suppressed production of prostanoids, (4) reduced PGE 2-mediated contraction, and (5) reduced superoxide generation. Within the timescale studied here, losartan did not change the protein expressions of endothelial NO synthase, COX1, or COX2 in mesenteric arteries from either OLETF or LETO rats. Losartan thus normalizes vascular dysfunction in this type 2 diabetic model, and the above effects may contribute to the reduction of adverse cardiovascular events seen in diabetic patients treated with angiotensin II receptor blockers.

Effects of Chronic Treatment With the CB1 Antagonist, Rimonabant on the Blood Pressure, and Vascular Reactivity of Obese Zucker Rats

Rimonabant (RM) is a cannabinoid CB1 receptor antagonist useful in the treatment of obesity associated cardiovascular risk factors. Since cannabinoids are vasoactive compounds, the aim of this study is to evaluate the effect of chronic treatment with RM on systolic blood pressure (SBP), and endothelial and vascular reactivity. Obese Zucker rats (OZRs) and their lean counterparts were orally treated during 20 weeks with either RM (10 mg/kg/day). Endothelial and vascular function was assessed in aorta and small mesenteric arteries (SMAs) by concentration response curves to acetylcholine (ACh) and phenylephrine (Phe), respectively. Participation of nitric oxide (NO) was evaluated by incubation with the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) and cyclooxygenase (COX)-derived products involvement was analyzed by incubation with indomethacin (INDO). Plasma lipid profile, insulin and adiponectin were also analyzed. Sympathetic activity was evaluated by urinary excretion of noradrenaline. As expected, RM decreased body weight gain and enhanced adiponectin concentration. Insulin resistance and sympathetic activity were also decreased. The increase in SBP observed in OZRs was reduced by treatment with RM. Aortae and SMAs from OZRs exhibited lower contractile response to Phe, being this effect prevented by RM administration. Although ACh-induced response and NO participation remained unaltered with obesity, enhanced COX-derived constrictor products were found in OZRs. RM treatment neither altered endothelium-dependent relaxation nor L-NAME-sensitive component of the response. Nevertheless, it was able to regulate COX-derived vasoactive products participation. Those effects may contribute to explain some of the cardiovascular protective actions elicited by this drug.

Ovariectomy increases the formation of prostanoids and modulates their role in acetylcholine-induced relaxation and nitric oxide release in the rat aorta

This study examines the effect of ovarian function on thromboxane A(2) (TXA(2)), prostaglandin (PG) I(2), PGF(2alpha), and PGE(2) release as well as the role of these substances in nitric oxide (NO) release and acetylcholine (ACh)-mediated relaxation. Aortic segments from ovariectomized and control female Sprague-Dawley rats were used. Cyclooxygenase (COX-1 and COX-2) expression was studied. ACh-induced relaxation was analysed in the absence and presence of the COX-2 inhibitor NS-398, the TXA(2) synthesis inhibitor furegrelate, the PGI(2) synthesis inhibitor tranylcypromine (TCP), or the thromboxane-prostanoid receptor antagonist SQ-29548. TXA(2), PGI(2), PGF(2alpha), and PGE(2) release was measured, and the vasomotor effect of exogenous TXA(2), PGI(2,) PGF(2alpha), and PGE(2) was assessed. Basal and ACh-induced NO release in the absence and presence of NS-398, furegrelate, TCP, or TCP plus furegrelate was studied. Ovariectomy did not alter or increased COX-1 or COX-2 expression, respectively. NS-398 decreased, and furegrelate did not change, the ACh-induced relaxation in arteries from both groups. SQ29,548 decreased the ACh-induced relaxation only in aortas from ovariectomized rats. TCP decreased the ACh-induced relaxation in both groups, and furegrelate or SQ29,548 totally restored that response only in aortas from control rats. Ovariectomy increased the ACh-induced TXA(2), PGI(2), and PGE(2) release and the contractile responses induced by exogenous TXA(2), PGF(2alpha), or PGE(2), while it decreased the PGI(2)-induced vasodilator response. In aortas from control rats, NS-398 did not alter the ACh-induced NO release, and furegrelate, TCP, or TCP plus furegrelate increased that release. In arteries from ovariectomized rats, NS-398, furegrelate, TCP, or TCP plus furegrelate decreased the ACh-induced NO release. Despite the prevalence of vasoconstrictor prostanoids derived from COX-2 in aortas from ovariectomized rats, the ACh-induced relaxation is maintained, probably as consequence of the positive regulation that prostanoids exert on eNOS activity.

Eicosapentaenoic Acid Improves Imbalance between Vasodilator and Vasoconstrictor Actions of Endothelium-Derived Factors in Mesenteric Arteries from Rats at Chronic Stage of Type 2 Diabetes

Accumulating evidence demonstrates that dietary intake of n-3 polyunsaturated fatty acids (PUFAs) is associated with a reduced incidence of several cardiovascular diseases that involve endothelial dysfunction. However, the molecular mechanism remains unclear. We previously reported that mesenteric arteries from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats exhibit endothelial dysfunction, leading to an imbalance between endothelium-derived vasodilators [namely, nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)] and vasoconstrictors [endothelium-derived contracting factors (EDCFs)] [namely cyclooxygenase (COX)-derived prostanoids] (Am J Physiol Heart Circ Physiol 293:H1480-H1490, 2007). We hypothesized that treating OLETF rats with eicosapentaenoic acid (EPA), a major n-3 PUFA, may improve endothelial dysfunction by correcting this imbalance. In OLETF rats [compared with age-matched control Long-Evans Tokushima Otsuka (LETO) rats]: 1) acetylcholine (ACh)-induced (endothelium-dependent) relaxation was impaired, 2) NO- and EDHF-mediated relaxations and nitrite production were reduced, and 3) ACh-induced EDCF-mediated contraction, production of prostanoids, and the protein expressions of COX-1 and COX-2 were all increased. When OLETF rats received chronic EPA treatment long-term (300 mg/kg/day p.o. for 4 weeks), their isolated mesenteric arteries exhibited: 1) improvements in ACh-induced NO- and EDHF-mediated relaxations and COX-mediated contraction, 2) reduced EDCF- and arachidonic acid-induced contractions, 3) normalized NO metabolism, 4) suppressed production of prostanoids, 5) reduced COX-2 expression, and 6) reduced phosphoextracellular signal-regulated kinase (ERK) expression. Moreover, EPA treatment reduced both ERK2 and nuclear factor (NF)-kappaB activities in isolated OLETF aortas. We propose that EPA ameliorates endothelial dysfunction in OLETF rats by correcting the imbalance between endothelium-derived factors, at least partly, by inhibiting ERK, decreasing NF-kappaB activation, and reducing COX-2 expression.

ROLE OF NAOFEN, A NOVEL WD REPEAT‐CONTAINING PROTEIN, IN REDUCING NITRIC OXIDE‐INDUCED RELAXATION

1. Naofen, a novel WD40 repeat domain-containing protein, has recently been found in the intracellular compartment. The aim of the present study was to determine whether naofen affects thoracic aortic vascular reactivity in normotensive and hypertensive rats and whether naofen is present in the thoracic aorta. In addition, we examined whether naofen modulates acetylcholine (ACh)-stimulated nitric oxide (NO) release from the endothelium. 2. Immunohistochemistry showed greater naofen expression in endothelial cells in the DOCA-salt group compared with controls. There was increased naofen mRNA expression in deoxycorticosterone acetate (DOCA)-salt hypertensive rats compared with normotensive rats. 3. Acetylcholine-induced relaxation of rat aortic strips was decreased in DOCA-salt hypertensive rats compared with normotensive rats. Naofen-N- but not naofen-C-terminal protein caused a significant decrease in ACh-induced relaxation of aortic strips from normotensive rats. 4. Using a nitrite assay in a murine aortic endothelial cell line demonstrated that naofen-N-terminal protein, but not naofen-C-terminal protein, significantly reduced ACh-induced NO production, suggesting that naofen interferes with NO production. 5. Administration of naofen-N-terminal protein, but not naofen-C-terminal protein, significantly inhibited cyclohydrolase (GCH) I mRNA expression in a murine aortic endothelial cell line, suggesting that naofen-N-terminal protein interferes with NO synthesis by inhibiting GCH I mRNA expression. 6. The results of the present study suggest that naofen is present in vascular endothelial cells and has an inhibitory effect on ACh-induced relaxation under normotensive conditions. The findings reinforce the functional significance of naofen-N-terminal protein on rat vascular reactivity.