Endothelial Nitric Oxide Release Research Articles

Acute-on-chronic liver disease enhances phenylephrine-induced endothelial nitric oxide release in rat mesenteric resistance arteries through enhanced PKA, PI3K/AKT and cGMP signalling pathways

Acute-on-chronic liver disease is a clinical syndrome characterized by decompensated liver fibrosis, portal hypertension and splanchnic hyperdynamic circulation. We aimed to determine whether the alpha-1 agonist phenylephrine (Phe) facilitates endothelial nitric oxide (NO) release by mesenteric resistance arteries (MRA) in rats subjected to an experimental microsurgical obstructive liver cholestasis model (LC). Sham-operated (SO) and LC rats were maintained for eight postoperative weeks. Phe-induced vasoconstriction (in the presence/absence of the NO synthase –NOS- inhibitor L-NAME) and vasodilator response to NO donor DEA-NO were analysed. Phe-induced NO release was determined in the presence/absence of either H89 (protein kinase –PK- A inhibitor) or LY 294002 (PI3K inhibitor). PKA and PKG activities, alpha-1 adrenoceptor, endothelial NOS (eNOS), PI3K, AKT and soluble guanylate cyclase (sGC) subunit expressions, as well as eNOS and AKT phosphorylation, were determined. The results show that LC blunted Phe-induced vasoconstriction, and enhanced DEA-NO-induced vasodilation. L-NAME increased the Phe-induced contraction largely in LC animals. The Phe-induced NO release was greater in MRA from LC animals. Both H89 and LY 294002 reduced NO release in LC. Alpha-1 adrenoceptor, eNOS, PI3K and AKT expressions were unchanged, but sGC subunit expression, eNOS and AKT phosphorylation and the activities of PKA and PKG were higher in MRA from LC animals. In summary, these mechanisms may help maintaining splanchnic vasodilation and hypotension observed in decompensated LC.

Ethanol enhances endothelial ionic currents and nitric oxide release via intermediate-conductance calcium-activated potassium channel

AimsEthanol is known to induce NO release and coronary vasorelaxation. Evidence suggests that K+ channels, especially a Ca2+-activated K+ channel (KCa), may regulate endothelial NO production. We aimed to investigate the ethanol effect on K+ currents in human coronary artery endothelial cells (HCAECs), identify the K+ channel type/subtype and signaling pathway involved, and demonstrate the relevance to ethanol-induced NO release. Main methodsIonic currents of cultured HCAECs were studied using whole-cell patch clamp technique. NO production were measured using the fluorescent probe, 2,3-diaminonaphthalene. Key findingsWe found that ethanol significantly potentiated HCAEC current (maximal increase to 155.68 ± 18.93%, 20 mM ethanol, +80 mV; mean ± SEM, n = 9). Ethanol-induced current was significantly inhibited by blockers of IKCa or SKCa (intermediate- or small-conductance KCa), but not by blocking other K+ channels. When other known HCAEC channels were inhibited except IKCa, 20 mM ethanol significantly increased IKCa current to 198 ± 25.11% (n = 6), but it could not enhance SKCa current that was similarly isolated. Moreover, ethanol-induced NO release was prevented by blocking IKCa channel, adenosine A2A receptor (A2AR), Gs protein, or protein kinase A (PKA). SignificanceThis study was the first to demonstrate that acute ethanol exposure could activate endothelial IKCa channel, via A2AR-Gs-PKA signaling, leading to increased whole-cell current and NO release, which could be an important mechanism underlying ethanol-induced NO release and vasodilation.

Microparticle Responses to Aerobic Exercise and Meal Consumption in Healthy Men.

Microparticles (MP) are shed extracellular vesicles that express the prothrombotic tissue factor (TF). Aerobic exercise may reduce MP count and TF expression. This study investigated the impact of acute running or rest followed by standardized meal consumption on MP phenotypes and TF expression. Fifteen males (age, 22.9 ± 3.3 yr; body mass, 81.9 ± 11.4 kg; V˙O2max, 54.9 ± 6.5 mL·kg·min; mean ± SD) completed 1 h of running (70% V˙O2max) or rest at 9:00 AM and consumed a standardized meal (1170 kcal, 43% CHO, 17% PRO, 40% fat) at 10:45 AM. Venous blood samples were taken at 9:00 AM, 10:00 AM, and 11:30 AM. The MP concentration, diameter, phenotypes, and TF expression were assessed using nanoparticle tracking analysis and flow cytometry. Nanoparticle tracking analysis identified no changes in MP concentration or diameter in response to time or trial. Flow cytometry revealed total MP count increased from 9:00 AM to 10:00 AM (1.62 ± 2.28 to 1.74 ± 2.61 × 10 L, P = 0.016, effect size (η) = 0.105), but was unaffected by trial. TF platelet-derived MP % reduced from 9:00 AM to 10:00 AM (44.0% ± 21.2% to 21.5% ± 9.3%, P = 0.001, η = 0.582) after exercise only (control, 36.8% ± 18.2% to 34.9% ± 11.9%; P = 0.972). TF neutrophil-derived MP percentage reduced from 9:00 AM to 11:30 AM (42.3% ± 17.2% to 25.1% ± 14.9%; P = 0.048, η = 0.801) in the exercise trial only (control, 28.5% ± 15.7% to 32.2% ± 9.6%; P = 0.508). Running induced a significant reduction in %TF platelet and neutrophil MP, suggesting a transient reduction in cardiovascular risk via reduced TF-stimulated thrombosis. This requires further investigation over longer periods in cardiovascular disease populations.

Bupivacaine-induced contraction is attenuated by endothelial nitric oxide release modulated by activation of both stimulatory and inhibitory phosphorylation (Ser1177 and Thr495) of endothelial nitric oxide synthase

This study examined the mechanism associated with the endothelium-dependent attenuation of vasoconstriction induced by bupivacaine (BPV), with a particular focus on the upstream cellular signaling pathway of endothelial nitric oxide synthase (eNOS) phosphorylation induced by BPV in human umbilical vein endothelial cells (HUVECs). BPV concentration-response curves were investigated in the isolated rat aorta. The effects of Nω-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), methylene blue, calmidazolium, the Src kinase inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and the combination of L-arginine and L-NAME on BPV-induced contraction in endothelium-intact aorta preparations were examined. The effects of BPV alone and in combination with PP2 on the phosphorylation of eNOS (at Ser1177 or Thr495), caveolin-1 and Src kinase were examined in HUVECs. BPV-induced contraction was lower in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, methylene blue and calmidazolium increased BPV-induced contraction in endothelium-intact aortae, whereas PP2 alone and combined treatment with L-arginine and L-NAME inhibited BPV-induced contraction. Low-concentration BPV (30 µM) induced both stimulatory (Ser1177) and inhibitory (Thr495) phosphorylation of eNOS in HUVECs. However, high-concentration BPV (150 µM) induced only stimulatory (Ser1177) eNOS phosphorylation. Additionally, phosphorylation of Src kinase, caveolin-1 and inhibitory eNOS (Thr495) induced by low-concentration BPV was inhibited by PP2. These results suggest that contraction induced by low-concentration BPV is attenuated by endothelial nitric oxide release, which is modulated both stimulatory (Ser1177) and inhibitory eNOS phosphorylation (Thr495). BPV-induced phosphorylation of eNOS (Thr495) is indirectly mediated by an upstream cellular signaling pathway involving Src kinase (Tyr416) and caveolin-1 (Tyr14).

Hemolysis and Tricuspid Regurgitation Jet Velocity Predict Mortality in Patients with Sickle Cell Disease

IntroductionOver the last two decades, sickle cell disease has become a model of diffuse endotheliopathy. In 2004, Gladwin and colleagues demonstrated that elevated tricuspid regurgitation jet velocity (TRJ) was an independent predictor of mortality and that chronic red blood cell hemolysis underlies pulmonary vascular disease (Gladwin et. al. NEJM, PMID:14985486). Belhassen and colleagues demonstrated that flow-mediated dilation of the brachial artery (FMD) was decreased in patients with sickle cell disease, specifically shear-mediated vasodilation (Belhassen et. al. Blood, PMID:11238095). Our group demonstrated that plasma free hemoglobin links decreased FMD and elevated TRJ in patients with sickle cell disease and that chronic transfusion therapy improves FMD (Detterich et. al. Blood, PMID:26036801). We aimed to determine whether markers of vascular function predict the risk of mortality in a cohort of patients with sickle cell disease.MethodsThis was a single center, prospective cohort study of non-transfused and chronically transfused sickle cell anemia patients at Children's Hospital of Los Angeles between 2009 and 2012 aiming to assess vascular function in sickle cell disease. This study was approved by the Children's Hospital Los Angeles Institutional Review Board. We used FMD, a test of shear-mediated endothelial nitric oxide release as the model for vascular function. We simultaneously measured the regional hemoglobin oxygen saturation in the hand using near infrared spectroscopy (NIRS). We also measured microcirculatory perfusion using laser doppler flowmetry (LDF) over the skin of the nailbed. Echocardiography was performed on the same day to assess tricuspid regurgitation jet velocity and cardiac function. Markers of inflammation, cell count and hemolysis were also measured on the day of the study. We prospectively followed them until 2018 to assess our primary outcome, survival. All of the measurements were made pre-transfusion for chronically transfused subjects. Kaplan Meier and Cox-Proportional Hazards multivariate model was used to determine independent predictors of survival. All statistics were performed using Stata/IC version 14.0.ResultsWe enrolled 26 chronically transfused patients with SCD and 63 non-transfused SCD patients in a study assessing the effects of hemolysis on endothelial function. Overall mortality in our cohort was 20% with a median follow up time of 7.8 years (7.1-8.3 IQR). Median age was 24 years at the time of study (17-36 IQR). Median age at the time of death was 36 years(28-51 IQR) and at the last follow up for the alive patients was 30 years (25-41 IQR), p=0.01. In the patients who died, they had higher TRJ (median 270 vs 235, p=0.03) and had elevated plasma Hgb, median 26 vs 14, p=0.01. FMD, regional oxygen saturation and microcirculatory perfusion were not different between alive and deceased patients. There was no difference for inflammatory markers (white blood cell count, platelet count, high sensitivity C-reactive protein), hemoglobin S%/transfusion, or anemia (hemoglobin/hematocrit/reticulocytes) between alive or deceased patients. By Kaplan Meier analysis individual factors associated with mortality were TRJ>250 (p=0.04) and age>21 (p=0.02) (Fig 1 and Fig 2). By Cox-Proportional Hazards Model, both TR jet velocity and plasma hemoglobin remain significant after correcting for age (Table 1).ConclusionsIn our cohort, there are multiple abnormal markers of vascular function and elevated markers of hemolysis; however, the strongest predictors of mortality were age, plasma free hemoglobin and tricuspid regurgitation jet velocity. These findings support the association between hemolysis, pulmonary vascular disease and mortality, a controversial topic in sickle cell disease. [Display omitted] DisclosuresCoates:Vifor Pharma: Consultancy; Sangamo: Consultancy, Honoraria; Celgene Corp.: Consultancy; ApoPharma: Consultancy, Honoraria.

Nano-medicine and Vascular Endothelial Dysfunction: Options and Delivery Strategies.

The endothelium is a thin innermost layer of flat cells which release various mediators including endothelin-1 (ET-1), prostanoids, von Willebrand factor (vWF) and endothelium-derived relaxing factor (EDRF; nitric oxide) to regulate vascular tone. Endothelial nitric oxide synthase (eNOS) is a key enzyme that generates nitric oxide (NO). NO maintains vascular homeostasis and cardiac functions by influencing major vascular protective properties such as anti-platelet, anti-proliferative, anti-migratory, antioxidant and anti-inflammatory action in vessels. Abnormal endothelial production and release of NO lead to vascular endothelial dysfunction (VED) and further leads to pathogenesis in myocardial and other tissues. Numerous pharmacological agents such as angiotensin-converting enzyme inhibitors, statins, calcium channel blockers, ET-1 receptor antagonists, insulin sensitizers, antioxidants and supplements like tetrahydrobiopterin, arginine and folate have been implicated in the treatment of VED, but their therapeutic potency was restricted due to some unavoidable adverse effects. The new era with advances in nanotechnology and its ability to target a specific disease, nano-medicine explored an innovative gateway for advanced therapy for VED. The present commentary reveals the various available, pipeline nano-medicine, their interaction with endothelium and in other associated pathological conditions and their delivery strategies for target-specific treatment of VED.

The renal and blood pressure response to low sodium diet in P2X4 receptor knockout mice.

In the kidney, purinergic (P2) receptor‐mediated ATP signaling has been shown to be an important local regulator of epithelial sodium transport. Appropriate sodium regulation is crucial for blood pressure (BP) control and disturbances in sodium balance can lead to hypo‐ or hypertension. Links have already been established between P2 receptor signaling and the development of hypertension, attributed mainly to vascular and/or inflammatory effects. A transgenic mouse model with deletion of the P2X4 receptor (P2X4−/−) is known to have hypertension, which is thought to reflect endothelial dysfunction and impaired nitric oxide (NO) release. However, renal function in this model has not been characterized; moreover, studies in vitro have shown that the P2X4 receptor can regulate renal epithelial Na+ channel (ENaC) activity. Therefore, in the present study we investigated renal function and sodium handling in P2X4−/− mice, focusing on ENaC‐mediated Na+ reabsorption. We confirmed an elevated BP in P2X4−/− mice compared with wild‐type mice, but found that ENaC‐mediated Na+ reabsorption is no different from wild‐type and does not contribute to the raised BP observed in the knockout. However, when P2X4−/− mice were placed on a low sodium diet, BP normalized. Plasma aldosterone concentration tended to increase according to sodium restriction status in both genotypes; in contrast to wild‐types, P2X4−/− mice did not show an increase in functional ENaC activity. Thus, although the increased BP in P2X4−/− mice has been attributed to endothelial dysfunction and impaired NO release, there is also a sodium‐sensitive component.

Eicosapentaenoic acid inhibited oxidized HDL-induced loss of endothelial nitric oxide release as compared to fenofibrate or niacin in vitro

Aim: HDL has atheroprotective properties and preserves endothelial function, including nitric oxide (NO) release. Eicosapentaenoic acid (EPA), an omega-3 fatty acid, has been shown to reduce lipoprotein oxidation due to its distinct physico-chemical lipid interactions; however, the effect of such antioxidant properties on HDL function is not fully understood. In this study, we tested the effects of HDL on endothelial NO release under oxidative stress conditions following pretreatment with EPA or other triglyceride (TG)-lowering agents.

Progressive Improvement in Endothelial Nitric Oxide Release due to Synthase Dimer Formation with LDL Reductions below 70 mg/dL during Hyperglycemia

In diabetes, endothelial cell (EC) dysfunction is characterized by reduced nitric oxide (NO) release and increased nitroxidative stress due to NO synthase (eNOS) dimer uncoupling. Reductions in LDL improve EC function but the effect is not well understood at levels below the target of 70 mg/dL. We measured NO and peroxynitrite (ONOO−) release as well as eNOS dimer formation in ECs under hyperglycemic conditions following progressive reductions of LDL in vitro. Human umbilical vein endothelial cells (HUVECs) were incubated with LDL at concentrations ranging from 150 to <10 mg/dL with high glucose (250 mg/dL). Cells were stimulated with calcium ionophore, and assayed for NO and ONOO− release using porphyrinic sensors. eNOS dimer levels were measured using immunochemical approaches. Results showed that reductions in LDL levels had a disproportionate benefit on endothelial NO and ONOO− release below 70 mg/dL. A reduction in LDL levels from 70 to <10 mg/dL produced a 181% increase in NO release (163 ± 18 to 458 ± 34 nM; p<0.0001) and 116% decrease in ONOO− release (393 ± 27 to 182 ± 23 nM; p<0.0001), corresponding to a 513% increase in the NO-to-ONOO− release ratio (0.42 ± 0.04 to 2.56 ± 0.40; p<0.0001), a comprehensive indicator of EC function. These changes also correlated with increased eNOS dimer formation by 819%. By contrast, only a modest 27% change in EC function was observed as LDL levels were reduced from 150 to 70 mg/dL with regard to the NO-to-ONOO− release ratio. In the absence of hyperglycemia, the benefits of LDL lowering on EC function and eNOS dimerization were also very evident, but to a lesser extent. Thus, progressive improvements in endothelial NO bioavailability and eNOS dimer formation were observed at LDL levels below 70 mg/dL to <10 mg/dL. These in vitro data suggest that reductions in LDL to very low levels may be effective in reversing EC dysfunction for the diabetic patient. Disclosure R. Mason: Research Support; Self; Amarin Corporation, Amgen Inc., Pfizer Inc.. Consultant; Self; Amarin Corporation. Speaker's Bureau; Self; Pfizer Inc.. H.E. Dawoud: None. S. Sherratt: None. T. Malinski: None.

Abstract 043: Endothelial Adenosine Kinase Deficiency Ameliorates Diet-induced Insulin Resistance

Background: Obesity is associated with endothelial dysfunction characterized by reduced bioavailability of nitric oxide (NO). It is well known that adenosine regulates endothelial nitric oxide release and vasodilation. However, it is unclear whether intracellular adenosine is able to modulate endothelial nitric oxide production and diet-induced metabolic disorders. This study is to investigate the effect of elevated intracellular adenosine caused by adenosine kinase (ADK) deletion or knockdown on endothelial nitric oxide production and diet-induced insulin resistance as well as the pathways associated to this effect. Methods and results: Endothelial-specific ADK knockout mice fed a high-fat diet show decreased fasting blood glucose and insulin, suppressed adipose tissue inflammation and hepatic steatosis, increased skeletal muscle arteriole vasodilation in an eNOS dependent manner. Mechanistically, ADK knockdown in Human Umbilical Vein Endothelial Cells (HUVECs) with adenoviral ADK shRNA elevated both intracellular and extracellular adenosine, and consequently increased endothelial nitric oxide synthase (eNOS) expression and activation, resulting in an increase in NO production, insulin uptake and vasodilatory-stimulated phosphoprotein (VASP) phosphorylation. Treatment of HUVECs with adenosine A2b receptor antagonist MRS 1754 abolished ADK-knockdown induced eNOS expression. eNOS phosphorylation in ADK-knockdown HUVECs were not interfered with adenosine receptors antagonists. Conclusion: ADK knockdown-mediated elevation of intracellular adenosine in endothelial cells ameliorates diet-induced insulin resistance and metabolic disorders. This is attributed to an enhancement of NO production caused by increased eNOS activation and expression, the latter is regulated via endothelial adenosine A2b receptor.

Endothelin-1 Regulation of Exercise-Induced Changes in Flow: Dynamic Regulation of Vascular Tone.

Although endothelin (ET)-1 is a highly potent vasoconstrictor with considerable efficacy in numerous vascular beds, the role of endogenous ET-1 in the regulation of vascular tone remains unclear. The perspective that ET-1 plays little role in the on-going regulation of vascular tone at least under physiologic conditions is supported by findings that potential ET-1 constriction is minimized by the release of the vasodilator and ET-1 synthesis inhibitor, nitric oxide (NO). Indeed, ET-1 release and constriction is self-limited by ET-1-induced, endothelial ETB receptor-mediated release of NO. Moreover, even if the balance between ET-1 and NO were reversed as the result of lowered NO activity, as occurs in a number of pathophysiologies associated with endothelial dysfunction, the well-known resistance of ET-1 constriction to reversal (as determined with exogenous ET-1) precludes ET-1 in the dynamic, i.e., moment-to-moment, regulation of vascular tone. On the other hand, and as presently reviewed, findings of ET-1-dependent modulation of organ blood flow with exercise under physiologic conditions demonstrate the dynamic regulation of vascular tone by ET-1. We speculate that this regulation is mediated at least in part through changes in ET-1 synthesis/release caused by pulsatile flow-induced shear stress and NO.

Residual NO modulates contractile responses and membrane potential in isolated rat mesenteric arteries

Shear stress or vasocontriction causes endothelial nitric oxide (NO) release resulting in the regulation of vascular smooth muscle tone in small resistance arteries. Generation of NO is inhibited by nitric oxide synthase (NOS) inhibitors. In this study, we investigated the effect of residual NO, released even in the presence of NOS inhibitors, on the membrane depolarization and phenylephrine-induced contractions of smooth muscle. For this purpose, we used hydroxocobalamin (HC), an NO scavenger, in the presence of NOS inhibitiors, Nω-nitro- L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in mesenteric arteries isolated from rats. Phenylephrine (0,01–10 μM), an α1-adrenoceptor agonist, led to depolarisation and concentration-dependent contraction in mesenteric arteries. The depolarisation and contractile responses were augmented by L-NA or L-NAME. Hydroxocobalamine (HC) or carboxy-PTIO (c-PTIO) also caused to further increase the membrane depolarization and contractions induced by phenylephrine in the presence of NOS inhibitors. Chemical removal of endothelium by saponin, tyrosin kinase inhibitor erbstatin A, but not calmodulin inhibitor calmidazolium inhibited the additional membrane depolarisation and contractile responses induced by L-NA or L-NAME and L-NA or L-NAME plus HC. These findings show that residual NO modulates the contractile responses in isolated rat mesenteric arteries by exerting a tonic inhibitor effect on the depolarization and vasoconstriction induced by phenylephrine.

Effects of clopidogrel on inflammatory cytokines and adhesion molecules in human endothelial cells: Role of nitric oxide mediating pleiotropic effects.

Clopidogrel is commonly used in prevention and treatment of atherothrombosis. Some previous studies have suggested a pleiotropic effect of clopidogrel; however, when this drug causes platelet-independent effects on endothelial function remains unclear. To evaluate the influence of clopidogrel on inflammatory biomarkers and adhesion molecules in human endothelial cells and the role of nitric oxide (NO) in this process. TNF-α-induced human umbilical vein endothelial cells (HUVEC) were exposed to clopidogrel. Gene expression and protein expression of ICAM-1, P-selectin, IL-8, IL-6, and MCP-1 were evaluated by qPCR, flux cytometry, or milliplex technology. Expression of endothelial nitric oxide synthase (NOS3) and NO release were also evaluated. Influence of clopidogrel was further evaluated in NOS3 downregulated HUVEC by RNAi. Clopidogrel at 20μmol/L induced NO release in HUVEC after 24-hours treatment. Gene expressions of inflammatory markers IL-8 and MCP1 were reduced after clopidogrel treatment (P<.05); however, only MCP-1 remained reduced at protein level. IL-6 was not modified by clopidogrel treatment. Gene expression and protein expression of ICAM-1 were diminished by 24-hours clopidogrel exposure, whereas P-selectin was not modified. NOS3 downregulated HUVEC model revealed that ICAM-1 modification by clopidogrel is dependent of this via, whereas MCP-1 is modulated in an NO-independent form. Our results support new evidence for pleiotropic effects of clopidogrel on inflammation and endothelial function. Reduction in ICAM-1 and MCP-1 in human endothelium is an important extent of the use of this drug for treatment of cardiovascular diseases, and NO has an important role in this process.

Contractile properties of periosteal arterioles in the guinea-pig tibia.

The periosteal arterioles of the compact bone may play a critical role in bone growth. To explore the contractile properties of tibial arterioles, spontaneous and nerve-evoked constrictions were compared in preparations from 3-week-old and 1-year-old guinea-pigs. Changes in arteriole diameters were measured using video microscopy. Their innervation was investigated using fluorescence immunohistochemistry. Fifty per cent and 40% of tibial arterioles from 3-week-old and 1-year-old guinea-pigs, respectively, exhibited spontaneous phasic constrictions that were inhibited by 1μM nifedipine, 10μM cyclopiazonic acid or 100μM 2-APB. Nerve-evoked phasic constrictions in both age groups were largely suppressed by phentolamine (1μM), an α-adrenoceptor antagonist, or sympathetic neurotransmitter depletion using guanethidine (10μM) but were enhanced by spanttide (1μM), a substance P receptor antagonist, or L-nitro arginine (L-NA; 100μM), an inhibitor of nitric oxide synthase (NOS). Nerve-evoked constrictions in 1-year-old animals were smaller than those in younger animals but greatly enhanced by L-NA. Immunohistochemistry revealed sympathetic and substance P-positive primary afferent nerves running along the arterioles as well as endothelial NOS expression in both age groups. Spontaneous arteriolar constrictions appear to rely on both Ca2+ release from the sarcoplasmic reticulum and Ca2+ influx through L-type Ca2+ channels. Noradrenaline released from sympathetic nerves triggers arteriolar constriction, while substance P released from primary afferent nerves dilates the arterioles by releasing nitric oxide (NO), presumably from the endothelium. Thus, the enhanced endothelial NO release in adult guinea-pigs may be important to increase the blood supply to meet the increased metabolic demands during bone growth.

Progressive Reduction in LDL Levels Improves Endothelial Cell Nitric Oxide Release and Lowers Oxidative Stress in a Non-Linear Manner

Progressive Reduction in LDL Levels Improves Endothelial Cell Nitric Oxide Release and Lowers Oxidative Stress in a Non-Linear Manner

Endothelium-independent vasodilator effect of 2-nitro-1-phenyl-1-propanol on mesenteric resistance vessels in rats

2-Nitro-1-phenyl-1-propanol (NPP) is a nitro alcohol with vasodilator activity in the rat aorta. The present study investigated the vasodilator properties of NPP in small vessels of the mesenteric bed, which, contrary to the aorta, contains resistance vessels. Using myography, isometric contractions were recorded in rings of second- and third-order branches from the rat mesenteric artery. NPP relaxed mesenteric ring preparations that were contracted with phenylephrine, U-46619, and KCl (40mM), resulting in significantly different EC50 values (0.41μM [0.31–0.55μM], 0.16μM [0.10–0.24μM], and 4.50μM [1.86–10.81μM], respectively). NPP-induced vasodilation decreased as the extracellular K+ concentration increased. The pharmacological blockade of K+ channels with tetraethylammonium, BaCl2, CsCl, and apamin also blunted NPP-induced vasorelaxation. In contrast, NPP-induced vasodilation was resistant to indomethacin, L-NG-nitroarginine methyl ester (L-NAME), and endothelium removal, indicating that neither prostaglandins nor the endothelial release of nitric oxide is involved in the relaxant effects of NPP. Conversely, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL-12,330A), and H-89 reduced NPP-induced vasodilation. Under Ca2+-free conditions, NPP did not alter transient contractions that were evoked by caffeine, but it reduced transient contractions that were evoked by phenylephrine. In mesenteric rings that were loaded with the fluorescent Ca2+ indicator Fluo-4 AM and stimulated with phenylephrine, NPP blunted both contractions and fluorescence signals that were related to cytosolic Ca2+ levels. In conclusion, the vasodilatory actions NPP on mesenteric vessel resistance involved the participation of cyclic nucleotides and the opening of K+ channels.

Nebivolol versus other beta blockers in patients with hypertension and erectile dysfunction.

Erectile dysfunction (ED) impacts over 100 million men worldwide and occurs at a higher incidence in men with hypertension. Beta blockers are one of several antihypertensive drug classes associated with ED. Nebivolol is a beta blocker with vasodilating properties mediated through endothelial release of nitric oxide which facilitates penile erection. Thus, nebivolol may offer an advantage over other beta blockers in the patient with hypertension and ED. A literature search comparing nebivolol with other beta blockers identified four European studies of limited duration, with the longest study being 28 weeks. Survey scores for erectile function showed significant improvement in erectile function with nebivolol in two of the studies, while the other two studies showed erectile function did not significantly worsen with nebivolol as compared with other beta blocker agents. One study showed improved erectile function scores, possibly due to the presence of a Hawthorne effect. Based on this small sample of studies, nebivolol may be of use in the patient with or at risk of developing ED, when a practitioner specifically wants to use a beta blocker as add-on antihypertensive treatment.

Dexmedetomidine Inhibits Phenylephrine-induced Contractions via Alpha-1 Adrenoceptor Blockade and Nitric Oxide Release in Isolated Rat Aortae.

The goal of this in vitro study was to examine the effect of the alpha-2 adrenoceptor agonist dexmedetomidine on phenylephrine (alpha-1 adrenoceptor agonist)-induced contraction in isolated rat aortae and to elucidate the associated cellular mechanisms, with a particular focus on alpha-1 adrenoceptor antagonism. Dexmedetomidine dose-response curves were generated in isolated endothelium-intact and endothelium-denuded rat aortae precontracted with phenylephrine or 5-hydroxytryptamine. Endothelium-denuded aortic rings were pretreated with either dexmedetomidine or the reversible alpha-1 adrenoceptor antagonist phentolamine, followed by post-treatment with the irreversible alpha-1 adrenoceptor blocker phenoxybenzamine. Control rings were treated with phenoxybenzamine alone. All rings were repeatedly washed with Krebs solution to remove all pretreatment drugs, including phenoxybenzamine, phentolamine and dexmedetomidine. Phenylephrine dose-response curves were then generated. The effect of rauwolscine on the dexmedetomidine-mediated change in phenylephrine-induced endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells was examined using western blotting. The magnitude of the dexmedetomidine-mediated inhibition of phenylephrine-induced contraction was higher in endothelium-intact aortae than in endothelium-denuded aortae or endothelium-intact aortae treated with Nω-nitro-L-arginine methyl ester. However, dexmedetomidine did not significantly alter 5-hydroxytryptamine-induced contraction. In further experiments, prazosin attenuated dexmedetomidine-induced contraction. Additionally, pretreatment with either dexmedetomidine plus phenoxybenzamine or phentolamine plus phenoxybenzamine produced greater phenylephrine-induced contraction than phenoxybenzamine alone, suggesting that dexmedetomidine protects aortae from the alpha-1 adrenoceptor blockade induced by phenoxybenzamine. Rauwolscine attenuated the dexmedetomidine-mediated enhancement of phenylephrine-induced eNOS phosphorylation. Taken together, these results suggest that dexmedetomidine attenuates phenylephrine-induced contractions via alpha-1 adrenoceptor blockade and endothelial nitric oxide release in the isolated rat aorta.

IRAP inhibition using HFI419 prevents moderate to severe acetylcholine mediated vasoconstriction in a rabbit model

Coronary artery vasospasm (constriction) caused by reduced nitric oxide bioavailability leads to myocardial infarction. Reduced endothelial release of nitric oxide by the neurotransmitter acetylcholine, leads to paradoxical vasoconstriction as it binds to smooth muscle cell M3 receptors. Thus, inhibition of coronary artery vasospasm will improve clinical outcomes. Inhibition of insulin regulated aminopeptidase has been shown to improve vessel function, thus we tested the hypothesis that HFI419, an inhibitor of insulin regulated aminopeptidase, could reduce blood vessel constriction to acetylcholine. The abdominal aorta was excised from New Zealand white rabbits (n=15) and incubated with 3mM Hcy to induce vascular dysfunction in vitro for 1h. HFI419 was added 5min prior to assessment of vascular function by cumulative doses of acetylcholine. In some rings, vasoconstriction to acetylcholine was observed in aortic rings after pre-incubation with 3mM homocysteine. Incubation with HFI419 inhibited the vasoconstrictive response to acetylcholine, thus improving, but not normalizing, vascular function (11.5±8.9% relaxation vs 79.2±37% constriction, p<0.05). Similarly, in another group with mild vasoconstriction, HFI419 inhibited this effect (34.9±4.6% relaxation vs 11.1±5.2%, constriction, p<0.05). HFI419 had no effect on control aorta or aorta with mild aortic dysfunction. The present study shows that HFI419 prevents acetylcholine mediated vasoconstriction in dysfunctional blood vessels. HFI419 had no effect on normal vasodilation. Our results indicate a therapeutic potential of HFI419 in reducing coronary artery vasospasm.

Divergent outcomes of fructose consumption on exercise capacity of rats: friend or foe.

To test the hypothesis that high fructose (HF) consumption divergently affects exercise capability as a function of feeding duration, rats were fed a normal (as control) diet or a normal caloric diet with HF for 3, 6, 10, and 30 days, respectively, and then were run on a treadmill. Results show that running distance and work were significantly increased, which was associated with greater exercise oxygen consumption in rats fed HF for 3 (HF-3D) and 6 days, but were decreased in rats fed HF for 30 days (HF-30D) compared with rats in their respective control groups. Shear stress-induced vasodilation (SSID) in isolated plantaris muscle arterioles was significantly greater in the HF-3D group than the control group. The difference in SSID between the two groups was abolished by Nω-nitro-l-arginine methyl ester (L-NAME), suggesting a nitric oxide (NO)-mediated response. Expression of phosphorylated/activated endothelial NO synthase (eNOS) and release of nitrite/NO were significantly increased in vessels of animals in the HF-3D group than controls. In contrast, arterioles isolated from the hypertensive rats in the HF-30D group displayed significantly attenuated NO-mediated SSID accompanied with greater production of superoxide compared with vessels of control animals. Additionally, the NO-dependent modulation of myocardial oxygen consumption (MV̇o2) was also impaired in the HF-30D group, and was prevented by blocking superoxide production with apocynin, an inhibitor that also normalized the reduced SSID in the HF-30D group. In conclusion, short-term (3-6 days) HF feeding enhances exercise potential via an increase in endothelial sensitivity to shear stress, which stimulates eNOS to release NO, leading to better tissue perfusion and utilization of oxygen. However, long-term (30 days) HF feeding initiates endothelial dysfunction by superoxide-dependent mechanisms to compromise exercise performance.NEW & NOTEWORTHY The evidence that short-term fructose intake potentiates exercise capacity by nitric oxide-mediated mechanisms yields an optimal fructose feeding frame in which beneficial effects of fructose have been acquired while detrimental effects have not yet been manifested. This highlights the significance of exercise physiology in providing constructive regimens to improve physical performance.