Putative Nox2 inhibitors worsen homocysteine-induced impaired acetylcholine-mediated relaxation

Background and aimIncreased homocysteine (Hcy) is associated with coronary artery disease (CAD). Hcy increases reactive oxygen species (ROS) via NADPH oxidases (Nox), reducing acetylcholine-mediated vasorelaxation. We aimed to determine if putative Nox2 inhibitors prevent Hcy-impaired acetylcholine-mediated vasorelaxation.Methods and resultsNew Zealand White rabbit and wild-type (C57BL/6) and Nox2^−/− (NOX) mice aortic rings were mounted in organ baths. Rabbit rings were incubated with either apocynin (10 μM), gp91ds-tat (GP, 1 μM) or PhoxI2 (1 μM) and mice rings GP (1 μM) only. Some rabbit rings were incubated with 3 mM Hcy, before pre-contraction, followed by dose–response relaxation to acetylcholine (ACh; 0.01μM-10μM). In rabbit rings treated with Hcy and GP, O₂‾ donor pyrogallol (1 μM) or Akt activator SC79 (1 μM) was added 5 min before ACh. Mice rings were used to compare Nox2 deletion to normal acetylcholine-mediated relaxation.In rabbits, Hcy reduced acetylcholine-mediated relaxation vs. control (p < 0.0001). Treatment + Hcy reduced relaxation compared with treatment alone (p < 0.0001). Pyrogallol and SC79 reversed the response of GP + Hcy (p = 0.0001). In mice, Nox2 deletion reduced acetylcholine-mediated vasorelaxation. Rabbit tissue analysis revealed that Hcy reduced eNOS phosphorylation at Thr⁴⁹⁵ and increased eNOS phosphorylation at Ser¹¹⁷⁷; no further alteration at Thr⁴⁹⁵ was observed with GP. In contrast, GP prevented increased phosphorylation at Ser¹¹⁷⁷.ConclusionsApocynin, GP and PhoxI2 worsens acetylcholine-mediated vascular relaxation in rabbit aorta, which is supported by results from mouse Nox2 deletion data. These inhibitors worsen Hcy-induced vascular dysfunction, suggesting that current putative Nox2 inhibitors might not be useful in treating HHcy.     

桃叶珊瑚苷舒张血管作用机制研究

目的探讨桃叶珊瑚苷舒张血管的作用机制。方法去甲肾上腺素(10-6 mol/L)收缩内皮完整、去内皮、加入一氧化氮合酶抑制剂L-NAME孵育的大鼠胸主动脉血管后,加入累积浓度的桃叶珊瑚苷,检测血管张力变化。桃叶珊瑚苷孵育大鼠心脏微血管内皮细胞24 h,应用MTT法检测细胞活力,应用ELISA法检测细胞eNOS蛋白表达。结果桃叶珊瑚苷(0.5,5,20μmol/L)具有舒张内皮完整的大鼠胸主动脉血管的作用,去内皮和L-NAME (100μmol/L)能够抑制桃叶珊瑚苷舒张血管的作用。桃叶珊瑚苷(0.5,5,20μmol/L,24 h)具有促进大鼠心脏微血管内皮细胞eNOS蛋白表达的作用。结论桃叶珊瑚苷可能通过内皮依赖的NO途径发挥快速的舒张血管的作用,并且具有促进eNOS蛋白表达的作用。     

Reactive Oxygen Species Are Necessary for High Flow (Shear Stress)‐induced Diameter Enlargement of Rat Resistance Arteries

Objectives: Chronic increases in blood flow induce remodeling associated with increases in diameter and endothelium‐mediated dilation. Remodeling requires cell growth and migration, which may involve reactive oxygen species (ROS). Nevertheless, the role of ROS in flow‐mediated remodeling in resistance arteries is not known. Materials and Methods: Rat mesenteric resistance arteries (MRAs) were exposed to high flow (HF) by sequentially ligating second‐order MRAs in vivo. After three weeks, arteries were collected for structural, pharmacological, and biochemical analysis. Results: In HF arteries, luminal diameter (431±12 to 553±14 μm; n=10), endothelium (acetylcholine)‐mediated vasodilatation (61±6 to 77±6% relaxation) and NAD(P)H subunit (gp91phox and p67phox) expression levels, and ROS (dihydroethydine microphotography) and peroxynitrite (3‐nitro‐tyrosine) production were higher than in normal flow arteries. Acute ROS scavenging with tempol improved acetylcholine‐dependent relaxation (92±4% relaxation), confirming that ROS are produced in HF arteries. Chronic treatment with tempol prevented the increase in diameter, reduced ROS and peroxynitrite production, and improved endothelium‐mediated relaxation in HF arteries. Thus, ROS and NO were involved in HF‐induced diameter enlargement, possibly through the formation of peroxynitrite, while ROS reduced the increase in endothelium‐dependent relaxation. Conclusions: ROS production is necessary for flow‐mediated diameter enlargement of resistance arteries. However, ROS counteract, in part, the associated improvement in endothelium‐mediated relaxation.     

VASORELAXATION OF RAT THORACIC AORTA CAUSED BY 14-DEOXYANDROGRAPHOLIDE

1. The pharmacological effects of 14-deoxyandrographolide on rat isolated thoracic aorta were examined. 2. 14-Deoxyandrographolide (2.5-120 μmiol/L) inhibited contractions induced by phenylephrine (PE; 0.1 μmol/L) and high K⁺ (80mmol/L) in a concentration-dependent manner in endothelium-intact aorta. The effect was attenuated in endo-thelium-denuded aorta without modifying the maximal response. Like verapamil, 14-deoxyandrographolide produced a much greater vasorelaxant effect in aorta precontracted by KCI than by PE. 14-Deoxyandrographolide (20-60 μmol/L) also inhibited responses of the rat aorta to PE. 3. In Ca²⁺-free medium (KCI 55mmol/L), 14-deoxyandrographolide (20-80 μmol/L) antagonized Ca²⁺-induced vasocon-traction in a concentration-dependent manner and transient contractions induced by both caffeine (10mmol/L) and noradrenaline (1μmol/L) were suppressed or almost abolished by 14-deoxyandrographolide. 4. The vasorelaxant effect of 14-deoxyandrographolide was partially antagonized by N^G-nitro-L -arginine methyl ester (25 μmol/L), a specific and competitive nitric oxide synthase (NOS) inhibitor, and methylene blue (10 μmol/L), a soluble guanylate cyclase inhibitor, but was not affected by indomethacin (20 μmol/L), a cyclo-oxygenase inhibitor, or glibenclamide (10 μmol/L), an ATP-sensitive K⁺-channel blocker. 5. These results suggest that the vasorelaxant activity of 14-deoxyandrographolide may be mediated via the activation of NOS and guanylate cyclase, as well as the blockade of Ca²⁺ influx through both voltage- and receptor-operated Ca²⁺ channels.     

Regional differences in the vasorelaxant effects of nicorandil and amlodipine on isolated porcine coronary arteries

Summary— The vasorelaxant effects of nicorandil, a K⁺-channel opener, and amlodipine, a dihydropyridine-type Ca²⁺-channel blocker, were investigated on partially and maximally K⁺-depolarized ring preparations from the porcine left anterior descending coronary artery. By comparing vascular responses in the proximal and distal parts of the epicardial segment, the scope of the study was to evaluate regional differences in the action of nicorandil and amlodipine. Nicorandil (10 ⁷- 10^-4 M) shifted the K⁺ concentration-response curves to the right and depressed the maximal contractile responses in a concentration-dependent manner, consistent with K⁺-channel opening and secondary non-K⁺-channel opening mechanisms of action. Nicorandil had a significantly more potent relaxant effect in the proximal compared to the distal arterial rings contracted with 85 mM K⁺. Pretreatment with methylene blue (10^-5 M) did not significantly influence the regional difference in the action of nicorandil. Amlodipine (10⁹- 10^-6 M) had a significantly more potent and effective inhibitory and relaxant effect than nicorandil under the same conditions. In contrast to nicorandil, the effect of amlodipine was more prominent in the distal compared to the proximal vessel rings. The cumulative addition of extracellular Ca²⁺ exhibited a more potent contractile response in the distal rather than in the proximal rings. Nicorandil totally and amlodipine partly eliminated the contractile responses to the lowest concentration of Ca²⁺. The inhibitory effect of amlodipine on the contractile responses to higher Ca²⁺ concentrations was more pronounced than that of nicorandil. The results show that there are regional differences in the responsiveness of porcine coronary arteries to Ca²⁺, nicorandil and amlodipine. Our findings indicate that the regional difference in nicorandil-induced vasodilation was caused neither by the K⁺-channel opening nor by the nitrate-like mechanism of action, but could be due to a direct Ca²⁺-influx blocking effect of the drug.     

Endothelium-Dependent Relaxation Effect of Apocynum venetum Leaf Extract via Src/PI3K/Akt Signalling Pathway

Botanical herbs are consumed globally not only as an essential diet but also as medicines or as functional/recreational food supplements. The extract of the Apocynum venetum leaves (AVLE), also known as Luobuma, exerts its antihypertensive effect via dilating the blood vessels in an endothelium- and concentration-dependent manner with optimal effect seen at as low as 10 µg/mL. A commercial Luoboma “antihypertensive tea” is available commercially in the western province of China. The present study seeks to investigate the underlying cellular mechanisms of the nitric oxide (NO)-releasing property of AVLE in rat aortas and human umbilical vein endothelial cells (HUVECs). Endothelium-dependent relaxation induced by AVLE was assessed in organ chambers in the presence or absence of polyethyleneglycol catalase (PP2, 20 µM; inhibitor of Src kinase), wortmannin (30 nM) and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM; PI3 (phosphatidylinositol3)-Kinase inhibitor), N^G-nitro-l-arginine (L-NAME, 100 µM; endothelial NO synthase inhibitor (eNOS)) and ODQ (1 µM; soluble guanylyl cyclase inhibitor). Total nitrite and nitrate (NOx) level and protein expression of p-Akt and p-eNOS were measured. AVLE-induced endothelium-dependent relaxation was reduced by PP2, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and abolished by L-NAME and ODQ. AVLE significantly increased total NO_x level in rat aortas and in HUVECs compared to control. It also instigated phosphorylation of Akt and eNOS in cultured HUVECs in a concentration-dependent manner and this was markedly suppressed by PP2, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. AVLE also inhibited superoxide generated from both NADPH oxidase and xanthine/xanthine oxidase system. Taken together, AVLE causes endothelium-dependent NO mediated relaxations of rat aortas through Src/PI3K/Akt dependent NO signalling pathway and possesses superoxide scavenging activity.     

彩色多普勒观察中药坎离煎对慢性心力衰竭患者内皮依赖性血管舒张功能的影响

目的:观察慢性心力衰竭(congestive heart failure,CHF)患者坎离煎治疗前后的内皮依赖性血管舒张功能(endothelium-dependent dilation,EDD)的变化.方法:运用彩色多普勒超声观察中药坎离煎对35例CHF患者治疗前后的桡动脉反应性充血后血流介导的血管舒张幅度(flow-mediated dilation,FMD)、搏动指数(pulsatile index,PI)、阻力指数(resistance index,RI),计算基础血流量(baseline blood flow,BF).结果:治疗后FMD显著增高(治疗前后分别为7.06±2.58,9.26±3.19,P＜0.05),但BF、PI及RI均无明显改善.结论:运用彩色多普勒超声观察坎离煎治疗CHF患者EDD的变化,可为中医药治疗CHF的临床疗效评价提供客观依据.     

荞麦花叶总黄酮的舒张血管作用及其机制

目的探讨荞麦花叶总黄酮(TFBFL)对大鼠离体胸主动脉的舒张作用与机制。方法采用离体血管环灌流装置,经生物信号采集与分析系统测定血管环张力的变化;激光扫描共聚焦显微镜技术检测血管平滑肌细胞内钙浓度。结果在苯肾上腺素(phenylephrine,PE)10-6mmol.L-1或KCl60 mmol.L-1预收缩的保留内皮和去除内皮的血管环中,TFBFL均能够浓度依赖性的引起血管舒张;相对于去除内皮的血管环,TFBFL对保留内皮的血管环的舒张作用更明显。保留内皮的血管环用一氧化氮合酶抑制剂L-NAME(100mmol.L-1)预孵后,TFBFL诱导的血管舒张作用明显减弱。在无钙灌流液中,用KCl 60 mmol.L-1去极化去除内皮的血管环后,逐渐加入Ca2+,使血管环呈浓度依赖性收缩;TFBFL(0.8 mg.L-1)孵育10 min后可使CaCl2的量效曲线向下移位且可使PE在无钙灌流液中诱导的血管环最大收缩幅度明显降低。激光扫描共聚焦显微镜检测细胞内钙的结果表明,TFBFL浓度依赖性(0.4、0.8 mg.L-1)的抑制了静息状态平滑肌细胞质内钙浓度的升高。结论 TFBFL能够浓度依赖性舒张大鼠胸主动脉,其作用机制可能是降低细胞内游离Ca2+浓度;对于保留内皮的血管环,TFBFL也作用于血管内皮细胞,促进一氧化氮的释放,引起血管舒张。     

Therapeutic applications of carbon monoxide-releasing molecules

Carbon monoxide (CO), which is formed in mammalian cells through the oxidation of haem by the enzyme haem oxygenase, actively participates in the regulation of key intracellular functions. Emerging evidence reveals that an increased generation of haem oxygenase-derived CO plays a critical role in the resolution of inflammatory processes and alleviation of cardiovascular disorders. The authors have identified a novel class of substances, CO-releasing molecules (CO-RMs), which are capable of exerting a variety of pharmacological activities via the liberation of controlled amounts of CO in biological systems. A wide range of CO carriers containing manganese (CORM-1), ruthenium (CORM-2 and -3), boron (CORM-A1) and iron (CORM-F3) are currently being investigated to tailor therapeutic approaches for the prevention of vascular dysfunction, inflammation, tissue ischaemia and organ rejection.