Inhaled nitric oxide does not influence bleeding time or platelet function in healthy volunteers

BACKGROUND: Bleeding time has been reported to increase during gaseous nitric oxide (NO) inhalation in healthy volunteers and patients, and it has been speculated that inhaled NO inhibits platelet function. However, results have not been unanimous, and we have been unable to document any effects of inhaled NO on circulating platelets. MATERIALS AND METHODS: We performed a double-blind, placebo controlled cross-over study in which healthy volunteers (n = 15) inhaled NO (30 ppm, 30 min) or control gas. Aspirin (640 mg x 1 orally) was used as positive control on the third occasion (n = 14). Bleeding time was measured, and platelet function was determined flow cytometrically by measuring the expression of P-selectin on circulating platelets and locally activated platelets in wound blood. Skin perfusion close to the site for bleeding time incisions was assessed by laser Doppler flowmetry. RESULTS: Bleeding time was unaffected by NO, as there were slight increases during both NO and control inhalation (+20% and +14% respectively, P = 0.9). Similarly, NO inhalation had no effect on platelet P-selectin expression in either systemic or wound blood, or on skin perfusion. Aspirin pretreatment, on the other hand, prolonged bleeding time (P < 0.001) and decreased P-selectin expression of platelets in wound blood (P = 0.03). CONCLUSIONS: This first placebo-controlled study indicates that inhaled NO does not influence either bleeding time, platelet activity or skin perfusion. Thus, it is unlikely that treatment of critically ill patients with inhaled NO will aggravate haemostatic disturbances, which has previously been feared, by influencing platelet function.     

高血压患者血栓形成与一氧化氮及血小板P-选择素的关系研究

目的:探讨高血压患者的血栓形成前状态与血浆一氧化氮水平、血小板活化的关系。方法:检测30例高血压患者(高血压组)与30名正常健康人(对照组)的血小板P-选择素、血小板膜糖蛋白Ⅱb/Ⅲa、血浆一氧化氮、血栓素B2、6-酮-前列腺素F1α水平。体外应用一氧化氮合酶抑制药N-硝基-L-精氨酸甲基酯(N-nitro-L-argininemethylester,L-NAME),观察其对血小板P-选择素水平的影响及一氧化氮前体L-精氨酸预处理后L-NAME对血小板P-选择素表达的影响。结果:高血压组血浆一氧化氮水平明显低于对照组[(55±16)μmol/L比(78±15)μmol/L,P<0·05)];血小板P-选择素、血小板膜糖蛋白Ⅱb/Ⅲa、血栓素B2水平明显高于对照组(P<0·01,P<0·01,P<0·05),而6-酮-前列腺素F1α水平显著降低(P<0·05)。应用L-NAME抑制一氧化氮合酶后,血小板P-选择素的水平增加,L-精氨酸预处理可明显阻断这一作用。结论:高血压患者内皮功能异常,血浆一氧化氮水平低下,可能是血小板活化、容易形成血栓的原因之一。应用一氧化氮前体或升高血浆一氧化氮水平的药物对预防高血压患者的血栓形成可能有益。     

亚硝基谷胱甘肽对冰冻血小板聚集及一氧化氮含量的影响

本研究探讨亚硝基谷胱甘肽(GSNO)对冷冻血小板聚集及一氧化氮(NO)含量的影响。用血小板聚集仪对血小板的聚集率进行测定,用硝酸还原酶法对NO含量进行检测。结果表明,新鲜液态血小板的聚集率为(63.44±2.96)%,冷冻血小板的聚集率为(35.47±2.93)%,加入GSNO后的冷冻血小板聚集率为(24.43±3.07)%。32例正常献血者新鲜液态血小板的NO浓度为(31.59±16.88)μmol/L。32例冷冻血小板的NO浓度为(22.16±6.38)μmol/L,明显低于新鲜液态血小板组。32例加入GSNO的冷冻血小板NO浓度为(45.64±6.31)μmol/L,明显高于新鲜液态血小板组。结论:GSNO增加了冷冻血小板NO的浓度,抑制血小板的聚集,保持血小板的功能,可以用作冷冻保护剂。     

10周游泳运动对高血压大鼠血浆一氧化氮、血管性血友病因子、P-选择素含量的影响

目的:研究运动对自发性高血压大鼠内皮功能、血小板活化状态的影响。方法:10周龄,雄性自发性高血压大鼠(SHR)17只,随机分为对照组(8只)和运动组(9只)。运动组SHR进行为期10周,每周5次,每次60min的游泳运动训练。实验期间每两周测定SHR血压,10周运动后,测定血浆一氧化氮(NO)水平,血小板NO水平,血管性血友病因子(vWF)、P-选择素浓度的变化。结果:运动组大鼠血压较对照组显著下降,血浆和血小板NO水平显著上升,血浆vWF和P-选择素显著降低。结论:规则有氧运动能产生较平稳持续的降压效果,能明显改善高血压内皮功能和血小板活化状态,降低高血压血栓并发症的发生。     

Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP-1 phosphatase

BACKGROUND: Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by post-translational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined. OBJECTIVES: We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity. METHODS: Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation. RESULTS: We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose- and time-dependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, co-associated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity. CONCLUSIONS: Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1.     

一氧化氮在烧伤大鼠心脏损害中作用的研究

目的：研究一氧化氮（ＮＯ）在防治烧伤早期心肌缺血缺氧性损害中的变化及意义。方法：动态观察大鼠３０％体表面积Ⅲ度烧伤后血浆及心脏ＮＯ、内皮素（ＥＴ）含量变化，应用外源性ＮＯ供体ＳＩＮ１以观察其对烧伤早期心脏损害的影响。结果：大鼠烧伤后心脏明显受损，烧伤后血浆及心肌组织ＮＯ－２、ＥＴ含量明显升高，由于ＮＯ－２升高幅度相对较低，ＥＴ／ＮＯ－２比值也显著增加；烧伤后在液体复苏下应用ＳＩＮ１可在一定程度上增加心肌组织超氧化物歧化酶（ＳＯＤ）、Ｎａ＋Ｋ＋ＡＴＰ酶活性及ＡＴＰ、ＡＤＰ含量，减少丙二醛（ＭＤＡ）、ＡＭＰ含量，减轻心肌组织水肿。结论：烧伤后由于心脏内源性ＮＯ的相对不足，导致心脏血管收缩，造成心肌组织缺血缺氧性损害，继而发生能量代谢紊乱及脂质过氧化损害；补充外源性ＮＯ则可在一定程度上逆转烧伤后心脏损害。     

A role of the endothelial nitric oxide system in acute renal colic caused by ureteral stone

Background and aims

Endothelial nitric oxide synthase gene polymorphisms play a role in some pathophysiological processes. In this study, the possible effects of endothelial nitric oxide synthase gene polymorphisms on ureteral stone disease in patients who were admitted to the emergency department with severe pain due to renal colic are examined.

Molecular control of blood flow and angiogenesis: role of nitric oxide

Summary.  In the past decade, the importance of the vascular endothelium as a multifunctional regulator of vascular smooth muscle physiology and pathophysiology has been appreciated . Indeed, the endothelium responds to hemodynamic stimuli (pressure, shear stress and wall strain) and locally manufactured mediators (such as bradykinin, prostaglandins, angiotensin II and nitric oxide) that can influence blood flow, cell trafficking into tissue and angiogenesis. In this chapter, the importance of nitric oxide (NO) as a mediator of blood flow control, vascular permeability and angiogenesis will be discussed.     

A novel anti-ischemic nitric oxide donor (LA419) reduces thrombogenesis in healthy human subjects

BACKGROUND: Platelet and endothelial production of nitric oxide (NO) is known to be impaired in coronary artery disease patients. Compounds that release NO (e.g. nitrates) have antiplatelet effects, but at supratherapeutic doses with hypotensive side effects. OBJECTIVES: To investigate the antithrombotic effect on human blood of a novel NO donor (LA419) with known anti-ischemic properties but without hypotensive side effects and to compare with abciximab. PATIENTS/METHODS: Healthy subjects (n = 8; 32 +/- 3 years) received daily aspirin starting three days prior to the study day. Treatments (LA419 10 and 20 microm, and abciximab 4 microm) were added ex vivo to non-anticoagulated blood, and the antithrombotic properties were assessed by measuring changes in thrombus size from pretreatment baseline in the Badimon perfusion chamber at low and high shear rates. Platelet surface adhesion using a Cone and Platelet Analyzer (CPA) and platelet fibrinogen-receptor activation with flow cytometry were also evaluated. RESULTS: At low shear rates, LA419 displayed a reduction in thrombus area of 43% +/- 8% (10 microm) and 56% +/- 6% (20 microm), whereas at high shear rates the reductions were 44% +/- 3% (10 microm) and 62% +/- 6% (20 microm). Platelet surface adhesion with the CPA was also reduced. Abciximab exhibited a strong inhibitory effect on thrombus formation, platelet surface adhesion and fibrinogen receptor activation. CONCLUSIONS: The novel NO donor, LA419, shows a strong antithrombotic effect in human blood, which is comparable to abciximab, especially under high shear rate conditions. Our observations suggest that the availability of an NO donor could prove beneficial in the prevention of thrombotic complications of cardiovascular disease. Further clinical studies are warranted.     

Warming and response to contractile agents in calf cardiac vein: role of the nitric oxide

The effects of warming on the response to various contractile agents of calf cardiac vein were studied using 2.5-mm long cylindrical segments. Concentration-response curves for carbachol (10(-9)-3 x 10(-4) m), 5-hydroxytryptamine (5-HT; 10(-8)-3 x 10(-3)), potassium chloride (KCl; 10(-4)-5 x 10(-2) m) and calcium chloride (CaCl2; 10(-4)-10(-2)) were isometrically recorded at 37 and 41 degrees C (warming). During warming the sensitivity, but not the maximal response, of carbachol 5-HT, KCl, and CaCl2 was significantly higher than at 37 degrees C. Warming to 41 degrees C after treatment with NG-nitro-L arginine methyl esther (10(-5) m) did not modify the effect of warming. These results suggest that nitric oxide seems to have no role in the warming-induced responses in calf cardiac vein.     

辛伐他汀纳米粒通过调节诱导型一氧化氮合酶/内皮型一氧化氮合酶系统对小鼠脓毒症相关急性肺损伤的影响

目的探讨辛伐他汀纳米粒对脓毒症相关急性肺损伤小鼠肺组织中诱导型一氧化氮合酶（iNOS）/内皮型一氧化氮合酶（eNOS）平衡的调节以及对脓毒症小鼠预后的影响。 方法将90只C57 / BL6小鼠分为假手术组、脓毒症组、灌胃组、静脉制剂组及纳米粒组,每组各18只。采用盲肠结扎穿孔术（CLP）建立脓毒症小鼠模型;灌胃组小鼠通过灌胃针,给予辛伐他汀口服制剂灌胃治疗后进行CLP术;静脉制剂组及纳米粒制剂组小鼠CLP术后,立即分别通过尾静脉注射预配置好的辛伐他汀静脉制剂和辛伐他汀纳米粒制剂。其中每组12只小鼠用于7 d生存评估,另外6只用于24 h时间点标本采集。每24小时观察小鼠的生存情况,然后记算各组小鼠每日生存情况。采用苏木素-伊红（HE）染色观察5组小鼠病理变化并计算肺损伤病理评分,免疫组织化学法检测5组小鼠肺组织iNOS、eNOS表达水平。 结果Kaplan-Meier生存曲线结果显示,5组小鼠7 d生存情况比较,差异有统计学意义（χ² = 3.780,P < 0.001）。进一步两两比较发现,脓毒症组及灌胃组小鼠的7 d生存情况均较假手术组明显下降（P均< 0.001）,而纳米粒组小鼠的7 d生存情况显著优于脓毒症组（P = 0.001）。HE染色结果显示,假手术组小鼠肺组织未见明显病理征象;脓毒症组小鼠肺组织弥漫性中性粒细胞浸润、肺泡腔变小、肺泡间中隔增厚、肺间质弥漫性水肿、细胞排列紊乱、部分肺组织完整性遭破坏;灌胃组小鼠病理所示与脓毒症组相似;静脉制剂组及纳米粒组小鼠中性粒细胞渗出均较脓毒症组损伤减少、肺泡完整性较好、损伤程度较轻。5组小鼠肺损伤病理评分、iNOS及eNOS表达水平比较,差异均有统计学意义（F = 889.200、9.633、6.918,P均< 0.05）。进一步两两比较发现,脓毒症组小鼠的肺损伤病理评分、iNOS及eNOS表达水平与假手术组比较,差异均有统计学意义（P均< 0.05）;静脉制剂组及纳米粒组小鼠病理评分、iNOS及eNOS表达水平与脓毒症组比较,差异均有统计学意义（P均< 0.05）,且纳米粒组小鼠的肺损伤病理评分较静脉制剂组显著降低（P < 0.05）。 结论不同的辛伐他汀制剂具有不同的效应,其中纳米粒制剂对于脓毒症相关的肺损伤最具保护价值,建立eNOS与iNOS之间的平衡,可以成为具有保护效应的重要处理位点。     

Human endothelial cells bioactivate organic nitrates to nitric oxide: implications for the reinforcement of endothelial defence mechanisms

Abstract. Although in therapeutic use for more than a century, the mode of cellular action of organic nitrates remains incompletely understood. Despite ample experimental evidence from animal studies to show that nitrates are metabolized to NO in the vascular smooth muscle, direct demonstration of such an activity in human vascular cells is still lacking. Moreover, the role of the endothelium in modulating the pharmacodynamic action of nitrates is far from clear. We therefore aimed to investigate whether or not human endothelial cells are capable of bioactivating these drugs to NO and whether the amounts generated are sufficient to elicit any biological effects. Using cultured human umbilical vein endothelial cells (HUVECs) as an established model system a combination of three different methods was used to address this issue: (1) quantification of NO formation upon endothelial nitrate metabolism using the oxyhaemo-globin technique; (2) evaluation of the second messenger response using radioimmunoassay for cGMP; and (3) assessment of mechanism and extent of potentiation of the anti-aggregatory effect of nitrates in the presence of endothelial cells as a relevant bioassay. We now show that superfusion of cultured human endothelial cells on microcarrier beads with either glyceryl trinitrate (GTN) or isosorbide dinitrate (ISDN; both at 01.100 μmol L^-1) results in a concentration-dependent formation of NO. NO generation from isosorbide 5-mononitrate (IS-5-N) was below the detection limit. The amounts of NO produced (maximally 2–97 ± 0.98 pmoles NO min^-1 x mg protein with 100μmol L^-1 GTN; n= 8) were similar to those elicited upon challenge of the cells with 100nM bradykinin. NO formation from either organic nitrate was accompanied, in a concentration-dependent and methylene blue-inhibitable manner, by stimulation of endothelial soluble guany-lyl cyclase with consequent increases in the intracel-lular level of cGMP (maximally 32-fold over basal levels with ISDN), a significant portion of which was released into the extracellular space. Upon continuous 30 min superfusion or repeated application of high concentrations of GTN (100μmol L^-1) nitrate bioac-tivation to NO was subject to partial tachyphylaxis. Co-incubation of washed human platelets with HUVECs potentiated the anti-aggregatory action of nitrates in a cell number dependent and oxyhaemo-globin-sensitive manner and this effect, too, was accompanied by increases in intraplatelet cGMP levels. The potentiating effect was largely inhibited after blockade of sulfhydryl groups by pre-incubadon of HUVECs with N-ethylmaleimide and completely abrogated after pretreatment of cells with the tissue fixative glutaraldehyde. These results demonstrate that human endothelial cells are capable of bioactivating organic nitrates to NO by an enzymatic, apparently thiol-sensitive pathway, in quantities sufficient to influence endothelial and platelet function. Besides the well known vasorelaxant action of organic nitrates, which is mainly due to their metabolism in the smooth muscle compartment, these drugs may therefore be endowed with a hitherto underestimated potential to directly influence endothelial functions via the NO/ cGMP pathway. Through specific bioactivation in the endothelium itself organic nitrates can thus mimic and reinforce protective functions normally served by a functional endothelium such as the modulation of blood cell/vessel wall interactions and inhibition of cell proliferation.     

Regulation of nitric oxide generation by up-regulated arginase I in rat spinal cord injury

Recently, arginase is suggested to regulate nitric oxide production by competing with nitric oxide synthase for the same substrate, L-arginine, in experimental asthma. We investigated the role of arginase and its relationship to nitric oxide production after spinal cord injury. Rats were subjected to laminectomy and complete transection of their spinal cords (injury group) or laminectomy only (sham group). In the injury group, arginase I was increased in the macrophages at the transection edge, and the peak was observed 48 h after spinal cord injury. However, nitric oxide production decreased significantly in the injury group despite increased nitric oxide synthase2 mRNA expression compared with the sham group. We also demonstrated the reduction in L-arginine concentrations, which was inversely associated with changes in arginase activity. Therefore, arginase appeared to regulate nitric oxide production by consuming L-arginine. The regulation of arginase activity and L-arginine levels may improve nitroxidative stress and reduce tissue damage in spinal cord injury.     

Anticonvulsive effect of atorvastatin on pentylenetetrazole‐induced seizures in mice: the role of nitric oxide pathway

Atorvastatin has shown to possess neuroprotective, antiexcitotoxic, and antiepileptic effects besides its cholesterol‐lowering properties. Nitric oxide (NO) may be responsible for a group of these effects. In the present study, a model of clonic seizure induced by pentylenetetrazole (PTZ) in male NMRI mice was used to investigate the anticonvulsive effects of atorvastatin through NO‐dependent pathways. Atorvastatin (5 mg/kg) significantly increased the seizure threshold (P < 0.001). Moreover, L‐arginine (a precursor of NO) significantly (P < 0.01) potentiated the anticonvulsive effects of subeffective doses of atorvastatin (1 mg/kg). Finally, L‐NAME [L‐arginine methyl ester dihydrochloride], a nonspecific NO synthase inhibitor, completely abolished the anticonvulsive properties of atorvastatin. Our findings demonstrated the role of atorvastatin as an anticonvulsive agent and showed the effects to be mediated through NO‐related pathways.     

生脉饮对内毒素诱导急性肺损伤大鼠一氧化氮及其合酶的影响

目的:探讨一氧化氮(NO)和诱生型一氧化氮合酶(iNO S)在脂多糖(LPS)诱导大鼠急性肺损伤(AL I)中的作用及生脉饮对其的影响。方法:雄性W istar大鼠按随机数字表法分为对照组、AL I组、生脉饮组、地塞米松组。舌下静脉注射LPS复制AL I模型。观察大体标本、组织病理以及肺湿/干重比、支气管肺泡灌洗液中中性粒细胞比、蛋白含量、肺毛细血管通透性和肺泡通透性指数等生物学指标。测定血浆NO和肺组织匀浆iNO S活性。结果:与对照组比较,AL I组肺组织病理显示肺间质及肺泡有明显的损伤和细胞浸润,各生物学指标及NO和iNO S显著升高(P<0.05或P<0.01);与AL I组比较,地塞米松组和生脉饮组肺组织病理明显减轻,各生物学指标及NO、iNO S也相应下降(P<0.05或P<0.01)。结论:地塞米松和生脉饮两种药物对LPS诱导的AL I具有保护作用,其机制可能是通过抑制NO水平和iNO S活性实现。     

Physiologic role for“inducible”nitric oxide synthase:A new form of astrocytic-neuronal interface

长期以来,一氧化氮(NO)都被看作是影响兴奋性突触传递的非典型神经信使分子,其细胞来源尚不清楚.许多脑区中的神经元型一氧化氮合酶(nNOS)只在少量抑制性神经元中表达.众所周知,胶质细胞诱导型一氧化氮合酶(iNOS)不在正常大脑中表达,其在免疫刺激后可经转录介导上调.因此,iNOS调节正常神经功能的作用常被忽视.许多研...     

Effects of cooling and warming on 5-hydroxytryptamine- and acetylcholine-induced contractions of human umbilical vessels: role of nitric oxide

The effects of cooling (to 28 degrees C) and warming (to 41 degrees C) on the vasoconstrictions induced by 5-hydroxytryptamine (5-HT) and acetylcholine (ACh) and the role of nitric oxide in these effects were analyzed in human umbilical artery and vein. 5-HT (10(-9)-10(-4) M) and ACh (10(-9)-10(-4) M) induced concentration-dependent contractions at 37, 28 and 41 degrees C. During cooling, the sensitivity, but not the maximal response, of 5-HT and ACh was significantly higher than at 37 degrees C; and during warming, again the sensitivity, but not the maximal response, of both contractile agents was significantly lower than at 37 degrees C. Neither cooling to 28 degrees C nor warming to 41 degrees C, after treatment with N(G)-nitro-L-arginine methyl esther (L-NAME, 10(-4) M), modify the effect of temperature in both vessels. These results suggest that cooling- and warming-induced responses in human umbilical artery and vein are independent of nitric oxide.