Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect.     

Nitrosation of melatonin by nitric oxide and peroxynitrite

Peroxynitrite (ONOO-) is an endogenous molecule, formed by rapid coupling between *NO and O2*-. ONOO- is known to be a strong oxidant of thiols and metalloorganic compounds and also a nitrating agent of aromatic compounds such as tyrosine. However, its chemistry is not yet well elucidated under physiological conditions. Melatonin, which is an indole-amine produced by the pineal gland and other organs, has antioxidant properties. We show that melatonin reacts with ONOO- in phosphate-buffered solutions. We provide evidence of nitrosation and oxidation at the pyrrole nitrogen leading to 1-nitrosomelatonin and 1-hydroxymelatonin, these being the major reactions in aqueous phosphate-buffered solutions besides other aromatic hydroxylations and nitration. 4-Nitromelatonin is formed, but in small amounts. The kinetics of all transformations were strictly dependent on ONOO- decay, whereas yields varied with pH and the presence of CO2. The N-oxidation became competitive with nitrosation at pH 7.4, in medium containing a sufficient amount of CO2. A proposed mechanism involves the transient formation of melatonyl radical and ONOO* radical derived from ONOO- decay.     

Arginase modulates myocardial contractility by a nitric oxide synthase 1-dependent mechanism

Cardiac myocytes contain two constitutive NO synthase (NOS) isoforms with distinct spatial locations, which allows for isoform-specific regulation. One regulatory mechanism for NOS is substrate (l-arginine) bioavailability. We tested the hypothesis that arginase (Arg), which metabolizes l-arginine, constrains NOS activity in the cardiac myocyte in an isoform-specific manner. Arg activity was detected in both rat heart homogenates and isolated myocytes. Although both Arg I and II mRNA and protein were present in whole heart, Arg II alone was found in isolated myocytes. Arg inhibition with S-(2-boronoethyl)-l-cysteine (BEC) augmented Ca(2+)-dependent NOS activity and NO production in myocytes, which did not depend on extracellular l-arginine. Arg II coimmunoprecipited with NOS1 but not NOS3. Isolation of myocyte mitochondrial fractions in combination with immuno-electron microscopy demonstrates that Arg II is confined primarily to the mitochondria. Because NOS1 positively modulates myocardial contractility, we determined whether Arg inhibition would increase basal myocardial contractility. Consistent with our hypothesis, Arg inhibition increased basal contractility in isolated myocytes by a NOS-dependent mechanism. Both the Arg inhibitors N-hydroxy-nor-l-arginine and BEC dose-dependently increased basal contractility in rat myocytes, which was inhibited by both nonspecific and NOS1-specific NOS inhibitors N(G)-nitro-l-arginine methyl ester and S-methyl-l-thiocitrulline, respectively. Also, BEC increased contractility in isolated myocytes from WT and NOS3 but not NOS1 knockout mice. We conclude that mitochondrial Arg II negatively regulates NOS1 activity, most likely by limiting substrate availability in its microdomain. These findings have implications for therapy in pathophysiologic states such as aging and heart failure in which myocardial NO signaling is disrupted.     

Comparative pharmacology of nitric oxide and nitric oxide generators on cardiac contractility in mammalian species

Nitric oxide (NO) is a potent vasorelaxing agent at nanomolar concentrations. At low nanomolar concentrations, NO also inhibits platelet aggregation, attenuates leukocyte adherence to the vascular endothelium, and quenches superoxide radicals. At high nanomolar concentrations, NO attenuates smooth muscle cells growth and stimulates proliferation of vascular endothelial cells. However, even at micromolar concentrations, NO fails to significantly alter cardiac contractility in isolated rat or cat cardiac muscle. Moreover, -arginine, even at millimolar concentrations, fails to exert a decrease in cardiac contractility significantly greater than that produced by -arginine.     

Effect of insulin on myocardial contractility during canine endotoxin shock

During endotoxin shock the heart becomes less responsive to the stimulatory effect of insulin on glucose uptake. In the present study we sought to determine if the heart was also less responsive to the positive inotropic effect of insulin during non-cardiogenic endotoxin shock. Responses of the heart to insulin were assessed under conditions of hyperinsulinaemic (4U.min-1), euglycaemic clamp (INS). Adult mongrel dogs, weighing 20-25 kg, were anaesthetised and instrumented to measure differences in substrate concentrations between arterial and coronary sinus blood, circumflex artery blood flow (using electromagnetic flow probe), haemodynamic variables, and left ventricular posterior wall thickness (using sonomicrometry). The first derivative of left ventricular pressure with respect to time was measured and its maximal value (LV dP/dtmax) used as an index of cardiac performance. Myocardial contractility was measured using the end systolic pressure-dimension relationship. Endotoxin shock was induced by Salmonella typhimurium (1 mg.kg-1 intravenously), and resulted in depression of myocardial performance but increased contractility. INS caused a twofold elevation in myocardial glucose uptake in control animals while in endotoxin shocked dogs it was unable to elevate glucose uptake above the pre-endotoxin level. In control animals INS caused both increased cardiac contractility and performance. In the endotoxin shock group INS was unable to increase LVdP/dtmax above the basal, pre-endotoxin level and did not cause any significant change in myocardial contractility. We suggest that the heart becomes less responsive to the positive inotropic as well as metabolic effects of insulin during endotoxin shock. Changes in LV dP/dtmax can be attributed to the changing loading conditions that occur during endotoxin shock.     

多巴胺系统及L-精氨酸/一氧化氮通路在大鼠心肌肥大中的作用研究

目的 探讨多巴胺系统及L-精氨酸／一氧化氮（L—Arg／NO）通路在大鼠心肌肥大动物模型中的作用及相关机制。方法 采用腹主动脉缩窄术复制大鼠心肌肥大动物模型。将Wistar大鼠随机分为4组：①腹主动脉缩窄术（AC）组；②假手术（SO）组；③L-精氨酸（L—Arg）组；④左旋硝基精氨酸甲酯（L—NAME）组。通过心肌肥大指数，心肌组织胶原染色，心功能检测等方法分析各组大鼠心肌组织肥大状况：采用RT—PCR和Western blot结合图像分析系统，观察药物干预后，心肌组织中多巴胺受体D1、D2mRNA和蛋白质表达变化：借助紫外分光光度计，分析心肌组织匀浆中一氧化氮（NO）水平和一氧化氮合酶（NOS）活性。结果 AC组左心肥大明显．表现为室内压显著升高，胶原增多；与SO组比较，腹主动脉缩窄术后D1、D2mRNA和蛋白质表达均明显降低（P〈0.01），NO、NOS水平明显下降（P〈0.01）；应用NOS抑制剂L-NAME预处理能够促进肥大的发生。使用NO合成的前体L-Arg干预，则抑制肥大发生。结论 压力负荷加大能引起心肌肥大。其机制可能与NO合成减少有关：大鼠心肌细胞内存在多巴胺受体D1、D2mRNA和蛋白质表达，心肌肥厚时二者均明显减低。     

Nitric oxide regulation of myocardial contractility and calcium cycling: independent impact of neuronal and endothelial nitric oxide synthases

The mechanisms by which nitric oxide (NO) influences myocardial Ca2+ cycling remain controversial. Because NO synthases (NOS) have specific spatial localization in cardiac myocytes, we hypothesized that neuronal NOS (NOS1) found in cardiac sarcoplasmic reticulum (SR) preferentially regulates SR Ca2+ release and reuptake resulting in potentiation of the cardiac force-frequency response (FFR). Transesophageal pacing (660 to 840 bpm) in intact C57Bl/6 mice (WT) stimulated both contractility (dP/dtmax normalized to end-diastolic volume; dP/dt-EDV) by 51+/-5% (P<0.001) and lusitropy (tau; tau) by 20.3+/-2.0% (P<0.05). These responses were markedly attenuated in mice lacking NOS1 (NOS1-/-) (15+/-2% increase in dP/dt-EDV; P<0.001 versus WT; and no change in tau; P<0.01 versus WT). Isolated myocytes from NOS1-/- (approximately 2 months of age) also exhibited suppressed frequency-dependent sarcomere shortening and Ca2+ transients ([Ca2+]i) compared with WT. SR Ca2+ stores, a primary determinant of the FFR, increased at higher frequencies in WT (caffeine-induced [Ca2+]i at 4 Hz increased 107+/-23% above 1 Hz response) but not in NOS1-/- (13+/-26%; P<0.01 versus WT). In contrast, mice lacking NOS3 (NOS3-/-) had preserved FFR in vivo, as well as in isolated myocytes with parallel increases in sarcomere shortening, [Ca2+]i, and SR Ca2+ stores. NOS1-/- had increased SR Ca2+ ATPase and decreased phospholamban protein abundance, suggesting compensatory increases in SR reuptake mechanisms. Together these data demonstrate that NOS1 selectively regulates the cardiac FFR via influences over SR Ca2+ cycling. Thus, there is NOS isoform-specific regulation of different facets of rate-dependent excitation-contraction coupling; inactivation of NOS1 has the potential to contribute to the pathophysiology of states characterized by diminished frequency-dependent inotropic responses.     

一氧化氮对心肌梗死后心肌细胞凋亡的影响

目的研究不同浓度一氧化氮(NO)对心肌梗死后心肌组织抗氧化损伤能力(T-AOC)、bcl-2、bax蛋白表达及细胞凋亡的影响。方法新西兰兔32只,随机分为4组,(每组8只)。假手术组(Sham组)、心肌梗死组(MI组)、低剂量消心痛组(LISDN组,1.5mg·kg-1·d-1)、高剂量消心痛组(HISDN组,4.0mg·kg-1.d-1),皆3次/d灌胃。6周后取梗死灶边缘缺血心肌制作组织匀浆进行NO浓度(以NO2-/NO3-间接表示)、T-AOC测定,并作病理观察;免疫组化观察各组bcl-2、bax蛋白表达;TUNEL法比较其细胞凋亡程度。结果HISDN组心肌间质水肿,较多心肌纤维化与炎性细胞浸润,可见局灶性肌丝溶解,肌浆网明显扩张。而LISDN组病理改变较轻。与HISDN组比较,LISDN组心肌组织匀浆中NO浓度降低而T-AOC升高(P<0.05),bcl-2表达阳性率增高而bax表达率降低(P<0.01),细胞凋亡数低于HISDN组(P<0.01)。结论高浓度NO降低心肌梗死后心肌细胞T-AOC并增加细胞凋亡,而低浓度NO显示出更好保护作用。     

Lethal effect of cytokine-induced nitric oxide and peroxynitrite on cultured rat cardiac myocytes

We examined the cytotoxic effect of iNOS-generated NO in cultured cardiac myocytes treated with IL-1 beta, IFN- gamma and LPS. Treatment of the myocytes with cytokines for 48 h resulted in a marked NO production, a significant decline in cellular ATP content, and a significant increase in myocyte death with morphological characteristics of necrosis. Moreover, immunohistochemical examination showed that the cytokines caused nitrotyrosine formation in the injured myocytes. Uric acid and L-cysteine which have the ability to quench peroxynitrite significantly attenuated these cytokine-induced effects, although they did not alter NO production or the decline in cellular ATP. These data suggest that NO production induced by cytokines can not only cause deleterious effects in the myocardial energy balance but also induce myocytes necrosis, through the formation of peroxynitrite.     

去甲肾上腺素预处理对大鼠供心热休克蛋白70、一氧化氮及一氧化氮合酶表达的影响

目的:探讨去甲肾上腺素预处理是否可诱导心肌热休克蛋白70(HSP70)的合成,并研究其对供心一氧化氮(NO)、一氧化氮合酶(NOS)的影响,探讨去甲肾上腺素预处理心肌保护作用机制。方法:Wistar大鼠18只,分为2组:对照组(C,n=9),腹腔注射0.9%氧化钠注射液0.5 mL,24 h后取离体心脏灌注(Histidine-tryptophan-ketoglutarte,HTK)心脏保护液,4℃保存3 h后建立Langendorff离体心脏灌注模型,灌注(Krebs-Henseleit,K-H)液2 h;实验组(E,n=9)腹腔注射重酒石酸去甲肾上腺素(溶于0.9%氯化钠液中)3.1μmol/kg(0.53 mg/kg),腹腔注射24 h后取离体心脏,处理方法同C组。测定心肌HSP70、NO、NOS的含量以及相关生化指标并做统计学处理比较。结果:HSP70含量E组较C组明显增高(P<0.01),NO、NOS的含量E组较C组明显增多(P<0.01),生化指标E组明显优于C组。结论:去甲肾上腺素预处理能诱导供心心肌组织HSP70、NO、NOS高表达,其对供心具有明显的保护效应,并且其促进心肌NO、NOS的表达,这可能是去甲肾上腺素预处理发挥供心保护作用的机制之一。     

一氧化氮在心肌细胞凋亡中的作用

作者综述了一氧化氮 （NO）对心肌细胞凋亡的作用机制。NO对心肌细胞的作用有双重性 :既是组织损伤的中间介质 ,又在心肌缺血 /再灌注损伤中有保护心肌作用。它可通过氧化应激反应 ,作用于心肌细胞的一些蛋白质、酶类、细胞因子以及胞液的钙超载等途径来影响心肌细胞凋亡     

一氧化氮和内毒素在梗阻性黄疸致病机制方面的作用

梗阻性黄疸（obstructive jaundice）是临床常见的表现。梗阻性黄疸患者术后并发症和死亡率较高，常死于感染和败血症，提示机体术前的免疫功能受到了抑制。梗阻性黄疸可使网状内皮系统功能降低，细菌清除能力减弱。肠道来源的细菌和内毒素逃脱肝脏枯否细胞的监视，进入血液循环。有研究发现梗阻性黄疸常并发肠源性内毒素血症，内毒素激活枯否细胞，产生大量炎性介质，如一氧化氮（NO），肿瘤坏死因子α（TNFα）及氧自由基等。     

一氧化氮和内毒素在梗阻性黄疸致病机制方面的作用

梗阻性黄疸（obstructive jaundice，OJ）是临床常见的症状。梗阻性黄疸患者术后并发症和死亡率较高，常死于感染和败血症，提示机体术前的免疫功能受到了抑制。梗阻性黄疸可使网状内皮系统功能降低，细菌清除能力减弱。肠道来源的细菌和内毒素逃脱肝脏枯否细胞的监视，进入血液循环。有研究发现梗阻性黄疸常并发肠源性内毒素血症，内毒素激活枯否细胞，产生大量炎性介质，如一氧化氮（NO）、肿瘤坏死因子α（TNFα）及氧自由基等。     

Effects of aminoguanidine on nitric oxide production induced by inflammatory cytokines and endotoxin in cultured rat hepatocytes

AIM To study the effects of aminoguanidine(AG)and two L-arginine analogues N~ω-nitro-L-arginine methyl ester(L-NAME)and N~ω-nitro-L-arginine(L-NNA)on nitric oxide(NO)productioninduced by cytokines(TNF-α,IL-1β,and IFN-γ)and bacterial lipopolysaccharide(LPS) mixture(CM)in the cultured rat hepatocytes,andexamine their mechanisms action.METHODS Rat hepatocytes were incubatedwith AG,L-NAME,L-NNA,Actinomycin D(ActD)and dexamethasone in a medium containing CM(LPS plus TNF-α,IL-1β,and IFN-γ)for 24 h.NO     

绞股蓝总皂甙对注射内毒素家兔血流动力学和NO的影响

目的 :探讨绞股蓝总皂甙 （GPS）对注射内毒素的家兔血流动力学和 NO的影响。方法 :动物随机分 3组 :对照组 ,内毒素 （L PS）组及 GPS+L PS组。动物静脉麻醉后 ,取血测量 NO水平 ;通过二道生理记录仪和 SMUP- A型生物信号处理仪测量血流动力学指标。动物处死后 ,取血再次测量 NO水平。结果 :对照组实验前后各指标没有明显的变化 （P>0 .0 5 ）。 L PS组平均动脉血压 （MAP）、心率、心室内压峰值、± dp/ dtmax和心力环面积在实验后 2 h时没有出现明显的变化 ,但在实验后 4h、6 h时明显低于实验前和对照组 （P<0 .0 1） ,实验后 NO- 2 明显高于实验前。GPS组实验后 2 h,4h时 MAP、心率、心室内压峰值、± dp / dtmax和心力环面积与实验前及对照组比较 ,没有明显差异 （P>0 .0 5 ） ,但在 4h时明显高于 L PS组 （P<0 .0 1） ;实验后 6 h时 ,MAP、心率、心室内压峰值、± dp/ dtmax和心力环面积低于实验前和对照组 （P<0 .0 5 ） ,但高于 L PS组 （P<0 .0 5 ） ;实验后的 NO- 2 浓度高于实验前和对照组 （P<0 .0 5 ） ,但低于 L PS组实验后的 NO- 2 浓度 （P<0 .0 5 ）。结论 :绞股蓝总皂甙对注射内毒素的家兔血流动力学有保护作用和抑制一氧化氮大量释放的作用     

Protective and pathological roles of nitric oxide in endotoxin shock.

OBJECTIVE: The aim was to investigate the effects of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of both the constitutive (Ca2+ dependent) and inducible (Ca2+ independent) nitric oxide (NO) synthases, or of pretreatment with the glucocorticoid dexamethasone, an inhibitor of the induction of the Ca2+ independent NO synthase, on lipopolysaccharide induced shock in the anaesthetised rabbit. METHODS: Mean arterial blood pressure, and blood flow in the portal vein, hepatic artery, and hindquarter vascular beds were measured in 49 halothane anaesthetised New Zealand White rabbits given lipopolysaccharide (Salmonella minnesota, 500 micrograms.kg-1 intravenously). The effects of pre- or post-lipopolysaccharide treatment with L-NMMA (300 mg.kg-1 intravenously) and of pretreatment with dexamethasone (3 mg.kg-1 intravenously) were determined. The effect of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 300 micrograms.kg-1.h-1 intravenously) in animals treated with lipopolysaccharide and L-NMMA was also studied. RESULTS: Lipopolysaccharide elicited an initial transient fall in mean arterial pressure and decreases in blood flow in the vascular beds, followed by a progressive fall in mean arterial pressure. L-NMMA when given either before or after lipopolysaccharide markedly exacerbated its effects and resulted in severe hypotension, intense vasoconstriction, and increased mortality. Pretreatment with dexamethasone had no effect on the initial haemodynamic changes following lipopolysaccharide, but prevented the subsequent fall in mean arterial pressure observed in animals treated with lipopolysaccharide alone. Dexamethasone failed, however, to protect animals also treated with L-NMMA before lipopolysaccharide. Animals pretreated with L-NMMA and SNAP showed reduced haemodynamic changes when compared with controls (lipopolysaccharide only) or lipopolysaccharide and L-NMMA treated animals. CONCLUSIONS: Inhibition of both constitutive and inducible NO synthases during endotoxaemia is deleterious. This can be overcome by replacing NO intravenously with a donor of NO. Selective inhibition of the inducible NO synthase may, however, be beneficial in shock.     

阿托伐他汀对冠心病患者血清一氧化氮及一氧化氮合酶含量的影响

目的　观察阿托伐他汀对冠心病患者血清一氧化氮 (NO)及一氧化氮合酶 (NOS)含量水平的影响。方法　对用阿托伐他汀治疗的 79例冠心病患者依据是否合并高胆固醇血症分为两组 ,对其治疗前后血清 NO及 NOS含量水平进行对比分析。结果　不论是否合并高胆固醇血症的冠心病 ,阿托伐他汀均可升高其血清 NO及 NOS水平。结论　阿托伐他汀可通过调脂治疗抑制脂质的过氧化反应 ,保护血管内皮功能 ,但其保护内皮功能的作用不受患者是否存在高脂血症的影响 ,改善内皮功能 ,对冠心病的防治具有重要意义。     

Relaxin inhibits the activation of human neutrophils: involvement of the nitric oxide pathway

In animal models of inflammation, the pregnancy hormone relaxin was shown to reduce the recruitment of leukocytes, especially neutrophils, in inflamed tissues. The current study was designed to clarify whether relaxin could inhibit activation of isolated human neutrophils and, if so, whether the nitric oxide (NO) biosynthetic pathway was involved, as occurs in other relaxin targets. Human neutrophils were preincubated with 1, 10, and 100 nmol/liter porcine relaxin for 1 h before activation with N-formyl-Met-Leu-Phe (10 micromol/liter) or phorbol-12-myristate-13-acetate (0.1 micromol/liter). In selected experiments, the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA, 100 micromol/liter) was added to the samples 30 min before relaxin. In other experiments, chemically inactivated relaxin (10 nmol/liter) was substituted for authentic relaxin. Untreated, nonactivated neutrophils were the controls. Relaxin reduced significantly and in a concentration-dependent fashion the expression of the surface activation marker CD11b, as well as the generation of superoxide anion, the rise of intracellular Ca(2+), the release of cytoplasmic granules, and the chemotactic migration. These effects of relaxin were blunted by N(G)-monomethyl-L-arginine and could not be reproduced by inactivated relaxin. Relaxin also increased neutrophil inducible NO synthase expression and NO generation. This study provides evidence that relaxin inhibits the activation of human neutrophils stimulated by different proinflammatory agents. This novel property of relaxin could be of relevance in toning down maternal neutrophil activation during pregnancy, thereby counteracting the occurrence of pregnancy-related disorders such as preeclampsia, which is regarded as an excess maternal inflammatory response to pregnancy.