Dynamics of nitric oxide release in the cardiovascular system

The endothelium plays a critical role in maintaining vascular tone by releasing nitric oxide (NO). Endothelium derived NO diffuses to smooth muscles, triggering their relaxation. The dynamic of NO production is a determining factor in signal transduction. The present studies were designed to elucidate dynamics of NO release from normal and dysfunctional endothelium. The nanosensors (diameter 100-300 nm) exhibiting a response time better than 100 micros and detection limit of 1.0 x 10(-9) mol L(-1) were used for in vitro monitoring of NO release from single endothelial cells from the iliac artery of normotensive (WKY) rats, hypertensive (SHR) rats, and normal and cholesterolemic rabbits. Also, the dynamics and distribution of NO in left ventricular wall of rabbit heart were measured. The rate of NO release was much higher (1200 +/- 50 nmol L(-1) s(-1)) for WKY than for SHR (460 +/- 10 nmol L(-1) s(-1)). Also, the peak NO concentration was about three times higher for WKY than SHR. Similar decrease in the dynamics of NO release was observed for cholesterolemic rabbits. The dynamics of NO release changed dramatically along the wall of rabbit aorta, being highest (0.86 +/- 0.12 micromol L(-1)) for the ascending aorta, and lowest for the iliac aorta (0.48 +/- 0.15 micromol L(-1)). The distribution of NO in the left ventricular wall of rabbit heart was not uniform and varied from 1.23 +/- 0.20 micromol L(-1) (center) to 0.90 +/- 0.15 micromol L(-1) (apex). Both, the maximal concentration and the dynamics of NO release can be useful diagnostic tools in estimating the level of endothelial dysfunction and cardiovascular system efficiency.     

Thienopyridines: effects on cultured endothelial cells.

In cultured endothelial cells harvested from human umbilical vein (HUVEC) or bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) caused an increase in nitrite generation in HUVEC from basal 227 +/- 37 to 372 +/- 60 or to 325 +/- 33 pmoles per 10(6) cells, respectively, and in BAEC from basal 182 +/- 17 to 378 +/- 18 or to 423 +/- 66 pmoles per 106 cells (n = 6), respectively. Calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) next to 30 min incubation with BAEC increased release of 6-keto-PGF 1alpha from basal level of 9.4 +/- 1.8 to 96.2 +/- 5.1 or to 99.5 +/- 10.2 pmoles per 10(6) cells, respectively. The pretreatment with aspirin (300 microM) cut down this rise to 4.2 +/- 0.1 pmoles per 10(6) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7 +/- 3.3 nM (n = 53), 68.3 +/- 5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cultured endothelial cells calcium ionophore A 23187 (0.1 microM) produced net maximum rise in [Ca2+]i by 157 +/-27 nM (n = 16)[ ECV304], by 107 +/- 58 nM (n=4) [HUVEC], and by 231.0 +/- 41.3 nM (n = 8) [BAEC], respectively, while ticlopidine (30 microM) produced net maximum rise in [Ca2+]i by 30.0 +/- 3.2 nM (n=9)[ECV304], 48.8 +/- 15.6 nM (n = 4)[HUVEC] and 28.4 +/- 5.4 nM (n = 8)[BAEC], respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than that of calcium A 23187 but also its maximum appeared after a lag period that was 2 3 times longer than that for A23187. In ECV304 clopidogrel at concentrations of 10, 30 and 100 microM produced maximum increment of [Ca2+]i by 16.5 +/- 3.8 nM (n = 7), 47.0 +/- 6.9 nM (n = 8) and 67.2 +/- 8.3 nM (n = 8), respectively. Incubation of BAEC with A23187 (microM), ticlopidine or clopidogrel (100 microM) for 2 h did not influence viability of cultured endothelial cells. We claim that thienopyridines, independently of their delayed anti-platelet properties ex vivo do release NO and PGI2 from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be mediated by a rise in [Ca2+]i, however, this possibility has not been proved.     

Designing analogs of ticlopidine,a wall teichoic acid inhibitor,to avoid formation of its oxidative metabolites

The thienopyridine antiplatelet agent, ticlopidine and its analog, clopidogrel, have been shown to potentiate the action of β-lactam antibiotics, reversing the methicillin-resistance phenotype of methicillin-resistant Staphylococcus aureus (MRSA), in vitro. Interestingly, these thienopyridines inhibit the action of TarO, the first enzyme in the synthesis of wall teichoic acid, an important cell wall polymer in Gram-positive bacteria. In the human body, both ticlopidine and clopidogrel undergo a rapid P450-dependent oxidation into their respective antiplatelet-active metabolites, resulting in very low plasma concentrations of intact drug. Herein, a series of analogs of ticlopidine and clopidogrel that would avoid oxidative metabolism were designed, prepared and evaluated as inhibitors of TarO. Specifically, we replaced the P450-labile thiophene ring of ticlopidine and clopidogrel to a more stable phenyl group to generate 2-(2-chlorobenzyl)-1,2,3,4-tetrahydro-isoquinoline) (6) and (2-chloro-phenyl)-(3,4-dihydro-1H-isoquinolin-2-yl)-acetic acid methyl ester (22), respectively. The latter molecules displayed inhibitory activity against TarO and formed the basis of a library of analogs. Most synthesized compounds exhibited comparable efficacy to ticlopidine and clopidogrel. So far, it was introduction of a trifluoromethyl group to compound 6, to generate 2-(2-trifluoromethyl-benzyl)-1,2,3,4-tetrahydro-isoquinoline (13) that exhibited enhanced activity against TarO. Compound 13 represents a novel stable inhibitor of TarO with synergistic impact on β-lactam antibiotics against MRSA and low potential for P-450 metabolism.     

一氧化氮在血管紧张素Ⅱ激活蛋白激酶C中的作用

实验在培养新生大鼠心肌细胞中检测NO前体L－精氨酸（L－Arg）和NO供体硝普钠（SNP）对血管紧张素Ⅱ（AngⅡ）激活蛋白激酶C（PKC）的作用,以探讨心肌细胞PKC水平的信号转导途径,实验结果如下：（1）无血清DMEM培养心肌细胞24h后加入AngⅡ,PKC活性呈剂量依赖性增高;（2）培养基中加入L－Arg,PKC活性呈剂量依赖性降低;（3）用L－Arg100μmol/L进行预处理,30min后分别加入AngⅡ0.1μmol/L或PMA10μmol/L,PKC活性均明显降低,与单纯AngⅡ组和单纯PMA组相比均有显著性差异;用NOS抑制剂L－NAME预处理后,再加入L－Arg,可明显阻断L－Arg对上述两个效应的影响;（4）培养液中加入NO供体SNP,PKC活性呈剂量依赖性地降低;（5）用SNP10μmol／L预处理心肌细胞,5min后分别加入AngⅡ或PMA,PKC活性分别与单纯AngⅡ和单纯PMA组相比均明显降低。以上结果表明,AngⅡ能剂量依赖性激活PKC,而NO可剂量依赖性抑制PKC活性;NOS参与L－Arg抑制AngⅡ或PMA激活PKC的作用。这些观察提示,NO抑制AngⅡ对心肌细胞的作用可能是通过抑制PKC活性实现的,PKC可能是NO和AngⅡ在心肌细胞内信号转导的交汇点（cross talk）。     

Idoxifene对人乳内动脉的舒张作用

为了研究idoxifene(吲哚昔酚,一种新型雌激素受体调节剂)对人乳内动脉(human internal mammarya artery,HIMA)的舒张作用及其机制,采用离体血管灌流的方法,观察idoxifene对完整内皮和去内皮HIMA的舒张作用,以及L-NAME和美蓝(metllylene blue,MB)对这一过程的影响,并且和17β雌二醇(17β-estradiol,E2)的舒血管作用进行了比较。实验中观察到保留血管内皮时idoxifenc(0．01～10μmol／L)和E2(0．1～100μmol／I,)可以剂量依赖性地舒张血管,且血管对idoxifene的灵敏度比对E2高15倍左右,而去内皮时则无此作用;NO合成酶阻断剂L-NAME和鸟苷酸环化酶(guanylate cyclasc,GC)的抑制剂MB使idoxifene舒张HIMA的作用完全消失。上述结果表明：idoxifene能够剂量依赖性地舒张HIMA,而且比E2更有效。idoxifene的这种作用是通过NO-GC-cGMP通路实现的。     

Is clopidogrel superior to aspirin in secondary prevention of vascular disease?

The cornerstone in clinical evidence of the relative efficacy of thienopyridines (clopidogrel, ticlopidine) versus aspirin in the secondary prevention of vascular disease is the Clopidogrel versus Aspirin in Patients at Risk of Ischaemic Events trial. This trial showed a modest benefit in the reduction of vascular events by clopidogrel. The results differed according to qualifying disorder: myocardial infarction, -3.7%; ischaemic stroke, +7.3%; and peripheral arterial disease, +23.8% (P = 0.042). Similar results were found for ticlopidine after brain ischaemia. The safety of clopidogrel appears to be similar to that of aspirin and better than that of ticlopidine. However, the recent report of thrombotic thrombocytopenic purpura in association with clopidogrel causes concern.     

Human recombinant erythropoietin alters the flow-dependent vasodilatation of in vitro perfused rat mesenteric arteries with unbalanced endothelial endothelin-1 / nitric oxide ratio

Chronic use of human recombinant erythropoietin (r-HuEPO) is accompanied by serious vascular side effects related to the rise in blood viscosity and shear stress. We investigated the direct effects of r-HuEPO on endothelium and nitric oxide (NO)-dependent vasodilatation induced by shear stress of cannulated and pressurized rat mesenteric resistance arteries. Intravascular flow was increased in the presence or absence of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 10(-4) mol/L). In the presence of r-HuEPO, the flow-dependent vasodilatation was attenuated, while L-NAME completely inhibited it. The association of r-HuEPO and L-NAME caused a vasoconstriction in response to the rise in intravascular flow. Bosentan (10(-5) mol/L), an inhibitor of endothelin-1 (ET-1) receptors, corrected the attenuated vasodilatation observed with r-HuEPO and inhibited the vasoconstriction induced by flow in the presence of r-HuEPO and L-NAME. r-HuEPO and L-NAME exacerbated ET-1 vasoconstriction. At shear stress values of 2 and 14 dyn/cm(2) (1 dyn = 10(-5) N), cultured EA.hy926 endothelial cells incubated with r-HuEPO, L-NAME, or both released greater ET-1 than untreated cells. In conclusion, r-HuEPO diminishes flow-induced vasodilatation. This inhibitory effect seems to implicate ET-1 release. NO withdrawal exacerbates the vascular effects of ET-1 in the presence of r-HuEPO. These findings support the importance of a balanced endothelial ET-1:NO ratio to avoid the vasopressor effects of r-HuEPO.     

一氧化氮和K+通道参与乙酰胆碱引起的大鼠离体输精管平滑肌细胞超极化

本文旨在探讨大鼠新鲜离体输精管平滑肌细胞中乙酰胆碱（acetylcholine,ACh）引起超极化反应的机制,采用细胞内微电极记录技术和细胞内荧光标记技术研究ACh对大鼠输精管不同走行方向平滑肌细胞的作用。用尖端含0．1％碘化吡啶（propidium iodide,PI）的记录电极标记电生理记录后的平滑肌细胞,其中37个为外层纵行细胞,17个为内层环行细胞。它们的平均静息膜电位分别为（-53．56±3．88）mV和（-51．62±4．27）mV,膜输入阻抗分别为（2245．60±372．50）MQ和（2101．50±513．50）MQ。ACh引起的膜超极化反应是浓度依赖性的,EC50为36 μmol／L。ACh引起的超极化反应可被非选择性的毒草碱（muscarinic receptor,M）受体阻断剂阿托品（atropine,1 μmol／L）和选择性的M3受体阻断剂diphenylacetoxy-N-methylpiperidine-methiodide（DAMP,100nmol/L）阻断。ACh引起的超极化还能被一氧化氮合酶抑制剂L-硝基-精氨酸甲酯（N-nitro-L-arginine methylester,L．NAME,300μmol／L）阻断,并可被ATP敏感的钾通道阻断剂glipizide（5μmol／L）或内向整流钾通道阻断剂钡离子（50μmol／L）部分阻断。Glipizide和钡离子联合使用可完全阻断ACh引起的超极化反应。上述结果表明：ACh通过作用于大鼠输精管平滑肌细胞膜上的M3受体引起超极化反应,一氧化氮、ATP敏感性钾通道和内向整流钾通道参与了ACh引起的超极化反应。     

植物雌激素三羟异黄酮抑制麻醉大鼠颈动脉窦压力感受器反射

采用隔离灌流麻醉雄性大鼠颈动脉窦技术,观察了植物雌激素三羟异黄酮（genistein,GST）对颈动脉窦压力感受器反射的影响。其结果如下:（1）以GST（50μmol/L）隔离灌流颈动脉窦区时,压力感受器机能曲线向右上方移位,曲线最大斜率（peak slope,PS）由0.36±0.01降至0.23±0.01（P<0.001）;反射性血压下降幅度（reflex decrease,RD）由39.75±1.58降至27.00±0.60 mmHg（P<0.001）,阈压（TP）和饱和压（SP）分别从65.63±2.10和192.23±3.90升至82.05±1.95mmHg（P<0.001）和215.76±3.75mmHg（P<0.001）。其中RD、PS和TP呈明显的剂量依赖性;（2）用Ca2+通道激动剂Bay K 8644（500 nmol/L）预处理后,能取消GST（50 μmol/L）对压力感受器反射的抑制作用;（3）预先灌流NO合酶阻断剂（L-NAME,100μmol/L）,不能阻断GST（50μmol/L）对压力感受器反射的抑制效应。以上结果提示,GST可能通过阻断Ca2+通道途径而抑制大鼠颈动脉窦压力感受器反射,并非由血管内皮细胞释放NO所致。     

八肽胆囊收缩素对大鼠颈动脉窦压力感受器反射的抑制作用

在36只隔离灌流颈动脉窦区的麻醉大鼠上,观察了八肽胆囊收缩素(cholecystokinin octapepide,CCK-8)对颈动脉窦压力感受器反射的影响。其结果如下：(1)以CCK-8(0．1、0．5、1．0μmol／L)隔离灌流颈动脉窦区时,压力感受器机能曲线向右上方移位,曲线最大斜率(peak slope,PS)减小,反射性血压下降幅度(reflex decrease,RD)减少,阈压(threshold pressure,TP)和饱和压(saturation pressure,SP)均增高。其中RD、PS和TP呈明显的剂量依赖性;(2)用CCK-8的非特异性受体拮抗剂丙谷胺(100μmol／L)预处理后,能明显减弱CCK-8(0．50mol/L)对压力感受器反射的抑制;(3)预先灌流一氧化氮合酶(nitric oxide synthase,NOS)阻断剂(L-NAME,100μmol／L),不能阻断CCK-8(0．5μmol/L)对压力感受器反射的影响;(4)用Ca^2 通道激动剂Bay K 8644(500nmol／L)预处理后,也能明显减弱CCK-8(0．5μmol／L)对压力感受器反射的抑制作用。以上结果提示,CCK-8是通过作用于颈动脉窦压力感受器神经元末梢上的受体而起到抑制作用的,其机制可能为抑制了牵张敏感性通道,致使Ca^2 离子内流减少,而与内皮细胞释放NO无关。     

一氧化氮在17-β雌二醇抑制血管平滑肌细胞增殖和原癌基因c -fos表达中的作用

实验利用大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC)作为模型,观察17-β雌二醇（E2）对VSMC增殖和原癌基因c-fos表达的影响,并探讨VSMC源性一氧化氮（NO）在基中的作用,检测指标包括NO释放的测定,细胞计数、^3H-Tdr掺入,噻唑蓝（MTT）测定和c-fosmRNA表达,结果显示,E2（10^-12-10^-8mol/L)呈浓度依赖性地促进VSMC中NO的释放;10^-8mol/LE2能明显抑制10%小牛血清（FCS）和10^-7mol/L内皮素-1（ET-1）诱导的细胞增殖和DNA合成,E2的抑制作用均可被雌激素受体（ER）拮抗剂tamoxifen(10^-7mol/L)和一氧化氮合酶抑制剂L-NAME（10^-6mol/L)明显减轻;E2（10^-8mol/L)可明显抑制10^-7mol/LET-1诱导的VSMCc-fos表达,这种抑制作用可被L-NAME（10^-6mol/L)明显减轻,这些结果提示E2能抑制VSMC增殖和原癌基因c-fos表达,这种促进VSMC的NO释放密切相关,而且该作用至少部分通过ER介导。     

Transient receptor potential ankyrin 1 (TRPA1) channel activation by the thienopyridine-type drugs ticlopidine,clopidogrel, and prasugrel

Transient receptor potential A1 (TRPA1) is widely expressed throughout the human and animal organism, including the dorsal root ganglia as well as the bladder, stomach and small intestine. Here, we examined the effect of three platelet aggregation inhibitors on TRPA1: ticlopidine, clopidogrel and prasugrel. Utilising fluorometric Ca²⁺ influx analysis and electrophysiological whole cell measurements in TRPA1-expressing HEK293 and in human enterochromaffin-like QGP-1 cells, we found that ticlopidine, clopidogrel and prasugrel are direct activators of TRPA1. Although this polymodal channel commonly contributes to the perception of pain, temperature and chemical irritants, recent studies provide evidence for its involvement in the release of serotonin (5-HT) from enterochromaffin cells. Therefore, we further investigated the ability of ticlopidine, clopidogrel and prasugrel to stimulate 5-HT release from QGP-1 cells. We could determine 5-HT in supernatants from cultured QGP-1 cells upon treatment with ticlopidine and clopidogrel but not with prasugrel. These findings indicate that a robust TRPA1 activation by ticlopidine and clopidogrel correlates with the stimulatory effect on the secretion of 5-HT. As recipients of ticlopidine and clopidogrel frequently complain about gastrointestinal adverse events such as nausea, vomiting and diarrhoea, an activation of TRPA1 may contribute to adverse effects of such drugs in the digestive system.     

白藜芦醇对哇巴因所致豚鼠乳头状肌迟后去极化及触发活动的效应

研究旨在应用标准玻璃微电极技术,观察白藜芦醇对哇巴因所引起的离体豚鼠乳头状肌迟后去极化(delayed after depolarization,DAD)及触发活动(triggered activity,TA)的效应。结果显示：(1)预先给予白藜芦醇(30、60、120μmol／L)可剂量依赖性地抑制哇巴因所引起的乳头状肌DAD及TA;(2)预先应用L型钙通道开放剂Bay K8644(0．25μmol／L),可取消白藜芦醇的上述效应;(3)预先应用一氧化氮合酶抑制剂L-NAME(1mmol／L),对白藜芦醇的上述效应无影响;(4)单独应用17β-雌二醇(E2,5μmol／1．0或白藜芦醇(30μmol／L)对DAD及TA无明显影响,而联合应用相同剂量的E2和白藜芦醇则对DAD及TA产生明显的抑制效应;(5)预先应用雌激素受体拮抗剂他莫昔芬(10μmol／L)不能取消白藜芦醇对DAD及TA的抑制作用。以上结果表明,白藜芦醇具有抑制乳头状肌DAD及TA的作用,这一效应可能与其抑制钙离子内流有关,但此作用机制中NO和雌激素受体的作用并不显著。白藜芦醇这种抗心律失常作用对于心血管系统具有一定的保护意义。     

Adenosine and hypoxic dilation of rat coronary small arteries: roles of the ATP-sensitive potassium channel, endothelium, and nitric oxide

The aims of the study were to examine the roles of the ATP-sensitive potassium (K(ATP)) channel, the endothelium, and nitric oxide (NO) in the responses of rat coronary small arteries to adenosine and hypoxia. Segments of rat coronary vessel were investigated in vitro using pressure myography; all vessels studied developed stable spontaneous myogenic tone during equilibration. Glibenclamide (a K(ATP) channel inhibitor) reversed pinacidil but not 2-deoxyglucose-induced dilation. Both adenosine and hypoxia dilated the vessels, and glibenclamide did not reverse these responses. Endothelial removal or N(G)-nitro-L-arginine methyl ester (L-NAME) inhibited the dilation to adenosine by approximately 50%; subsequent addition of glibenclamide was without effect. Hypoxic dilation was completely inhibited by endothelium removal or L-NAME. We conclude that adenosine- and hypoxia-induced dilation of rat coronary arteries does not appear to involve the K(ATP) channel. Adenosine-induced dilation is partially and hypoxic dilation is completely dependent on endothelium-derived NO.     

一氧化氮抑制AngⅡ介导的心肌肥大反应的信号机制

本文主要利用培养的新生大鼠心肌细胞,从细胞学及分子生物学角度研究一氧化氮（NO）信号系统在AngⅡ介导的心肌肥大反应中的作用及机制。实验以心肌细胞蛋白合成速率、心房钠尿肽（ANP）的表达作为心肌肥大反应的指标,以硝酸盐及亚硝酸盐含量反映心肌细胞NO水平,以免疫印迹法测定MKP－1蛋白表达,以RT－PCR测定eNOS mRNA水平。结果发现：（1）L－精氨酸（L－Arg)10,100μmol/L分别增加心肌细胞NO水平16％及31％,L－Arg(100μmol/L）还可增加心肌细胞eNOS mRNA表达,其作用可被NOS抑制剂L－NAME所抑制;（2）L－Arg(100μmol/L）可降低AngⅡ(0.1μmol/L）诱导的心肌细胞ANP mRNA表达水平和蛋白合成速率,而在L－Arg处理之前用针对MKP－1的反义寡核苷酸转染心肌细胞,蛋白合成速率明显增加,可取消L－Arg的抑制作用,甚至超过AngⅡ组;（3）L－Arg(100μmol/L）明显增加MKP－1蛋白表达,比对照组增加225％,NOS抑制剂L－NAME及蛋白激酶G（PKG）抑制剂KT－5823皆可抑制L－Arg诱导的MKP－1蛋白表达,分别抑制88％、83％,而AngⅡ能增加L－Arg诱导的MKP－1的表达,较对照组增加365％,增强了L－Arg的作用。以上结果表明,NO抑制AngⅡ介导心肌肥大反应的机制可能是通过激活PKG,促进MKP－1的表达,从而增加MAPK去磷酸化实现的。     

17β-雌二醇抑制雄性大鼠颈动脉窦压力感受器反射

采用隔离灌流麻醉雄性大鼠颈动脉窦技术 ,观察了 17β 雌二醇 (E2 )对颈动脉窦压力感受器反射的影响。结果如下 :(1)以E2 (10 μmol/L)隔离灌流颈动脉窦区时 ,压力感受器机能曲线向右上方移位 ,曲线最大斜率(peakslope,PS)由 0 49± 0 0 3降至 0 2 5± 0 0 1(P <0 0 1) ,反射性血压下降幅度 (reflexdecrease,RD)由 7 37± 0 42kPa降至 3 49± 0 2 0kPa (P <0 0 0 1) ,阈压 (thresholdpressure ,TP)和饱和压 (saturationpressure ,SP)分别由 9 5 2±0 6 8kPa和 2 4 5 3± 0 48kPa增至 13 3± 0 11kPa (P <0 0 0 1)和 2 7 5 2± 0 2 0kPa (P <0 0 1) ,其中PS、RD、TP和SP呈明显的剂量依赖性 ;(2 )用雌激素受体阻断剂tamoxifen (1、5、10、30 μmol/L)预处理后 ,不能阻断E2 对压力感受器反射的抑制作用 ;(3)预先灌流NO合酶阻断剂 (L NAME ,10 0 μmol/L) ,可完全消除E2 (10 μmol/L)对压力感受器反射的抑制效应。以上结果表明 ,17β 雌二醇可通过非基因组机制抑制大鼠颈动脉窦压力感受器反射 ,其效应系E2 引起血管内皮细胞释放NO所致。     

白藜芦醇对离体豚鼠乳头状肌的电生理效应

本文旨在应用标准玻璃微电极技术,观察白藜芦醇对离体豚鼠乳头状肌的电生理效应。结果显示：(1)白藜芦醇(30、60、120μmol／L)可剂量依赖性地缩短乳头状肌细胞的动作电位时程;(2)对部分去极化的乳头状肌,白藜芦醇(60μmol／L)不仅缩短动作电位时程,而且降低动作电位的幅值和超射值,减慢零期最大上升速度;(3)用无钙K-H液灌流标本可完全取消白藜芦醇对乳头状肌细胞的作用：(4)钾通道开放剂四乙基氯化铵(TEA,20mmol／L),不能阻断白藜芦醇的电生理效应;(5)预先应用一氧化氮合酶抑制剂L-NAME(1mmol／L),对白藜芦醇的上述效应无影响。以上结果表明,白藜芦醇可缩短正常乳头状肌细胞动作电位时程,这一效应可能与其抑制钙离子内流有关,但此作用机制中NO的作用并不显著。     

Interaction between L-arginine: no system and cyclooxygenase metabolic products of arachidonic acid in coronary autoregulation.

Coronary autoregulation (CA) is the intrinsic ability of the heart to maintain its nutritive blood supply constant over a wide range of perfusion pressure. This phenomenon is regulated through several control mechanisms, while metabolic and myogenic control mechanism have dominant effects. In last few years, endothelial control mechanism, which is part of metabolic control, was intensive investigated. Dominant topic of endothelial-investigation was bioregulatory L-arginine: NO system, with his effective product--nitric oxide (NO). On the other hand, cyclooxygenase metabolic pathway products of arachidonic acid plays an important role in the control of vasomotor tone of coronary arteries. For this purpose, the aim of our study was to evaluate role of L-arginine: NO system, cyclooxygenase metabolites of arachidonic acid, as well as, their interactions in the control of CA of the isolated rat heart.. In our study rat hearts autoregulate CF between 50 and 90 cm H2O of CPP. Basal release (at 60 cm H2O) of NO (as nitrite), cAMP, cGMP and HX+X (i.e. adenosine) amounted to 2.85+/-0.25 nmol/min/g wt, 29.45+/-2.22 pmol/min/g wt, 0.43+/-0.08 pmol/min/g wt and 37.50+/-2.89 nmol/min/g wt respectively. Release of NO, cAMP and cGMP were strictly parallel with CPP-CF curve, while release of adenosine (i.e. HX + X) was an inverse function of perfusion pressure. Inhibition of NOS (L-NAME, 30 micromol/l) significantly widened autoregulatory range (40-100 cm H2O), with significant reduction in CF and NO- and cGMP release, while release of cAMP was completely reversed in the presence of L-NAME. However, inhibition of cyclooxygenase didn't influence autoregulatory range, with similar changes of NO- and cAMP-release and completely inversed values of released adenosine. When L-NAME an indomethacin (an nonspecific COX-inhibitor), 3 micromol/l where added together, they exhibit interactions between these two enzymatic systems. Namely, when L-NAME was added first, indomethacin didn't influence hemodynamic effects of NOS-inhibitor. On the other hand, when COX-inhibitor was added first, L-NAME widened autoregulatory range in small manner as after control autoregulatory experiments (40-90 cm H2O). All hemodynamic changes were followed with similar changes in NO-release, what suggest that exist interaction between L-arginine: NO system and COX-metabolites in the regulation of coronary autoregulation.     

Functional characterization of the mechanisms underlying bradykinin-induced relaxation in the isolated rat carotid artery

This work aimed to functionally characterize the mechanisms underlying the relaxation induced by bradykinin (BK) in the rat carotid artery. Vascular reactivity experiments, using standard muscle bath procedures, showed that BK (0.1 nmol/L-3 mumol/L) induced relaxation of phenylephrine-pre-contracted rings in a concentration-dependent manner. Endothelial removal strongly attenuated BK-induced relaxation. HOE-140, the selective antagonist of bradykinin B(2) receptors concentration-dependently reduced the relaxation induced by BK. Pre-incubation of endothelium-intact rings with L-NAME (100 micromol/L), a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (100 micromol/L), a selective inhibitor of the eNOS or 7-nitroindazole (100 micromol/L), the selective inhibitor of nNOS, reduced BK-induced relaxation. Conversely, 1400 W (10 nmol/L), a selective inhibitor of iNOS, did not alter the relaxation induced by BK. Surprisingly, indomethacin (10 micromol/L) a non-selective inhibitor of cyclooxygenase (COX) increased BK-induced relaxation in endothelium-intact but not denuded rings. Neither SQ29548 (3 micromol/L), a competitive antagonist of PGH(2)/TXA(2) receptors nor AH6809 (10 micromol/L), an antagonist of PGF(2alpha) receptors significantly altered the relaxation induced by BK in endothelium-intact rings. The combination of SQ29548 and AH6809 increased BK-induced relaxation. The present study shows that the vasorelaxant action displayed by BK in the rat carotid is mediated by endothelial B(2) receptors and the activation of the NO pathway. The major finding of this work is that it demonstrated functionally that endothelial-derived vasoconstrictor prostanoids (probably PGH(2), TXA(2) and PGF(2alpha)) counteract the vasorelaxant action displayed by BK.     

一氧化碳对大鼠离体肺动脉的舒张作用

本研究观察了一氧化碳 (CO)对离体大鼠肺动脉的舒张作用。制备Wistar大鼠肺动脉环 ,作出ACh浓度效应曲线之后 ,肺动脉环用一氧化氮合成酶抑制剂L NAME 3 0 μmol/L (n =10 )或血红素氧化酶抑制剂ZnPPIX 10μmol/L +L NAME 3 0 μmol/L (n =10 )孵育 3 0min ,再制备一个ACh的浓度效应曲线 ,观察ZnPPIX对ACh的浓度效应曲线的影响。另取一组肺动脉环 ,分为内皮完整组和去内皮组 ,观察外源性CO对肺动脉环张力的影响。结果表明 ,用L NAME孵育后 ,ACh的血管舒张反应受抑 ,最大抑制率为 5 0 4± 9 2 % ;用ZnPPIX +L NAME孵育后 ,ACh的血管舒张反应进一步受抑 ,最大抑制率为 84 4± 11 2 %。外源性CO无论对内皮完整组还是去内皮组肺动脉都有舒张作用。本研究提示 ,ZnPPIX可抑制ACh的内皮依赖性肺动脉舒张反应 ,CO是一个内皮源性的血管舒张因子 ,外源性CO可舒张肺动脉