甲基苯丙胺条件性位置偏爱大鼠中枢神经系统中一氧化氮合酶表达增加

目的·· :探讨甲基苯丙胺条件性位置偏爱大鼠中枢神经系统中 ,一氧化氮合酶 (NOS)表达变化。方法·· :建立大鼠甲基苯丙胺条件性位置偏爱模型 ,采用还原型辅酶Ⅱ -黄递酶组织化学染色方法对生理盐水组和甲基苯丙胺组大鼠不同脑区NOS阳性神经元数目进行比较。结果·· :与生理盐水相比 ,0.25 -1mg·kg-1甲基苯丙胺在大鼠中可以产生显著的条件性位置偏爱效应 ,1mg·kg-1 甲基苯丙胺组大鼠的伏核壳区、杏仁核、腹侧被盖区和大脑脚区域中NOS阳性细胞数目明显增加。结论·· :一氧化氮在甲基苯丙胺的条件性位置偏爱效应中发挥重要作用     

阿片类药物对诱导型NO合酶稳定表达神经细胞受体介导AC-cAMP系统的影响

目的：利用稳定表达诱导型ＮＯ合酶的ＮＧＬＮＣＸｉＮＯＳ细胞,观察阿片类药物对受体介导ＡＣｃＡＭＰ系统的影响。方法：竞争性蛋白结合法和完整细胞受体结合实验测定ｃＡＭＰ含量和受体结合力。结果：阿片类药物短时程作用抑制ＡＣ活性,受阿片受体介导的、百日咳毒素敏感的Ｇ蛋白信号通路调节。阿片类药物长时程作用和纳洛酮急性戒断引起受体脱敏,呈时间和剂量依赖性,伴有受体下调。结论：成功地建立阿片耐受和依赖细胞模型,可用于研究ＡＣｃＡＭＰ系统和ＮＯｃＧＭＰ系统在阿片耐受和成瘾中的调节。     

EFFECT OF HOMOCYSTEINE ON THE PRODUCTION OF NITRIC OXIDE IN ENDOTHELIAL CELLS

1. Homocysteine decreased the release of nitric oxide in cultured human umbilical vein endothelial cells. 2. Homocysteine did not affect constitutive and inducible nitric oxide synthase activity.     

INHIBITION OF ENDOTHELIAL NITRIC OXIDE BIOSYNTHESIS BY N-NITRO-l-ARGININE

1. The actions of N-nitro-L-arginine (NOLA) on the release of nitric oxide (NO) from arterial endothelial cells was studied in rat isolated thoracic aortic rings and by bioassay of NO derived from cultured bovine aortic endothelial cells. 2. NOLA (3-10 mumol/L) caused concentration-dependent inhibition of acetylcholine-induced relaxation of phenylephrine-contracted rat aortic rings, which is dependent on the release of NO from the endothelium. The inhibitory actions of NOLA could be prevented by pre- and co-incubation with L-arginine (1 mmol/L). 3. Endothelium-independent relaxation induced by sodium nitroprusside was not affected by NOLA. 4. The release of NO from bovine aortic endothelial cells, induced by bradykinin (10 nmol/L), was detected by bioassay on pre-contracted rabbit aortic strips. NOLA (1-3 mumol/L, given through the cell column) reduced or abolished the release of NO, but did not affect relaxations of the bioassay tissues induced by glyceryl trinitrate or authentic NO. 5. These data indicate that NOLA potently inhibits the biosynthesis of NO from L-arginine, and thus prevents its release from arterial endothelial cells. It may be a useful pharmacological tool for probing the significance of NO biosynthesis in cardiovascular function.     

年龄及人参皂甙 Rg1 对大鼠大脑皮层 NO 释放的影响

探讨了NO与衰老的关系及与Rg1抗衰老机制的关系。用Griess法,³H-L-精氨酸转化法分别研究大鼠大脑皮层细胞NO含量及NOS活性,观察其随龄变化及人参皂甙Rg1(Rg1)对老年鼠NO及NOS的影响。实验表明,成年鼠(9月龄)与青年鼠(3月龄)NO含量及NOS活性无显著性差异。老年鼠(27月龄)脑皮层NO含量明显高于青年鼠和成年鼠,NOS活性也明显增高。老年鼠给予Rg1后可显著减少大脑皮层NO含量和降低NOS活性。结果提示,NO与衰老关系密切,Rg1的抗衰老作用与其对NOS活性的抑制有关。     

CONTROL OF CORONARY BLOOD FLOW BY ENDOTHELIAL RELEASE OF NITRIC OXIDE

1. Nitric oxide (NO) is released from vascular endothelium following conversion of l-arginine to l-citrulline by calcium-calmodulin-dependent ‘constitutive’ NO-synthase. 2. Nitric oxide release occurs under basal conditions, in response to chemical stimuli (acetylcholine, bradykinin, thrombin, prostacyclin, serotonin, etc.) and in response to changes in shear stress (effects of blood velocity on vascular endothelium). 3. Analogues of l-arginine inhibit NO and are widely used to study the effects of NO on the cardiovascular system: in intact animals, these inhibitors cause vasoconstriction, leading to an increase in arterial blood pressure (ABP) and bradycardia. 4. Bradycardia induced by NO inhibitors is due, in part, to baroreceptor activity following the increase in ABP and in part to a direct effect on the sino-atrial node. 5. In the intact animals and isolated perfused heart, NO inhibitors cause coronary vasoconstriction and hence a reduction in basal coronary flow. This effect, however, is not seen in isolated coronary vessels. 6. From experiments in which ABP did not change, NO does not appear to have an important role in regulating coronary vasomotor tone under basal conditions. 7. Nitric oxide appears to be involved in the duration of reactive hyperaemia following coronary vascular occlusion but is not involved to any significant extent in the peak amplitude of hyperaemia. 8. Responses to vasodilator stimuli which do not involve NO in the initiation of the vasodilation may be prolonged by the effect of increased blood flow (shear stress) which releases NO and potentiates hyperaemia.     

NON-SPECIFIC INHIBITORS OF NITRIC OXIDE SYNTHASE CAUSE MYOCARDIAL NECROSIS IN THE RAT

1. To study the effect of acute nitric oxide (NO) inhibition on the rat heart both in vitro and in vivo, male Wistar rats received a single bolus injection of saline, N^ω-nitro-L-arginine methyl ester (L-NAME; 0.5,1.5,5.0,15.0 and 45.0 mg/kg) and D-NAME (45.0mg/kg). 2. Animals were killed 72 h after the bolus injection of L-NAME and the hearts were removed and studied under light microscopy. In other groups of animals, saline, L-NAME and D-NAME were administered as above and the mean arterial blood pressure (MABP/carotid) was recorded. Furthermore, L-NAME was also administered in the drinking water (20 mg/kg per day) for 72 h and animals were then killed and their hearts evaluated as described above. Hearts of control animals were perfused in vitro and coronary flow was measured following saline, L-NAME (45 μg/heart) and D-NAME (45 μ/heart). 3. Areas of necrosis were observed in the left ventricle of animals that had received L-NAME at 5.0, 15.0 and 45.0 mg/kg. Also, only doses higher than 1.5 mg/kg caused an important increase in MABP. The frequency and extent of the lesions paralleled the dose of L-NAME administered and no lesions were observed in D-NAME- and saline-treated animals. 4. The oral administration of L-NAME also caused myocardial lesions similar to those described above, but the frequency and extent of these lesions were more discrete compared with those observed following 5.0 mg/kg, i.v., L-NAME. 5. Bolus injection of L-NAME into control rat hearts in vitro resulted in a small and transient fall in coronary flow (17.2 ± 1.4 and 12.2 ± 1.2 mL/min before and after L-NAME administration, respectively) within 30 s and this was followed 4.5 min later by a further (11.5 ± 1.6 mL/min) decrease. The administration of D-NAME to control hearts caused no change in coronary flow. 6. In conclusion, the acute inhibition of NO biosynthesis by L-NAME causes myocardial necrosis. Both high levels of MABP and a small but significant reduction in coronary flow (associated or not) can be responsible for the lesions we found.     

ENDOTOXIN ALTERS THE SYSTEMIC DISPOSITION OF NITRIC OXIDE SYNTHASE INHIBITORS IN THE AWAKE SHEEP

1. We evaluated the haemodynamic effects and systemic disposition of the nitric oxide synthase (NOS) inhibitor N^L-nitro-L-arginine (NOLA) after intravenous (i.v.) administration of two different doses (5 and 20 mg/kg) in awake healthy sheep and awake sheep given a continuous i.v. infusion of endotoxin (lipopolysaccharide, 12 ng/kg per h, i.v., for 18 h). In addition, we determined the systemic disposition of another NOS inhibitor, N^L-nitro-L-arginine methylester (L-NAME; 20 mg/kg, i.v.) in awake healthy sheep only. 2. A^rL-Nitro-L-arginine produced a dose-dependent decrease in heart rate (HR) and cardiac output (CO) together with a dose-dependent increase in mean arterial pressure (MAP) and peripheral vascular resistance (PVR) when compared to baseline. In endotoxic sheep NOLA produced a greater increase in MAP and mean pulmonary arterial pressure (MPAP). 3. In healthy sheep there was a dose-related increase in total body clearance (CI) of NOLA. The CI increased from 0.028 L/min after the lower dose to 0.032 L/min after the higher dose. The infusion of endotoxin caused an increase in CI of NOLA to 0.040 and 0.047 L/min, respectively, and a decrease in plasma slow half-life (t_1/2 from 825 to 546 min and from 780 to 453 min, respectively. 4. N^L-Nitro-L-arginine methylester was rapidly cleared from the plasma with a slow half-life of approximately 7.5 min and there was a simultaneous appearance of NOLA in the plasma. 5. These results support the view that nitric oxide has a significant role in regulating vascular tone in healthy and endotoxic sheep and indicate that the increases in CI of NOLA with an increase in its dose and the presence of endotoxin will be important in influencing appropriate dosage regimens in clinical studies.     

ROLE OF NITRIC OXIDE AND ENDOTHELIUM IN THE FLOW-INDUCED DILATION OF RAT CORONARY ARTERIES UNDER TWO PRECONSTRICTION CONDITIONS

1. Pressure-induced tone and flow-induced dilations were studied in a rat perfused epicardial coronary artery mounted in an arteriograph. Spontaneous tone was assessed in arteries submitted either to 60 or 90 mmHg intraluminal pressure either under control conditions, after incubation with NG-nitro-L-arginine methyl ester (L-NAME; 100 mumol/L) or after endothelial denudation. Flow-induced dilation was quantified under these conditions in preparations either submitted to 60 mmHg and preconstricted with 10 mumol/L 5-hydroxytryptamine (5-HT) or exhibiting spontaneous tone at 90 mmHg. 2. Spontaneous tone was greater at 90 mmHg compared with tone obtained at 60 mmHg (21 +/- 2 vs 10 +/- 2% reduction of the fully dilated diameter after sodium nitroprusside incubation, respectively). Incubation with L-NAME or removal of the endothelium significantly increased spontaneous tone at both pressures compared with control. 3. In arteries submitted to 60 mmHg and preconstricted with 10 mumol/L 5-HT, flow (0-800 microL/min) induced a continuous dilation (maximal value 63 +/- 4%). As a function of flow, shear stress first increased and then plateaued at values of approximately 76 +/- 6 dyn/cm2. After L-NAME incubation or endothelial denudation, the flow-induced dilation was reduced to the same extent and was obtained for higher values of shear stress (172 +/- 14 and 150 +/- 14 dyn/cm2, respectively). 4. In arteries exhibiting spontaneous tone, starting flow led, first, to a constriction followed by a dilation up to 76 +/- 4% of the initial tone. Incubation with L-NAME greatly altered flow-induced dilation. Endothelium removal further reduced the dilation obtained for very high values of shear stress (up to 300 dyn/cm2). 5. The present study shows that different patterns of vasodilation induced by flow can be observed, depending on the initial vasoconstrictor stimulus. In 5-HT-preconstricted arteries, flow-induced dilation appears to be fully dependent on the synthesis and release of nitric oxide. In arteries with spontaneous tone, a vasoconstrictor substance could be released for low values of flow. Nitric oxide is mainly, but not exclusively, responsible for the vasodilation. For both experimental conditions, removal of the endothelium greatly reduced the response, but a dilation was still observed.     

INHIBITION OF NITRIC OXIDE SYNTHASE SPECIFICALLY ENHANCES ADRENERGIC VASOCONSTRICTION IN RABBITS

1. The effect of inhibition of nitric oxide biosynthesis using N-nitro-L-arginine (NOLA) was examined in conscious rabbits and rabbit isolated aortae. 2. In autonomically blocked conscious rabbits intravenous infusion of NOLA (15 mg/kg) significantly increased arterial pressure and hindlimb vascular resistance but did not affect heart rate. Depressor and hindlimb vasodilator responses to acetylcholine (3-12 micrograms/kg per min) were significantly attenuated in the presence of NOLA. In contrast, NOLA significantly enhanced responses to intravenous infusion of glyceryl trinitrate (10-40 micrograms/kg per min) in vivo. 3. Infusion of noradrenaline (1-4 micrograms/kg per min) or the release of neuronal noradrenaline in response to the infusion of tyramine (80-320 micrograms/kg per min) increased arterial pressure and hindlimb vascular resistance in autonomically blocked conscious rabbits. After the administration of NOLA, the vasoconstrictor responses to both noradrenaline and tyramine were significantly enhanced. 4. In isolated rabbit aortae, NOLA (10 mumol/L) significantly impaired relaxant responses to acetylcholine but did not affect responses to glyceryl trinitrate. NOLA enhanced contractile responses to the adrenoceptor agonists noradrenaline and phenylephrine but did not affect the contractile responses to the thromboxane-mimetic U46619. 5. These data indicate that in autonomically blocked conscious rabbits, NOLA causes systemic vasoconstriction, impairs dilator responses to acetylcholine and enhances dilator responses to glyceryl trinitrate. In addition, NOLA enhances constrictor responses to both exogenous and neuronally-released noradrenaline. These results suggest that nitric oxide is important in the regulation of normal vascular tone and in the modulation of vascular responses to vasodilator and vasoconstrictor agents.     

EFFECT OF INHIBITION OF NITRIC OXIDE SYNTHASE AND GUANYLATE CYCLASE ON HYDRALAZINE-INDUCED VASODILATATION OF THE HUMAN FETAL PLACENTAL CIRCULATION

1. The vasodilator effects of hydralazine in vitro, using the Krebs’ perfused human placental lobule was studied. Single placental lobules were bilaterally perfused (maternal and fetal sides 5 mL/min each, 95% O₂, 5% CO₂, 37°C) and changes in fetal arterial pressure (FAP) and venous outflow (VO) were recorded. 2. Submaximal vasoconstriction was induced by KCl (20–50 mmol/L), which increased basal FAP from 22.8 ± 1.7 to 91.3 ± 3.9 mmHg (n = 9 , P<0.001), and decreased VO from 4.1 ± 0.6 to 0.2 ± 0.1 mL/min (n = 6 , P<0.01). 3. Hydralazine caused vasodilatation (IC₅₀ 1.9 mmol/L, n = 9) and increased VO in the presence of KCl-induced vasoconstriction. 4. Infusion of N^ω-nitro-L-arginine (100 μmol/ L) to block nitric oxide synthase caused the basal FAP to increase from 30.9 ± 5.9 to 47.4 ± 6.7 (n = 6 , P<0.05) and significantly potentiated hydralazine-induced vasodilatation (n = 7 , P<0.05). 5. The soluble guanylate cyclase inhibitor LY 83583 (6-anilino-5,8-quinolinedione) (1 μmol/L) significantly antagonized the vasodilatation produced by hydralazine (n = 5 , P<0.05). 6. Thus, hydralazine appears to activate guanylate cyclase, leading to increased cyclic GMP in fetal arterial vascular smooth muscle to cause vasorelaxation. No evidence was obtained to suggest that hydralazine exerted its action by either releasing nitric oxide from endothelial cells in the placenta or acting as a nitric oxide donor.     

REGULATION OF LEUKOCYTE FUNCTION BY NITRIC OXIDE DONORS: THE EFFECT OF S-NITROSO-THIOL COMPLEXES

The pulmonary toxicity of particles is often studied using a single intratracheal instillation of the material. It was hypothesized that smaller multiple intratracheal administrations of silica would result in differences in pulmonary responses as compared to a single large intratracheal administration. In the first of a series of experiments, the pulmonary responses in male F344 rats to a single intratracheal instillation of crystalline silica (5 mg/100 g body weight) given on d 0 were compared with those resulting from 5 consecutive daily intratracheal administrations of the dust (1 mg/100 g body weight/d) with the initial dose given on d 0. Controls received saline intratracheally. In the second experiment, the dose was reduced to 1 mg/100 g body weight for the single-dose protocol and 0.2 mg/100 g body weight/d for 5 consecutive days for the multiple-dose protocol. In both experiments, responses were assessed on d 14. In the third experiment, the doses were the same as the first experiment, but the responses were assessed on d 28. The indices of toxicity were cellular differentials recovered by bronchoalveolar lavage, which is an index of inflammation, and the level of albumin in the bronchoalveolar lavage fluid, a measure of damage to the capillary-epithelial barrier. At the higher dose of silica, similar levels of inflammation and lung damage were evident in both dosing protocols. Less severe responses occurred at the lower dose. The comparative pattern between the single and multiple dosing protocols was similar in all three experiments. Since only minor differences were noted in the pulmonary responses when the responses to the single- and multiple-dose protocols were compared, data indicate that the multiple-dose protocol does not offer any advantages over the single-dose protocol.     

INTRARENAL LOCALIZATION OF NITRIC OXIDE SYNTHASE ISOFORMS AND SOLUBLE GUANYLYL CYCLASE

1. Nitric oxide (NO) plays an important role in the regulation of renal function. To date, five isoforms of NO synthase (NOS) and four subunits of soluble guanylyl cyclase have been cloned. The kidney contains four isoforms of NOS and all sub-units of soluble guanylyl cyclase. 2. This review focuses on the intrarenal location of the isoforms of NOS and the subunits of soluble guanylyl cyclase.     

一氧化氮合酶抑制剂和增强剂的高通量筛选

目的建立一氧化氮合酶(NOS)活性的高通量检测方法，筛选调节NOS活性的药物。方法通过NADPH荧光值的变化，间接反映NOS活性。通过对反应体系的优化，调整各反应底物(NADPH，L-Arg，NOS)及抑制剂(L-NNA)的浓度，建立高通量筛选模型并对5 600个样品进行筛选。结果方法简便、容易操作、灵敏度高、结果稳定，发现了一批对NOS具有抑制或增强作用的化合物。结论此法适用于高通量药物筛选。     

EXPRESSION AND REGULATION OF THE SODIUM–CALCIUM EXCHANGER IN CARDIAC MICROVASCULAR ENDOTHELIAL CELLS

1. The sodium-calcium exchanger (NCX) plays an important role in Ca2+ homeostasis. In the heart, NCX participates in the control of contraction and relaxation and in large vessel endothelial cells some data suggest that NCX could influence nitric oxide (NO) generation. In this context, the cardiac microvasculature has received considerable attention as a mediator of myocardial performance, via the release of paracrine acting factors such as NO. Therefore, the aim of the current study was to characterize NCX expression and regulation in cardiac microvascular endothelial cells (CMEC). The NCX expression was also examined in neonatal ventricular cardiomyocytes where aspects of its function and regulation have been well characterized. 2. The presence of functional NCX in CMEC was confirmed by the presence of a consistent rise in intracellular Ca2+ concentration ([Ca2+]i) in response to removal of extracellular Na+. Furthermore, NCX mRNA expression was readily detectable in CMEC. 3. In order to examine the role of possible physiological regulators of NCX expression, the effect of intracellular Ca2+ loading, caused by 24 h exposure to 10 mumol/L ouabain, was investigated. In Ca(2+)-loaded CMEC, there was a substantially greater rise in [Ca2+]i during exposure to Na(+)-free buffer: 33 +/- 6 versus 124 +/- 25 nmol/L% (P < 0.05), consistent with increased protein expression. Consistent with these findings, northern blot analysis confirmed the presence of a two-fold increase in NCX mRNA in these cells. 4. These data indicate the presence of functional NCX in CMEC and identify [Ca2+]i as a potential physiological regulator of expression.