Role of poly(ADP-ribose) polymerase activation in endotoxin-induced cardiac collapse in rodents

Reactive oxygen and nitrogen species are overproduced in the cardiovascular system during circulatory shock. Oxidant-induced cell injury involves the activation of poly(ADP-ribose) polymerase (PARP). Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with the new potent phenanthridinone PARP inhibitor PJ34 [the hydrochloride salt of N-(oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide], we studied whether the impaired cardiac function in endotoxic shock is dependent upon the PARP pathway. Escherichia coli endotoxin (lipopolysaccharide, LPS) at 55 mg/kg, i.p., induced a severe depression of the systolic and diastolic contractile function, tachycardia, and a reduction in mean arterial blood pressure in both rats and mice. Treatment with PJ34 significantly improved cardiac function and increased the survival of rodents. In addition, LPS-induced depression of left ventricular performance was significantly less pronounced in PARP-1 knockout mice (PARP(-/-)) as compared with their wild-type littermates (PARP(+/+)). Thus, PARP activation in the cardiovascular system is an important contributory factor to the cardiac collapse and death associated with endotoxin shock.     

Role of nitrosative stress and poly(ADP-ribose) polymerase activation in diabetic vascular dysfunction

Complications of diabetes rather than the primary disease itself pose the most challenging aspects of diabetic patient management. Diabetic vascular dysfunction represents a problem of great clinical importance underlying the development of many of the complications including retinopathy, neuropathy and the increased risk of stroke, hypertension and myocardial infarction. Hyperglycaemia stimulates many cellular pathways, which result in oxidative stress, including increased production of advanced glycosylated end products, protein kinase C activation, and polyol pathway flux. Endothelial cells produce nitric oxide constitutively to regulate normal vascular tone; the combination of this nitric oxide with the hyperglycaemia-induced superoxide formation results in the production of reactive nitrogen species such as peroxynitrite. This nitrosative stress results in many damaging cellular effects, but it is these effects on DNA, which are the most damaging to the cell function; nitrosative stress induces DNA single stand breaks and leads to over-activation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP). PARP activation contributes to endothelial cell dysfunction and appears to be the central mediator in all the mechanisms by which hyperglycaemia-induces diabetic vascular dysfunction. This review focuses on the mechanism by which hyperglycaemia induces nitrosative stress and the role PARP activation plays in diabetic vascular dysfunction.     

The role of nitric oxide, superoxide and peroxynitrite in the anti-arrhythmic effects of preconditioning and peroxynitrite infusion in anaesthetized dogs

Background and purpose:

Both ischaemia preconditioning (PC) and the intracoronary infusion of peroxynitrite (PN) suppress ischaemia and reperfusion (I/R)-induced arrhythmias and the generation of nitrotyrosine (NT, a marker of PN). However, it is still unclear whether this latter effect is due to a reduction in nitric oxide (NO) or superoxide (O₂⁻) production.

Experimental approach:

Dogs anaesthetized with chloralose and urethane were infused, twice for 5 min, with either saline (control) or 100 nM PN, or subjected to similar periods of occlusion (PC), 5 min prior to a 25 min occlusion and reperfusion of the left anterior descending coronary artery. Severities of ischaemia and ventricular arrhythmias, as well as changes in the coronary sinus nitrate/nitrite (NOx) levels were assessed throughout the experiment. The production of myocardial NOx, O₂⁻ and NT was determined following reperfusion.

Key results:

Both PC and PN markedly suppressed the I/R-induced ventricular arrhythmias, compared to the controls, and increased NOx levels during coronary artery occlusion. Reperfusion induced almost the same increases in NOx levels in all groups, but superoxide production and, consequently, the generation of NT were significantly less in PC- and PN-treated dogs than in controls.

Conclusions and implications:

Since both PC and the administration of PN enhanced NOx levels during I/R, the attenuation of endogenous PN formation in these dogs is primarily due to a reduction in the amount of O₂ produced. Thus, the anti-arrhythmic effect of PC and PN can almost certainly be attributed to the preservation of NO availability during myocardial ischaemia.     

四逆汤对失血性休克家兔一氧化氮(NO)和一氧化氮合酶(NOS)的影响

目的：探讨中药四逆汤对失血性休克家兔一氧化氮的影响。方法：采用动物分组对照实验,测量不同状态下动物血浆一氧化氮和一氧化氮合酶(NOS)的变化。结果：与休克前比较,休克组的家兔血浆一氧化氮水平各时段均明显提高(P＜0．01),而治疗组仅在治疗后30min家兔一氧化氮及NOS水平明显提高(P＜0．01),其他时段与休克前比较,未见显著差异(P＜0．01),且休克后lh、4h、8h休克组一氧化氮水平显著高于治疗组(P＜0．01)。经药物治疗的失血性休克家兔血浆一氧化氮水平明显降低。     

Beneficial effects of 3-aminobenzamide,an inhibitor of poly (ADP-ribose) synthetase in a rat model of splanchnic artery occlusion and reperfusion

1. Peroxynitrite, a potent cytotoxic oxidant formed by the reaction of nitric oxide with superoxide anion, and hydroxyl radical, formed in the iron-catalysed Fenton reaction, are important mediators of reperfusion injury. In in vitro studies, DNA single strand breakage, triggered by peroxynitrite or by hydroxyl radical, activates the nuclear enzyme poly (ADP-ribose) synthetase (PARS), with consequent cytotoxic effects. Using 3-aminobenzamide, an inhibitor of PARS, we investigated the role of PARS in the pathogenesis of splanchnic artery occlusion shock.
2. Splanchnic artery occlusion and reperfusion shock (SAO/R) was induced in rats by clamping both the superior mesenteric artery and the coeliac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies.
3. SAO/R rats developed a significant fall in mean arterial blood pressure, significant increase of tissue myeloperoxidase activity and marked histological injury to the distal ileum. SAO/R was also associated with a significant mortality (0% survival at 2 h after reperfusion).
4. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO/R rats, starting early after reperfusion, but not during ischaemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific ‘footprint'' of peroxynitrite, in the necrotic ileum in shocked rats, as measured at 60 min after the start of reperfusion.
5. In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively).
6. In a separate set of studies, using a 4000 Dalton fluorescent dextran tracer, we investigated the changes in epithelial permeability associated with SAO/R. Ten minutes of reperfusion, after 30 min of splanchnic artery ischaemia, resulted in a marked increase in epithelial permeability.
7. There was a significant increase in PARS activity in the intestinal epithelial cells, as measured 10 min after reperfusion ex vivo. 3-Aminobenzamide, a pharmacological inhibitor of PARS (applied at 10 mg kg⁻¹, i.v., 5 min before reperfusion, followed by an infusion of 10 mg kg⁻¹ h⁻¹), significantly reduced ischaemia/reperfusion injury in the bowel, as evaluated by histological examination. Also it significantly improved mean arterial blood pressure, improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and reduced the reperfusion-induced increase in epithelial permeability.
8. 3-Aminobenzamide also prevented the infiltration of neutrophils into the reperfused intestine, as evidenced by reduced myeloperoxidase activity. It improved the histological status of the reperfused tissues, reduced the production of peroxynitrite in the late phase of reperfusion and improved survival.
9. In conclusion, our study demonstrates that the PARS inhibitor 3-aminobenzamide exerts multiple protective effects in splanchnic artery occlusion/reperfusion shock. We suggest that peroxynitrite and/or hydroxyl radical, produced during the reperfusion phase, trigger DNA strand breakage, PARS activation and subsequent cellular dysfunction. The vascular endothelium is likely to represent an important cellular site of protection by 3-aminobenzamide in SAO shock.

     

Nitric oxide production and endothelium-dependent vasorelaxation induced by wine polyphenols in rat aorta

1. The aim of this work was to investigate the mechanism of vasorelaxation induced by red wine polyphenolic compounds (RWPC) and two defined polyphenols contained in wine, leucocyanidol and catechin. The role of the endothelium, especially endothelium-derived nitric oxide (NO), was also investigated.
2. Relaxation produced by polyphenols was studied in rat aortic rings with and without functional endothelium, pre-contracted to the same extent with noradrenaline (0.3 and 0.1 μM, respectively). RWPC and leucocyanidol, but not catechin, produced complete relaxation of vessels with and without endothelium. However, 1000 fold higher concentrations were needed to relax endothelium-denuded rings compared to those with functional endothelium.
3. High concentrations of catechin (in the range of 10⁻¹ g l⁻¹) only produced partial relaxation (maximum 30%) and had the same potency in rings with and without endothelium.
4. The NO synthase inhibitor, N^ω-nitro-L-arginine-methyl-ester (L-NAME, 300 μM) completely abolished the endothelium-dependent but not the endothelium-independent relaxations produced by all of the polyphenolic compounds.
5. In contrast to superoxide dismutase (SOD, 100 u ml⁻¹), neither RWPC nor leucocyanidol affected the concentration-response curve for the NO donor, SIN-1 (3-morpholino-sydnonimine) which also produces superoxide anion (O₂⁻).
6. In aortic rings with endothelium, RWPC (10⁻² g l⁻¹) produced a 7 fold increase in the basal production of guanosine 3′ : 5′-cyclic monophosphate (cyclic GMP) which was prevented by L-NAME (300 μM).
7. Electron paramagnetic resonance (e.p.r.) spectroscopy studies with Fe²⁺-diethyldithiocarbamate as an NO spin trap demonstrated that RWPC and leucocyanidol increased NO levels in rat thoracic aorta about 2 fold. This NO production was entirely dependent on the presence of the endothelium and was abolished by L-NAME (300 μM).
8. These results show that RWPC and leucocyanidol, but not the structurally closely related polyphenol catechin, induced endothelium-dependent relaxation in the rat aorta. They indicate that this effect results from enhanced synthesis of NO rather than enhanced biological activity of NO or protection against breakdown by O₂⁻. It is concluded that some polyphenols, with specific structure, contained in wine possess potent endothelium-dependent vasorelaxing activity.

     

洛伐他汀对低密度脂蛋白损伤血管内皮的保护作用

目的：探讨洛伐他汀对低密度脂蛋白所致血管内皮功能损伤的保护作用及可能的机制．方法：一次性从大鼠舌下静脉注射天然低密度脂蛋白(n-LDL 4mg／kg),在舌下静脉注射n-LDL之前大鼠腹腔注射洛伐他汀(2或4mg／kg),每天一次,连续五天．注射n-LDL后48小时检测乙酰胆碱诱导的血管内皮依赖性舒张(EDR)及血清一氧化氮(N0)、丙二醛(MDA)的含量和超氧化物歧化酶(SOD)的活性．结果：一次注射n-LDL导致了EDR、血清NO水平及SOD活性明显降低,MDA的浓度明显增高。预先给予洛伐他汀能明显减轻LDL引起的EDR的抑制和血清NO水平及SOD活性的降低,减少MDA的生成,左旋硝基精氨酸(L-NNA)减弱洛伐他汀对血管内皮的保护作用．结论：洛伐他汀对LDL损伤的血管内皮具有保护作用,可能与保护内皮依赖性松弛因子和抗氧化作用有关。     

Cigarette smoking in men and vascular responsiveness

AIMS: Smoking is a major risk factor for developing atherosclerosis. In order to understand the vascular abnormalities observed in smokers, we investigated vascular responsiveness in cigarette smokers. METHODS: We performed two consecutive matched group comparative studies to investigate vascular responsiveness using venous occlusion plethysmography. The mean effects of three incremental doses of each vasoactive agent are presented. Both studies compared smokers with nonsmokers. RESULTS: The first investigated 68 subjects (smokers = 29; mean +/- s.d. ages; 24 +/- 6 vs 25 +/- 5 years; P = NS) and found smoking was associated with a significant blunting of the flow ratio between treated and untreated arms to endothelium-dependent vasodilatation to acetylcholine (mean +/- s.d., nonsmokers vs smokers) 4.07 +/- 2.18 vs 3.42 +/- 1.79 (P = 0.04, 95% CI 0.02, 1.12). By contrast, there was no significant difference in the responses to the endothelium-independent vasodilators sodium nitroprusside and verapamil. Smoking was also associated with a significant impairment in endothelium-dependent vasoconstriction induced by monomethyl-L-arginine (L-NMMA) 0.78 +/- 0.22 vs 0.87 +/- 0.21 (P = 0.006, 95% CI -0.14, -0.02) and a trend to blunted endothelium-independent vasoconstrictor responses to noradrenaline. In the second study we investigated the response to angiotensin I and II in 23 subjects (smokers = 12; mean +/- s.d. ages; 34 +/- 10 vs 32 +/- 11 years). There was significant impairment in smokers of the mean vasoconstrictor response to angiotensin I 0.51 +/- 0.15 vs 0.59 +/- 0.16 (nonsmokers vs smokers; P = 0.003, 95% CI -0.13, -0.03) and a nonsignificant trend towards impairment of the response to angiotensin II. CONCLUSIONS: Cigarette smoking in male volunteers is associated with blunted basal and stimulated nitric oxide bioactivity. Endothelial independent vasodilator responses (to nitroprusside and verapamil) were unaltered in smokers. A defect in the vasoconstrictor response to angiotensin I was also seen.     

Requirement of intracellular calcium mobilization for peroxynitrite-induced poly(ADP-ribose) synthetase activation and cytotoxicity.

Peroxynitrite is a cytotoxic oxidant produced during shock, ischemia reperfusion, and inflammation. The cellular events mediating the cytotoxic effect of peroxynitrite include activation of poly(ADP-ribose) synthetase, inhibition of mitochondrial respiration, and activation of caspase-3. The aim of the present study was to investigate the role of intracellular calcium mobilization in the necrotic and apoptotic cell death induced by peroxynitrite. Peroxynitrite, in a low, pathophysiologically relevant concentration (20 microM), induces rapid (1 to 3 min) Ca(2+) mobilization in thymocytes. Inhibition of this early calcium signaling by cell-permeable Ca(2+) chelators [EGTA-acetoxymethyl ester (AM), 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N , N',N'-tetraacetic acid-tetra-AM] abolished cytotoxicity as measured by propidium iodide uptake. Intracellular Ca(2+) chelators also inhibited DNA single-strand breakage and activation of poly(ADP-ribose) synthase (PARS), which is a major mediator of cell necrosis in the current model. Intracellular Ca(2+) chelators also protected PARS-deficient thymocytes from peroxynitrite cytotoxicity, providing evidence for a PARS-independent, Ca(2+)-dependent cytotoxic pathway. Chelation of intracellular Ca(2+) blocked the peroxynitrite-induced decrease of mitochondrial membrane potential, secondary superoxide production, and mitochondrial membrane damage. Peroxynitrite-induced internucleosomal DNA cleavage was increased on BAPTA-AM pretreatment in the wild-type cells but decreased in the PARS-deficient cells. Two other apoptotic parameters (phosphatidylserine exposure and caspase 3 activation) were inhibited by BAPTA-AM in both the wild-type and the PARS-deficient thymocytes. Our findings provide evidence for the pivotal role of an early Ca(2+) signaling in peroxynitrite cytotoxicity.     

Role of nitrosative stress and poly(ADP-ribose) polymerase activation in myocardial reperfusion injury

Ischemia and reperfusion injury leads to a complex pathophysiological process, which in turn results in the generation of free radicals. Peroxynitrite, a highly reactive species causes DNA single strand breaks, which activates the nuclear enzyme, poly (ADP-ribose) polymerase (PARP). The activation of PARP leads to an energy consuming inefficient repair cycle with subsequent depletion of NAD(+) and ATP pools and necrotic cell death. The present review overviews the pathophysiological role of the peroxynitrite-PARP pathway in cardiac ischemia/reperfusion injury with special reference to the therapeutic potential of PARP inhibitors in the treatment of this disease.     

AMP-activated protein kinase activation as a strategy for protecting vascular endothelial function

1. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase involved in the regulation of cellular and organismal metabolism. AMPK has a heterotrimeric structure, consisting of a catalytic alpha-subunit and regulatory beta- and gamma-subunits, each of which has two or more isoforms that are differentially expressed in various tissues and that arise from distinct genes. The AMPK system acts as a sensor of cellular energy status that is conserved in all eukaryotic cells. In addition, AMPK is activated by physiological stimuli and oxidants. 2. The importance of AMPK in cardiovascular functions is best demonstrated by recent studies showing that widely used drugs, including statins, metformin and rosiglitazone, execute cardiovascular protective effects at least partly through the activation of AMPK. As a consequence, AMPK has been proposed as a candidate target for therapeutic intervention in the treatment of both Type 2 diabetes and metabolic syndrome owing to its central role in the regulation of energy balance; it may also have a role in weight control. 3. In the present brief review, we summarize the recent progress of AMPK signalling and regulation focusing on vascular endothelial cells. We further hypothesize that AMPK is a dual sensor for energy and redox status within a cell and AMPK may be a therapeutic target for protecting vascular endothelial function.     

Inhibition of poly(ADP-ribose) polymerase prevents vascular hyporesponsiveness induced by lipopolysaccharide in isolated rat aorta

Recent studies clearly show that there is a relationship between endotoxemia and impaired vascular responsiveness. The aim of this study was to investigate whether treatment with the new potent PARP inhibitor PJ34 could prevent the vascular hyporesponsiveness induced by lipopolysaccharide (LPS). Endotoxemia was induced in rats by LPS injection (20 mgkg-1, i.p.). Administration of LPS caused a decrease in mean blood pressure and an increase in heart rate. In endothelium-denuded rings of thoracic aorta from untreated rats, contractile responses to KCl and phenylephrine decreased after LPS injection. Furthermore, there was a significant loss of endothelium-dependent vasodilatation in response to acetylcholine in LPS-treated rats. The animals pretreated with PJ34 (10 mgkg-1, i.p., 30 min before LPS injection), the effect of LPS on vascular responsiveness was lower than the untreated ones. Pretreating the animals with PJ34 before the LPS challenge prevented the decline in mean blood pressure. However, this did not result in significant changes to the heart rate. The inhibitory effect of LPS treatment on both KCl- and phenylephrine-induced contraction responses was significantly antagonized by PJ34. Additionally, pretreatment of the rats with PJ34 attenuated the LPS-induced endothelial dysfunction in endothelium-intact aorta rings. This study demonstrates that PARP activation in the vascular system is an important contributory factor to the impaired vascular responsiveness associated with endotoxic shock. Hence, the pharmacological inhibition of PARP pathway might be an effective intervention to prevent endotoxin-induced vascular hyporesponsiveness.     

Role for poly(ADP-ribose) polymerase activation in diabetic nephropathy, neuropathy and retinopathy

Chronic complications of diabetes mellitus e.a. diabetic nephropathy, neuropathy and retinopathy develop in at least 30-50% of patients with both Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetes, and are the major cause of increased morbidity and mortality. The ultimate consequences of diabetes complications include renal failure, foot ulceration and amputation, and blindness. The magnitude of the problem and its economic impact make extremely important to understand the natural history of chronic diabetes complications and to identify more successful preventive and therapeutic options. The pathogenesis of diabetes complications involves multiple mechanisms. The importance of vascular component is well recognized in diabetic retinopathy, which is primarily a vascular disease, as well as diabetic nephropathy developing as a result of complex interplay between hemodynamic and metabolic factors. The importance of vascular versus non-vascular mechanisms in the pathogenesis of diabetic neuropathy remains a subject of debate. Studies in animal and cell culture models revealed that such mechanisms as increased aldose reductase activity, non-enzymatic glycation/glycoxidation, activation of protein kinase C, impaired growth factor support, enhanced oxidative/nitrosative stress, and its downstream effectors such as mitogen-activated protein kinase activation, inflammatory response, endothelin-1 overexpression and impaired Ca(++) signaling, play an important role in all three tissue-targets for diabetes complications i.e. kidney, retina and peripheral nerve. Evidence for important role of the downstream effector of free radical and oxidant-induced DNA injury, poly(ADP-ribose) polymerase activation, is emerging. This review describes recent studies addressing the role for poly(ADP-ribose) polymerase activation in diabetic nephropathy, neuropathy and retinopathy.     

Interaction between superoxide anion and nitric oxide in the regulation of vascular endothelial function

1. Nitric oxide (NO)-mediated, endothelium-dependent vasodilator function in rat aortic smooth muscle was investigated in an in vitro model of endogenous vascular superoxide anion stress, generated by pretreatment with the Cu/Zn superoxide dismutase (SOD, EC 1.15.1.1) inhibitor, diethyldithiocarbamate (DETCA).
2. Contraction to noradrenaline (NA, 1 nM–1 μM) in endothelium-intact vessels was augmented after a 30 min pretreatment with DETCA (10 mM) followed by 30 min washout. This effect was abolished by N^G-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) and removal of the endothelium and partially reversed by exogenous Cu/Zn SOD (200 u ml⁻¹).
3. Endothelium- and basal NO-dependent vasorelaxation to the phosphodiesterase (PDE) type V inhibitor ONO-1505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquinazoline methanesulphonate) (0.1–10 μM) was inhibited after DETCA (10 mM) pretreatment. In addition, the ability of L-NAME (0.3 mM) to enhance established contractile tone was effectively absent.
4. In contrast, DETCA pretreatment did not significantly affect vasorelaxation to acetylcholine (ACh, 1 nM–3 μM) or S-nitroso-N-acetyl penicillamine (SNAP, 0.03–30 μM). However, L-NAME (0.3 mM) unmasked an inhibitory effect of DETCA pretreatment on vasorelaxation to SNAP in endothelium-intact vessels while markedly potentiating vasorelaxation to SNAP in control tissue.
5. L-NAME (0.3 mM)- and exogenous catalase (200 u ml⁻¹)-sensitive vasorelaxation to exogenous Cu/Zn SOD (200 u ml⁻¹) was greater after DETCA (10 mM) pretreatment in endothelium-intact aortic rings. This difference was abolished by catalase (200 u ml⁻¹).
6. In conclusion, tissue Cu/Zn SOD inhibition elicited a selective lesion in basal endothelial function in rat isolated aortic smooth muscle, consistent with the inactivation of basal NO by superoxide anion. The resulting leftward shift in nitrovasodilator reactivity, due to the loss of the tonic depression by basal NO, is likely to mask the inhibitory effect of superoxide anion on agonist-stimulated endothelial function and nitrovasodilator-derived NO, thereby accounting for the differential pattern of endothelial dysfunction after DETCA pretreatment.

     

Endothelium-independent relaxation induced by histamine in human dorsal penile artery

1. In vitro preparations of human dorsal penile arteries were used to evaluate the effect of histamine and to characterize the histamine receptors involved in the response. 2. Cumulative administration of histamine induced a concentration-dependent relaxation in precontracted arteries. The H1 receptor agonist 2-pyridylethylamine induced a biphasic response: contraction followed by dilation. The H2 receptor agonist dimaprit produced a marked relaxation. Mepyramine, a histamine H1 receptor antagonist, led to a slight but statistically significant change in the pD2 value corresponding to the relaxant phase of the H1 receptor agonist and the histamine curve. The H2 receptor antagonist cimetidine induced a marked shift in the dimaprit concentration-response curve without affecting the maximum response. Incubation with cimetidine led to a considerable loss in the sensitivity of the arteries to histamine and in the maximum relaxation. Combined treatment with histamine H1 and H2 receptor antagonists resulted in an additional displacement compared with the effect of each antagonist alone on the histamine response. The effects observed using a histamine H3 receptor agonist and antagonist suggest that the involvement of this receptor is unlikely. 3. Removal of the endothelium was unable to reverse the histamine response. Pretreatment with NG-nitro-L-arginine methyl ester, L-arginine and indomethacin had no effect on the histamine control curve. 4. In conclusion, the vasodilation of human dorsal penile artery induced by histamine seems to be mainly mediated by muscular histamine H2 receptors, without the intervention of key intracellular mediators, such nitric oxide or relaxant prostanoids. A minor population of relaxant histamine H1 receptors cannot be excluded.     

Role of adventitial nitric oxide in vascular hyporeactivity induced by lipopolysaccharide in rat aorta

This study was designed to elucidate the role of the adventitia in NO-mediated vascular effects of lipopolysaccharide (LPS). After incubation of rat aorta with LPS, the adventitia generated 3.5 times more nitrite plus nitrate than a corresponding segment of media. Control media covered by adventitia from LPS-treated aortic rings exhibited a 4 fold elevated level of cyclic GMP. Medial layers from LPS-treated aortic rings (like LPS-treated adventitia-intact rings) exhibited a decrease in sensitivity to noradrenaline (NA) that was reversed by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (1 μM) or N^ω-nitro-L-arginine methylester (0.3 mM). However, in contrast to LPS-treated adventitia-intact rings, medial layers showed no reduction in maximal contraction to NA and virtually no relaxation to L-arginine. These data indicate that in blood vessels exposed to LPS, the adventitia is a more powerful source of NO than the media. The adventitia-derived NO can reach soluble guanylyl cyclase in the medial layer and contribute greatly to vascular hyporeactivity and L-arginine-induced relaxation observed in blood vessels exposed to LPS.     

心肌缺血预适应对血管内皮生长因子水平的影响及其临床意义

目的：探讨心肌缺血预适应（IP）对围术期血浆血管内皮生长因子（VEGF）水平的影响及其临床意义,方法：20例择期心脏手术患者随机均分为两组,对照组常规建立体外循环（CPB）,阻断升主动脉后经主动脉根部顺灌4℃St THomas′停博液,IP组实施3分钟缺血,5分钟再灌注后余同对照组,于不同时点取桡动脉血2ml,采用酶联免疫吸附（ELISA）法测定VEGF浓度,结果：对照组在主动脉阻断30分钟时VEGF浓度开始上升,术后6小时达到高峰（P＜0．01）,24小时仍明显高于正常（P＜0．01）,IP组在IP结束时VEGF浓度开始上升,术后6小时达到高峰（P＜0．01）,24小时仍明显高于正常（P＜0．01）,IP组在IP结束时VEGF水平迅速升高（P＜0．01）,主动脉阻断30分钟时达高峰（P＜0．01）,主动脉开放30分钟时开始下降,术后24小时已基本降至正常,结论IP可诱导VEGF生成增加,这可能也是IP的心肌保护机制之一。     

The gastrointestinal peptide obestatin induces vascular relaxation via specific activation of endothelium-dependent NO signalling

BACKGROUND AND PURPOSE

Obestatin is a recently discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation.

EXPERIMENTAL APPROACH

Cumulative relaxation responses to obestatin peptides were assessed in rat isolated aorta and mesenteric artery (n≥ 8) in the presence and absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC).

KEY RESULTS

Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1–23) were greater than those to obestatin(1–10) and obestatin(11–23). Obestatin(1–23)-induced relaxation was attenuated by endothelial denudation, l-NAME (NOS inhibitor), high extracellular K⁺, GDP-β-S (G-protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK_Ca blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked GPCR, PI3K/PKB, Ca²⁺-dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K⁺. Supporting data from BAEC indicated that nitrite production, intracellular Ca²⁺ and PKB phosphorylation were increased after exposure to obestatin(1–23). Relaxations to obestatin(1–23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarizing factors (EDHFs) and combined SK_Ca/IK_Ca blockade, suggesting that EDHF-mediated pathways were not involved.

CONCLUSIONS AND IMPLICATIONS

Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterized by endothelial dysfunction and cardiovascular complications.     

Anti-atherogenic effect of statins: role of nitric oxide,peroxynitrite and haem oxygenase-1

Background and purpose:

The pleiotropic effects of HMG-CoA inhibitors (statins), which include anti-inflammation, antioxidation and immunomodulation, are not yet fully understood. The present study was designed to elucidate the role of nitric oxide (NO), peroxynitrite (ONOO⁻) and haem oxygenase-1 (HO-1) in the anti-atherogenic effect of statins.

Experimental approach:

Normal and atherosclerotic New Zealand rabbits were treated with atorvastatin or simvastatin in the presence or absence of inhibitors and promoters of endothelial nitric oxide synthase (eNOS) and HO-1. NO and ONOO⁻ released from isolated aortae by calcium ionophore were measured with nanosensors placed 6 ± 2 nm from aortic endothelium. Expression of eNOS and HO-1 protein, HO activity, plasma malondialdehyde (MDA) and vessel wall thickness were also measured.

Key results:

Hypercholesterolaemia decreased eNOS expression by 31 ± 3%, decreased NO (230 ± 16 vs. 433 ± 17 nmol·L⁻¹ control) and increased cytotoxic ONOO⁻ (299 ± 15 vs. 187 ± 11 nmol·L⁻¹ control). The concentration ratio of [NO]/[ONOO⁻] decreased from 2.3 ± 0.1 (normal) to 0.7 ± 0.1 indicating an increase of nitroxidative stress in atherosclerotic endothelium. Expression of HO-1 protein increased by 20 ± 8% in atherosclerosis and further increased (about 30%) after treatment with statins. Statins partially restored the [NO]/[ONOO⁻] balance (1.5 ± 0.1 for atorvastatin and 1.4 ± 0.1 simvastatin), decreased MDA and wall thickening. Promoters of eNOS and HO-1 (L-arginine and haemin) ameliorated the [NO]/[ONOO⁻] ratio while their inhibitors (L-NAME or tin-protoporphyrin) showed no improvement in these ratio.

Conclusions and implications:

Atherosclerosis induced an endothelial [NO]/[ONOO⁻] balance indicative of endothelial dysfunction. Statins showed anti-atherosclerotic effects mediated by HO-1/eNOS, restoring the [NO]/[ONOO⁻] imbalance and reducing lipid peroxidation.