Vascular and anti-oxidant actions of flavonols and flavones

1. Flavonols and flavones are plant-derived polyphenolic compounds that are commonly consumed in the diet. Epidemiological studies indicating that high dietary intake of flavonols reduces the risk of mortality due to coronary heart disease have provoked interest in the mechanism of this cardioprotective effect. 2. We have investigated the structure-activity relationships of a range of flavonols and flavones with regard to their vascular relaxant and anti-oxidant activity. In rat isolated thoracic aorta, the synthetic flavonol 3',4'-dihydroxyflavonol (DiOHF) was found to be a significantly more potent vasorelaxant than the naturally occurring compounds chrysin, apigenin, luteolin, quercetin and fisetin. Similarly, DiOHF was significantly more potent than those compounds in the inhibition of calcium-induced contraction of the rat aorta. 3. 3',4'-Dihydroxyflavonol was also found to significantly inhibit superoxide radical generation in a cell-free system in the presence of xanthine/xanthine oxidase or by rat isolated aorta in the presence of NADPH. In the presence of oxidant stress generated by pyrogallol or xanthine/xanthine oxidase, endothelium-dependent relaxation of rat aortic rings was impaired. 3',4'-Dihydroxyflavonol was able to significantly improve endothelium-dependent relaxation in the presence of those oxygen radical generators. 4. In addition, DiOHF was found to significantly improve dilatation in the rat hindquarters vasculature after exposure to ischaemia and reperfusion. 3',4'-Dihydroxyflavonol was found to be equally effective whether applied before ischaemia or during ischaemia just before reperfusion. 5. In conclusion, DiOHF is an effective vasodilator and anti-oxidant that is able to prevent vascular reperfusion injury. We suggest that DiOHF may be useful as an adjunct to thrombolytic therapy in the management of reperfusion injury.     

Protective effects of curcumin against ischaemia/reperfusion insult in rat forebrain.

Oxidative stress is believed to be implicated in the pathogenesis of postischaemic cerebral injury. Many antioxidants were shown to be neuroprotective in experimental models of cerebral ischaemia/reperfusion (I/R). The present study was designed to investigate the potential protective effects of curcumin (CUR) against I/R insult in rat forebrain. The model adopted was that of surgically-induced forebrain ischaemia, performed by means of bilateral common carotid artery occlusion (BCCAO) for 1 h, followed by reperfusion for another 1h. The effects of a single i.p. dose of CUR (50, 100 or 200 mg kg(-1)), administered 0.5 h after the onset of ischaemia, were investigated by assessing oxidative stress-related biochemical parameters in rat forebrain. CUR, at the highest dose level (200 mg kg(-1)), decreased the I/R-induced elevated xanthine oxidase (XO) activity, superoxide anion (O(2)*(-)) production, malondialdehyde (MDA) level and glutathione peroxidase (GPx), superoxide dismutase (SOD), and lactate dehydrogenase (LDH) activities. On the other hand, CUR did not affect the declined reduced glutathione (GSH) content due to I/R insult. Worth mentioning is that the activity of catalase (CAT) did not change in response to either I/R insult or drug treatment. In conclusion, CUR was found to protect rat forebrain against I/R insult. These protective effects may be attributed to its antioxidant properties and/or its inhibitory effects on xanthine dehydrogenase/xanthine oxidase (XD/XO) conversion and resultant O(2)*(-) production.     

3',4'-Dihydroxyflavonol reduces infarct size and injury associated with myocardial ischaemia and reperfusion in sheep

1 The antioxidant properties of flavonols in vivo and their potential benefits in myocardial ischaemia/reperfusion (I/R) injury have been little investigated. We evaluated the ability of a synthetic flavonol, 3',4'-dihydroxyflavonol (DiOHF) to scavenge superoxide in post-I/R myocardium and to prevent myocardial I/R injury. 2 Anaesthetized sheep were studied in four groups (n=5-6): control, ischaemic preconditioning (IPC), vehicle and DiOHF (before reperfusion, 5 mg kg(-1), i.v.). The left anterior descending coronary artery was occluded distal to the second diagonal branch for 1 h followed by 2 h of reperfusion. Infarct size, myocardial function, NADPH-activated superoxide generation and biochemical markers of injury were measured. 3 DiOHF (10(-8)-10(-4) m) incubated in vitro with post-I/R myocardium from the vehicle group suppressed superoxide production dose-dependently. 4 DiOHF administered in vivo also significantly reduced superoxide generation in vitro. DiOHF and IPC markedly reduced infarct size, which was 73+/-2% of the area at risk in vehicle, 50+/-4% in DiOHF, 75+/-5% in control and 44+/-4% in IPC. Post-I/R segment shortening within the ischaemic zone was greater in DiOHF (2.3+/-0.7%; P<0.01) and IPC (1.7+/-0.5%; P<0.01) than those in corresponding controls (-1.7+/-0.4; -2.1+/-0.4%). 5 DiOHF and IPC improved coronary blood flow to the ischaemic area and preserved higher levels of nitric oxide metabolites in the venous outflow from the ischaemic zone. 6 DiOHF attenuated superoxide production in post-I/R myocardium, and significantly reduced infarct size and injury following I/R in anaesthetized sheep. The extent of protection by DiOHF is comparable to that afforded by IPC. Thus, DiOHF has clinical potential for improving recovery from acute myocardial infarction and other ischaemic syndromes.     

内源性阿片肽介导大鼠后肢缺血预适应的保护作用

研究了内源性阿片肽介导大鼠后肢缺血预适应的保护作用. 后肢缺血2 h，乙酰胆碱（ACh）诱导的血管内皮依赖性舒张性反应明显下降. 缺血预适应（缺血5 min，再灌5 min，重复3 次）能显著减弱长时间缺血对ACh舒血管效应的抑制作用，这种保护作用可被纳洛酮（3 mg·kg^-1）取消. 预先给予吗啡（300 μg·kg^-1）也能产生与缺血预适应相同的血管内皮保护作用. 然而，预先用辣椒素（50 mg·kg^-1）耗竭降钙素基因相关肽后，吗啡的保护作用被取消. 结果提示，内源性阿片肽介导大鼠后肢缺血预适应的血管保护作用，其机理可能涉及内源性降钙素基因相关肽.     

Kinetics of the inhibition of xanthine dehydrogenase and of the reversible and irreversible forms of xanthine oxidase by silibinin and bendazac

Xanthine oxidase exists in vivo predominantly as a NAD⁺-dependent dehydrogenase form (xanthine dehydrogenase) which can be transformed into oxygen-dependent oxidase forms as a result of sulfhydryl oxidation (reversible xanthine oxidase) or proteolysis (irreversible xanthine oxidase). Xanthine oxidase has been hypothesized to be a potential source of oxygen-derived free radicals during reperfusion of ischemic tissues. Xanthine dehydrogenase was purified from rat liver and converted into reversible xanthine oxidase by heating at 37 °C and into irreversible xanthine oxidase by proteolysis with trypsin. Silibinin and bendazac are compounds used in therapeutics and to which free radical scavenging properties were ascribed. The effects of the compounds silibinin and bendazac on the different forms of the enzyme were studied. Silibinin inhibited all the forms of the enzyme but bendazac inhibited only reversible and irreversible xanthine oxidase. The inhibitions seem to be mixed non-competitive-competitive. The authors discuss the hypothesis that selective inhibitors of xanthine oxidase, preventing the interruption of uric acid formation, may have some advantage over the inhibitors of both xanthine dehydrogenase and xanthine oxidase in the treatment and prevention of situations such as ischemia and reperfusion syndromes.     

The contribution of the pyrogallol moiety to the superoxide radical scavenging activity of flavonoids

Sixteen flavonoids including flavonols, flavones, flavanonol and catechins, and five aromatic compounds were examined for their ability to scavenge superoxide radical (O2-*) generated enzymatically in a xanthin-xanthinoxidase system and non-enzymatically in a phenazine methosulfate-NADH system. Pyrogallol, gallic acid and its ester, were much more efficient in scavenging O2-* than catechol. The superiority of pyrogallol over catechol in the flavonoidal nucleus is apparent from the much higher O2-* scavenging activity of myricetin and epigallocatechin, which contain 3',4',5'-trihydroxyl substitution in the B-ring, compared to quercetin and epicatechin, which contain 3',4'-dihydroxyl substitution, respectively. The strong O2-* scavenging ability of pyrogallol appears to function even in the A-ring, as in baicalein, and also in the form of a pyrogalloyl ester at the C-3 position in the C-ring, as in epicatechin gallate and epigallocatechin gallate. It can be concluded that the pyrogallol moiety is an active component of flavonoids for displaying high O2-* scavenging activity. Flavonoids and aromatics were also examined to correlate their O2-* scavenging activity with their oxidizability, which was measured on the basis of electrochemical redox potential and the reducing ability of the Cu2+ ion. Aromatics such as pyrogallol, gallic acid and its ester, and flavonoids such as baicalein, epicatechin gallate and epigallocatechin gallate, in which the O2-* scavenging activity is enhanced by the presence of a pyrogallol moiety which does not belong to the B-ring, reduced the correlation between the higher O2-* scavenging activity and the lower redox potential. The O2-* scavenging activity was well correlated with the Cu2+ reducing ability of flavonoids and aromatics.     

Role of superoxide dismutase in in vivo and in vitro nitrate tolerance.

We assessed whether pharmacological inhibition of CuZn-superoxide dismutase (SOD) mimics the molecular mechanism of either in vitro or in vivo nitrovasodilator tolerance. In endothelium-intact aortic rings from in vivo tolerant rabbits the GTN- and acetylcholine (ACh)-induced maximal relaxation was attenuated by 36 and 23%, respectively. In vitro treatment of control rings with GTN (1 h 10 microM) similarly attenuated the vasorelaxant response to GTN, but not to ACh. Formation of superoxide radicals (*O2-) in endothelium-intact rings (lucigenin-chemiluminescence) increased 2.5 fold in in vivo tolerance, but significantly decreased in in vitro tolerance. The membrane associated NADH oxidase activity was increased 2.5 fold in homogenates of in vivo tolerant aortae, but was not changed in in vitro tolerant aorta. Conversely, SOD activity and protein expression was halved in in vivo tolerance, but SOD activity was not altered by in vitro tolerance. The *O2- scavenger tiron (10 mM) effectively restored the vasorelaxant response to GTN in in vivo tolerant aortic rings, but not the reduced response to GTN in in vitro tolerant rings. Pretreatment (1 h) of vessels with diethyldithiocarbamate (DETC; 10 mM) attenuated vasorelaxant responses to GTN and ACh, increased vascular *O2- production, and inhibited SOD activity in vessel homogenates to a similar degree as observed in in vivo tolerance. DETC-treatment of in vivo-tolerant vessels induced an additional increase in *O2- production. Increased *O2- production in in vivo nitrate tolerant aorta is associated with activation of vascular NADH oxidase and inactivation of CuZnSOD. Therefore, in vivo tolerance can be mimicked by in vitro inhibition of CuZnSOD, but not by in vitro exposure to GTN, which does not affect vascular *O2- production, NADH oxidase and CuZnSOD.     

The possible implication of trans-Resveratrol in the cardioprotective effects of long-term moderate wine consumption.

trans-Resveratrol (t-RESV; 1-10 microM), a phenolic component of wines, had no effect on phenylephrine-(PE; 1 microM) and high KCl-(60 mM) induced contractions in endothelium-denuded rat aortic rings. However, it relaxed the contractile response produced by these vasoconstrictor agents in intact rat aorta. The vasorelaxing effects of t-RESV were completely inhibited by N(G)-nitro-L-arginine (L-NOARG; 0.1 mM) and methylene blue (10 microM), but they were unaffected by atropine (10 microM) and yohimbine (1 microM). The reversal effect produced by L-NOARG was antagonized by L-arginine but not by D-arginine (0.1 mM). t-RESV (1-10 microM) did not significantly modify rat aorta constitutive nitric-oxide synthase activity. However, this natural compound decreased NADH/NADPH oxidase activity in rat aortic homogenates. In addition, t-RESV (1-10 microM) was ineffective in scavenging superoxide anions (O(2)*) generated enzymatically by a hypoxanthine/xanthine oxidase (HX/XO) system and/or to inhibit XO. The above data demonstrate that the characteristic endothelium-dependent vasorelaxant effect of t-RESV in rat aorta seems to be caused by the inhibition of vascular NADH/NADPH oxidase and the subsequent decrease of basal cellular O(2)* generation and, therefore, of NO biotransformation. Under the assumption that t-RESV exhibits a similar behavior in human blood vessels and bearing in mind that an overactivity of NADH/NADPH oxidase has been found in a number of cardiovascular pathologies, the results obtained in this work suggest that t-RESV could play an important role in the cardioprotective effects induced by the long-term moderate wine consumption.     

Does luminol chemiluminescence detect free radical scavengers?

Thiol compounds have been reported to abolish hypoxanthine/xanthine oxidase induced luminol chemiluminescence and this effect has been attributed to scavenging of superoxide (O2-)/(H2O2) produced from hypoxanthine/xanthine oxidase. Yet other workers have reported that thiol compounds have shown little, if any, reactivity towards O2-/H2O2. The aim of this study was to examine the discrepancy between these two sets of findings further. Captopril (a thiol angiotensin-converting enzyme (ACE) inhibitor) and MPG (a simple thiol) were observed to abolish hypoxanthine/xanthine oxidase induced chemiluminescence. The reactivity of captopril and MPG towards O2-/H2O2 was then determined by measurement of thiol oxidation in captopril and MPG after their incubation with hypoxanthine/xanthine oxidase. Incubation (at 10 min, 37 degrees C) with 4 mM hypoxanthine/0.03 u ml-1 xanthine oxidase resulted in 7% and 20% thiol oxidation in captopril and MPG (at 1 mM) respectively. Captopril and MPG, therefore, appeared to be ineffective scavengers of oxidants produced by hypoxanthine/xanthine oxidase. Captopril and MPG also did not affect urate production or oxygen consumption by xanthine oxidase which indicated that captopril and MPG quench luminol chemiluminescence by a mechanism that excludes the inhibition of xanthine oxidase. Hypoxanthine/xanthine oxidase induced luminol chemiluminescence may, therefore, be an unsuitable method for measuring free radical scavenging activity by drugs.     

In vitro studies on the activity of japanese kampo herbal medicines Oren-Gedoku-To (TJ-15) and Toki-Shakuyaku-San (TJ-23) as scavengers of free radicals

Oren-Gedoku-To (TJ-15) inhibited enzymatically (NADPH or CumOOH) and non-enzymatically (Fe-ascorbate) induced lipid peroxidation in rat liver microsomes as assessed by TBA-reactive product accumulation. Toki-Shakuyaku-San (TJ-23) had little effect on either system. The protective effect of TJ-15 against lipid peroxidation was not dependent upon the presence of microsomal drug-metabolizing activity and could not be fully accounted for by its action on microsomal electron transfer, as evaluated by studying the kinetics of cytochrome c reduction. Both TJ-15 and TJ-23 reduced the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), indicating their free radical scavenging ability. The reactivity of TJ-15 was comparable with that of standard ascorbate, while the activity of TJ-23 was approximately 10 times lower. In a free solution TJ-15 and TJ-23 effectively scavenged OH* radicals, as indicated by the inhibition of ethylene production from 2-keto-4-methiolbutyric acid (KMBA), and O2*- anion radicals, as assessed by the inhibitory effect on the rate of nitro blue tetrazolium (NBT) reduction by the O2*- generating system xanthine/xanthine oxidase.     

The beneficial effect of resveratrol on rat bladder contractility and oxidant damage following ischemia/reperfusion

OBJECTIVE: The present study aimed to investigate the possible beneficial activities of resveratrol (3,5,4'-trans-trihydroxystilbene), a natural phytoalexin, on contractility and oxidant damage after ischemia/reperfusion (I/R) of the rat urinary bladder. MATERIALS AND METHODS: The abdominal aorta of Sprague-Dawley rats was occluded for 60 min to induce ischemia and then allowed 60 min of reperfusion. Resveratrol (10 mg/kg) or saline was administered intraperitoneally 15 min before ischemia and immediately before reperfusion. In the sham-operated group, the abdominal aorta was left intact and the animals were treated with resveratrol or saline. The bladder samples were either used for functional studies or stored for biochemical assays. RESULTS: In the I/R group, the isometric contractile responses of the bladder strips to carbachol (CCh; 10(-8)-10(-4) mol/l) were lower than those of the control group and were reversed by treatment with resveratrol. Histological evaluation revealed loss of urothelial cells, detachment and loss of urothelial cells and local ulcerated areas and severe inflammatory cell infiltration in the untreated I/R group, and regeneration of luminal mucosa and a significant decrease in the density of the inflammatory cell population in the resveratrol-treated I/R group. Lipid peroxidation and the myeloperoxidase activity of the bladder tissues in the I/R group were higher than in the sham-operated group. Resveratrol treatment in the I/R group decreased these parameters compared with I/R alone. Similarly, the significant decrease in tissue glutathione level in the I/R group compared with controls was also prevented by resveratrol. Conclusion: Treatment with resveratrol almost completely reversed the low contractile responses of the rat urinary bladder to CCh and prevented oxidative tissue damage following I/R.     

Radical scavenging properties of novel benzopyran derivatives, TA248 and TA276, and effects of the compounds on ischemic/reperfused myocardium in dogs

Characteristics of novel benzopyran derivatives, TA248 and TA276, and their effects on myocardial contraction in ischemic/reperfused hearts in dogs were examined. TA248 and TA276 inhibited NADPH-dependent lipid peroxidation induced by Fe(3+) in the rat brain homogenate. Both compounds reduced *O(2-) produced by xanthine-xanthine oxidase system in a dose-dependent manner. TA276 scavenged.OH generated by Fenton reaction in a dose-dependent manner. TA248 also inhibited the.OH production, but the effect was neither complete nor dose dependent. Myocardial contraction was assessed as segment shortening of the left ventricular wall in pentobarbital-anesthetized open-chest dogs. The segment shortening was decreased by the left anterior descending coronary artery ligation (ischemia) and returned by release of the ligated artery (reperfusion). The segment shortening did not recover fully during reperfusion. Either TA248 or TA276 injected 10 min before ischemia improved the recovery of myocardial contraction during reperfusion. Both compounds preserved the level of ATP in the 60-min reperfused myocardium. However, the level of lipid peroxides was not changed by TA248 and TA276. TA248 and TA276 may protect myocardium against ischemic/reperfusion insult, partly because of their free radical scavenging activity, but no significant change in myocardial lipid peroxide level was observed.     

Neuroprotective effects of resveratrol on ischemic injury mediated by improving brain energy metabolism and alleviating oxidative stress in rats

In this study, we investigated whether resveratrol could protect against ischemic injury by improving brain energy metabolism and alleviating oxidative stress. Male rats were divided into three groups: sham operation, ischemia treatment, and ischemia combined with resveratrol treatment (resveratrol-treated group, 30 mg/kg intraperitoneally for 7 days). Cerebral ischemia was induced by using the model of middle cerebral artery occlusion. The dialysates in hypothalamus were obtained by brain microdialysis technique. The effects of resveratrol on neurologic functions and histopathologic changes were evaluated. The levels of ATP, ADP, AMP, adenosine, inosine, hypoxanthine and xanthine in microdialysate were monitored by HPLC analysis. The levels of malondialdehyde and the activities of xanthine oxidase in brain tissues were analyzed in three groups. This study shows that the ischemic infarcts were significantly reduced and neurological functions were improved in resveratrol-treated group compared to ischemia group. The analysis results show that resveratrol treatments remarkably enhanced the level of glucose, ATP and energy charge; decreased the levels of lactate during I/R period. Resveratrol treatments significantly increased the basal levels of adesonine and inosine, inhibited the elevations of hypoxanthine and xanthine levels and remarkably decreased xanthine oxidase activity and malondialdehyde levels. This study provides in vivo evidence that resveratrol could exert neuroprotective effect against ischemia injury by improving brain energy metabolism and alleviating oxidative stress via inhibiting xanthine oxidase activity and preventing the production of hypoxanthine, xanthine and oxygen radicals during ischemia/reperfusion.     

Inhibiton of NF-kappaB activation during ischemia reduces hepatic ischemia/reperfusion injury in rats

The aim of this study was to determine whether nuclear factor-kappaB (NF-kappaB) inhibitors are efficient against hepatic ischemia/reperfusion (I/R) injury. We previously demonstrated that xanthine oxidase-derived reactive oxygen species activate NF-kappaB during ischemia. However, the role of NF-kappaB activation during ischemia in post-reperfusion injury remains unclear. Therefore, while we examined the effects of NF-kappaB inhibitors, sulfasalazine and pyrrolidinedithiocarbamate on hepatic I/R injury using a rat lobar hepatic I/R model, we estimated the relationship between NF-kappaB activation during ischemia and following hepatic damage caused by reperfusion. The portal vein and the hepatic artery were clamped for 1 hr followed by reperfusion for up to 24 hr. NF-kappaB activation was determined by Western blot analysis. NF-kappaB activation was observed in the ischemic lobe of the liver, and the activation was prevented by pre-administration with NF-kappaB inhibitors. Although the serum ALT level, hepatic MPO activity and BSP clearance, as an index of hepatic injury, were increased after reperfusion, the increase was attenuated by pre-administration with NF-kappaB inhibitors. These findings suggest that NF-kappaB activation during ischemia is relevant to hepatic I/R injury. Moreover, we first showed that pre-administration with NF-kappaB inhibitors is effective against hepatic I/R injury.     

Requirement for endothelium-derived nitric oxide in vasodilation produced by stimulation of cholinergic nerves in rat hindquarters.

1. We aimed to determine whether nitric oxide (NO) and/or the endothelium is involved in cholinergic neurogenic vasodilatation in the rat isolated hindquarters. 2. The abdominal aorta was cannulated for perfusion of the rat hindquarters with Krebs bicarbonate solution containing phenylephrine, to induce basal constrictor tone. In the presence of noradrenergic neurone blockade with guanethidine (200 mg kg-1, i.p.) electrical stimulation of peri-aortic nerves induced frequency-dependent decreases in hindquarters perfusion pressure, indicating vasodilatation. Both the endothelium-dependent vasodilator, acetylcholine (ACh) and the endothelium-independent vasodilator, sodium nitroprusside (SNP) induced dose-dependent decreases in perfusion pressure. In each experiment, responses to either nerve stimulation, ACh or SNP were recorded before and after treatment with saline vehicle, atropine (1 microM), NG-nitro-L-arginine (L-NOARG, 100 microM), L-arginine (1 mM), L-arginine plus L-NOARG, or 3-3 cholamidopropyl dimethylammonio 1-propanesulphonate (CHAPS, 30 mg). Hindquarters dilatation after each treatment was expressed as a percentage of the control response. 3. Following treatment with saline, responses to nerve stimulation and ACh were 99 +/- 9% and 107 +/- 10% of control, respectively demonstrating the reproducibility of these responses. Nerve stimulation-induced dilation was abolished by atropine (0 +/- 0% of control, P < 0.05) or reduced to 14 +/- 10% of control by NO synthase inhibition with L-NOARG (P < 0.05). Dilator responses to ACh were also abolished by atropine (0 +/- 0% of control, P < 0.05) or inhibited by L-NOARG (59 +/- 10% of control, P < 0.05), indicating that the neurogenic dilatation is cholinergic and is mediated by NO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Wogonin inhibits excitotoxic and oxidative neuronal damage in primary cultured rat cortical cells

The present study evaluated effects of wogonin (5,7-dihydroxy-8-methoxyflavone) on excitotoxic and oxidative stress-induced neuronal damage in primary cultured rat cortical cells. Wogonin was shown to inhibit the excitotoxicity induced by glutamate or N-methyl-D-aspartic acid, whereas it showed no effects on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- or kainate-induced toxicity. In addition, wogonin inhibited the oxidative neuronal damage induced by H(2)O(2), xanthine/xanthine oxidase, and by a glutathione depleting agent D,L-buthionine [S,R]-sulfoximine. Furthermore, wogonin dramatically inhibited lipid peroxidation initiated by Fe(2+) and L-ascorbic acid in rat brain homogenates. It also exhibited 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Taken together, these results demonstrate that wogonin exhibits neuroprotective actions in cultured cortical cells by inhibiting excitotoxicity and various types of oxidative stress-induced damage, and that its antioxidant actions with radical scavenging activity may contribute, at least in part, to the neuroprotective effects.     

Functional and analytical evidence for scavenging of oxygen radicals by L-arginine

L-Arginine, the substrate of nitric oxide synthase, is known to exert favorable effects in the prevention and treatment of cardiovascular diseases. In several conditions, including atherosclerosis and ischemia/reperfusion, where oxygen metabolites are thought to mediate endothelial and myocardial injury, L-arginine has protective effects. Here we studied the mechanisms by which L-arginine protects against oxygen radical-induced myocardial injury. Buffer-perfused rat hearts were subjected to oxygen radicals generated by electrolysis or to hypoxanthine and xanthine oxidase, which generates superoxide anions (O(2)). Both sources of radicals impaired myocardial contractility, whereas L-arginine prevented the impairment. The observation that D-arginine as well as nitric oxide synthase inhibitors, such as N(G)-nitro-L-arginine but not glycine, had similar cardioprotective effects indicated that the protection might be due to a direct chemical interaction of L-arginine and its derivatives with oxygen radicals. In support, L-arginine and the derivatives prevented the formation of O(2) as determined by sensitive standard methods, whereas glycine did not. The radical scavenging activity of L-arginine and derivatives was dose-dependent, with an apparent rate constant of approximately 4.8 x 10(3) M s(-1) for the reaction of L-arginine with O(2) as determined by electron paramagnetic resonance spectroscopy using 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONE-H) as spin trap. In summary, the results of this study demonstrate protective effects of L-arginine against oxygen radical-induced cardiac injury by free radical scavenging.     

Ischaemia enhances the role of Ca2+-activated K+ channels in endothelium-dependent and nitric oxide-mediated dilatation of the rat hindquarters vasculature

1. We compared the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) and tetraethylammonium (TEA), a blocker of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, on vasodilator responses to endothelium-dependent (acetylcholine; ACh) and -independent (sodium nitroprusside; SNP) vasodilators. The mechanism of the vasodilator responses was determined in rat hindquarters under normal conditions (sham ischaemia) and after 2 h ischaemia followed by reperfusion with physiological saline. 2. In sham ischaemia, the responses to ACh were significantly reduced by L-NOARG (1 mmol/L) and TEA (1 mmol/L) and there was a further reduction in response the presence of both agents. Dilator responses to SNP were significantly enhanced by L-NOARG, whereas TEA did not alter the SNP-induced vasodilatation when given either alone or in the presence of L-NOARG. 3. After ischaemia, L-NOARG caused a similar inhibition of ACh-induced dilatation to that observed in sham ischaemia. However, TEA alone or combined with L-NOARG caused a significantly greater inhibition of the ACh-induced vasodilatation after ischaemia than observed in the sham ischaemia group. Tetraethylammonium alone did not affect the responses to SNP, but it did attenuate the enhanced dilatation observed in the presence of L-NOARG. 4. In the rat hindquarters vasculature, both nitric oxide and the opening of TEA-sensitive K(+) channels contribute to ACh-induced endothelium-dependent dilatation. In addition, a TEA-sensitive mechanism was not involved in the SNP-induced dilatation under normal conditions but, after ischaemia, if there is a further inhibition of endogenous nitric oxide by L-NOARG, exogenous nitric oxide causes dilatation that is sensitive, in part, to TEA. Thus, the contribution of the opening of BK(Ca) channels to endothelium-dependent vasodilatation assumes greater importance after ischaemia and reperfusion. This may reflect an increased ability of nitric oxide or cGMP to open BK(Ca) channels after ischaemia.     

Taurine inhibits ischemia／reperfusion-induced compartment syndrome in rabbits

Aim: To investigate effects of taurine on ischemia/reperfusion (I/R)-induced compartment syndrome in rabbit hind limbs. Methods: Rabbits underwent femoral artery occlusion after ligation of branches from terminal aorta to femoral artery. After a 7-h ischemia, reperfusion was established with the use of heparinized polyethylene shunts. Rabbits received taurine (1 g/kg) or normal saline (control) by iv infusion 10 min before shunt placement. During reperfusion, anterior compartment pressure (ACP) was monitored continuously in the left lower extremity. Gastrocnemius muscle triphenyltetrazolium chloride (TTC) level, taurine content and myeloperoxidase activity were assayed. Oxidative stress was induced in the in vitro gastrocnemius muscle slices by free radical generating systems (FRGS), and the malondialdehyde content was measured in presence or absence of taurine. Results: After 7 h of ischemia, none of the parameters that we measured were different from those before ischemia, except that TTC reduction decreased by 80%. In the control group, after 2 h of reperfusion, ACP increased 4.5-fold, and gastrocnemius muscle taurine content was reduced by 33%. In taurine-treated animals, at 2 h reperfusion, the mean arterial blood pressure and heart rate were increased, by 6% and 10%. ACP decreased by 39%, muscle edema decreased by 16%, TTC reduction increased by 150%, and lactate dehydrogenase decreased by 36% compared to control group. Plasma and muscle taurine content increased by 70% and 88%, respectively. In the taurine-treated group, at 2 h reperfusion, plasma malondialdehyde and conjugated diene content were decreased by 38% and 23%, respectively, and muscle malondialdehyde and conjugated diene content decreased by 22% and 30%, respectively compared to the control group. At 2 h reperfusion, myeloperoxidase activity was increased 3.5-fold in control animals. In the in vitro study, taurine decreased malondialdehyde content in muscle slices incubate dwith hypochlorous acid in a dose-dependent manner, but there was no change when incubated with hydrogen peroxide and xanthine oxidase. Conclusion: Treatment with taurine inhibited I/R-induced compartment syndrome by at least in part attenuating oxidative stress injury induced by I/R, suggesting clinical application of taurine might be a new strategy for the prevention and treatment of compartment syndrome.     

Hydrogen peroxide is an endothelium-dependent contracting factor in rat renal artery

In addition to endothelium-derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone by releasing endothelium-derived contracting factor(s) (EDCF), but the identity of EDCF remains obscure. We studied here the involvement of hydrogen peroxide (H2O2) in endothelium-dependent contraction (EDC) of rat renal artery to acetylcholine (ACh). ACh (10(-6), 10(-5), and 10(-4) M) induced a transient contraction of rat renal artery with intact endothelium in a concentration-related manner, but not in the artery with endothelium removed. In phenylephrine-precontracted renal arteries, ACh induced an endothelium-dependent relaxation response at lower concentrations (10(-8)-10(-6) M), and a relaxation followed by a contraction at higher concentrations (10(-5) M). Inhibition of nitric oxide synthase by N(omega)-nitro-L-arginine (10(-4) M) enhanced the EDC to ACh. Catalase (1000 U ml(-1)) reduced the EDC to ACh. H2O2 (10(-6), 10(-5), and 10(-4) M) induced a similar transient contraction of the renal arteries as ACh, but in an endothelium-independent manner. Inhibition of NAD(P)H oxidase and cyclooxygenase by diphenylliodonium chloride and diclofenac greatly attenuated ACh-induced EDC, while inhibition of xanthine oxidase (allopurinol) and cytochrome P450 monooxygenase (17-octadecynoic acid) did not affect the contraction. Antagonist of thromboxane A2 and prostaglandin H2 receptors (SQ 29548) and thromboxane A2 synthase inhibitor (furegrelate) attenuated the contraction to ACh and to H2O2. In isolated endothelial cells, ACh (10(-5) M) induced a transient H2O2 production detected with a fluorescence dye sensitive to H2O2 (2',7'-dichlorofluorescein diacetate). The peak concentration of H2O2 was 5.1 x 10(-4) M at 3 min and was prevented by catalase. Taken together, these results show that ACh triggers H2O2 production through NAD(P)H oxidase activation in the endothelial cells, and that ACh and H2O2 share the same signaling pathway in causing smooth muscle contraction. Therefore, H2O2 is most likely the EDCF in rat renal artery in response to ACh stimulation.