Enhanced postischemic ATP repletion by pharmacological inhibition of nucleoside washout and catabolism

We tested the hypothesis that inhibition of adenosine transport by dipyridamole and inhibition of adenosine deamination by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) prevents nucleoside loss and stimulates postischemic ATP-repletion. In an open chest canine model, dipyridamole (0.5 mg/kg/h) and EHNA (5 mg/kg/h) were infused intra-atrially during a coronary occlusion period of 45 min and a reperfusion period of 180 min. Transmural needle biopsies, obtained during the ischemic period and within the reperfusion period, were analyzed using high performance liquid chromatography for adenine nucleotides and adenosine, inosine, xanthine, and hypoxanthine as well as creatine phosphate. During ischemia and under the influence of dipyridamole plus EHNA, 56% of the catabolized adenine nucleotides were recovered stoichiometrically as adenosine, whereas in the untreated group less than 10% of the nucleotides were recovered as adenosine because of rapid deamination to inosine. In the control group, ATP levels decreased during ischemia from control values of 5.25 +/- 0.28 microns/g to 2.01 +/- 0.18 microns/g. In the group treated with dipyridamole and EHNA, ATP levels fell to 2.2 +/- 0.22 microns/g but rose to 3.22 +/- 0.29 microns/g within 180 min of reperfusion, whereas in the untreated control group tissue levels of ATP did not increase. However, a significant proportion of the adenosine accumulated during ischemia under the influence of dipyridamole plus EHNA was not used for the restoration of the ATP level during reperfusion. A significant amount of adenosine was probably trapped in the interstitial space and could not be transported back into the myocytes in the presence of dipyridamole during reperfusion. In both groups, creatine phosphate levels were restored to normal levels during reperfusion.     

Cerebroprotective effect of resveratrol through antioxidant and anti-inflammatory effects in diabetic rats

Oxidative stress and inflammation have been implicated in cerebral ischemia/reperfusion injury and complication of diabetes. The present study was designed to evaluate whether resveratrol has cerebroprotective action through antioxidant and anti-inflammatory actions in diabetic rats. Bilateral common carotid artery occlusion (30 min) and reperfusion (4 h) was employed to induce cerebral infarction in diabetic Wistar rats. Diabetes was induced by streptozocine (50 mg/kg) intraperitoneally at once. Diabetic animals were divided into groups as: normal, sham, ischemia–reperfusion, and resveratrol-treated (5, 10, 20, and 30 mg/kg). These were used for estimation of cerebral infarction. Furthermore, 20 mg/kg dose was selected for estimation of oxidative stress markers (malondialdehyde, superoxide dismutase, and catalase). Inflammatory markers like TNF-α, IL-6, IL-10, and myeloperoxidase were estimated and histological characters were studied. Resveratrol produced dose-dependent reduction in percent cerebral infarction. With resveratrol of 20 mg/kg dose, levels of oxidative stress markers and inflammatory markers like malondialdehyde, TNF-α, IL-6, and myeloperoxidase were reduced and there was a significant increase in the levels of antioxidant and anti-inflammatory markers like catalase, superoxide dismutase, and IL-10. In the present study, we found that mechanism(s) responsible for the cerebroprotective effect of resveratrol in the diabetic rat brain involves antioxidant and anti-inflammatory actions.     

Effect of TEI-6720, a xanthine oxidase inhibitor, on the nucleoside transport in the lung cancer cell line A549

To examine the effect of 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid (TEI-6720), an inhibitor of xanthine oxidase, on purine metabolism in the lung cancer cell line A549, the activities of adenosine deaminase, purine nucleoside phosphorylase, adenine phosphoribosyltransferase, hypoxanthine guanine phosphoribosyltransferase, xanthine oxidase, and guanase together with pyrimidine nucleoside phosphorylase were measured with or without the addition of TEI-6720, and the extracellular concentrations of hypoxanthine, xanthine, inosine, uracil, and uridine were measured after the addition of inosine or uridine to the incubation medium with or without TEI-6720. Moreover, the Na-independent nucleoside transport was determined in A549 cells with or without TEI-6720. TEI-6720 inhibited the activity of xanthine oxidase in A549 cells, but did not affect other enzymes. During incubation, TEI-6720 not only prevented a decrease in the inosine concentration in inosine-containing medium, but also a decrease in the uridine concentration in uridine-containing medium. Furthermore, the Na-independent transport of uridine was inhibited by TEI-6720 with a K(i) value of 4.1 micromol/l. These results indicate that TEI-6720 is an inhibitor of the Na-independent nucleoside transport of uridine and inosine, as well as xanthine oxidase.     

三七总皂甙对鸡胚神经细胞缺氧及大鼠脑缺血再灌注损伤的作用

观察了三七总皂甙（ＰＮＳ）对氰化钠引起鸡胚脑神经细胞缺氧和大鼠全脑缺血纹状体细胞外嘌呤类代谢产物的影响。结果表明：ＰＮＳ（５０和１００ｍｇ·Ｌ－１）明显延缓缺氧２ｈ神经细胞的能量耗竭，促进再给氧期细胞内高能磷酸化合物的合成。大鼠脑缺血前给予ＰＮＳ（２００ｍｇ·ｋｇ－１，ｉｐ）能明显降低缺血和再灌注期纹状体细胞外液次黄嘌呤，黄嘌呤和肌苷含量的升高。提示：ＰＮＳ对神经细胞氰化钠损伤及大鼠脑缺血再灌注损伤具有保护作用，其机理可能与改善能量代谢有关。     

丁基苯酞对全脑缺血大鼠的纹状体细胞外液嘌呤类代谢物含量的影响

采用大鼠4动脉阻断的全脑缺血模型,用推挽式微灌流方法,用HPLC-UV检测缺血及重灌期纹状体灌流液中腺苷(Ade)、肌苷(Ino)、次黄嘌呤(Hyp)和黄嘌呤(Xan)的含量。结果表明,ip丁基苯酞20或40mg·kg^-1均能显著降低大鼠全脑缺血纹状体细胞外液Ade,Ino,Hyp和Xan的升高。但对假手术组动物则无影响。Hyp和Xan是产生氧自由基的底物。丁基苯酞这一抑制作用提示它对缺血性神经元的损伤有保护作用。     

Anticonvulsant doses of inosine result in brain levels sufficient to inhibit [3H] diazepam binding

Several purines have been shown to be competitive inhibitors of [³H] diazepam binding. Inosine has also been shown to have benzodiazepine-like neurophysiologic, pharmacologic and behavioral effects, and to partially inhibit caffeine-induced seizures in mice. Using presumptive therapeutic doses of inosine, levels were determined in mouse brain at various times following injection. Inosine and hypoxanthine concentrations in brain increased several fold following inosine administration, indicating that inosine permeated the blood-brain barrier. The levels of inosine and hypoxanthine attained in brain were sufficient to inhibit by more than 50% the GABA-stimulated [³H] diazepam binding. These data suggest that that the anticonvulsant properties of inosine are related to its interaction with the benzodiazepine receptor.     

大鼠脑缺血损伤后MDA含量和SOD活性变化及胡黄连苷Ⅱ干预作用

目的通过正交试验优化胡黄连苷Ⅱ治疗大鼠脑缺血损伤的最佳剂量和时间窗。方法应用双侧颈总动脉结扎法(BCCAO)建立大鼠前脑缺血模型,按照正交试验设计分组,经腹腔注射胡黄连苷Ⅱ干预治疗,硫代巴比妥酸法测定血清和脑组织中脂质过氧化物丙二醛(MDA)的含量。黄嘌呤氧化酶法检测血清和脑组织中超氧化物歧化酶(SOD)的活性。结果胡黄连苷Ⅱ治疗脑缺血损伤的最佳效果,根据血清和脑组织MDA含量分析,均为脑缺血1.5h腹腔注射10mg/kg体质量。根据血清和脑组织SOD活性分析,均为脑缺血1.5h腹腔注射20mg/kg体质量。结论从用药剂量最小化和治疗时间窗最大化的角度综合评价,胡黄连苷Ⅱ治疗脑缺血损伤的最佳治疗时间窗和剂量为脑缺血1.5 h腹腔注射10-20mg/kg体质量。     

Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys

Reactive oxygen species have been implicated in the pathophysiology of renal ischemia reperfusion injury. Antioxidants including polyphenolics have been found to protect renal cells from the cellular injury induced by ischemia and reperfusion. Resveratrol, a stilbene polyphenol found in grapes and red wine, has recently been found to protect isolated rat heart from ischemia reperfusion injury. This study was sought to determine if resveratrol could also protect renal cells from ischemic injury. Male Wistar rats were treated with control, resveratrol (0.23 microg/kg), vehicle used to solubilize resveratrol, and resveratrol plus L-NAME (15 mg/kg body wt), a nitric oxide blocker. Our results demonstrated that resveratrol administration reduced the mortality of ischemic rats from 50% to 10% and renal damage was reduced as indicated by histologic examination and serum creatinine level. The short-term administration of resveratrol also inhibited renal lipid peroxidation induced by ischemia and reperfusion both in cortex and in medulla. Electron paramagnetic resonance detected an increased formation of nitric oxide in the resveratrol-treated kidney that was reduced to the baseline value after treating the rats with L-NAME in addition to resveratrol. The results suggest that resveratrol reduced the renal ischemia reperfusion injury through a nitric oxide-dependent mechanism.     

Protective effect of resveratrol on oxidative damage in male and female stroke-prone spontaneously hypertensive rats

1. In the present study, we examined the effect of resveratrol (3,4',5-trihydroxystilbene), a phytoestrogen found in the skins of most grapes, on oxidative DNA damage in male and female stroke-prone spontaneously hypertensive rats (SHRSP). 2. Five-week-old male and female SHRSP were divided into control and resveratrol groups. The resveratrol group was given 1 mg/kg per day, orally, resveratrol by gastric intubation once a day. 3. Following an 8 week feeding period, the levels of 8-hydroxydeoxyguanosine (8-OHdG), produced from deoxyguanosine under conditions of oxidative stress, in the urine of male and female resveratrol-treated SHRSP were significantly lower than that in control SHRSP. 4. The urine of resveratrol-treated male and female SHRSP had lower levels of hydroperoxide compared with control SHRSP, but the difference was not significant. 5. Treatment with resveratrol resulted in a 25 and 30% reduction in plasma glycated albumin in male and female SHRSP, respectively, compared with controls. 6. Gender differences for SHRSP with regard to 8-OHdG, hydroperoxide and glycated albumin levels were not confirmed, resveratrol having similar protective effects on male and female SHRSP. 7. These results indicate that dietary resveratrol: (i) plays a role in suppressing oxidative DNA damage and glycoxidative stress in vivo; and (ii) has similar protective effects in both male and female SHRSP, suggesting that the direct effects of this phytoestrogen on oxidative stress in vivo are not sexually dimorphic.     

灯盏花乙素对超氧阴离子引起的大鼠脑突触体氧化应激的保护作用

目的：研究灯盏花乙素对超氧阴离子引起的大鼠脑突触体氧化应激的保护作用．方法：采用与黄嘌呤(0．3mmol／L)和黄嘌呤氧化酶(0．02U)体系在37℃下孵育30min,建立大鼠脑突触体超氧阴离子氧化损伤模型．通过测定脂质过氧化产物丙二醛评价脂质过氧化程度．通过脂溶性荧光探针DPH的各向异性判断突触体膜的流动性．胞内钙离子的测定采用荧光光度法,以Fura2-AM为荧光探针．测定ATP酶解释放的无机磷确定Na~ ／K~ -ATP酶的活性．结果：超氧阴离子使大鼠脑突触体的脂质过氧化产物丙二醛及胞内钙离子浓度显著上升,突触体的膜流动性和Na~ ／K~ -ATP酶的活性则显著下降,预先加入灯盏花乙素(25-100μmol／L)则能显著缓解超氧阴离子引起的氧化性损伤,表现为丙二醛的水平和胞内钙离子浓度下降,膜流动性增加及Na~ ／K~ -ATP酶活性的恢复．结论：灯盏花乙素对超氧阴离子引起的大鼠脑突触体氧化应激具有良好的保护作用．     

Effect of trimetazidine on the nucleotide profile in rat kidney with ischemia-reperfusion injury.

Ischemia-reperfusion injury is often responsible for delayed graft function after transplantation. Trimetazidine (TMZ) is an antioxidant agent used to protect grafts from ischemia-reperfusion injury. The aim of the study was to examine the effect of TMZ on nucleotide profile in rat kidney with ischemia-reperfusion injury. The study was carried out on Wistar rats divided into two groups: animals treated with TMZ and control group receiving placebo. TMZ 10mg/kg/day was administrated for 30 days. Concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (Ado), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), guanosine (Guo), inosine monophosphate (IMP), inosine (Ino), hypoxanthine (Hyp), xanthine (Xan), uric acid (UA), uridine (Urd), nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) were determined in kidney tissues after ischemia-reperfusion using HPLC. The total adenine nucleotide concentration (TAN) and adenylate energy charge (AEC) were also determined. Moreover the kidneys were evaluated histologically. Tissue concentrations of ATP, ADP, AMP, TAN and AEC were significantly increased in kidneys from rats treated with TMZ in comparison with rats receiving placebo. Concentrations of products of nucleotide degradation: inosine (Ino), guanosine (Guo) and uridine (Urd), as well as oxypurines: Hyp and Xan, were significantly decreased in rats treated with trimetazidine. Moreover, significantly less pronounced acute tubular necrosis was observed in kidneys of rats treated with TMZ. These results suggest that trimetazidine protects against dephosphorylation of nucleotides and ischemic damage.     

Plasma xanthine oxidase and resistance to hypoxia: effect of purines and alcohol administration.

The administration of allopurinol significantly increased resistance to repeated hypobaric hypoxia, while hypoxanthine decreased it. The administration of adenine or inosine (25 mg/kg) was without effect. The results show pathogenic significance of xanthine oxidase-dependent production of free oxygen radicals in posthypoxic damage. In other experiments, the administration of 50% ethanol (12 ml/kg) by gastric catheter increased plasma xanthine oxidase activity in both rats and hamsters.     

The beneficial effect of ATP-MgCl(2) on hepatic ischemia/reperfusion-induced mitochondrial dysfunction

The present study was undertaken to determine whether ATP-MgCl(2) administration in rats could protect hepatic mitochondrial function and improve energy metabolism during hepatic ischemia and subsequent reperfusion. Global hepatic ischemia was produced for 60 min followed by reperfusion. The rats then received 0.5 ml of saline or ATP-MgCl(2) intravenously. In saline-treated ischemic rats, serum alanine-aminotransferase levels peaked at 5 h. The aminotransferase level was significantly reduced in the ATP-MgCl(2) treatment group. The wet weight-to-dry weight ratio of the liver was significantly increased by ischemia/reperfusion. ATP-MgCl(2) treatment minimized the increase in this ratio. The ketone body ratio in blood, which reflects the mitochondrial free NAD(+)/NADH ratio, decreased after ischemia and at 1 h following reperfusion. This decrease was somewhat improved by ATP-MgCl(2) infusion. At 1 and 5 h after reperfusion, mitochondrial monoamine oxidase and glutamate dehydrogenase activities decreased. ATP-MgCl(2) infusion following ischemia restored the lost activities. Hepatic ATP levels in saline-treated rats were found to be 50% lower 5 h following reperfusion; however, treatment with ATP-MgCl(2) resulted in significantly higher ATP levels and energy charge. The accumulation of purine catabolites in ischemic tissues was reduced during reperfusion. ATP-MgCl(2) infusion resulted in accumulation of adenosine in reperfused liver. Mitochondrial lipid peroxidation was elevated in the saline-treated ischemic group, but this elevation was inhibited by ATP-MgCl(2) infusion. The present results lead us to conclude that the amelioration of liver function which occurs with ATP-MgCl(2) infusion following ischemia may be mediated through improvement in ischemia-induced mitochondrial energy metabolism.     

Nifedipine reduces adenine nucleotide breakdown in ischemic rat heart

An ATP-sparing effect has been demonstrated for a number of calcium antagonists. Nifedipine probably has a similar action, but data supporting this view are limited. Therefore we decided to study the effect of nifedipine on high-energy phosphate (and carbohydrate) metabolism in the ischemic rat heart. Langendorff preparations were made ischemic for less than 15 min. The reduction in coronary flow was 60 or 70%. Apex displacement during ischemia, a measure of contractility, was comparable for nifedipine-treated and untreated hearts. Ischemia caused a considerable release of the AMP catabolites adenosine, inosine and (hypo)xanthine, and of lactate. Nifedipine (10–100 μg/l) prevented this in a dose-dependent way. The highest dose reduced the release of purines and lactate by 90% (P<0.01) and 60% (P<0.001), respectively. The drug acted in a similar way during reperfusion. Due to ischemia, the adenylate energy charge (ATP+0.5 ADP)/(ATP+ADP+AMP), decreased 15% (P<0.001); nifedipine at a concentration of 100 μg/l prevented this decrease (P<0.05). We conclude that nifedipine exerts a benificial effect on myocardial adenine nucleotide metabolism during ischemia and reperfusion.     

AMG-1对小鼠和大鼠缺血脑能量代谢及神经细胞损伤的影响

本文观察AMG-1对小鼠断头全脑缺血时能量代谢及大鼠大脑中动脉阻断(MCAO)后行为和病理改变影响。小鼠sc AMG-1 1～10mg/kg 30min后,能有效减少脑缺血后乳酸(LA)堆积,ATP和磷酸肌酸(PCr)的耗竭。sc AMG-1 5mg/kg和尼英地平0.5 mg/kg,可减轻大鼠MCAO后的神经症状和神经细胞缺血性损害。AMG-1的这一改善脑缺血作用与尼莫地平近似。     

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.     

人参皂苷保护小鼠精原细胞氧化损伤的研究

目的观察人参皂苷对活性氧引起的小鼠睾丸生殖细胞氧化损伤的保护作用。方法利用体外培养的小鼠精原细胞建立氧化应激模型,通过检测生殖细胞活性、脂质过氧化产物丙二醛(MDA)生成、超氧化物歧化酶(SOD)活性和谷胱甘肽(GSH)水平评价人参皂苷对精原细胞氧化损伤的缓解作用。结果次黄嘌呤/黄嘌呤氧化酶(HX/XO)体系产生的活性氧可引起生殖细胞活性降低、MDA的生成量增加、SOD活性和GSH水平降低,而添加人参皂苷(10mg·L-1)能恢复HX/XO引起的生殖细胞活性、SOD活性和GSH水平的下降以及MDA生成的增加。结论人参皂苷可通过抗氧化作用保护活性氧引起的小鼠精原细胞氧化损伤。     

小鼠脑缺血后的能量代谢改变和药物的作用

应用部分结扎小鼠颈总动脉(包括迷走神经)及小鼠断头法引起脑缺血后,脑组织的ATP和磷酸肌酸明显降低,乳酸明显升高。部分结扎颈动脉出现四肢无力、转圈及昏睡等症状,其严重程度与脑能量代谢改变相平行。皮下注射尼莫地平、硝苯吡啶、尼卡地平和三七皂甙对脑缺血有一定保护作用。苯巴比妥钠能改善正常和脑缺血小鼠的脑能量代谢,人参皂甙Rb₁可降低正常小鼠脑乳酸含量。     

二乙基二硫代氨基甲酸钠对沙土鼠脑缺血再灌注损伤的保护作用

蒙古沙土鼠脑缺血６０ｍｉｎ后，脑组织能量物质ＡＴＰ、ＡＤＰ明显下降，且有可检测的次黄嘌呤产生；氧在含量上有趋势。再灌注５ｍｉｎ，氧自由基含量显著增加；灌注３０ｍｉｎ时，ＡＴＰ、ＡＤＰ有所回，ＨＸ降至无法检测ＯＦＲ降于缺血组水平。