二氮嗪对大鼠心肌线粒体膜电位及呼吸功能的影响

目的 :研究二氮嗪对大鼠心肌线粒体功能的影响 ,以探讨其心肌保护作用的可能机理。方法 :以罗丹明 12 3为荧光探针测定线粒体膜电位 ,氧电极法测线粒体呼吸 ,观察二氮嗪对正常大鼠心肌线粒体跨膜电位及呼吸的影响。结果 :5 0 μmol·L-1的二氮嗪可引起线粒体膜电位去极化 ,此影响可被钾通道阻滞剂消除 ;二氮嗪 10 0 μmol·L-1可降低大鼠心肌线粒体琥珀酸链的R3、R4速率 ,但不影响呼吸控制率。结论 :二氮嗪可调节大鼠心肌线粒体功能 ,这与其抗心肌缺血保护作用有关。     

心肌线粒体ATP敏感性钾通道开放保护线粒体结构和功能

目的　评价线粒体ATP敏感性钾通道 (mitoKATP)开放剂二氮嗪对缺氧心肌线粒体结构和功能的影响。方法　异丙肾上腺素 (ISO)皮下注射诱发大鼠心肌缺氧损伤 ,观察二氮嗪对线粒体呼吸控制比 (RCR)、膜流动性、磷脂酶A2 和磷脂含量的影响。结果　ISO皮下注射诱发大鼠心肌缺氧损伤后 ,与对照组比较线粒体RCR降低了 2 0 8% (P <0 0 1) ,膜流动性降低了 9 1% (P <0 0 5 ) ,磷脂酶A2 活性增加了14 4 5 % (P <0 0 1) ,磷脂含量下降了 37 5 % (P <0 0 5 ) ,二氮嗪预防性给药后可改善RCR、膜流动性和磷脂含量。结论　二氮嗪可保护心肌缺氧损伤后线粒体膜结构和功能的完整     

开放线粒体KATP通道对心肌细胞氧化应激损伤的保护作用

目的观察ATP敏感性钾通道(KATP)开放剂二氮嗪对心肌细胞氧化应激损伤的保护作用,并探讨其作用机制.方法采用过氧化氢(500 μmol·L-1)诱导法制备大鼠培养心肌细胞氧化应激损伤模型,通过检测培养液中乳酸脱氢酶以及用流式细胞仪结合罗丹明-123和碘化丙啶双标记法检测线粒体膜电位和细胞存活状态,观察二氮嗪(100 μmol·L-1)对氧化应激损伤的保护作用.结果二氮嗪预处理后,细胞培养液中乳酸脱氢酶活性较过氧化氢处理组显著降低(P＜0.01),细胞存活率升高,并可减少氧化应激造成的线粒体膜电位的丢失,其作用可被线粒体KATP通道阻断剂5-羟基癸酸酯所拮抗.结论二氮嗪对过氧化氢造成的培养心肌细胞氧化应激损伤具有保护作用,其可能通过激活线粒体KATP通道介导.     

MitoKATP通道开放对重症急性胰腺炎大鼠心肌保护的实验研究

目的：探讨线粒体ATP敏感性钾通道（MitoKATP通道）开放对重症急性胰腺炎（SAP）心肌的保护作用及机制。方法：清洁级雄性SD大鼠36只随机分为4组：K组假手术;M组建立SAP大鼠模型;D组和H组分别给予连续腹腔注射二氮嗪(DZ)、DZ+5-羟基葵酸盐（5-HD）3d后建立SAP大鼠模型。观察4组大鼠术后24 h血清肌酸激酶同工酶（CK-MB）、乳酸脱氢酶（LDH）水平;分光光度法测定心肌组织Na+-K+-ATPase活性;流式细胞仪检测心肌细胞线粒体线粒体膜电位(?ψm)变化;原位末端标记（TUNEL）法检测心肌细胞凋亡情况;留取部分心脏、胰腺组织行病理学检查。结果：M组CK-MB、LDH水平、心脏病理评分及凋亡指数较D组升高,Na+-K+ -ATPase活性及?ψm较D组降低,差异均有统计学意义（P < 0.01）,但M组上述指标与H组比较差异均无统计学意义（P > 0.05）。结论：MitoKATP通道开放对SAP引起的心肌损伤有保护作用。     

二氮嗪预处理对体内脏器的保护作用及机制研究进展

二氮嗪(Diazoxide),别名:降压嗪、氯甲苯噻嗪,由于其能有效的松弛动脉平滑肌,降低外周血管阻力而降低血压,临床主要用于抢救高血压危象、作为线粒体ATP敏感钾通道(mitoKATP通道)开放剂的二氮嗪,可以减轻心肌,脑,肝脏等器官的缺血再灌注损伤,起到重要的保护作用,本文对其研究新进展综述如下.     

通心络胶囊对缺血大鼠脑细胞超微结构及线粒体呼吸功能的保护作用

目的：研究通心络胶囊对脑缺血大鼠脑细胞超微结构及脑线粒体呼吸功能的影响。方法：采用线栓法制作大鼠大脑中动脉缺血模型，透射电镜观察缺血后脑组织神经元超微结构的改变．检测线粒体内的NAD及FAD氧化呼吸链R3、R4、RCR、P／O等评价呼吸功能的指标，观察通心络（1．0、2．0mg／kg）对上述结构和指标的影响。结果：电镜照片显示通心络能改善脑组织神经元在缺血后的结构破坏情况；通心络治疗组大鼠线粒体呼吸功能明显改善。结论：通心络对缺血脑细胞有明显保护作用．机制与其能改善线粒体呼吸功能有关。     

失血性休克时细胞线粒体呼吸功能变化及中药防治研究

目的：研究失血性休克时细胞线粒体呼吸功能变化及防治方法。方法：将24只Wistar大鼠随机均分为假手术组、模型组和治疗组，模型组和治疗组颈动脉快速放血造成失血性休克模型，维持1．5h后复苏。假手术组动物除不放血及复苏外其他操作同模型组。治疗组于实验前2d开始及放血前4h各给予静脉注射中药益气活血解毒汤1mL／kg干预治疗。于复苏后4h处死动物，测定肝匀浆内毒素和线粒体呼吸功能。结果：与假手术组比较，模型组肝脏内毒素水平明显升高（P＜0．01），而Ⅲ态呼吸耗氧速率（RS3）和呼吸控制率（RCR）下降显著（P＜0．01）。与模型组比较，治疗组肝脏内毒素的升高及RS3、RCR的降低明显减轻（P＜0．05）。结论：大鼠失血性休克复苏后肝线粒体呼吸功能严重受损，以RS3下降为主，中药益气活血解毒汤治疗可明显改善线粒体功能。     

线粒体KATP开放剂二氮嗪促进星形胶质细胞摄取谷氨酸

目的研究线粒体ATP敏感性钾通道(mitochondrial ATP-sensitive potassium channel, mitoKATP)开放剂二氮嗪(diazoxide)对星形胶质细胞摄取谷氨酸(glutamate)的影响.方法取新生大鼠脑星形胶质细胞作原代培养,用液体闪烁计数仪测定[3H]-D,L-谷氨酸的摄入量判断细胞的谷氨酸摄取功能.结果二氮嗪呈浓度依赖性地促进星形胶质细胞摄取谷氨酸,且能抑制1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP+)对星形胶质细胞摄取谷氨酸的损伤作用;浓度在100μmol·L-1以上时,二氮嗪可完全逆转MPP+对星形胶质细胞摄取谷氨酸的抑制作用;二氮嗪的上述作用可被选择性mito KATP 阻断剂5-羟基癸酸 (5-hydroxydecanoate,5-HD)拮抗.结论二氮嗪通过开放mitoKATP增强星形胶质细胞谷氨酸转运体(glutamate transporters, GluTs)的功能.     

慢性肾衰大鼠脑组织线粒体呼吸功能变化及氯沙坦的干预

目的：研究慢性肾功能衰竭（chronic renal failure,CRF）模型大鼠脑组织线粒体呼吸功能和细胞凋亡相关蛋白质的变化,并探讨氯沙坦的干预作用。方法：雄性SD大鼠24只,随机分为假手术组（Sham）、病理组（Nx）和氯沙坦干预组（Lst）,行5/6肾切除手术,建立慢性肾衰模型,Lst组于饮水中给予氯沙坦（50 mg·L-1）治疗。给药结束后,测定SCr、BUN水平及脑组织MDA的含量和SOD的活性,氧电极法检测脑组织线粒体呼吸3态氧耗率（ST3）、4态氧耗率（ST4）和呼吸控制率（RCR）,免疫印迹法观察Caspase-3、Cleaved caspase-3、Cleaved caspase-9、Bcl-2蛋白表达变化。结果：与Sham组相比,Nx组SCr、BUN含量显著升高（P<0.01）,MDA含量显著升高（P<0.01）,SOD活性显著下降（P<0.01）,ST3、RCR显著降低（P<0.01）,ST4显著升高（P<0.05）,Cleaved caspase-3、Cleaved caspase-9蛋白水平显著升高（P<0.01、P<0.05）,Caspase-3、Bcl-2蛋白水平显著降低（P<0.01）。与Nx组相比, Lst组SCr、BUN含量显著降低（P<0.01）,MDA含量显著下降（P<0.01）,ST3、RCR显著升高（P<0.01）,Cleaved caspase-3、Cleaved caspase-9蛋白水平明显降低（P<0.01）,Caspase-3、Bcl-2蛋白水平明显升高（P<0.01）。结论：慢性肾衰可造成大鼠脑组织氧化损伤,损害线粒体呼吸功能,促进神经细胞凋亡。氯沙坦通过减轻氧化应激损伤,改善线粒体呼吸功能,减少细胞凋亡,发挥神经保护作用。     

银杏酸对大鼠肝脏线粒体的损伤作用

目的：在分离制备的大鼠卧脏线粒体上，检验银杏酸（GAs）的损伤作用。方法：银杏酸（6-烃基水杨酸衍生物）从银杏外种皮中通过提取、分离、纯化而制备，并通过液一质联机鉴定。采用其中两个主要单体C13：0（GA13）和C15：1（GA15）进行研究。采用分步差速离心法制备大鼠肝脏线粒体。以线粒体呼吸功能和胞浆及线粒体标志酶（乳酸脱氢酶和柠檬酸合酶）活件来评估线粒体制备质量。采用Clark氧电极法测定线粒体呼吸功能（以RCR和ADP／O值为指标），采用分光光度法测定线粒体通透性孔道开放（mPTP），采用荧光指示剂Rhodamine123测定线粒体膜电位（△ψm），采用MTT法评价线粒体活力。结果：GAs可呈剂量依赖性地抑制线粒体的呼吸功能，并显示对氧化磷酸化有解偶联作用。GA13和GA15可剂量依赖性地诱导mPTP的开放（EC50值分别为2．75μM和3．20μM），且均被mPTP拮抗剂CysA特异性抑制。     

Diazoxide acts more as a PKC-epsilon activator, and indirectly activates the mitochondrial K(ATP) channel conferring cardioprotection against hypoxic injury

BACKGROUND AND PURPOSE: Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoK(ATP) channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoK(ATP) channel in diazoxide-induced cardioprotection. EXPERIMENTAL APPROACH: In H9c2 cells and neonatal rat cardiomyocytes, PKC-epsilon activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca(2+) and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay. KEY RESULTS: Diazoxide (100 microM) induced translocation of PKC-epsilon from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-epsilon by either epsilonV1-2 or dominant negative mutant PKC-epsilon (PKC-epsilon KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either epsilonV1-2 or PKC-epsilon KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-epsilon induced by diazoxide. Transfection with wild type PKC-epsilon mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by epsilonV1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca(2+), mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by epsilonV1-2 or 5-HD. CONCLUSIONS AND IMPLICATIONS: Diazoxide induced isoform-specific translocation of PKC-epsilon as an upstream signaling molecule for the mitoK(ATP) channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway.     

Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells

Several drugs used in the treatment of chronic peripheral ischaemic and venous diseases, i.e. aescine, Cyclo 3, Ginkor Fort, hydroxyethylrutosides, naftidrofuryl, naphthoquinone and procyanidolic oligomers, were tested on the mitochondrial respiratory activity. The results show that all these drugs protected human endothelial cells against the hypoxia-induced decrease in ATP content. In addition, they all induced a concentration-dependent increase in respiratory control ratio (RCR) of liver mitochondria pre-incubated with the drugs for 60 min. The drugs were divided into two groups according to their effects. The first group (A), comprising aescine, Ginkor Fort, naftidrofuryl and naphthoquinone, increased RCR by decreasing state 4 respiration rate. The second group of drugs (B), comprising hydroxyethylrutosides, procyanidolic oligomers and Cyclo 3, increased RCR by increasing state 3 respiration rate. The drugs of group A were able to prevent the inhibition of complexes I and III respectively by amytal and antimycin A while the first two drugs of group B increased adenine nucleotide translocase activity. Cyclo 3 inhibited the carbonylcyanide m-chlorophenyl hydrazone (mCCP)-induced uncoupling of mitochondrial respiration. None of these seven drugs could protect complexes IV and V, respectively, from inhibition by cyanide and oligomycin. When tested on endothelial cells the drugs of group A, in contrast to group B, prevented the decrease in ATP content induced by amytal or antimycin A. The present results suggest that the protective effects on mitochondrial respiration activity by these venotropic drugs may explain their protective effect on the cellular ATP content in ischaemic conditions and some of their beneficial therapeutic effect in chronic vascular diseases.     

Elucidation of mitochondrial effects by tetrahydroaminoacridine (tacrine) in rat, dog, monkey and human hepatic parenchymal cells

Tetrahydroaminoacridine (tacrine) causes morphological and functional changes in the endoplasmic reticulum, ribosomes, and mitochondria in the liver of humans and animals. In order to investigate species differences as well as to understand the morphological changes, we examined the effects of tacrine on respiration and electron transport in mitochondria isolated from rat, dog, monkey, and human liver. Tacrine produced significantly decreased respiratory control ratios (RCR) in all species at concentrations ranging from 5 to 25 μg/ml. Human mitochondria were more sensitive to tacrine effects with RCR decreased 24% at 5 μg/ml while other species were unaffected at this concentration. The tacrine effects were characterized by increased hepatic mitochondrial State 4 respiration in rats and decreased State 3 respiration in humans. Mitochondria from aged rats were more sensitive to the effects of tacrine than mitochondria from young animals, with significantly decreased RCR at 10 μg/ml in aged rats while mitochondria from young rats were unaffected at this concentration. Concomitant with the respiratory changes, mitochondrial DNA synthesis was impaired. Since tacrine undergoes extensive biotransformation, we also explored the possibility that metabolites could exert detrimental effects. The ranking order of potency for decreasing RCR caused by monohydroxylated metabolites was: tacrine >4-OH and 7-OH >2-OH, 1-OH, and velnacrine with the latter group of metabolites having no effect on mitochondrial respiration at concentrations up to 50 μg/ml. In vivo administration of 20 mg/kg tacrine to rats for up to 20 days caused a paradoxical increase in RCR and P/O on Day 1 and decreased RCR on Days 9 and 20, the later findings being consistent with in vitro data. From these data we propose that tacrine does not necessarily have to be metabolized to exert effects on mitochondria at different sites in the electron transport chain that differ among species. These effects are exacerbated in mitochondria from older animals and humans appear to be more sensitive than the laboratory animals studied. Received: 23 September 1997 / Accepted: 17 November 1997     

维生素C对缺氧大鼠心肌线粒体功能和ATP含量的影响

AIM: To observe the effects of large dose of vitamin C (Vc) on myocardial mitochondrial function, ATP content, and myocardial structure in acute and chronic hypoxic rats. METHODS: Rats were exposed to a simulated altitude 4000 m (barometric pressure = 43 kPa) for 3 and 30 d. Vc (0.75 g.kg-1.d-1) was injected i.p. The heart mitochondrial respiratory function were determined by Clark-type O2 electrode; mitochondrial membrane fluidity (MMF) were assayed through fluorescence polarizative method; the contents of ATP, ADP, and AMP in myocardial tissue were measured with HPLC. RESULTS: After administration of Vc, the ATP content was increased from 35 +/- 3 mg.g-1 to 53 +/- 3 mg.g-1 in acute hypoxic rats (P < 0.01), from 42 +/- 4 mg.g-1 to 48 +/- 3 mg.g-1 in chronic hypoxic rats (P < 0.01); Pa, O2 was increased from 7.2 +/- 1.4 kPa to 9.5 +/- 1.2 kPa in acute hypoxic rats (P < 0.01); mitochondrial respiratory control rate (RCR) was increased from 2.1 +/- 0.6 to 4.7 +/- 0.5 in acute hypoxic rats (P < 0.01), and from 3.3 +/- 0.7 to 4.5 +/- 0.6 in chronic hypoxic rats (P < 0.01); MMF was increased in acute and chronic hypoxic rats (P < 0.05); the degree of myocardial necrosis in vitamin C preventive rats was attenuated as compared with those of acute hypoxic rats. CONCLUSION: Vc is effective on improving myocardial energy metabolism and protecting against myocardial structural injury in hypoxic rats.     

Cardioprotective effects of (2S,3R,4S)-N'-benzyl-N"-cyano-N-(3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-benzopyran-4-yl)-guanidine (KR-31372) in rats and dogs

The cardioprotective effects of (2S,3R,4S)-N'-benzyl- N"-cyano-N-(3,4-dihydro-2-dimethoxymethyl-3-hydro- xy-2-methyl-6-nitro-2H-benzopyran-4-yl)-guanidine (KR-31372) were evaluated against ischemic/reperfusion injury in isolated rat hearts in vitro and in anesthetized rats and dogs in vivo. In isolated perfused rat hearts subjected to a 30-min global ischemia/30-min reperfusion, KR-31372 (1-10 microM) significantly improved severe contracture (end-diastolic pressure and time to contracture), markedly reduced reperfusion lactate dehydrogenase release, and enhanced the recovery of reperfusion contractile function (left ventricular developed pressure and double product) in a concentration-dependent manner compared with the vehicle-treated group. In anesthetized rats subjected to a 45-min coronary occlusion and a 90-min reperfusion, intravenous KR-31372 dose-dependently reduced infarct size from 58.6% to 48.5, 48.1 and 39.6% at 0.3, 1.0 and 3.0 mg/kg, respectively (p < 0.05). In anesthetized beagle dogs that underwent a 1.5-hour occlusion followed by a 5-hour reperfusion, KR-31372 (2 mg/kg, i.v.) markedly reduced infarct size from 57.0% in controls to 28.0% (p < 0.05). The cardioprotective effects of KR-31372 on contractile function in globally ischemic rat hearts and on reperfusion injury in anesthetized rats were significantly reversed by pretreatment with selective adenosine triphosphate-sensitive potassium (K(ATP)) channel blockers, sodium 5-hydroxydecanoate and glibenclamide. Taken together, these results indicate that KR-31372 possesses potent cardioprotective effects in rats and dogs and its effects may be mediated by activation of mitochondrial K(ATP) channels.     

Differential effects of zidovudine and zidovudine triphosphate on mitochondrial permeability transition and oxidative phosphorylation

1. The effects of zidovudine (ZDV) and zidovudine triphosphate (ZDV-3P) on Ca²⁺-induced mitochondrial permeability transition (MPT), respiratory control ratio (RCR) and ATP synthesis have been investigated on isolated rat liver mitochondria.
2. ZDV slightly but significantly decreased RCR and ATP synthesis but was ineffective in inhibiting MPT. In contrast, ZDV-3P did not alter RCR and ATP synthesis but strongly inhibited MPT (IC₅₀=3.0±0.9 μM).
3. The effect of ZDV-3P on mitochondrial swelling required a preincubation time. When incubated 10 min with mitochondria, ZDV-3P (8 μM) totally inhibited the rate of swelling.
4. ADP, ATP and atractyloside, which are agents known to interact with the mitochondrial adenine nucleotide carrier (ANC), antagonized the effect of ZDV-3P on mitochondrial swelling. Indeed, the IC₅₀ value of ZDV-3P increased from 3.0 to 17.4, 93.6 and 66.5 μM, in the presence of 20 μM, ADP, ATP or atractyloside, respectively.
5. ZDV-3P did not displace [³H]-ATP from its mitochondrial binding site(s) whereas ADP and atractyloside did, suggesting that ZDV-3P and [³H]-ATP do not share the same binding sites.
6. ZDV-3P did not affect either mitochondrial respiration or ATP synthesis but inhibited Ca²⁺-dependent mitochondrial swelling. It was concluded that mitochondrial toxic effects observed during the chronic administration of ZDV cannot be related to its active metabolite (ZDV-3P).