菟丝子水提物对衰老模型大鼠心肌线粒体呼吸链酶复合体活性的影响

目的 探讨菟丝子水提物对大鼠心肌线粒体呼吸链酶复合体活性的影响.方法 将Wistar大鼠30只,随机分为青年对照组,衰老模型组,模型给药组,制作D-半乳糖衰老模型,模型给药组同时灌服菟丝子水提物.观察各组大鼠心肌线粒体丙二醛(MDA)的含量、超氧化物歧化酶(SOD)的活性以及呼吸链酶复合体Ⅰ、Ⅳ活性的变化.结果 菟丝子水提物可提高心肌线粒体SOD的活性、降低MDA的含量;并且使呼吸链酶复合体Ⅰ、Ⅳ活性明显升高.结论 菟丝子水提物能提高心肌线粒体抗氧化能力,改善线粒体能量代谢障碍,维护线粒体功能.     

二氮嗪对大鼠缺血再灌注损伤脑组织线粒体ATP酶活性的影响

目的 探讨线粒体ATP敏感性钾通道开放剂二氮嗪对大鼠局灶性脑缺血再灌注损伤脑组织线粒体ATP酶活性的影响.方法 采用改良线栓法建立大鼠局灶性大脑中动脉缺血再灌注损伤模型.将21只Wistar雄性大鼠随机分为假手术组(N组)、缺血再灌注组(IR组)、二氮嗪干预组(DZ组).缺血1 h再灌注24 h后留取标本,测定脑组织线粒体Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性的变化.结果 与假手术组比较,缺血再灌注组Na+-K+-ATPase、Ca2+-Mg2+-ATP酶活性明显降低(P<0.05);二氮嗪干预组Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性均较缺血再灌注组有不同程度的提高(P<0.05).结论 二氮嗪预处理能通过提高脑组织线粒体Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性,减轻脑缺血再灌注损伤,保护神经元线粒体的功能,有效维持大脑能量代谢,发挥脑保护作用.     

肉苁蓉多糖对衰老大鼠肝线粒体保护作用的研究

目的观察肉苁蓉多糖对D-半乳糖致衰大鼠肝线粒体氧化损伤的保护作用。方法采用D-半乳糖所致衰老模型大鼠,灌服肉苁蓉多糖6 w,测定肝脏Ca2＋-ATP酶活性、肝线粒体丙二醛（MDA）含量、磷脂酶A2（PLA2）活性、膜流动性及呼吸链复合体Ⅰ＋Ⅲ、Ⅱ＋Ⅲ活性。结果肉苁蓉多糖显著提高衰老模型大鼠肝脏Ca2＋-ATP酶活性、肝线粒体膜流动性及呼吸链复合体Ⅰ＋Ⅲ、Ⅱ＋Ⅲ活性,显著降低肝线粒体MDA含量、PLA2活性。结论肉苁蓉多糖能提高衰老模型大鼠肝线粒体抗氧化能力,改善线粒体能量代谢,具有抗衰老作用。     

五子衍宗丸及其拆方对老年大鼠骨骼肌线粒体DNA氧化损伤的影响

目的：研究五子衍宗丸及其拆方对老年大鼠骨骼肌线粒体DNA缺失,线粒体呼吸链酶复合体及ATP合成的影响,方法：用聚合酶链反应（PCR）,酶动力学和生物发光技术进行检测,结果：老年大鼠骨骼肌线粒体DNA缺失较青幼年大鼠明显守多,线粒体呼吸链复合酶IV活力及ATP合成量较青年大鼠明显减少,五子衍宗丸及其拆方的枸禁枸札子,菟丝可减少老年大鼠骨骼肌线粒体DNA缺失（P＜0．01）,提高线粒体呼吸链复合I酶IV活力（P＜0．05）,五子衍宗丸还可提高老年大鼠骨骼肌线粒体ATP的合成（P＜0．05）,结论．：五子衍宗,枸杞子和菟丝子对老年大鼠线粒体DNA的氧化损伤有保护作用。     

Ghrelin reduces voltage-gated potassium currents in GH3 cells via cyclic GMP pathways

Ghrelin is an endogeneous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca²⁺ concentration ([Ca²⁺]_i), which is determined by Ca²⁺ influx and release from intracellular Ca²⁺ storage sites. Ca²⁺ influx is via voltage-gated Ca²⁺ channels, which are activated by cell depolarization. Membrane potential is mainly determined by transmembrane K⁺ channels. The present study investigates the in vitro effect of ghrelin on membrane voltage-gated K⁺ channels in the GH₃ rat somatotrope cell line. Nystatin-perforated patch clamp recording was used to record K⁺ currents under voltage-clamp conditions. In the presence of Co²⁺ (1 mM, Ca²⁺ channel blocker) and tetrodotoxin (1 μM, Na⁺ channel blocker) in the bath solution, two types of voltage-gated K⁺ currents were characterized on the basis of their biophysical kinetics and pharmacological properties. We observed that transient K⁺ current (I _A) represented a significant proportion of total K⁺ currents in some cells, whereas delayed rectifier K⁺ current (I _K) existed in all cells. The application of ghrelin (10 nM) reversibly and significantly decreased the amplitude of both I _A and I _K currents to 48% and 64% of control, respectively. Application of apamin (1 μM, SK channel blocker) or charybdotoxin (1 μM, BK channel blocker) did not alter the K⁺ current or the response to ghrelin. The ghrelin-induced reduction in K⁺ currents was not affected by PKC and PKA inhibitors. KT5823, a specific PKG inhibitor, totally abolished the K⁺ current response to ghrelin. These results suggest that ghrelininduced reduction of voltage-gated K⁺ currents in GH₃ cells is mediated through a PKG-dependent pathway. A decrease in voltage-gated K⁺ currents may increase the frequency, duration, and amplitude of action potentials and contribute to GH secretion from somatotropes.     

CGRP对大鼠全脑缺血再灌注ATP酶活性的影响

目的研究降钙素基因相关肽(CGRP)对大鼠全脑缺血再灌注(I/R)脑组织ATP酶活性变化的影响及神经保护机制.方法45只SD大鼠,随机分为假手术组、对照组、CGRP)组.采用四血管阻断方法制备SD大鼠全脑I/R模型,定磷比色法测定全脑I/R及全脑I/R CGRP治疗大鼠海马Na ,K -ATP、Ca2 -ATP酶活性.结果与假手术组比较,大鼠全脑I/R后海马Na ,K -ATP、Ca2 -ATP酶活性降低.CGRP对I/R后海马Na ,K -ATP、Ca2 -ATP酶活性降低有明显的抑制作用.结论CGRP对大鼠全脑I/R脑组织损伤有保护作用.     

骨髓增生异常综合征线粒体呼吸链酶复合体的变化

目的:研究骨髓增生异常综合征(MDS)患者骨髓单个核细胞线粒体呼吸链的功能变化并分析其与MDS的关系.方法:测定26例MDS患者与10例骨髓象正常者的单个核细胞线粒体呼吸链酶复合体Ⅰ、Ⅲ、Ⅳ的活性.结果:MDS患者呼吸链酶复合体Ⅰ、Ⅲ、Ⅳ的活性明显低于对照组(P＜0.05);线粒体呼吸链酶复合体Ⅰ、Ⅲ的活性在RAEB-Ⅰ、RAEB-Ⅱ组与RA、RARS组的差异无统计学意义(均P＞0.05),而酶复合体Ⅳ的活性在RAEB-Ⅰ、RAEB-Ⅱ组较RA、RARS组高,差异有统计学意义(P＜0.05).结论:线粒体呼吸链酶复合体的活性在MDS患者中降低,可能与MDS的病态造血及无效造血有关.     

白藜芦醇对老年大鼠心肌细胞线粒体细胞色素C氧化酶表达的影响

目的探讨白藜芦醇对老年大鼠心肌细胞线粒体的抗氧化作用以及对呼吸链酶复合体Ⅳ(CoⅣ)活性和表达的影响。方法 SD大鼠30只随机分为青年组,老年组和用药组,用药组每日灌胃白藜芦醇70 mg/kg,共4 w,检测各组大鼠心肌线粒体丙二醛(MDA)的含量、超氧化物歧化酶(Mn-SOD)的活性、呼吸链酶CoⅣ活性及其mRNA表达的变化。结果白藜芦醇可提高心肌线粒体Mn-SOD的活性、降低MDA的含量,并且使呼吸链酶CoⅣ活性增加、CoⅣmRNA的表达降低。结论白藜芦醇能提高心肌线粒体抗氧化能力,改善线粒体能量代谢障碍,维护线粒体功能,为老年人心肌功能改善药物的开发奠定实验基础。     

ATP酶在细菌潜生体相关的IBS大鼠模型肠黏膜中的变化

目的:研究细菌潜生体相关的肠易激综合征(irritable bowel syndrome,IBS)大鼠模型肠黏膜ATP酶的变化.方法:Wistar大鼠分为正常对照组和细菌潜生体相关的IBS大鼠模型组.采用无机磷法检测大鼠回盲部黏膜ATP酶活性,高效液相色谱法检测回盲部黏膜细胞的腺苷酸能荷及ATP与总腺苷酸库比值,MTT法检测回盲部黏膜呼吸酶活性.结果:与正常对照组相比较,IBS大鼠模型组回盲部肠黏膜Na+-K+-ATP酶(22.44±5.54 vs14.20±3.03,P<0.01),Ca2+-Mg2+-ATP酶均显著下降(16.46±1.86 vs 10.63±1.78,P<0.01),ATP显著降低(0.96±0.18 v s 0.48±0.20,P<0.01),同时呼吸酶活性显著变化(0.50±0.07vs 0.21±0.05,P<0.01).结论:IBS大鼠回盲部黏膜ATP酶活性显著降低,与能量代谢降低相关,这可能是引起肠黏膜屏障功能受损的重要原因,与IBS病因相关.     

还原型谷胱甘肽合抗震止痉胶囊对帕金森病细胞保护作用的实验研究

目的 :观察还原型谷胱甘肽 (GSH)合抗震止痉胶囊对帕金森病 (PD )大鼠模型的异常行为、黑质抗氧化系统、线粒体呼吸链功能的影响 ,并探讨其作用机制。方法 :应用 6-羟基多巴胺立体定向注射制作PD大鼠模型。将 2 4只大鼠随机分为 3组 :模型组、GSH合抗震止痉胶囊组、假手术组 ,每组 8只 ,分别给予相应处理 ,共45d ,给药前后均进行行为学测试 ,并测定各组大鼠黑质区GSH -Px、MDA、活性氧及线粒体呼吸链酶复合体Ⅰ水平。结果 :①GSH合抗震止痉胶囊可明显改善大鼠旋转行为(P <0 .0 5 ) ;②GSH合抗震止痉胶囊能减轻黑质区氧化应激损伤 ;③GSH合抗震止痉胶囊能增强黑质呼吸链酶复合体Ⅰ活性。结论 :GSH合抗震止痉胶囊能部分改善大鼠旋转行为 ,减轻PD模型大鼠黑质区氧化应激损伤 ,并对线粒体呼吸链具有一定保护作用。     

辣椒素减轻肾缺血再灌注损伤的线粒体相关机制

目的探讨辣椒素对大鼠肾缺血再灌注损伤的保护作用及线粒体相关作用机制。方法将50只雄性SD大鼠分成假手术组、肾缺血再灌注损伤组和辣椒素低、中、高剂量组。采用夹闭双侧肾蒂构建肾缺血再灌注损伤模型。肾缺血45 min,再灌注24 h,过量麻醉法处死大鼠,收集肾脏和血清。检测血清肌酐(SCr)、血尿素氮(BUN)、肾脏组织病理形态和细胞凋亡,测定线粒体三磷酸腺苷(ATP)和丙二醛(MDA)含量以及Ca~(2+)-ATP酶、Na~+-K~+-ATP酶、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)和超氧化物歧化酶(SOD)活性。结果辣椒素干预可减少SCr和BUN含量,降低肾组织病理改变和细胞凋亡,增加线粒体Ca~(2+)-ATP酶、Na~+-K~+-ATP酶、CAT、GPx和SOD酶活性以及ATP的含量,但减少MDA的水平。结论辣椒素对肾缺血再灌注损伤有保护作用,其作用呈浓度效应,机制与抑制线粒体脂质过氧化相关。     

Mitochondrial Ca2+ flux and respiratory enzyme activity decline are early events in cardiomyocyte response to H2O2

Oxidative stress is involved in mitochondrial apoptosis, and plays a critical role in ischemic heart disease and cardiac failure. Exposure of cardiomyocytes to H₂O₂ leads to oxidative stress and mitochondrial dysfunction. In this study, we investigated the temporal order of mitochondrial-related events in the neonatal rat cardiomyocyte response to H₂O₂ treatment. At times ranging from 10 to 90 min after H₂O₂ treatment, levels were determined for respiratory complexes I, II, IV and V, and citrate synthase activities, mitochondrial Ca²⁺ flux, intracellular oxidation, mitochondrial membrane potential and apoptotic progression. Complexes II and IV activity levels were significantly reduced within 20 min of H₂O₂ exposure while complexes I and V, and citrate synthase were unaffected. Mitochondrial membrane potential declined after 20 and 60 min of H₂O₂ exposure while intracellular oxidation, declining complex I activity and apoptotic progression were detectable only after 60 min. Measurement of mitochondrial Ca²⁺ ([Ca²⁺]_m) using rhodamine 2 detected an early accumulation of [Ca²⁺]_m occurring between 5 and 10 min. Pretreatment of cardiomyocytes with either ruthenium red or cyclosporin A abrogated the H₂O₂-induced decline in complexes II and IV activities, indicating that [Ca²⁺]_m flux and onset of mitochondrial permeability transition pore opening likely precede the observed early enzymatic decline. Our findings suggest that [Ca²⁺]_m flux represents an early pivotal event in H₂O₂-induced cardiomyocyte damage, preceding and presumably leading to reduced mitochondrial respiratory activity levels followed by accumulation of intracellular oxidation, mitochondrial membrane depolarization and apoptotic progression concomitant with declining complex I activity.     

Ovariectomy alters energy metabolism in rat striatum: effect of supplementation with soy diet rich in isoflavones

In the present study we investigated the effect of ovariectomy on some parameters of energy metabolism, namely Na⁺,K⁺-ATPase and pyruvate kinase activities, as well as the mitochondrial respiratory chain enzymes activities succinate dehydrogenase, complex II and cytochrome c oxidase in rat striatum. The influence of soy diet rich in isoflavones on the effects elicited by ovariectomy on enzyme activities was also evaluated. Female adult Wistar rats were assigned to one of the following groups: sham (submitted to surgery without removal of the ovaries) and ovariectomized. Seven days after surgery animals were fed for 30 days on a special diet with soy protein or a standard diet with casein (control). Rats were sacrificed after treatment and the striatum was dissected. Results showed that rats subjected to ovariectomy presented a significant increase in Na⁺,K⁺-ATPase, succinate dehydrogenase and complex II activities. Treatment with isoflavones-rich soy diet was able to reverse the increase of Na⁺,K⁺-ATPase activity, but was not effective in reversing the changes caused by ovariectomy on succinate dehydrogenase and complex II activities. Since ovariectomy mimics postmenopausal changes, our findings suggest that dysfunction of brain energy metabolism may be related to neurological symptoms observed in some postmenopausal women.     

Kinetic characteristics of K-activated para-nitrophenylphosphatase in isolated adult dog heart myocytes

K⁺-activated para-nitrophenylphosphatase (K⁺-pNPPase) was characterized kinetically in isolated adult dog heart myocytes. The results show that: (i) the K_m and V_max for K⁺ activation were increased by increasing concentrations of Mg²⁺ and para-nitrophenylphosphate (pNPP); the highest V_max/K_m value was obtained at 5 m Mg²⁺, 5 m pNPP and 10 m K⁺; (ii) the optimal molar ratio of Mg²⁺ to pNPP was 1 when the concentration of K⁺ was 10 m and that of pNPP was higher than 1.25 m ; (iii) when the molar ratio of Mg²⁺ to pNPP was kept at 1 and the K⁺ concentration was 10 m , increases in Mg²⁺ and pNPP concentrations increased the enzyme activity sigmoidally with a Hill coefficient of 1.7 and a S_0.5 value of 2.3 m ; (iv) Na⁺ at low concentrations (< 10 m ) activated, while Na⁺ at high concentrations (> 10 m ) inhibited K⁺-pNPPase activity when K⁺ concentrations were greater than 1 m ; (v) Ca²⁺ at low concentrations (0.1 to 0.2 m ) inhibited K⁺-pNPPase activity by competing with K⁺, while Ca²⁺ at high concentrations (0.5 to 1.0 m ) inhibited K⁺-pNPPase activity by mixed (both competitive and non-competitive) competition with K⁺; (vi) ouabain inhibited K⁺-pNPPase activity with a Hill coefficient of 0.59 and a S_0.5 value of 4.8 × 10⁻⁷ ; the inhibitory effect of ouabain (< 10⁻⁶ ) was decreased in the presence of Ca²⁺ (0.1–0.5 m ). These results demonstrate that isolated adult heart myocytes possess K⁺-pNPPase activity which can be used as a sensitive and specific probe for studies of the myocardial sodium pump ATPase enzyme system.     

The release of calcium from heart mitochondria by sodium

The release of Ca²⁺ from heart mitochondria could be induced by addition of 20 to 50 mm Na⁺ to the incubation medium. Of the other cations tested, K⁺, Rb⁺, Cs⁺, and Mg²⁺ were without effect, whereas some release was induced by Li⁺. In the presence of ruthenium red, which prevented the re-binding of the released Ca²⁺, 2 to 5 mm Na⁺ were sufficient to produce a measurable release of Ca²⁺. The amount of Ca²⁺ freed from the mitochondria was proportional to the amount of Na⁺ added, and to the concentration of Ca²⁺ present in the mitochondria. The release appeared to be a biphasic process, and in the first 15 s up to 3 nmol Ca²⁺ per mg of mitochondrial protein could be dissociated from the mitochondria.The phenomenon was more evident at pH 6.5 than under slightly alkaline conditions. The significance of this release in the process of heart contraction is discussed.     

Altered Arachidonic Acid Metabolism Contributes to the Failure of Dopamine to Inhibit Na+, K+-ATPase in Kidney of Spontaneously Hypertensive Rats

Dopamine decreases tubular sodium reabsorption in part by inhibition of Na⁺, K⁺-ATPase activity in renal proximal tubules. The signaling mechanism involved in dopamine-mediated inhibition of Na⁺, K⁺-ATPase is known to be defective in spontaneously hypertensive animals. The present study was designed to evaluate the role of phospholipase A2 (PLA2) and its metabolic pathway in dopamine-induced inhibition of Na⁺, K⁺-ATPase in renal proximal tubules from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Renal proximal tubular suspensions were prepared and Na⁺, K⁺-ATPase activity was measured as ouabain-sensitive adenosine triphosphate hydrolysis. Dopamine inhibited Na⁺,K⁺-ATPase activity in a concentration (1 nM - 10 μM)-dependent manner in WKY rats while it failed to inhibit the enzyme activity in SHR. Dopamine (10μM)-induced inhibition of Na⁺,K⁺-ATPase activity in WKY rats was significantly blocked by mepacrine (10 μM), a PLA2 inhibitor, suggesting the involvement of PLA2 in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. Arachidonic acid (a product released by PLA2 action) inhibited Na⁺,K⁺-ATPase in a concentration-dependent (1-100 μM) manner in WKY rats while the inhibition in SHR was significantly attenuated (IC₅₀: 7.5 and 80 μM in WKY rats and SHR, respectively). Furthermore, lower concentrations of arachidonic acid stimulated (30% at 1 μM) Na⁺,K⁺-ATPase activity in SHR. This suggests a defect in the metabolism of arachidonic acid in SHR. Proadifen (10 μM), an inhibitor of cytochrome P-450 monoxygenase (an arachidonic acid metabolizing enzyme) significantly blocked the inhibition produced by arachidonic acid in WKY rats and abolished the difference in arachidonic acid inhibition of Na⁺,K⁺-ATPase between WKY rats and SHR. These data suggest that PLA2 is involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase and altered arachidonic acid metabolism may contribute to reduced dopaminergic inhibition of Na⁺,K⁺-ATPase activity in spontaneously hypertensive rats.     

Ganoderma lucidum (Fr.) P. Karst enhances activities of heart mitochondrial enzymes and respiratory chain complexes in the aged rat

Aging is associated with increased oxidative damage at multiple cellular levels, decline in cellular energy production and enhanced free radical status. The effect of the medicinal mushroom, Ganoderma lucidum on the activities of tricarboxylic acid (Krebs) cycle enzymes and mitochondrial complexes I–IV of the electron transport chain in aged rats were investigated. The activity of Krebs cycle enzymes, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV were determined in heart of aged male Wistar rats orally administrated with 70% ethanolic extract (50 and 250 mg/kg) of G. lucidum. DL-α-lipoic acid (100 mg/kg) was taken as the positive control. Administration of the G. lucidum, once daily for 15 days, was significantly (P < 0.05) effective to enhance the Krebs cycle dehydrogenases, and mitochondrial electron transport chain complex IV activities in aged rats. The profound activity of the extract can be correlated to the significant antioxidant property of G. lucidum. The results of the study revealed that G. lucidum is effective to ameliorate the age associated decline of cellular energy status.     

Early appearance of age-associated deterioration in mitochondrial function of diaphragm and heart in rats treated with doxorubicin

Age-associated deterioration of mitochondrial energy transduction seems to be a major contributory factor to age-related decline in organ function. Free radicals are likely to be involved in the age-related decline in mitochondrial function. This study was designed to elucidate whether or not doxorubicin, a radical generating drug that was administered to 7-week-old rats, affects age-associated mitochondrial functional changes in diaphragm, heart, and liver. Mitochondria from each tissue were prepared from rats aged 7, 13, 20, 28, 35, and 55 weeks, and the activities of four complexes in the mitochondrial energy transduction system were measured enzymatically. In diaphragm mitochondria of the control group, the complex I activity in 28-week-old rats declined to 82% of the activity in rats aged 7 weeks, and the complex IV activity in 55-week-old rats declined to 70% of the activity in rats aged 7 weeks. On the contrary, a significant decrease in the activity of complex I in rats aged 20 weeks (84%) and that of complex IV in rats aged 35 weeks (86%) were observed in the doxorubicin-treated group. In heart mitochondria, age-related changes in activities of complexes I and IV did not appear in rats aged up to 55 weeks, whereas significant decreases in the activities of complexes I (78%) and IV (90%) were observed in rats aged 35 weeks in the doxorubicin group. Age-related changes in liver mitochondria were not found in rats aged up to 55 weeks, and no deleterious effects of doxorubicin were observed in liver mitochondrial function. From these results, the early appearance of aging effects on mitochondrial function was observed in rats treated with doxorubicin particularly in postmitotic cells.     

An effect of the cardiotoxic protein volvatoxin a on the function and structure of heart muscle cells

Volvatoxin-A, the heat labile cardiotoxin present in the mushroom Volvariella volvacea, causes a competitive, dose and time-dependent inhibition of the Ca²⁺-accumulating activity of a sarcoplasmic-reticulum rich microsomal fraction isolated from guinea pig ventricular muscle. The inhibition is accompanied by an activation of the Ca²⁺-dependent ATPase enzyme. Concentrations of toxin which inhibit the Ca²⁺-transporting activity of the microsomes render them leaky to Ca²⁺, but do not effect the rate of incorporation of P³². Ten μg/ml toxin failed to alter the activity of the Na⁺ + K⁺-activated, ouabain sensitive ATPase enzyme. It damaged the fine morphology of the mitochondria, inhibited the ability of isolated mitochondria to accumulate Ca²⁺, and had little effect on the ability of isolated plasma membranes to bind Ca²⁺. These findings may explain why volvatoxin A increases the diastolic resting tension in heart muscle.