UCP2 and ANT differently modulate proton-leak in brain mitochondria of long-term hyperglycemic and recurrent hypoglycemic rats

A growing body of evidence suggests that mitochondrial proton-leak functions as a regulator of reactive oxygen species production and its modulation may limit oxidative injury to tissues. The main purpose of this work was to characterize the proton-leak of brain cortical mitochondria from long-term hyperglycemic and insulin-induced recurrent hypoglycemic rats through the modulation of the uncoupling protein 2 (UCP2) and adenine nucleotide translocator (ANT). Streptozotocin-induced diabetic rats were treated subcutaneously with twice-daily insulin injections during 2 weeks to induce the hypoglycemic episodes. No differences in the basal proton-leak, UCP2 and ANT protein levels were observed between the experimental groups. Mitochondria from recurrent hypoglycemic rats presented a decrease in proton-leak in the presence of GDP, a specific UCP2 inhibitor, while an increase in proton-leak was observed in the presence of linoleic acid, a proton-leak activator, this effect being reverted by the simultaneous addition of GDP. Mitochondria from long-term hyperglycemic rats showed an enhanced susceptibility to ANT modulation as demonstrated by the complete inhibition of basal and linoleic acid-induced proton-leak caused by the ANT specific inhibitor carboxyatractyloside. Our results show that recurrent-hypoglycemia renders mitochondria more susceptible to UCPs modulation while the proton-leak of long-term hyperglycemic rats is mainly modulated by ANT, which suggest that brain cortical mitochondria have distinct adaptation mechanisms in face of different metabolic insults.     

A comparative study of the inhibitory effects of purine nucleotides and carboxyatractylate on the uncoupling protein-3 and adenine nucleotide translocase

Uncoupling proteins (UCPs) mediate fatty acid-induced proton cycling in mitochondria, which is stimulated by superoxide and inhibited by GDP. Fatty acid anions can also be transported by adenine nucleotide translocase (ANT), thus resulting in the uncoupling of oxidative phosphorylation. In the present work, an attempt was made to distinguish between the protonophoric activity of UCP3 and that of ANT using inhibition analysis. This study was carried out using mitochondria from skeletal muscles of hibernating Yakut ground squirrel, which have a significant level of UCP3 mRNA. We found that millimolar concentrations of GDP, which is considered to be a specific inhibitor of UCPs, slightly recoupled the mitochondrial respiration and restored the membrane potential. Addition of the specific ANT inhibitor CAT (carboxyatractylate), in micromolar concentration, prior to GDP prevented its recoupling effect. Moreover, GDP and ADP exhibited a competitive kinetic behavior with respect to ANT. In brown adipose tissue, CAT did not prevent the UCP1-iduced increase in chloride permeability and the inhibitory effect of GDP, thus confirming the inability of CAT to affect UCP1. These results allow us to conclude that the recoupling effect of purine nucleotides on skeletal muscle mitochondria of hibernating ground squirrels can be explained by interaction of the nucleotides with ANT, whereas UCP3 is not involved in the process.     

A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling

Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production. We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT). Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP). The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice. The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT. Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production.     

GDP and carboxyatractylate inhibit 4-hydroxynonenal-activated proton conductance to differing degrees in mitochondria from skeletal muscle and heart

The lipid peroxidation product 4-hydroxynonenal (HNE) increases the proton conductance of the inner mitochondrial membrane through effects on uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT); however, the relative contribution of the two carriers to these effects is unclear. To clarify this we isolated mitochondria from skeletal muscle and heart of wild-type and Ucp3 knockout (Ucp3KO) mice. To increase UCP3 expression, some mice were i.p. injected with LPS (12 mg/kg body weight). In spite of the increased UCP3 expression levels, basal proton conductance did not change. HNE increased the proton conductance of skeletal muscle and heart mitochondria. In skeletal muscle, this increase was lower in Ucp3KO mice and higher in LPS-treated wild-type mice, and was partially abolished by GDP (UCPs inhibitor) and completely abolished by carboxyatractylate (ANT inhibitor) or addition of both inhibitors. GDP had no effect on HNE-induced conductance in heart mitochondria, but carboxyatractylate or administration of both inhibitors had a partial effect. GDP-mediated inhibition of HNE-activated proton conductance in skeletal muscle mitochondria was not observed in Ucp3KO mice, indicating that GDP is specific for UCP3, at least in muscle. Carboxyatractylate was able to inhibit UCP3, probably through an indirect mechanism. Our results are consistent with the conclusion that, in skeletal muscle, HNE-induced increase in proton conductance is mediated by UCP3 (30%) and ANT, whereas in the heart the increase is mediated by ANT and other carriers, possibly including UCP3.     

Quantitative evaluation of the effects of mitochondrial permeability transition pore modifiers on accumulation of calcium phosphate: comparison of rat liver and brain mitochondria

Mitochondria play a critical role in some forms of apoptosis, and the Ca(2+)-dependent permeability transition (PT) is a key initiator of this process. We quantitatively examined major control mechanisms of PT in rat brain (RBM) and liver (RLM) mitochondria. Compared with RLM, RBM were less sensitive to cyclosporin A (CsA), but the combined action of CsA+ADP was much more pronounced in RBM. Carboxyatractyloside abrogated the effects of all mPTP inhibitors in RBM but not in RLM, where the effects of CsA were not reduced. Estimated H(+)/Ca(2+) ratios were 0.81+/-0.01 for RLM and 0.84-0.93 for RBM, suggesting that Ca(2+) and Pi were sequestered in the matrix as CaHPO(4) and Ca(3)(PO(4))(2) salts, and that RBM sequester more CaPi as the least soluble salt. We conclude that: (1) RBM and RLM differ in their baseline behavior of the PT and in their responses to PT modifiers, and (2) PT modifiers can be functionally divided into those which directly affect the mitochondrial PT pore and are not energy-dependent (CsA, free Ca(2+), ADP(ex), and Mg(2+)), and those which affect the energy-dependent calcium phosphate sequestration process (ADP(mt), CATR, local anesthetics). We also conclude that ANT affects PT by changing mitochondrial capacity for energization.     

Involvement of the mitochondrial calcium uniporter in cardioprotection by ischemic preconditioning

The objective of the present study was to determine whether the mitochondrial calcium uniporter plays a role in the cardioprotection induced by ischemic preconditioning (IPC). Isolated rat hearts were subjected to 30 min of regional ischemia by ligation of the left anterior descending artery followed by 120 min of reperfusion. IPC was achieved by two 5-min periods of global ischemia separated by 5 min of reperfusion. IPC reduced the infarct size and lactate dehydrogenase release in coronary effluent, which was associated with improved recovery of left ventricular contractility. Treatment with ruthenium red (RR, 5 μM), an inhibitor of the uniporter, or with Ru360 (10 μM), a highly specific uniporter inhibitor, provided cardioprotective effects like those of IPC. The cardioprotection induced by IPC was abolished by spermine (20 μM), an activator of the uniporter. Cyclosporin A (CsA, 0.2 μM), an inhibitor of the mitochondrial permeability transition pore, reversed the effects caused by spermine. In mitochondria isolated from untreated hearts, both Ru360 (10 μM) and RR (1 μM) decreased pore opening, while spermine (20 μM) increased pore opening which was blocked by CsA (0.2 μM). In mitochondria from preconditioned hearts, the opening of the pore was inhibited, but this inhibition did not occur in the mitochondria from hearts treated with IPC plus spermine. These results indicate that the mitochondrial calcium uniporter is involved in the cardioprotection conferred by ischemic preconditioning.     

Redox regulation of the mitochondrial K(ATP) channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

4-hydroxy-2-nonenal and uncoupling proteins: an approach for regulation of mitochondrial ROS production

One factor that has the potential to regulate reactive oxygen species (ROS) generation is the mild uncoupling of oxidative phosphorylation, i.e. proton (H(+)) leak across the mitochondrial inner membrane. Proton leak has been shown to attenuate ROS generation, whereas ROS and their derivatives (such as superoxide and hydroxynonenal) have been shown to induce H(+) leak through uncoupling proteins (UCPs). This suggests the existence of a feedback loop between ROS and H(+) leak mediated through UCPs. Although the physiological functions of the new UCPs, such as UCP2 and UCP3, are still not established, extensive data support the idea that these mitochondrial carrier proteins are involved in the control of ROS generation. The molecular basis of both ROS generation and hydroxynonenal-induced uncoupling through UCPs is reviewed and the consequences of their interaction for protection against excessive ROS production at the expense of energy production is discussed.     

Adenine nucleotide translocator promotes oxidative phosphorylation and mild uncoupling in mitochondria after dexamethasone treatment

The composition of the mitochondrial inner membrane and uncoupling protein [such as adenine nucleotide translocator (ANT)] contents are the main factors involved in the energy-wasting proton leak. This leak is increased by glucocorticoid treatment under nonphosphorylating conditions. The aim of this study was to investigate mechanisms involved in glucocorticoid-induced proton leak and to evaluate the consequences in more physiological conditions (between states 4 and 3). Isolated liver mitochondria, obtained from dexamethasone-treated rats (1.5 mg.kg(-1).day(-1)), were studied by polarography, Western blotting, and high-performance thin-layer chromatography. We confirmed that dexamethasone treatment in rats induces a proton leak in state 4 that is associated with an increased ANT content, although without any change in membrane surface or lipid composition. Between states 4 and 3, dexamethasone stimulates ATP synthesis by increasing both the mitochondrial ANT and F1-F0 ATP synthase content. In conclusion, dexamethasone increases mitochondrial capacity to generate ATP by modifying ANT and ATP synthase. The side effect is an increased leak in nonphosphorylating conditions.     

The permeability transition pore complex: another view

Mitochondria play a critical role in initiating both apoptotic and necrotic cell death. A major player in this process is the mitochondrial permeability transition pore (MPTP), a non-specific pore, permeant to any molecule of < 1.5 kDa, that opens in the inner mitochondrial membrane under conditions of elevated matrix [Ca(2+)], especially when this is accompanied by oxidative stress and depleted adenine nucleotides. Opening of the MPTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis. In addition mitochondria become depolarised causing inhibition of oxidative phosphorylation and stimulation of ATP hydrolysis. Pore opening is inhibited by cyclosporin A analogues with the same affinity as they inhibit the peptidyl-prolyl cis-trans isomerase activity of mitochondrial cyclophilin (CyP-D). These data and the observation that different ligands of the adenine nucleotide translocase (ANT) can either stimulate or inhibit pore opening led to the proposal that the MPTP is formed by a Ca-triggered conformational change of the ANT that is facilitated by the binding of CyP-D. Our model is able to explain the mode of action of a wide range of known modulators of the MPTP that exert their effects by changing the binding affinity of the ANT for CyP-D, Ca(2+) or adenine nucleotides. The extensive evidence for this model from our own and other laboratories is presented, including reconstitution studies that demonstrate the minimum configuration of the MPTP to require neither the voltage activated anion channel (VDAC or porin) nor any other outer membrane protein. However, other proteins including Bcl-2, BAX and virus-derived proteins may interact with the ANT to regulate the MPTP. Recent data suggest that oxidative cross-linking of two matrix facing cysteine residues on the ANT (Cys(56) and Cys(159)) plays a key role in regulating the MPTP. Adenine nucleotide binding to the ANT is inhibited by Cys(159) modification whilst oxidation of Cys(56) increases CyP-D binding to the ANT, probably at Pro(61).     

Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins.     

Mitochondrial uncoupling,ROS generation and cardioprotection

Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase —a process known as proton leak— generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis.     

Energization-dependent endogenous activation of proton conductance in skeletal muscle mitochondria

Leak of protons into the mitochondrial matrix during substrate oxidation partially uncouples electron transport from phosphorylation of ADP, but the functions and source of basal and inducible proton leak in vivo remain controversial. In the present study we describe an endogenous activation of proton conductance in mitochondria isolated from rat and mouse skeletal muscle following addition of respiratory substrate. This endogenous activation increased with time, required a high membrane potential and was diminished by high concentrations of serum albumin. Inhibition of this endogenous activation by GDP [classically considered specific for UCPs (uncoupling proteins)], carboxyatractylate and bongkrekate (considered specific for the adenine nucleotide translocase) was examined in skeletal muscle mitochondria from wild-type and Ucp3-knockout mice. Proton conductance through endogenously activated UCP3 was calculated as the difference in leak between mitochondria from wild-type and Ucp3-knockout mice, and was found to be inhibited by carboxyatractylate and bongkrekate, but not GDP. Proton conductance in mitochondria from Ucp3-knockout mice was strongly inhibited by carboxyatractylate, bongkrekate and partially by GDP. We conclude the following: (i) at high protonmotive force, an endogenously generated activator stimulates proton conductance catalysed partly by UCP3 and partly by the adenine nucleotide translocase; (ii) GDP is not a specific inhibitor of UCP3, but also inhibits proton translocation by the adenine nucleotide translocase; and (iii) the inhibition of UCP3 by carboxyatractylate and bongkrekate is likely to be indirect, acting through the adenine nucleotide translocase.     

棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白活性及质子漏的影响

本文旨在通过观察棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白(uncoupling proteins,UCPs)活性的影响及脑线粒体质子漏与膜电位的改变,探讨UCPs在介导游离脂肪酸对低氧时线粒体氧化磷酸化功能改变中的作用.将SpragueDawley大鼠随机分为对照组、急性低氧组和慢性低氧组.低氧大鼠于低压舱内模拟海拔5 000 m高原23 h/d作低氧暴露,分别连续低氧3 d和30 d.用差速密度梯度离心法提取脑线粒体,[3H-GTP法测定UCPs含量与活性,TPMP 电极与Clark氧电极结合法测量线粒体质子漏,罗丹明123荧光法测定线粒体膜电位.结果显示,低氧使脑线粒体内UCPs含量与活性升高、质子漏增加、线粒体膜电位降低;同时,低氧暴露降低脑线粒体对棕榈酸的反应性,UCPs活性的改变率低于对照组,且线粒体UCPs含量、质子漏、膜电位变化率亦出现相同趋势.线粒体质子漏与反映UCPs活性的Kd值呈线性负相关(P<0.01 r=-0.906),与反映UCPs含量的Bmax呈线性正相关(P<0.01,r=0.856),与膜电位呈线性负相关(P<0.01,r=-0.880).以上结果提示,低氧导致的脑线粒体质子漏增加及膜电位降低与线粒体内UCPs活性升高有关,同时低氧暴露能降低脑线粒体对棕榈酸的反应性,提示在高原低氧环境下,游离脂肪酸升高在维持线粒体能量代谢中起着自身保护和调节机制.     

Acute knockdown of uncoupling protein-2 increases uncoupling via the adenine nucleotide transporter and decreases oxidative stress in diabetic kidneys

Increased O(2) metabolism resulting in chronic hypoxia is common in models of endstage renal disease. Mitochondrial uncoupling increases O(2) consumption but the ensuing reduction in mitochondrial membrane potential may limit excessive oxidative stress. The present study addressed the hypothesis that mitochondrial uncoupling regulates mitochondria function and oxidative stress in the diabetic kidney. Isolated mitochondria from kidney cortex of control and streptozotocin-induced diabetic rats were studied before and after siRNA knockdown of uncoupling protein-2 (UCP-2). Diabetes resulted in increased UCP-2 protein expression and UCP-2-mediated uncoupling, but normal mitochondria membrane potential. This uncoupling was inhibited by GDP, which also increased the membrane potential. siRNA reduced UCP-2 protein expression in controls and diabetics (-30-50%), but paradoxically further increased uncoupling and markedly reduced the membrane potential. This siRNA mediated uncoupling was unaffected by GDP but was blocked by ADP and carboxyatractylate (CAT). Mitochondria membrane potential after UCP-2 siRNA was unaffected by GDP but increased by CAT. This demonstrated that further increased mitochondria uncoupling after siRNA towards UCP-2 is mediated through the adenine nucleotide transporter (ANT). The increased oxidative stress in the diabetic kidney, manifested as increased thiobarbituric acids, was reduced by knocking down UCP-2 whereas whole-body oxidative stress, manifested as increased circulating malondialdehyde, remained unaffected. All parameters investigated were unaffected by scrambled siRNA. In conclusion, mitochondrial uncoupling via UCP-2 regulates mitochondria membrane potential in diabetes. However, blockade of the diabetes-induced upregulation of UCP- 2 results in excessive uncoupling and reduced oxidative stress in the kidney via activation of ANT.     

Activation of Xenopus eggs by the kinase inhibitor 6-DMAP suggests a differential regulation of cyclin B and p39(mos) proteolysis

The molecular mode of action of arsenic, a therapeutic agent employed in the treatment of acute promyelocytic leukemia, has been elusive. Here we provide evidence that arsenic compounds may act on mitochondria to induce apoptosis. Arsenite induces apoptosis accompanied by a loss of the mitochondrial transmembrane potential (Delta Psim). Inhibition of caspases prevents the arsenite-induced nuclear DNA loss, but has no effect on the Delta Psim dissipation and cytolysis induced by arsenite. In contrast, Bcl-2 expression induced by gene transfer prevents all hallmarks of arsenite-induced cell death, including the Delta Psim collapse. PK11195, a ligand of the mitochondrial benzodiazepine receptor, neutralizes this Bcl-2 effect. Mitochondria are required in a cell-free system to mediate arsenite-induced nuclear apoptosis. Arsenite causes the release of an apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space. This effect is prevented by the permeability transition (PT) pore inhibitor cyclosporin A, as well as by Bcl-2, which is known to function as an endogenous PT pore antagonist. Arsenite also opens the purified, reconstituted PT pore in vitro in a cyclosporin A- and Bcl-2-inhibitible fashion. Altogether these data suggest that arsenite can induce apoptosis via a direct effect on the mitochondrial PT pore.     

The antiestrogen 4-hydroxytamoxifen protects against isotretinoin-induced permeability transition and bioenergetic dysfunction of liver mitochondria: comparison with tamoxifen

The combination of isotretinoin (13-cis-retinoic acid) with antiestrogens seems to be a promising strategy for cancer chemotherapy. The aim of the study was to evaluate the effects of isotretinoin alone or in combination with 4-hydroxytamoxifen (OHTAM) and with its prodrug tamoxifen (TAM), on the functions of rat liver mitochondria, i.e., mitochondrial permeability transition (MPT), bioenergetic functions and adenine nucleotide translocase (ANT). Isotretinoin (5 nmol/mg protein) induced the Ca²⁺-dependent MPT pore opening in mitochondria energized with succinate, which was prevented by OHTAM, cyclosporine A, TAM and ANT ligands. When mitochondria were energized with glutamate/malate and in the absence of added Ca²⁺ isotretinoin decreased the state 3 respiration, the ATP levels, the active ANT content and increased the lag phase of the phosphorylation cycle, demonstrating that isotretinoin decreased the mitochondrial phosphorylation efficiency. These changes of isotretinoin in bioenergetic parameters were not significant in the presence of succinate. The effects of isotretinoin at 5 nmol/mg protein on the Ca²⁺-dependent MPT and phosphorylative efficacy may be related with interactions with the ANT. Above 10 nmol/mg protein isotretinoin strongly diminished the active ANT content, decreased the Δψ, inhibited the complex I and induced proton leak through the Fo fraction of complex V. The combination of OHTAM with isotretinoin only induced significant changes in the energy production systems at concentrations ≥5 nmol isotretinoin/mg protein. Therefore, our results suggest that isotretinoin-associated liver toxicity is possibly related with mitochondrial dysfunctions and that the combination with OHTAM may contribute to decrease its toxicity.     

Effects of pharmacological preconditioning by emodin/oleanolic acid treatment and/or ischemic preconditioning on mitochondrial antioxidant components as well as the susceptibility to ischemia-reperfusion injury in rat hearts

Using an ex vivo rat heart model of ischemia-reperfusion (I-R) injury, we examined the effect of pharmacological preconditioning by chronic treatment with emodin (EMD)/oleanolic acid (OA) at low dose (25 μ mol/kg/day × 15) and/or ischemic preconditioning (IPC) (4 cycles of 5 min ischemia followed by 5 min of reperfusion) on myocardial I-R injury. The results indicated that EMD/OA pretreatment, IPC, or their combinations (EMD+IPC and OA+IPC) protected against myocardial I-R injury, as assessed by lactate dehydrogenase leakage and contractile force recovery. The cardioprotection was associated with a differential enhancement in mitochondrial antioxidant components. The combined EMD/OA and IPC pretreatment produced cardioprotective action in a semi-additive manner. This suggested that EMD/OA pretreatment and IPC protected against myocardial I-R injury via a similar but not identical biochemical mechanism.     

UCP2-dependent proton leak in isolated mammalian mitochondria.

A role for uncoupling protein (UCP) homologues in mediating the proton leak in mammalian mitochondria is controversial. We subjected insulinoma (INS-1) cells to adenoviral expression of UCP2 or UCP1 and assessed the proton leak as the kinetic relationship between oxygen use and the inner mitochondrial membrane potential. Cells were infected with different amounts of rat UCP2, and, in other experiments, with either UCP2 or UCP1. The relative molar expression of these subtypes was quantified through comparison with histidine-tagged UCP1 or UCP2 proteins engineered by expression in Escherichia coli. Adenoviral infection with UCP2, compared with beta-galactosidase, resulted in a dose-dependent shift in kinetics indicating increased H(+) flux at any given membrane potential. UCP1 also enhanced H(+) flux, but, on a relative molar basis, the overexpression of the endogenous protein, UCP2, was more potent than UCP1. These results were not due to nonspecific overexpression of mitochondrial protein since UCP1 activity was inhibited by GDP and because overexpression of another membrane carrier protein, the oxoglutarate malate carrier had no effect. UCP2-mediated H(+) conduction was not GDP sensitive. These data suggest that the UCP homologue, UCP2, mediates the proton leak in mitochondria of a mammalian cell wherein UCP2 is the native subtype.     

低压缺氧对大鼠脑线粒体腺苷酸转运体特性的影响

本文探讨低压缺氧对大鼠脑线粒体内膜腺苷酸转运体（adenine nucleotide translocator,ANT）转运特性的影响。实验将雄性Wistar大鼠随机分为常氧对照组和缺氧组,后者分别连续暴露于模拟5000m高原1、5、15、30d（23h／d）。分别于平原和模拟4000m高原断头处死动物,分离脑线粒体,用抑制剂终止法测定线粒体对。H-ADP的转运效率,抑制剂滴定法测定ANT密度,HPLC测定线粒体内腺苷酸含量。结果显示：缺氧后ANT转运活性均明显低于常氧组,缺氧不同天数线粒体内膜ANT分布密度无显著改变,线粒体内（ATP＋ADP）含量下降与转运活性变化一致。以上观察结果表明,低压缺氧暴露可显著抑制ANT转运活性,降低能量产生和利用的周转率,但不改变ANT密度,提示ANT活性改变是低压缺氧时细胞能量代谢障碍的重要机制。