Sequential ultrastructural and biochemical changes induced in vivo by the hepatotoxic microcystins in liver of the phytoplanktivorous silver carp Hypophthalmichthys molitrix

Up to now, in vivo studies on the toxic effects of microcystins (MCs) on the ultrastructures of fish liver have been very limited. The phytoplanktivorous silver carp was injected i.p. with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 microg MC-LReq. kg(-1) body weight, showing a time-dependent ultrastructural change in liver as well as significant increases in enzyme activity of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). We observed for the first time the occurrence of a large amount of activated secondary lysosomes, which might be an adaptive mechanism to eliminate or lessen cell damage caused by MCs through lysosome activation. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS2, respectively. MCs concentration in the liver reached the maximum (114.20 microg g(-1) dry weight) after 3 h post-injection, and then rapidly dropped to 7.57 microg g(-1) dry weight at 48 h, indicating a depuration of 99% accumulated MC-LReq. On the other hand, a decrease trend in glutathione (GSH) concentration was observed in the liver of silver carp while the activity of glutathione S-transferase (GST) increased significantly after injection. The high tolerance of silver carp to MCs might be due to the high basic GSH level in their liver, and/or an increased GSH synthesis.     

The effect of taurine on the activity of transport ATPases and of energy metabolism enzymes in different tissues of rats with acute hypoxic hypoxia]

The membrane activity of Na+, K(+)-ATPase, Mg2+, Ca(2+)-ATPase, mitochondrial NAD-isocitrate dehydrogenase, mitochondrial and cytosolic L-glycerol-3-phosphate dehydrogenase was determined in the liver and brain of Wistar rats under acute hypoxic hypoxia against the background of preventive taurine administration. It was shown that preliminary taurine treatment prevented a decrease of hypoxia in activity of Na+. K(+)-ATPase and mitochondrial calcium-dependent enzymes, mostly in the liver. Changes in the intracellular calcium content and biomembrane structure have been discussed as the mechanisms of the taurine effect on the enzymes' activity.     

11,12.环氧二十碳三烯酸预处理与后处理对缺血/再灌注大鼠心肌的保护作用

采用Langendorff离体灌流装置,通过停灌40 min/复灌30 min复制大鼠心肌缺血/再灌注(ischemia/reperfusion,IR)损伤模型,观察11,12-环氧二十碳三烯酸(11,12-epoxyeicosatrienoic acid,11,12-EET)预处理和后处理对心肌线粒体功能以及心功能的影响,探讨11,12-EET顸处理和后处理对IR大鼠心肌的作用及其机制.将30只Sprague-Dawley大鼠随机分为对照组、IR组、EET预处理组(Pre-EET)、EET后处理组(Post-EET),每组6只.除对照组外,其它各组全心缺血40 min,再灌注30 min.监测左心室内压差(ALVP)和左心室内压升降的最大变化率(±dp/dtmax)等心功能指标,测定灌流液中乳酸脱氢酶(1actate dehydrogenase,LDH)的活性.灌流结束后,测定心肌线粒体琥珀酸脱氢酶(succinate dehydrogenase,SDH)、Ca"ATPase、Na - K -ATPase活性以及心肌超氧化物歧化酶(superoxide dismutase,SOD)活性、丙二醛(malondialdehyde,MDA)含量.结果显示:(1)与IR组相比,Pre-EET组及Post.EET组Na -K -ATPase和SDH活性均增强,Ca2 -ATPase活性均减弱,有显著性差异(P<0.05);而Pre-EET与Post-EET组间没有显著性差异.(2)与IR组相比,Pre-EET组及Post-EET组心功能明显改善,LDH漏出显著减少,心肌SOD活性明显增强,MDA含量明显降低,有显著性差异(P<0.05);而Pre-EET与Post-EET组间没有显著性差异.结果表明,11,12-EET预处理及后处理均可通过上调心肌线粒体Na -K -ATPase、SDH活性以及下调Ca2 -ATPase活性改善线粒体功能和心肌能量代谢,拮抗心肌IR损伤;11,12-EET预处理及后处理还可通过提高心肌SOD活性、降低MDA含量改善IR心肌的氧化应激.     

On the properties of calcium-induced permeability transition in neonatal heart mitochondria

Permeability transition was examined in heart mitochondria isolated from neonate rats. We found that these mitochondria were more susceptible to Ca(2+)-induced membrane leakiness than mitochondria from adult rats. In K(+) containing medium, at 25?°C, mitochondria were unable to accumulate Ca(2+). Conversely, in Na(+) containing medium, mitochondria accumulated effectively Ca(2+). At 15?°C mitochondria accumulated Ca(2+) regardless of the presence of K(+). Kinetics of Ca(2+) accumulation showed a similar Vmax as that of adult mitochondria. Lipid milieu of inner membrane contained more unsaturated fatty acids than adult mitochondria. Aconitase inhibition and high thiobarbituric acid-reactive substances (TBARS) indicate that oxidative stress caused mitochondrial damage. In addition, proteomics analysis showed that there is a considerable diminution of succinate dehydrogenase C and subunit 4 of cytochrome oxidase in neonate mitochondria. Our proposal is that dysfunction of the respiratory chain makes neonate mitochondria more susceptible to damage by oxidative stress.     

Toxicity of PCB 1232 on mitochondria of fish Arius caelatus (Valenciennes)

Mitochondrial proteins and phospholipids were estimated and SDH, Na(+)-K(+)-ATPase and Mg(2+)-ATPase activities were analysed in the gill, liver and heart tissues of PCB 1232 (sublethal doses) treated fish A. caelatus. Protein and phospholipids were found to be decreased significantly and SDH, Na(+)-K(+)-ATPase, Mg(2+)-ATPase and other enzyme systems displayed an inverse relationship with PCB dosage. Statistical analysis was carried out to indicate the relationship between sublethal doses of varying concentration and the activities of the enzyme systems involved in energy metabolism. The studies indicated impairment in mitochondrial functions.     

Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart

The opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD(+) glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca(2+) to isolated rat heart mitochondria resulted in a profound decrease in their NAD(+) content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD(+) depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD(+) available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD(+) content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD(+), which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 microm CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD(+) contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD(+), which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart.     

ATPase activity of rat liver mitochondria in acute tetrachloromethane poisoning

The ATPase activity (proton ATPase) of rat liver mitochondria was studied 2, 24, 28, 96 and 168 h after acute tetrachloromethane poisoning. It is established that the tetrachloromethane poisoning. It is established that the tetrachloromethane poisoning is accompanied by a considerable activation of mitochondrial H+-ATPase and a decrease of the DNP and Ca+, Na+ and K+ activating influence on it. Maximum changes in the H+-ATPase activity is observed 24 h after poisoning. Changes in the H+-ATPase properties are accompanied by a fall in the alpha-ketoglutarate dehydrogenase and succinate dehydrogenase activities and by disturbance of the liver mitochondria contractile properties. The electrochemical membrane potential of the mitochondria under the effect of tetrachloromethane is supposed to be reduced due to a primary damage of the phospholipid matrix of the coupling membrane and an increase in its proton conductivity.     

Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria

There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondrial K(ATP) channel opening is approximately 30 nmol. min(-1). mg(-1), a very low rate that will depolarize by only 1-2 mV. However, this increase in K(+) influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial K(ATP) channels has little direct effect on respiration, membrane potential, or Ca(2+) uptake but has important effects on matrix and intermembrane space volumes.     

Effects of microcystins on human polymorphonuclear leukocytes

Microcystins (MCs) are cyclic heptapeptides produced by cyanobacteria present in water contaminated reservoirs. Reported toxic effects for microcystins are liver injury and tumour promotion. In this study, we evaluated the effects of two MCs, MC-LR and [Asp(3)]-MC-LR, on human neutrophil (PMN). We observed that even at concentrations lower than that recommended by World Health Organization for chronic exposure (0.1 nM), MCs affect human PMN. Both MCs have chemotactic activity, induce the production of reactive oxygen species, and increase phagocytosis of Candida albicans. MC-LR also increased C. albicans killing. The effect of MCs on PMN provides support for a damage process mediated by PMN and oxidative stress, and may explain liver injury and tumour promotion associated to long-term MCs exposures.     

The effect of lysophosphatidylcholine on the activity of various mitochondrial enzymes.

The influence of lysophosphatidylcholine (LPC) on H(+)-ATPase, cytochrome oxidase (COX), glycerolphosphate dehydrogenase (GPDH) and malate dehydrogenase (MDH) was followed. The activities of H(+)-ATPase and COX increased with increasing LPC concentration up to 0.5 mg/mg protein when maxima were achieved. This activatory effect is LPC-specific, because Lubrol-treated or frozen-thawed mitochondria showed lower activities of these enzymes. H(+)-ATPase was not influenced by higher concentration of LPC, while COX activity decreased with increasing amount of LPC. The activity of GPDH decreased at very low concentration of LPC and was not further modified at higher LPC concentration. In an attempt to find the concentration of LPC necessary for a complete permeabilization of inner mitochondrial membrane we followed the influence of lysolipid on the release of MDH activity from the mitochondrial matrix. The full activity of this enzyme was obtained with a concentration 0.75 mg LPC/mg protein indicating that mitochondria were completely broken. Our data indicate that LPC significantly affects activity of enzymes connected with mitochondrial membrane and can be useful for evaluation of the importance of phospholipid microenvironment for the enzyme function.     

Stimulatory effect of regucalcin on ATP-dependent Ca(2+) uptake activity in rat liver mitochondria

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver mitochondria was investigated. The presence of regucalcin (0.1, 0.25, and 0.5 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the mitochondria. Ruthenium red (10(-5) M) or lanthanum chloride (10(-4) M), an inhibitor of mitochondrial Ca(2+) uptake, completely inhibited regucalcin (0.25 microM)-increased mitochondrial Ca(2+)-ATPase activity and (45)Ca(2+) uptake. The effect of regucalcin (0.25 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of digitonin (10(-2)%), a solubilizing reagent of membranous lipids, or vanadate (10(-5) M), an inhibitor of phosphorylation of ATPase. The activatory effect of regucalcin (0.25 microM) on Ca(2+)-ATPase activity was not further enhanced in the presence of dithiothreitol (2.5 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme, or calmodulin (0.60 microM), a modulator protein of Ca(2+) action that could increase mitochondrial Ca(2+)-ATPase activity. The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver mitochondria, and that the protein may act on an active site (SH group)-related to phosphorylation of mitochondrial Ca(2+)-ATPase.     

Subfractionation of rat liver plasma membrane. Uneven distribution of plasma membrane-bound enzymes on the liver cell surface.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A

Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Calcium and magnesium ions as effectors of adipose-tissue pyruvate dehydrogenase phosphate phosphatase

The metal-ion requirement of extracted and partially purified pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat-pads was investigated with pig heart pyruvate dehydrogenase [(32)P]phosphate as substrate. The enzyme required Mg(2+) (K(m) 0.5mm) and was activated additionally by Ca(2+) (K(m) 1mum) or Sr(2+) and inhibited by Ni(2+). Isolated fat-cell mitochondria, like liver mitochondria, possess a respiration- or ATP-linked Ca(2+)-uptake system which is inhibited by Ruthenium Red, by uncouplers when linked to respiration, and by oligomycin when linked to ATP. Depletion of fat-cell mitochondria of 75% of their total magnesium content and of 94% of their total calcium content by incubation with the bivalent-metal ionophore A23187 leads to complete loss of pyruvate dehydrogenase phosphate phosphatase activity. Restoration of full activity required addition of both MgCl(2) and CaCl(2). SrCl(2) could replace CaCl(2) (but not MgCl(2)) and NiCl(2) was inhibitory. The metal-ion requirement of the phosphatase within mitochondria was thus equivalent to that of the extracted enzyme. Insulin activation of pyruvate dehydrogenase in rat epididymal fat-pads was not accompanied by any measurable increase in the activity of the phosphatase in extracts of the tissue when either endogenous substrate or (32)P-labelled pig heart substrate was used for assay. The activation of pyruvate dehydrogenase in fat-pads by insulin was inhibited by Ruthenium Red (which may inhibit cell and mitochondrial uptake of Ca(2+)) and by MnCl(2) and NiCl(2) (which may inhibit cell uptake of Ca(2+)). It is concluded that Mg(2+) and Ca(2+) are cofactors for pyruvate dehydrogenase phosphate phosphatase and that an increased mitochondrial uptake of Ca(2+) might contribute to the activation of pyruvate dehydrogenase by insulin.     

Acrolein is a mitochondrial toxin: effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Acrolein is an air pollutant from cigarette smoking and other pollutions and also a by-product of lipid peroxidation. Studies have demonstrated that acrolein causes cytotoxicity and genotoxicity, including liver damage and death of hepatocytes. However, the toxic effects and the underlying mechanisms of acrolein on mitochondria, especially, on liver mitochondria, have not been well studied. In the present study, we investigated the toxic effects and mechanisms of acrolein on mitochondria isolated from rat liver by examining mitochondrial respiration, dehydrogenases, complex I, II, III, IV and V, permeability transition, and protein oxidation. Acrolein incubation (10-1000 microM, or 0.02-2 micromol/mg protein) with mitochondria caused dose-dependent inhibition of NADH- and succinate-linked mitochondrial respiration chain, change of mitochondrial permeability transition, increase in protein carbonyls, and selective enzyme inhibition of mitochondrial complex I, II, pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, but no effects on mitochondrial complex III, IV, V and malate dehydrogenase. These results suggest that acrolein is a mitochondrial toxin and that mitochondrial dysfunction caused by acrolein may play an important role in acrolein toxicity such as hepatotoxicity and also smoking-related diseases.     

The use of the Ca2(+)-sensitive intramitochondrial dehydrogenases and entrapped fura-2 to study Sr2+ and Ba2+ transport across the inner membrane of mammalian mitochondria

In extracts of rat heart mitochondria, Sr2+ mimicked the activatory effects of Ca2+ on the Ca2(+)-sensitive intramitochondrial enzymes, pyruvate dehydrogenase phosphate phosphatase, isocitrate dehydrogenase (NAD+), and 2-oxoglutarate dehydrogenase, but at about tenfold higher concentrations (effective range approximately 1-100 muM) in each case. Ba2+ had no effect on extracted phosphatase, but did mimic the effect of Ca2+ on the other two enzymes with effective concentration ranges similar to those of Sr2+; as with Ca2+ and Sr2+, effective Ba2+ ranges were slightly (2-3-fold) raised by increases in ATP/ADP. In intact uncoupled rat heart mitochondria, the effects of Sr2+ and Ba2+ on the pyruvate and 2-oxoglutarate dehydrogenases were essentially similar to their effects in extracts. In fully coupled rat heart or liver mitochondria, the effective concentration ranges of extramitochondrial Sr2+, leading to activation of the matrix enzymes, were always approximately tenfold higher than those for Ca2+ under all conditions. Ba2+ did not affect pyruvate dehydrogenase in coupled mitochondria, but was shown to activate 2-oxoglutarate dehydrogenase in heart or liver mitochondria, and also isocitrate dehydrogenase (NAD+) in the latter; effective concentration ranges for extramitochondrial Ba2+ were approximately 100-fold greater than those for Ca2+, and like those for Ca2+ and Sr2+, were affected markedly by Mg2+ and spermine (which inhibit and promote mitochondrial Ca2+ uptake, respectively) but, in contrast to Ca2+ and Sr2+, they were hardly affected at all by Na+ (which promotes mitochondrial Ca2+ egress). Ba2+ effects were also blocked by ruthenium red (an inhibitor of mitochondrial Ca2+ uptake), but not so effectively as its blockage of the effects of Sr2+ and Ca2+. Ba2+ and Sr2+ both mimicked the inhibitory effects of extramitochondrial Ca2+ on the Na+/Ca2+ exchanger, but only Sr2+ could mimic Ca2+ in exchanging for internal Ca2+ by this mechanism. Both Sr2+ and Ba2+ changed the fluorescent properties of fura-2 or indo-1 in a similar manner to Ca2+, but with higher kd values. In fura-2-loaded rat heart mitochondria, increases in matrix Sr2+ and Ba2+ and the effects of the transport effectors could be readily demonstrated.     

Zn2+ inhibits alpha-ketoglutarate-stimulated mitochondrial respiration and the isolated alpha-ketoglutarate dehydrogenase complex

Intracellular free Zn(2+) is elevated in a variety of pathological conditions, including ischemia-reperfusion injury and Alzheimer's disease. Impairment of mitochondrial respiration is also associated with these pathological conditions. To test whether elevated Zn(2+) and impaired respiration might be linked, respiration of isolated rat liver mitochondria was measured after addition of Zn(2+). Zn(2+) inhibition (K(i)(app) = approximately 1 micrometer) was observed for respiration stimulated by alpha-ketoglutarate at concentrations well within the range of intracellular Zn(2+) reported for cultured hepatocytes. The bc(1) complex is inhibited by Zn(2+) (Link, T. A., and von Jagow, G. (1995) J. Biol. Chem. 270, 25001-25006). However, respiration stimulated by succinate (K(i)(app) = approximately 6 micrometer) was less sensitive to Zn(2+), indicating the existence of a mitochondrial target for Zn(2+) upstream from bc(1) complex. Purified pig heart alpha-ketoglutarate dehydrogenase complex was strongly inhibited by Zn(2+) (K(i)(app) = 0.37 +/- 0.05 micrometer). Glutamate dehydrogenase was more resistant (K(i)(app) = 6 micrometer), malate dehydrogenase was unaffected, and succinate dehydrogenase was stimulated by Zn(2+). Zn(2+) inhibition of alpha-ketoglutarate dehydrogenase complex required enzyme cycling and was reversed by EDTA. Reversibility was inversely related to the duration of exposure and the concentration of Zn(2+). Physiological free Zn(2+) may modulate hepatic mitochondrial respiration by reversible inhibition of the alpha-ketoglutarate dehydrogenase complex. In contrast, extreme or chronic elevation of intracellular Zn(2+) could contribute to persistent reductions in mitochondrial respiration that have been observed in Zn(2+)-rich diseased tissues.     

Calcium-dependent spontaneously reversible remodeling of brain mitochondria

An exposure of cultured hippocampal neurons expressing mitochondrially targeted enhanced yellow fluorescent protein to excitotoxic glutamate resulted in reversible mitochondrial remodeling that in many instances could be interpreted as swelling. Remodeling was not evident if glutamate receptors were blocked with MK801, if Ca(2+) was omitted or substituted for Sr(2+) in the bath solution, if neurons were treated with carbonylcyanide p-trifluoromethoxyphenylhydrazone to depolarize mitochondria, or if neurons were pretreated with cyclosporin A or N-methyl-4-isoleucine-cyclosporin (NIM811) to inhibit the mitochondrial permeability transition. In the experiments with isolated brain synaptic or nonsynaptic mitochondria, Ca(2+) triggered transient, spontaneously reversible cyclosporin A-sensitive swelling closely resembling remodeling of organelles in cultured neurons. The swelling was accompanied by the release of cytochrome c, Smac/DIABLO, Omi/HtrA2, and AIF but not endonuclease G. Depolarization with carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the Ca(2+) uniporter with Ru360 prevented rapid onset of the swelling. Sr(2+) depolarized mitochondria but failed to induce swelling. Neither inhibitors of the large conductance Ca(2+)-activated K(+) channel (charybdotoxin, iberiotoxin, quinine, and Ba(2+)) nor inhibitors of the mitochondrial ATP-sensitive K(+) channel (5-hydroxydecanoate and glibenclamide) suppressed swelling. Quinine, dicyclohexylcarbodiimide, and Mg(2+), inhibitors of the mitochondrial K(+)/H(+) exchanger, as well as external alkalization inhibited a recovery phase of the reversible swelling. In contrast to brain mitochondria, liver and heart mitochondria challenged with Ca(2+) experienced sustained swelling without spontaneous recovery. The proposed model suggests an involvement of the Ca(2+)-dependent transient K(+) influx into the matrix causing mitochondrial swelling followed by activation of the K(+)/H(+) exchanger leading to spontaneous mitochondrial contraction both in situ and in vitro.