Peculiarities of functioning of liver mitochondria of the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> and the common frog <Emphasis Type="Italic">Rana temporaria</Emphasis> at periods of suppression and activation of energy metabolism

The work dealt with study of mitochondria in reversible metabolic suppression of heap-tocytes of the river lamprey Lampetra fluviatilis in the course of prespawning starvation and of liver mitochondria of the common frog Rana temporaria during hibernation and activity. In winter the metabolic depression of lamprey hepatocytes, unlike that of frog hepatocytes, has been found to be due to deactivation of complex I of the electron transport mitochondrial chain, a low rate of NAD-dependent substrate oxidation, a low content of adenine nucleotide content, and a high degree of mitochondrial membrane permeability to H⁺ and other monovalent ions (KCl^–, K⁺). The mitochondrial membrane permeability decreases in the presence of ethyleneglycoldiamineethyltetraacetic acid (EGTA), cyclosporine A (CsA), adenosine-5′-diphosphate (ADP), and Mg²⁺. These facts indicate the presence in these mitochondria of the Ca²⁺-dependent unspecific pore in the low-conductance state. Histological studies showed the lamprey and the frog to have principal differences in use of energy substrates at the period of metabolic depression. Lampreys utilize predominantly lipids, whereas frogs—glycogen. The clearly pronounced activation of lipid consumption is observed at the spring period before spawning and death of lamprey. Possible causes of metabolic depression are discussed as well as similarity and difference in behavior of mitochondria of cyclostomes and amphibians throughout depression and activity.     

Bioenergetic parameters of lamprey and frog liver mitochondria during metabolic depression and activity

The objective of this study is to elucidate the role of mitochondria in reversible metabolic depression of hepatocytes of the Baltic lamprey (Lampetra fluviatilis) taking place in the last year of its life cycle and to compare their main bioenergetic parameters with those of the frog (Rana temporaria) and the white outbred mouse (Mus musculus). Using isolated mitochondria as a model, we have revealed significant seasonal variations in the main bioenergetic parameters of the lamprey liver. These changes indicate that the metabolic depression is mediated by prolonged reversible alterations of mitochondrial functions, which manifest in low activity of the mitochondrial respiratory chain, low oxidative phosphorylation, low content of mitochondrial adenine nucleotides, high level of reduced mitochondrial pyridine nucleotides and leaky mitochondrial membranes observed in winter. The enhanced ion membrane permeability of winter lamprey liver mitochondria is found to be sensitive to EGTA and to cyclosporine A in combination with ADP and Mg²⁺ and is likely mediated opening the mitochondrial permeability transition pore in its low conductance state. The sharp activation of oxidation and phosphorylation in the lamprey liver mitochondria followed by spawning and death of the animal is observed in spring. The possible causes of the phenomenon and the differences obtained between lamprey, frog and mouse are under discussion.     

Effect of cadmium ions on the respiration, cation transport and morphology of the liver mitochondria in the rat and the lamprey

The influence of Cd2+ on the function and structure of liver mitochondria of rats and lamprey (Lampetra fluviatilis L.) has been studied in vitro. It is shown that Cd2+ can penetrate into the mitochondrial matrix due to Ca2+-transport mechanism. Being stored in the mitochondria, Cd2+ inhibits respiration and an energy dependent transport of penetrating cations (Cs+-valinomycin), and disturbs passive permeability of the inner mitochondrial membrane for monovalent cations and H+. The effect of Cd2+ on the lamprey liver mitochondria is more pronounced than in the case of rats.     

Reversible metabolic depression in lamprey hepatocytes during prespawning migration: dynamics of mitochondrial membrane potential

The lamprey (Lampetra fluviatilis L.) is an extant representative of the ancient vertebrate group of Agnathans. During the prespawning migration (the river period of life from autumn until spring) lamprey hepatocytes exhibit widely different energy states: a high-energy state in autumn and spring, corresponding to a normal physiological standard, and a low-energy state in winter, which is provoked by prolonged starvation and profound metabolic arrest. In spring the restoration of energy status (return to an active state) is associated with hormonally induced lipolysis of the lipid droplets stored in the cells. Lamprey hepatocytes demonstrate an aerobic metabolism based on oxidation of free fatty acids. The dynamics of mitochondrial membrane potential (MMP) were measured throughout the prespawning migration. Pharmacological inhibition of the electron transport chain decreased the MMP and caused extensive depletion of cellular ATP without loss of cell viability. The potential molecular mechanisms responsible for winter metabolic depression in lamprey hepatocytes are discussed.     

Participation of ADP/ATP- and Aspartate/Glutamate Antiporters in the Uncoupling Action of Fatty Acids in Liver Mitochondria of the Frog Rana temporaria

Data are presented on molecular mechanisms of uncoupling of oxidative phosphorylation by fatty acids (laurate) in liver mitochondria of one of the poikilothermal animals, the frog Rana temporaria. It has been shown that the uncoupling action of laurate in frog liver mitochondria, like in those of mammals, occurs with participation of protein carriers of anions of the inner mitochondrial membrane, ADP/ATP- and aspartate/glutamate antiporters. At the same time, in frog liver mitochondria the uncoupling activity of laurate is lower than in liver mitochondria of mammals (white mice). Seasonal differences in the laurate uncoupling activity in frog liver mitochondria are revealed: it is much lower in April, than in January, the season of metabolic depression. This difference is due to that in January the degree of participation of the aspartate/glutamate antiporter in the uncoupling is considerably decreased.     

Tissue-specific depression of mitochondrial proton leak and substrate oxidation in hibernating arctic ground squirrels

A significant proportion of standard metabolic rate is devoted to driving mitochondrial proton leak, and this futile cycle may be a site of metabolic control during hibernation. To determine if the proton leak pathway is decreased during metabolic depression related to hibernation, mitochondria were isolated from liver and skeletal muscle of nonhibernating (active) and hibernating arctic ground squirrels (Spermophilus parryii). At an assay temperature of 37 degrees C, state 3 and state 4 respiration rates and state 4 membrane potential were significantly depressed in liver mitochondria isolated from hibernators. In contrast, state 3 and state 4 respiration rates and membrane potentials were unchanged during hibernation in skeletal muscle mitochondria. The decrease in oxygen consumption of liver mitochondria was achieved by reduced activity of the set of reactions generating the proton gradient but not by a lowered proton permeability. These results suggest that mitochondrial proton conductance is unchanged during hibernation and that the reduced metabolism in hibernators is a partial consequence of tissue-specific depression of substrate oxidation.     

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria

Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 microM CAT and 75 microM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 microM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 microM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.     

Mitochondrial and lysosomal pathways of lamprey (Lampetra fluviatilis L.) hepatocyte death

Mechanisms of mitochondrial and lysosomal pathways of natural death of lamprey hepatocytes are described at the spring period of the prespawn migration. The mitochondrial pathway (release of cytochrome c from mitochondria into cytosol and activation of caspases) is realized by the classic scheme of apoptosis. Comparatively recently, the lysosomal pathway of cell death associated with cathepsin B activation has been revealed in cells in pathologies, specifically in obstruction of gallbladder and bile ducts. A peculiarity of lamprey hepatocytes consists in that in the adult animal liver there takes place biliary atresia (the absence of gallbladder and bile ducts. Thereby, the lamprey hepatocytes are an excellent object for study of this new pathway of cell death. We have revealed development of the mitochondrial and the lysosomal pathways of cell death of lamprey hepatocytes.     

Reversible metabolic depression in hepatocytes of lamprey (Lampetra fluviatilis) during pre-spawning: regulation by substrate availability

The regulation of oxidative metabolism in hepatocytes of lampreys (Lampetra fluviatilis) during the freshwater pre-spawning period of their life cycle was studied. The energy metabolism in these cells is characterized by a simplified scheme, where glycolytic ATP production is insignificant and fatty acids are the major respiratory substrates. Seasonal changes in aerobic cell metabolism include a considerable reversible depression of metabolic rate in lamprey hepatocytes during the winter months of the pre-spawning period. The depression is characterized by a more than twofold decrease in hepatocyte endogenous respiration rate, a reduction of oxidative phosphorylation and drop in cellular ATP content. The addition of fatty acids to the hepatocyte incubation medium prevents the decrease in the metabolic rate. In spring, before spawning, a marked activation of energy metabolism in lamprey hepatocytes is found. These observations support the conclusion that the regulation of lamprey hepatocyte energy metabolism is realized through the availability of fatty acids for oxidation.     

Metabolic depression during aestivation does not involve remodelling of membrane fatty acids in two Australian frogs

Changes in membrane lipid composition (membrane remodelling) have been associated with metabolic depression in some aestivating snails but has not been studied in aestivating frogs. This study examined the membrane phospholipid composition of two Australian aestivating frog species Cyclorana alboguttata and Cyclorana australis. The results showed no major membrane remodelling of tissue in either frog species, or in mitochondria of C. alboguttata due to aestivation. Mitochondrial membrane remodelling was not investigated in C. australis. Where investigated in C. alboguttata, total protein and phospholipid content, and citrate synthase (CS) and cytochrome c oxidase (CCO) activities in tissues and mitochondria mostly did not change with aestivation in liver. In skeletal muscle, however, CS and CCO activities, mitochondrial and tissue phospholipids, and mitochondrial protein decreased with aestivation. These decreases in muscle indicate that skeletal muscle mitochondrial content may decrease during aestivation. Na⁺K⁺ATPase activity of both frog species showed no effect of aestivation. In C. alboguttata different fat diets had a major effect on both tissue and mitochondrial phospholipid composition indicating an ability to remodel membrane composition that is not utilised in aestivation. Therefore, changes in lipid composition associated with some aestivating snails do not occur during aestivation in these Australian frogs.     

Permeability transition in rat liver mitochondria is modulated by the ATP-Mg/Pi carrier

Mitochondrial permeability transition, due to opening of the permeability transition pore (PTP), is triggered by Ca2+ in conjunction with an inducing agent such as phosphate. However, incubation of rat liver mitochondria in the presence of low micromolar concentrations of Ca2+ and millimolar concentrations of phosphate is known to also cause net efflux of matrix adenine nucleotides via the ATP-Mg/Pi carrier. This raises the possibility that adenine nucleotide depletion through this mechanism contributes to mitochondrial permeability transition. Results of this study show that phosphate-induced opening of the mitochondrial PTP is, at least in part, secondary to depletion of the intramitochondrial adenine nucleotide content via the ATP-Mg/Pi carrier. Delaying net adenine nucleotide efflux from mitochondria also delays the onset of phosphate-induced PTP opening. Moreover, mitochondria that are depleted of matrix adenine nucleotides via the ATP-Mg/Pi carrier show highly increased susceptibility to swelling induced by high Ca2+ concentration, atractyloside, and the prooxidant tert-butylhydroperoxide. Thus the ATPMg/Pi carrier, by regulating the matrix adenine nucleotide content, can modulate the sensitivity of rat liver mitochondria to undergo permeability transition. This has important implications for hepatocytes under cellular conditions in which the intramitochondrial adenine nucleotide pool size is depleted, such as in hypoxia or ischemia, or during reperfusion when the mitochondria are exposed to increased oxidative stress.     

Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca²⁺ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca²⁺-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca²⁺. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.     

Detection and functional role of local gradients of H+-ions on the intracellular mitochondrial membrane with covalently linked pH-probe

The study is devoted to the registration of local H+ gradients on the inner membrane of mitochondria under conditions of H+ pump functioning were recorded. By using a covalently linked pH probe (fluorescein isothiocyanate), a local increase in the activity of hydrogen ions on the outer face of the inner mitochondrial membrane in the presence of the respiration substrate at increased permeability of the membrane for K+ was registered. It was also found that the buffer capacity of medium affects the respiration rate of completely uncoupled mitochondria; a change in respiration rate strictly correlates with changes in local H+ gradients on the mitochondrial membrane. It was concluded that local gradients of H+ activity can control the rate of functioning of H+ pumps. It was shown that, under certain conditions, the system of H+ pumps incorporated into succinate oxidase of mitochondria functions as a nonliner system.     

硫化氢对低温胁迫下甜樱桃柱头和子房线粒体功能的影响

为探索低温胁迫下外源硫化氢(H₂S)对甜樱桃花的柱头和子房线粒体功能的影响,本研究以甜樱桃品种‘早大果’花枝为试材,在-2 ℃低温下喷施0.05 mmol·L^-1硫氢化钠(NaHS,H₂S供体)和15 μmmol·L^-1 次牛磺酸(HT、H₂S清除剂),测定柱头和子房线粒体中活性氧、抗氧化酶和线粒体膜通透性转换孔(MPTP)开放程度、膜流动性、膜电位和细胞色素(Cyt c/a)比值变化。结果表明: 低温胁迫导致线粒体内过氧化氢(H₂O₂)和丙二醛(MDA)含量显著增加,线粒体MPTP明显增大,膜流动性降低,膜电位和线粒体Cyt c/a吸光度比值、膜H⁺-ATPase活性显著下降,线粒体结构受到损伤。低温胁迫下,外施0.05 mmol·L^-1 NaHS可显著降低低温胁迫下柱头和子房线粒体H₂O₂和MDA含量,在较长时间内维持较高的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性,减小线粒体MPTP开放程度,增强线粒体膜流动性,提高线粒体膜电位、Cyt c/a值和膜H⁺-ATPase活性;NaHS清除剂HT则抵消NaHS对上述参数的影响。综上所述,外源H₂S可以提高低温胁迫下甜樱桃柱头和子房线粒体抗氧化酶活性,减少H₂O₂和MDA积累,提高膜H⁺-ATPase活性,稳定线粒体膜结构和功能,进而缓解低温胁迫对花器官的伤害。     

Hepatitis B virus HBx protein localizes to mitochondria in primary rat hepatocytes and modulates mitochondrial membrane potential

Over 350 million people are chronically infected with hepatitis B virus (HBV), and a significant number of chronically infected individuals develop primary liver cancer. HBV encodes seven viral proteins, including the nonstructural X (HBx) protein. The results of studies with immortalized or transformed cells and with HBx-transgenic mice demonstrated that HBx can interact with mitochondria. However, no studies with normal hepatocytes have characterized the precise mitochondrial localization of HBx or the effect of HBx on mitochondrial physiology. We have used cultured primary rat hepatocytes as a model system to characterize the mitochondrial localization of HBx and the effect of HBx expression on mitochondrial physiology. We now show that a fraction of HBx colocalizes with density-gradient-purified mitochondria and associates with the outer mitochondrial membrane. We also demonstrate that HBx regulates mitochondrial membrane potential in hepatocytes and that this function of HBx varies depending on the status of NF-kappaB activity. In primary rat hepatocytes, HBx activation of NF-kappaB prevented mitochondrial membrane depolarization; however, when NF-kappaB activity was inhibited, HBx induced membrane depolarization through modulation of the mitochondrial permeability transition pore. Collectively, these results define potential pathways through which HBx may act in order to modulate mitochondrial physiology, thereby altering many cellular activities and ultimately contributing to the development of HBV-associated liver cancer.     

Confocal microscopy analysis of the activity of mitochondria contained within the 'mitochondrial cloud' during oogenesis in Xenopus laevis.

We have used ratiometric confocal microscopy and three fluorescence techniques to study the distribution and activity of mitochondria in frog oocytes during the early stages of oogenesis. Mitochondria in frog oocytes during oogenesis were characterised by a high ratio in the 'mitochondrial cloud' and perinuclear region and a low ratio in mitochondria freely dispersed within the cytoplasm. We tested whether the high ratio visualised by the three techniques represented mitochondrial membrane potential by perturbing the mitochondrial membrane potential. Carbonyl cyanide p-(trifluoromethyl)phenylhydrazone (FCCP) caused the immediate destruction of the membrane potential, and consequent loss of fluorescence from the membrane-potential-sensitive confocal channel. In contrast, nigericin caused an increase in membrane potential represented by a steady increase in fluorescence ratio. These data demonstrate that mitochondrial activity can be measured during oogenesis in frog oocytes, and suggest that the mitochondrial cloud and perinuclear regions are characterised by highly active mitochondria.     

Calcium- and phosphate-dependent release and loading of glutathione by liver mitochondria

The status of glutathione (GSH) was studied in isolated rat liver mitochondria under conditions which induce a permeability transition. This transition, which is inhibited by cyclosporin A (CyA), requires the presence of Ca2+ and an inducing agent such as near physiological levels (3 mM) of inorganic phosphate (Pi). The transition is characterized by an increased inner membrane permeability to some low molecular weight solutes and by large amplitude swelling under some experimental conditions. Addition of 70 microM Ca2+ and 3 mM Pi to mitochondria resulted in mitochondrial swelling and extensive release of GSH that was recovered in the extramitochondrial medium as GSH. Both swelling and the efflux of mitochondrial GSH were prevented by CyA. Incubation of mitochondria in the presence of Ca2+, Pi, and GSH followed by addition of CyA provided a mechanism to load mitochondria with exogenous GSH that was greater than the rate of uptake by untreated mitochondria. Thus, GSH efflux from mitochondria may occur under toxicological and pathological conditions in which mitochondria are exposed to elevated Ca2+ in the presence of near physiological concentrations of Pi through a nonspecific pore. Cyclical opening and closing of the pore could also provide a mechanism for uptake of GSH by mitochondria.     

Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.     

Mitochondrial damage and its role in causing hepatocyte injury during stimulation of lipid peroxidation by iron nitriloacetate.

Incubation of isolated rat hepatocytes with 0.1 mM iron nitrilotriacetic acid (FeNTA) caused a rapid rise in lipid peroxidation followed by a substantial increase in trypan blue staining and lactate dehydrogenase release, but did not affect the protein and non-protein thiol content of the cells. Hepatocyte death was preceded by the decline of mitochondrial membrane potential, as assayed by rhodamine 123 uptake, and by the depletion of cellular ATP. Chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid or inhibition of Ca2+ cycling within the mitochondria by LaCl3 or cyclosporin A did not prevent the decline of rhodamine 123 uptake. On the other hand, a dramatic increase in the conjugated diene content was observed in mitochondria isolated from FeNTA-treated hepatocytes. Oxidative damage of mitochondria was accompanied by the leakage of matrix enzymes glutamic oxalacetic aminotransferase (GOT) and glutamate dehydrogenase (GLDH). The addition of the antioxidant N,N'-diphenylphenylene diamine (DPPD) completely prevented GOT and GLDH leakage, inhibition of rhodamine 123 uptake, and ATP depletion induced by FeNTA, indicating that Ca(2+)-independent alterations of mitochondrial membrane permeability consequent to lipid peroxidation were responsible for the loss of mitochondrial membrane potential. DPPD addition also protected against hepatocyte death. Similarly hepatocytes prepared from fed rats were found to be more resistant than those obtained from starved rats toward ATP depletion and cell death caused by FeNTA, in spite of undergoing a comparable mitochondrial injury. A similar protection was also observed following fructose supplementation of hepatocytes isolated from starved rats, indicating that the decline of ATP was critical for the development of FeNTA toxicity. From these results it was concluded that FeNTA-induced peroxidation of mitochondrial membranes impaired the electrochemical potential of these organelles and led to ATP depletion which was critical for the development of irreversible cell injury.     

Permeability of inner mitochondrial membrane and oxidative stress

The mechanism of increase in the inner membrane permeability induced by Ca2+ plus Pi, diamide and hydroperoxides has been analyzed. (1) The permeability increase is antagonized by oligomycin and favoured by atractyloside. The promoting effect of atractyloside is strongly reduced if the mitochondria are simultaneously treated with oligomycin. (2) Addition of the free-radical scavenger, butylhydroxytoluene, results in a complete protection of the membrane with respect to the permeability increase. (3) Although membrane damage and depression of the GSH concentration are often associated, there is no direct correlation between extent of membrane damage and concentration of reduced glutathione. Abolition of the permeability increase by butylhydroxytoluene or by oligomycin is not accompanied by maintenance of a high GSH concentration in the presence of diamide or hydroperoxides. The membrane damage induced by Ca2+ plus Pi is not accompanied by a depression of the GSH concentration. (4) It is proposed that a variety of processes causing an increased permeability of the inner mitochondrial membrane merge into some ultimate common steps involving the action of oxygen radicals.