Exacerbated responses to oxidative stress by an Na+ load in isolated nerve terminals: the role of ATP depletion and rise of [Ca2+](i)

We have explored the consequences of a [Na 1 ] i load and oxidative stress in isolated nerve terminals. The Na 1 load was achieved by veratridine (5–40 m M ), which allows Na 1 entry via a voltage-operated Na 1 channel, and oxidative stress was induced by hydrogen peroxide (0.1–0.5 m M ). Remarkably, nei- ther the [Na 1 ] i load nor exposure to H 2 O 2 had any major effect on [Ca 2 1 ] i , mitochondrial membrane potential ( D c m), or ATP level. However, the combination of an Na 1 load and oxidative stress caused ATP depletion, a collapse of D c m, and a pro- gressive deregulation of [Ca 2 1 ] i and [Na 1 ] i homeostasis. The decrease in the ATP level was unrelated to an increase in [Ca 2 1 ] i and paralleled the rise in [Na 1 ] i . The loss of D c m was prevented in the absence of Ca 2 1 but unaltered in the presence of cyclosporin A. We conclude that the increased ATP con- sumption by the Na,K–ATPase that results from a modest [Na 1 ] i load places an additional demand on mitochondria met- abolically compromised by an oxidative stress, which are un- able to produce a sufficient amount of ATP to fuel the ATP- driven ion pumps. This results in a deregulation of [Na 1 ] i and [Ca 2 1 ] i , and as a result of the latter, collapse of D c m. The vicious cycle generated in the combined presence of Na 1 load and oxidative stress could be an important factor in the neuro- nal injury produced by ischemia or excitotoxicity, in which the oxidative insult is superimposed on a disturbed Na 1 homeostasis

Bioenergetikai változások glutamát excitotoxicitásban és oxidatív stresszben = Bioenergetical changes in glutamate excitotoxicity and oxidative stress

Munkánk egyfelől az ischemia és számos neurodegenerativ betegség pathomechanizmusában meghatározó szerepet játszó oxidativ stressz akut hatásaival, másfelől az oxidativ stressz keletkezésével kapcsolatban hozott uj eredményeket. Megállapitások: 1) Enyhe oxidativ stressz potencirozza a Na-terhelésre bekövetkező glutamat felszabadulást és csökkenti a gluthation mennyiségét izolált idegvégződésekben. 2) Az oxidativ stressz gátolja a pH regulációt és a mitokondriumok kalcium háztartást reguláló szerepét az agy-vér gátat alkotó kapillárisokból tenyésztett endothel sejtekben. 3) A mitokondriális légzési lánc komponensei közül a ROS keletkezésében a meghatározó a komplex I gátlása, amely már kis mértékű gátlás esetén olyan mennyiségű ROS képzéssel jár, hogy gátolja az endogén akonitázt. 4) A légési lánc mellett fontos általunk leirt új forrása a ROS keletkezésnek az alpha-ketoglutarát enzim, amely normál katalitikus működése során szuperoxidot és hidrogén peroxidot termel, amit a NADH/NAD arány regulál. 5) Agykéregből származó sejttenyészeten a glutamat excitotoxikus hatásában szerepet játszanak a transient receptor potential csatornák és a pathomechanizmus fontos része a sejtek NADH szintjének csökkenése. 6) Megtörtént az eddig ismeretlen funkciójú kalcium-aktiválta nem-szelektiv kation csatornák részletes jellemzése agyi kapilláris endothel sejt tenyészetben. | The findings of this research project show, on one hand, sensitive processes that are impaired by oxidative stress in neurons and rat brain capillary endothelial cells (RBCE), on the other hand, mechanisms involved in the generation of oxidative stress. Major findings: i) Hydrogen peroxide potentiate the release of glutamate induced by a sodium load in isolated nerve terminals. ii) Oxidative stress impairs the intracellular pH regulation and the mitochondrial calcium regulation in RBCE cells. iii) Physiologically the most relevant site of reactive oxygen species (ROS) production in the respiratory chain is at complex I; when this complex is inhibited by 25-30 %, relevant to Parkinson?s disease, ROS is generated in sufficient amount to inhibit endogenous mitochondrial aconitase. iv) In addition to the respiratory chain, alpha-ketoglutarate dehydrogenase could be a significant source of ROS generation in mitochondria under conditions when the NADH/NAD ratio is increased (ischemia, Parkinson?s disease). v) Transient receptor potential (TRP) channels are involved in the delayed calcium deregulation (DCD) associated with glutamate excitotoxicity in cortical cell cultures. DCD is accompanied by a significant drop in the intracellular NADH level. vi) Calcium-activated non-selective cation channels, which are abundant in RBCE cells, have been characterized; the gating characteristics as well as the regulation by nucleotides have been described in details

Az intermedier anyagcsere és a szabadgyökképzés kapcsolata izolált idegvégkészülékekben és mikroglia sejtekben. = Correlation between intermediary metabolism and free radical formation in isolated nerve endings and in microglial cells

Izolált idegvégkészülékeken végzett kísérleteink alapján valószínűsítettük, hogy citrát-kör alfa-ketoglutarát dehidrogenáz enzimének szerepe lehet a reaktív oxigénszármazékok (ROS) képzésében. Ezt a feltételezést izolált enzimen végzett vizsgálatokkal megerősítettük. Megállapítottuk, hogy a ROS képzést a hipoxiás körülmények között észlelhető NADH/NAD arány növekedés erősen stimulálja. Vizsgáltuk a NADH-t termelő citrát-köri szubsztrátokkal, illetve a szukcináttal légző mitokondriumok ROS-képző tulajdonságait, a mitokondrium membránpotenciál és a ROS termelés közötti összefüggést. Rámutattunk, hogy a kvalitatíven is különböző irodalmi eredmények oka a preparátumok eltérő membránpotenciál-ja. Karakterizáltuk a mitokondrium-citoszól kapcsolatban fontos szerepet játszó glicerofoszfát inga működése során az alfa-glicerofoszfát (a-GP) mitokondriális oxidációja során képződő hidrogén peroxid keletkezési mechanizmusát, és megállapítottuk, hogy az a-GP ROS-képzése történhet mind reverz elektron transzporttal, mind pedig magán az alfa-glicerofoszfát dehidrogenáz (a-GPDH) enzimen. Kimutattuk, hogy az a-GPDH-n történő ROS-képzés jelentősen stimulálható a citoplazmatikus [Ca2+] fiziológiás tartományban történő emelésével. Megállapítottuk, hogy az idegvégkészülékekben "in situ" elhelyezkedő mitokondriumok ROS képzése a membránpotenciál csökkentésével nem befolyásolható, mely eredmény nem támasztja alá a membránpotenciál csökkentésére alapozott neuroprotekció elméletét. | Our group demonstrated that Krebs cycle enzyme alpha-ketoglutarate dehydrogenase (a-KGDH) was able to generate H2O2 and thus, could be a source of reactive oxygen species (ROS) in mitochondria. ROS production was stimulated by the high NADH/NAD ratio mimicking hypoxic conditions. Relationship between ROS formation and mitochondrial membrane potential (mMP) was studied in isolated mitochondria respiring on NADH-linked substrates and succinate. The rate of H2O2 formation with NADH-linked substrates was sensitive to changes in mMP only in highly polarized mitochondria, while mMP could determine both the mechanism and the intensity of ROS generation in succinate-supported mitochondria. Alpha-glycerophosphate (a-GP) shuttle has a role in the oxidation of cytosolic NADH in mitochondria. We suggest that oxidation of a-GP leads to ROS generation both by reverse electron transport (RET), and by the direct ROS formation on the a-GP-dehydrogenase (a-GPDH) enzyme. In a-GP-supported mitochondria activation of a-GPDH by Ca2+ leads to an accelerated RET-mediated ROS generation as well as to a stimulated ROS production on the a-GPDH. In nerve terminals we tested whether ROS generation by "in situ" mitochondria, functioning in a normal cytosolic environment and oxidize glucose-derived substrates, is also dependent on changes in mMP. Basal ROS generation by "in situ" mitochondria is not sensitive to changes in mMP, challenging the rational of the "mild uncoupling" theory for neuroprotection

Human 2-Oxoglutarate Dehydrogenase Complex E1 Component Forms a Thiamin-derived Radical by Aerobic Oxidation of the Enamine Intermediate.

Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2alpha-hydroxy)-gamma-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2alpha-hydroxy)-gamma-carboxypropylidene thiamin diphosphate (the "ThDP-enamine"/C2alpha-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an "off-pathway" side reaction comprising less than 1% of the "on-pathway" reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease

Methylene Blue Bridges the Inhibition and Produces Unusual Respiratory Changes in Complex III-Inhibited Mitochondria. Studies on Rats, Mice and Guinea Pigs

Methylene blue (MB) is used in human therapy in various pathological conditions. Its effects in neurodegenerative disease models are promising. MB acts on multiple cellular targets and mechanisms, but many of its potential beneficial effects are ascribed to be mitochondrial. Ac-cording to the “alternative electron transport” hypothesis, MB is capable of donating electrons to cytochrome c bypassing complex I and III. As a consequence of this, the deleterious effects of the inhibitors of complex I and III can be ameliorated by MB. Recently, the beneficial effects of MB ex-erted on complex III-inhibited mitochondria were debated. In the present contribution, several pieces of evidence are provided towards that MB is able to reduce cytochrome c and improve bio-energetic parameters, like respiration and membrane potential, in mitochondria treated with com-plex III inhibitors, either antimycin or myxothiazol. These conclusions were drawn from meas-urements for mitochondrial oxygen consumption, membrane potential, NAD(P)H steady state, MB uptake and MB-cytochrome c oxidoreduction. In the presence of MB and complex III inhibitors, unusual respiratory reactions, like decreased oxygen consumption as a response to ADP addition as well as stimulation of respiration upon administration of inhibitors of ATP synthase or ANT, were observed. Qualitatively identical results were obtained in three rodent species. The actual metabolic status of mitochondria is well reflected in the distribution of MB amongst various com-partments of this organelle

Versatility of microglial bioenergetic machinery under starving conditions

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear. In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary - and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies. All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation. It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism

Elektron transzfer rendszerek élettani szerepe = The physiological role of electron transfer systems

Fagocitákban leírtuk a NADPH oxidázt szabályozó két különböző GTPáz aktiváló fehérje szabályozását és a kísérő K+ transzport baktérium ölő hatását. Agyi mitokondriumokban (mito) a légzési lánc I. komplexének szubsztrátjai membránpotenciál (Em) függően reaktív oxigénszármazékot (ROS) képeznek. Az alfa-glicerofoszfát (aGP) ROS-t képez az I. komplexen és az aGP-dehidrogenáz enzimen, utóbbit a Ca2+ aktivája. Idegvégződésekben a mito ROS képzését az Em nem befolyásolja. A mito-k elektromos szincíciumot képeznek, de a Ca2+ diffúziója korlátozott. Alacsony O2.- szint a Ca2+ -mobilizáló agonista Ca2+ jel képző hatását glomerulóza sejtben gátolja. A ROS támadáspontja a belső raktárból történő Ca2+ felszabadulás. UV hatására a mito Ca2+ felvétele is csökkent. Angiotenzin II -vel ingerelt H295R sejtben a mito Ca2+ jel képzés sebessége a mito és az endoplazmás retikulum (ER) közelségével korrelál. A p38 MAPK és az újtípusú PKC izoformák egyidejű gátlása a Ca2+ jelnek a citoszolból a mito-ba történő áttevődését gátolja és a fenti korrelációt megszünteti. Az ER lumenében a tiol/diszulfid rendszertől elkülönülő NAD(P)+/NAD(P)H rendszer működik. Redox állapotát a glukóz-6-foszfát transzporter és az intraluminális oxidoreduktázok határozzák meg. A redukált állapot fenntartása szükséges a glukokortikoidok prereceptoriális aktiválásához, s egyes sejtekben antiapoptotikus hatású. Jellemeztük az ER szulfát transzporterét, valamint a transzlokon peptid csatorna anion permeabilitását. | We described in phagocytes the regulation of two GTPase activating proteins, terminating the activity of plasmalemmal NADPH oxidase and the role of K+ movements in bacterial killing. In brain mitochondria complex I dependent substrates show a membrane potential (Em) dependent reactive oxygen species (ROS) formation. ROS production by alpha-glycerophosphate (aGP) occured at complex I and on the aGP-dehydrogenase enzyme. The latter is activited by Ca2+. Mitochondria form an electric syntitium but the diffusion of Ca2+ is limited. In glomerulosa cells, at low [O2.-] angiotensin-induced Ca2+ signalling is attenuated, the site of ROS action is Ca2+ release from the internal stores. The rate of mitochondrial Ca2+ uptake in angiotensin-stimulated cells correlates with the vicinity of the mitochondrion and the endoplasmic reticulum (ER). Simultaneous activation of p38 MAPK and the novel isoforms of PKC attenuates the transfer of cytosolic Ca2+ signal into the mitochondria and abolishes this correlation. In the ER we observed a novel NAD(P)+/NAD(P)H system different from the thiol/disulphide system. Its reduced state is tuned by the glucose-6-phosphate transporter and the luminal oxidoreductases and is required for the prereceptorial activation of glucocorticoids. We have characterized the sulphate transport in the ER, and the contribution of the translocon peptide channel to the membrane permeation of small anions

The Effect of Cyclophilin D Depletion on Liver Regeneration Following Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy

AIM: Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) is a modification of two-stage hepatectomy profitable for patients with inoperable hepatic tumors by standard techniques. Unfortunately, initially poor postoperative outcome was associated with ALPPS, in which mitochondrial dysfunction played an essential role. Inhibition of cyclophilins has been already proposed to be efficient as a mitochondrial therapy in liver diseases. To investigate the effect of Cyclophilin D (CypD) depletion on mitochondrial function, biogenesis and liver regeneration following ALPPS a CypD knockout (KO) mice model was created. METHODS: Male wild type (WT) (n = 30) and CypD KO (n = 30) mice underwent ALPPS procedure. Animals were terminated pre-operatively and 24, 48, 72 or 168 h after the operation. Mitochondrial functional studies and proteomic analysis were performed. Regeneration rate and mitotic activity were assessed. RESULTS: The CypD KO group displayed improved mitochondrial function, as both ATP production (P < 0.001) and oxygen consumption (P < 0.05) were increased compared to the WT group. The level of mitochondrial biogenesis coordinator peroxisome proliferator-activated receptor γ co-activator 1-α (PGC1-α) was also elevated in the CypD KO group (P < 0.001), which resulted in the induction of the mitochondrial oxidative phosphorylation system. Liver growth increased in the CypD KO group compared to the WT group (P < 0.001). CONCLUSIONS: Our study demonstrates the beneficial effect of CypD depletion on the mitochondrial vulnerability following ALPPS. Based on our results we propose that CypD inhibition should be further investigated as a possible mitochondrial therapy following ALPPS

Novel mitochondrial transition pore inhibitor N‐methyl‐4‐isoleucine cyclosporin is a new therapeutic option in acute pancreatitis

Bile acids, ethanol and fatty acids affect pancreatic ductal fluid and bicarbonate secretion via mitochondrial damage, ATP depletion and calcium overload. •Pancreatitis-inducing factors open the membrane transition pore (mPTP) channel via cyclophilin D activation in acinar cells, causing calcium overload and cell death; genetic or pharmacological inhibition of mPTP improves the outcome of acute pancreatitis in animal models. •Here we show that genetic and pharmacological inhibition of mPTP protects mitochondrial homeostasis and cell function evoked by pancreatitis-inducing factors in pancreatic ductal cells. •The results also show that the novel cyclosporin A derivative NIM811 protects mitochondrial function in acinar and ductal cells, and it preserves bicarbonate transport mechanisms in pancreatic ductal cells. •We found that NIM811 is highly effective in different experimental pancreatitis models and has no side-effects. NIM811 is a highly suitable compound to be tested in clinical trials