Agmatine prevents the Ca2+-dependent induction of permeability transition in rat brain mitochondria

The arginine metabolite agmatine is able to protect brain mitochondria against the drop in energy capacity by the Ca(2+)-dependent induction of permeability transition (MPT) in rat brain mitochondria. At normal levels, the amine maintains the respiratory control index and ADP/O ratio and prevents mitochondrial colloid-osmotic swelling and any electrical potential (DeltaPsi) drop. MPT is due to oxidative stress induced by the interaction of Ca(2+) with the mitochondrial membrane, leading to the production of hydrogen peroxide and, subsequently, other reactive oxygen species (ROS) such as hydroxyl radicals. This production of ROS induces oxidation of sulfhydryl groups, in particular those of two critical cysteines, most probably located on adenine nucleotide translocase, and also oxidation of pyridine nucleotides, resulting in transition pore opening. The protective effect of agmatine is attributable to a scavenging effect on the most toxic ROS, i.e., the hydroxyl radical, thus preventing oxidative stress and consequent bioenergetic collapse

Interactions of melatonin with mammalian mitochondria. Reducer of energy capacity and amplifier of permeability transition.

Melatonin, a metabolic product of the amino acid tryptophan, induces a dose-dependent energy drop correlated with a decrease in the oxidative phosphorylation process in isolated rat liver mitochondria. This effect involves a gradual decrease in the respiratory control index and significant alterations in the state 4/state 3 transition of membrane potential (ΔΨ). Melatonin, alone, does not affect the insulating properties of the inner membrane but, in the presence of supraphysiological Ca2+, induces a ΔΨ drop and colloid-osmotic mitochondrial swelling. These events are sensitive to cyclosporin A and the inhibitors of Ca2+ transport, indicative of the induction or amplification of the mitochondrial permeability transition. This phenomenon is triggered by oxidative stress induced by melatonin and Ca2+, with the generation of hydrogen peroxide and the consequent oxidation of sulfydryl groups, glutathione and pyridine nucleotides. In addition, melatonin, again in the presence of Ca2+, can also induce substantial release of cytochrome C and AIF (apoptosis-inducing factor), thus revealing its potential as a pro-apoptotic agent

Effect of peroxides on spermine transport in rat brain and liver mitochondria.

The polyamine spermine is transported into the matrix of various types of mitochondria by a specific uniporter system identified as a protein channel. This mechanism is regulated by the membrane potential; other regulatory effectors are unknown. This study analyzes the transport of spermine in the presence of peroxides in both isolated rat liver and brain mitochondria, in order to evaluate the involvement of the redox state in this mechanism, and to compare its effect in both types of mitochondria. In liver mitochondria peroxides are able to inhibit spermine transport. This effect is indicative of redox regulation by the transporter, probably due to the presence of critical thiol groups along the transport pathway, or in close association with it, with different accessibility for the peroxides and performing different functions. In brain mitochondria, peroxides have several effects, supporting the hypothesis of a different regulation of spermine transport. The fact that peroxovanadate can inhibit tyrosine phosphatases in brain mitochondria suggests that mitochondrial spermine transport is regulated by tyrosine phosphorylation in this organ. In this regard, the evaluation of spermine transport in the presence of Src inhibitors suggests the involvement of Src family kinases in this process. It is possible that phosphorylation sites for Src kinases are present in the channel pathway and have an inhibitory effect on spermine transport under regulation by Src kinases. The results of this study suggest that the activity of the spermine transporter probably depends on the redox and/or tyrosine phosphorylation state of mitochondria, and that its regulation may be different in distinct organs

Aprovechamiento de hojas secas de ajo para la elaboración de pellets combustibles

El término pellet (también pelet) es una denominación genérica, utilizada para referirse a pequeñas porciones de material aglomerado o comprimido de diferentes materiales. Puede referirse a un pellet de madera, cada una de las pequeñas porciones de materia vegetal comprimida utilizadas como combustible al que se le puede agregar otros materiales descartes de industrias. Se trata de un biocombustible densificado.EEA La ConsultaFil: Burba, Jose Luis. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria La Consulta; ArgentinaFil: Grancara, Nancy. Instituto de Enseñanza Superior 9-010 "Rosario Vera Peñaloza". Coordinación Área Producción Agro Forestal ; ArgentinaFil: Castillo, Javier Valerio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria La Consulta; ArgentinaFil: Lanzavechia, Silvina Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria La Consulta; Argentin

Further characterization of agmatine binding to mitochondrial membranes: involvement of imidazoline I2 receptor.

Agmatine, a divalent diamine with two positive charges at physiological pH, is transported into the matrix of liver mitochondria by an energy-dependent mechanism, the driving force of which is the electrical membrane potential. Its binding to mitochondrial membranes is studied by applying a thermodynamic treatment of ligand-receptor interactions on the analyses of Scatchard and Hill. The presence of two mono-coordinated binding sites S(1) and S(2), with a negative influence of S(2) on S(1), has been demonstrated. The calculated binding energy is characteristic for weak interactions. S(1) exhibits a lower binding capacity and higher binding affinity both of about two orders of magnitude than S(2). Experiments with idazoxan, a ligand of the mitochondrial imidazoline receptor I(2), demonstrate that S(1) site is localized on this receptor while S(2) is localized on the transport system. S(1) would act as a sensor of exogenous agmatine concentration, thus modulating the transport of the amine by its binding to S(2)

Interactions of melatonin with mammalian mitochondria. Reducer of energy capacity and amplifier of permeability transition.

Melatonin, a metabolic product of the amino acid tryptophan, induces a dose-dependent energy drop correlated with a decrease in the oxidative phosphorylation process in isolated rat liver mitochondria. This effect involves a gradual decrease in the respiratory control index and significant alterations in the state 4/state 3 transition of membrane potential (ΔΨ). Melatonin, alone, does not affect the insulating properties of the inner membrane but, in the presence of supraphysiological Ca2+, induces a ΔΨ drop and colloid-osmotic mitochondrial swelling. These events are sensitive to cyclosporin A and the inhibitors of Ca2+ transport, indicative of the induction or amplification of the mitochondrial permeability transition. This phenomenon is triggered by oxidative stress induced by melatonin and Ca2+, with the generation of hydrogen peroxide and the consequent oxidation of sulfydryl groups, glutathione and pyridine nucleotides. In addition, melatonin, again in the presence of Ca2+, can also induce substantial release of cytochrome C and AIF (apoptosis-inducing factor), thus revealing its potential as a pro-apoptotic agent

Bidirectional fluxes of spermine across the mitochondrial membrane.

The polyamine spermine is transported into the mitochondrial matrix by an electrophoretic mechanism having as driving force the negative electrical membrane potential (DW). The presence of phosphate increases spermine uptake by reducingDpH and enhancingDW. The transport system is a specific uniporter constituted by a protein channel exhibiting two asymmetric energy barriers with the spermine binding site located in the energy well between the two barriers. Although spermine transport is electrophoretic in origin, its accumulation does not follow the Nernst equation for the presence of an efflux pathway. Spermine efflux may be induced by different agents, such as FCCP, antimycin A and mersalyl, able to completely or partially reduce theDWvalue and, consequently, suppress or weaken the force necessary to maintain spermine in the matrix. However this efflux may also take place in normal conditions when the electrophoretic accumulation of the polycationic polyamine induces a sufficient drop inDWable to trigger the efflux pathway. The release of the polyamine is most probably electroneutral in origin and can take place in exchange with protons or in symport with phosphate anion. The activity of both the uptake and efflux pathways induces a continuous cycling of spermine across the mitochondrial membrane, the rate of which may be prominent in imposing the concentrations of spermine in the inner and outer compartment. Thus, this event has a significant role on mitochondrial permeability transition modulation and consequently on the triggering of intrinsic apoptosis

Different behaviour of liver and brain mitochondria in Permeability Transition: role of biogenic monoamines

Biologically active amines are a class of compounds synthesized by normal metabolic processes in living organisms. They are classified as biogenic amines (serotonin, agmatine, tyramine, histamine, dopamine, phenylethylamine, tryptamine and catecholamines), or polyamines (putrescine, spermidine and spermine). Biogenic amines can act as neurotransmitters and display various other physiological functions throughout the organism. The degradation of these molecules is catalyzed by monoamine oxidases (MAOs) A and B, isoenzymes localized on outer mitochondrial membrane, which induce an oxidative deamination. This reaction leads to the production of hydrogen peroxide and aldehydes, which are then oxidized into acids or converted into alcohols or glycols. In particular hydrogen peroxide can trigger the formation of other reactive oxygen species (ROS) and induce mitochondrial damages and apoptosis. Considering that biogenic amines can undergo these catabolic reactions, the possible effects of them, or of their products, on different types of mitochondria, were studied. The aim of this work was to study the action of monoamines as regulator of mitochondrial functions in isolated rat mitochondria from different organs: liver, brain, heart, and kidney. The first part of the work focused on the action of these amines on mitochondrial permeability transition induction. They induce a collapse of and a strong amplification of swelling in rat isolated mitochondria of liver (RLM), heart (RHM) and kidney (RKM). Furthermore they oxidize thiol groups and pyridine nucleotides. These observations support the hypothesis that monoamines are amplifiers of mitochondrial permeability transition (MPT), inducing an oxidative stress, through the generation of H2O2, which is most probably the agent responsible of MPT occurrence. Instead, in isolated rat brain mitochondria (RBM), the amines do not amplify the swelling and do not alter the partial drop of induced by Ca2+, even if they oxidize thiol groups and pyridine nucleotides. These results led us to hypothesize the existence in RBM of a mechanism of MPT pore opening different from that present in the other mitochondria. In the second part of the study it is reported the serotonin uptake by mitochondria with characterization of the transport system. Experimental evidences suggesting that aldehyde is the possible accumulated species are reported. Finally, in the third part of the work, in order to better define the process that triggers MPT in RBM we have investigated the role of signaling pathways, in particular the possible involvement of P-Tyr-phosphorilated proteins since it has been reported that this type of mitochondria contains Tyr-kinases of the Src-family. We found, on the one hand, that a variety of tyrosine-kinase inhibitors do not affect the process while the “Inhibitor Tyr-phosphatases Cocktail 2”, and the known phosphatase inhibitor sodium-pervanadate reduce the occurrence of MPT in parallel with an increase of the P-Tyr level of some proteins, in particular of proteins of apparent M.W. of 160, 72 and 35 kDa. Experiments are in progress to define, first of all, the identity of the P-Tyr-Protein involved in this process and then the characteristics and physiological significance of this phenomenon. In conclusion the obtained results show an important role of monoamines in mitochondria that depends on the tissues and their specific physiological processes. Furthermore two different mechanisms seem to be involved in MPT. In RLM the opening of permeability transition pore appears to require oxidation of thiol groups and the MPT amplification seems to depend on the oxidative stress induced by the reactive oxygen species produced by monoamine oxidation. In RBM the pore opening seems to involve two different mechanisms: in addition to the oxidative stress also the Tyr-phosphorylation of some proteins whose nature is under investigation.Le amine biologicamente attive sono una classe di composti sintetizzati negli organismi viventi da normali processi metabolici. Esse sono classificate come amine biogene: serotonina, agmatina, tiramina, istamina, dopamina, feniletilamina, triptamina e catecolamine, o poliamine : putrescina, spermidina e spermina. Le amine biogene possono agire come neurotrasmettitori e dimostrano altre funzioni fisiologiche in diversi organi. La degradazione di queste molecole è catalizzata dalle monoamino-ossidasi (MAO) A e B, isoenzimi localizzati sulla membrana esterna mitocondriale, che inducono una deaminazione ossidativa. Questa reazione porta alla produzione di perossido di idrogeno e delle corrispondenti aldeidi, che vengono poi ossidate ad acidi o ridotte ad alcoli o glicoli. In particolare, il perossido di idrogeno può innescare la formazione di altre specie reattive dell'ossigeno (ROS) e indurre danni mitocondriali e apoptosi. Considerando che le amine biogene possono subire queste reazioni cataboliche, i loro possibili effetti, o quelli dei loro prodotti di ossidazione, sono stati studiati sui diversi tipi di mitocondri. Lo scopo di questo studio è stato quello di studiare l'azione delle monoamine come regolatrici delle funzioni mitocondriali nei mitocondri isolati da differenti organi di ratto: fegato, cervello, cuore e reni. La prima parte del lavoro si concentra su l'azione di queste ammine sull'induzione di transizione di permeabilità mitocondriale. Esse producono un crollo del potenziale elettrico di membrana (ΔΨ) e una forte amplificazione dello swelling di mitocondri isolati di fegato (RLM), cuore (RHM) e reni (RKM) di ratto. Inoltre le amine ossidano i gruppi tiolici e i nucleotidi piridinici. Queste osservazioni supportano l'ipotesi che le monoamine siano amplificatrici della transizione di permeabilità mitocondriale (MPT), inducendo uno stress ossidativo, attraverso la generazione di H2O2. Quest’ultimo composto sembra essere, molto probabilmente, l'agente responsabile dell’induzione della MPT. Invece, nei mitocondri di cervello di ratto (RBM), le amine non amplificano lo swelling e non alterano il parziale calo di ΔΨ indotti dal Ca2+, nonostante i gruppi tiolici e i piridin nucleotidi vengano ossidati come nei RLM. Questi risultati ci hanno portato ad ipotizzare l'esistenza nei RBM di un meccanismo di apertura del poro di transizione diverso da quello presente negli altri tipi mitocondriali. Nella seconda parte dello studio è riportato il trasporto della serotonina nei mitocondri con la caratterizzazione del sistema di trasporto. Evidenze sperimentali suggeriscono che sia l’aldeide derivata dalle monoamine la possibile specie accumulata. Infine, nella terza parte del lavoro, al fine di definire meglio il processo che innesca la MPT nei RBM, abbiamo studiato il ruolo delle vie di trasmissione del messaggio, in particolare il possibile coinvolgimento di proteine tirosin fosforilate, anche in base al fatto che è stato riportato che questo tipo di mitocondri contiene Tyr-chinasi della famiglia Src. Abbiamo trovato, da un lato, che una serie di inibitori delle tirosin-chinasi non influenzano la MPT, mentre l’”Inhibitor Tyr-phosphatases Cocktail 2”, e il noto inibitore delle fosfatasi pervanadato riducono l'insorgenza di tale processo in parallelo con un aumento del livello P-Tyr di alcune proteine, in particolare, proteine di un apparente massa molecolare 160, 72 e 35 KDa. Esperimenti sono in corso per definire, prima di tutto, l'identità delle proteine fosforilate in tirosina coinvolte in questo processo e quindi le caratteristiche e il significato fisiologico di questo fenomeno. In conclusione i risultati ottenuti mostrano un ruolo importante delle monoamine nei mitocondri che dipende dai tipi di tessuto e dai loro specifici processi fisiologici. Inoltre sembrano essere coinvolti nel processo della MPT due diversi meccanismi. Nei RLM l'apertura del poro di transizione della permeabilità sembra richiedere l'ossidazione dei gruppi tiolici e l'amplificazione della MPT sembra dipendere dallo stress ossidativo indotto da specie reattive dell'ossigeno prodotte dall'ossidazione delle monoamine. Nei RBM l'apertura del poro sembra invece dipendere da due diversi meccanismi: oltre che dallo stress ossidativo anche dalla fosforilazione tirosinica di alcune proteine sulla cui natura si sta attualmente indagando