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

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)

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

Pathophysiological implications of mitochondrial oxidative stress mediated by mitochondriotropic agents and polyamines: The role of tyrosine phosphorylation

Mitochondria, once merely considered as the "powerhouse" of cells, as they generate more than 90 % of cellular ATP, are now known to play a central role in many metabolic processes, including oxidative stress and apoptosis. More than 40 known human diseases are the result of excessive production of reactive oxygen species (ROS), bioenergetic collapse and dysregulated apoptosis. Mitochondria are the main source of ROS in cells, due to the activity of the respiratory chain. In normal physiological conditions, ROS generation is limited by the anti-oxidant enzymatic systems in mitochondria. However, disregulation of the activity of these enzymes or interaction of respiratory complexes with mitochondriotropic agents may lead to a rise in ROS concentrations, resulting in oxidative stress, mitochondrial permeability transition (MPT) induction and triggering of the apoptotic pathway. ROS concentration is also increased by the activity of amine oxidases located inside and outside mitochondria, with oxidation of biogenic amines and polyamines. However, it should also be recalled that, depending on its concentration, the polyamine spermine can also protect against stress caused by ROS scavenging. In higher organisms, cell signaling pathways are the main regulators in energy production, since they act at the level of mitochondrial oxidative phosphorylation and participate in the induction of the MPT. Thus, respiratory complexes, ATP synthase and transition pore components are the targets of tyrosine kinases and phosphatases. Increased ROS may also regulate the tyrosine phosphorylation of target proteins by activating Src kinases or phosphatases, preventing or inducing a number of pathological states

Mechanism and Pathophysiological Role of Polyamine Transport in Mammalian Mitochondria. Answer to Debated Questions

Mitochondria are known to be the main players in important mitochondrial bioenergetic functions such as ATP synthesis, thermoregulatory energy dissipation, Ca2+ transport, generation of reactive oxygen species and mediation of intrinsic apoptosis. Naturally occurring polyamines, due to their high pka are almost completely protonated at physiological pH and behave as polycations in their interactions with mitochondrial membranes. Thanks to these interactions, polyamines are transported electrophoretically into the mitochondrial matrix, where they exhibit a number of effects of significant importance for the above-mentioned mitochondrial functions, particularly inner membrane permeability transition (MPT). This event is closely correlated with the intrinsic occurrence of apoptosis, so that the effect of polyamine interactions with mitochondria has important implications in the pathophysiological consequences of inducing apoptosis, i.e., protection against cancer and neurodegenerative diseases. This review also provides some answers to the old debated problems regarding the possible interactions of polyamines with mitochondrial DNA, overcoming of the Born charging energy by spermine, the \u394\u3a8 threshold value for polyamine transport, and protection of MPT by spermine in in vivo conditions. In conclusion, the old question: \u201cWhat do polyamines do?\u201d is partially solved

Mitochondrial oxidative stress induced by Ca 2+ and monoamines: Different behaviour of liver and brain mitochondria in undergoing permeability transition

Mitochondrial permeability transition (MPT) is correlated with the opening of a nonspecific pore, the socalled transition pore, that triggers bidirectional traffic of inorganic solutes and metabolites across the mitochondrial membrane. This phenomenon is caused by supraphysiological Ca 2+ concentrations and by other compounds leading to oxidative stress, while cyclosporin A, ADP, bongkrekic acid, antioxidant agents and naturally occurring polyamines strongly inhibit it. The effects of polyamines, including the diamine agmatine, have been widely studied in several types of mitochondria. The effects of monoamines on MPT have to date, been less well-studied, even if they are involved in a variety of neurological and neuroendocrine processes. This study shows that in rat liver mitochondria (RLM), monoamines such as tyramine, serotonin and dopamine amplify the swelling induced by calcium, and increase the oxidation of thiol groups and the production of hydrogen peroxide, effects that are counteracted by the above-mentioned inhibitors. In rat brain mitochondria (RBM), the monoamines do not amplify calcium-induced swelling, even if they demonstrate increases in the extent of oxidation of thiol groups and hydrogen peroxide production. In these mitochondria, the antioxidants are not at all or scarcely effective in suppressing mitochondrial swelling. In conclusion, we hypothesize that different mechanisms induce the MPT in the two different types of mitochondria evaluated. Calcium and monoamines induce oxidative stress in RLM, which in turn appears to induce and amplify MPT. This process is not apparent in RBM, where MPT seems resistant to oxidative stress. © Springer-Verlag 2011

Milestones and recent discoveries on cell death mediated by mitochondria and their interactions with biologically active amines

Mitochondria represent cell “powerhouses,” being involved in energy transduction from the electrochemical gradient to ATP synthesis. The morphology of their cell types may change, according to various metabolic processes or osmotic pressure. A new morphology of the inner membrane and mitochondrial cristae, significantly different from the previous one, has been proposed for the inner membrane and mitochondrial cristae, based on the technique of electron tomography. Mitochondrial Ca2+ transport (the transporter has been isolated) generates reactive oxygen species and induces the mitochondrial permeability transition of both inner and outer mitochondrial membranes, leading to induction of necrosis and apoptosis. In the mitochondria of several cell types (liver, kidney, and heart), mitochondrial oxidative stress is an essential step in the induction of cell death, although not in brain, in which the phenomenon is caused by a different mechanism

Mitochondrial Permeability Transition as Target of Anticancer Drugs

Mitochondria are the cell powerhouses but also contain the mechanisms leading to cell death. Many signals converge on mitochondria to cause the permeabilization of mitochondrial membranes by the mitochondrial permeability transition (MPT) induction and the opening of transition pores (PTPs). These events cause loss of ionic homeostasis, matrix swelling, outer membrane rupture leading to pro-apoptotic factors release, and impairment of bioenergetics functions. The molecular mechanism underlying MPT induction is not completely elucidated however, a growing body of evidence supports the concept that pharmacological induction of PTPs in mitochondria of neoplastic cells is an effective and promising strategy for therapeutic approaches against cancer. The first part of this article presented as a review also evidences the main constituents of PTP and several compounds targeting them for inducing the phenomenon. The second part of the article regards the recent experimental development in the field, in particular, the effects of peniocerol (PEN), a sterol isolated from the root of Mirtillocactus geometrizans, at cellular and mitochondrial level. PEN exhibits a cytotoxic activity on some human tumor cell lines, whose mechanism is attributable to the oxidation of critical thiols located on adenine nucleotide translocase, the protein mainly involved in PTP. This event in the presence of Ca2+ induces the MPT with the release of the pro-apoptotic factors cytochrome c and apoptosis inducing factor. These observations evidence that PEN may trigger both the caspase-dependent and caspase-independent apoptotic pathways. This characteristic renders PEN a very interesting compound that could be developed to obtain more effective antiproliferative agents targeting mitochondria for anticancer therap

Potential anticancer application of polyamine oxidation products formed by amine oxidase: a new therapeutic approach

The polyamines spermine, spermidine and putrescine are ubiquitous cell components. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper-containing amine oxidases. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. In tumors, polyamines and amine oxidases are increased as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H(2)O(2) and aldehydes produced by the reaction. This study demonstrated that multidrug-resistant (MDR) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding wild-type (WT) ones to H(2)O(2) and aldehydes, the products of BSAO-catalyzed oxidation of spermine. Transmission electron microscopy (TEM) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. Increasing the incubation temperature from 37 to 42A degrees C enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumor growth, particularly well if the enzyme has been conjugated to a biocompatible hydrogel polymers. Since both wild-type and MDR cancer cells after pre-treatment with MDL 72527, a lysosomotropic compound, are sensitized to subsequent exposure to BSAO/spermine, it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumor cells. It is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting