INVOLVEMENT OF BIOGENIC ACTIVE AMINES IN MITOCHONDRIAL MEMBRANE PERMEABILIZATION AND PRO-APOPTOTIC FACTORS RELEASE

Activation of the intrinsic pathway of apoptosis is related to the permeabilization of mitochondrial membranes. This complex process is far away from being completely understood but, generally, could be divided into two steps that, depending on the experimental conditions, can be linked or independent: the permeabilization of the mitochondrial outer membrane and the opening of the mitochondrial permeability transition pore. Both processes are tightly regulated by different components that attract increasing interest as promising targets for several diseases treatment, in particular cancer. A large number of mitochondrial permeability transition pore-targeting agents, indeed, are under studies because the mitochondrial apoptotic machinery in cancer cells is structurally and functionally different from that of normal cells. Thus, mitochondrial-targeting agents are expected to have a tumor selectivity. In this scenario, a family of compounds known as biologically active amines acquires increasing importance. These amines, among which the most-known spermine, spermidine, putrescine and agmatine, are polycationic molecules at physiological pH, naturally present in almost all living species where they exert an essential role for cell growth and differentiation. It is to note that the polyamine biosynthetic pathway is very active in cancer cells and that high polyamines concentrations are often present in rapidly dividing tumor cells and growing tissues. Moreover the deregulation of polyamine metabolism may induce apoptosis. Thus, their primary role in regulating proliferation and cell death led scientists to investigate their role at a mitochondrial level. The main target of these amines seems to be the mitochondrial permeability transition: spermine, spermidine and putrescine share an inhibitory action whereas agmatine acts (in liver) as an inducer or an inhibitor, depending on the concentrations used. Furthermore in mitochondria two specific mitochondrial transporters have been detected: one for spermine, spermidine, and putrescine and one for agmatine. The aim of this work is to study the action of biologically active amines as regulators of mitochondrial membrane permeabilization and pro-apoptotic factors release in isolated rat liver mitochondria. In particular their interaction with specific membrane structures is analyzed, trying to elucidate their mechanism of action. The first part of the work evidences the agmatine double effect on the permeability transition process. In particular the attention is focused on agmatine inhibiting action. Despite its protective effect on the mitochondrial permeability transition, the amine is able to induce the release of some pro-apoptotic factors. A possible explanation relates to the induction of the mitochondrial outer membrane permeabilization. A further confirmation of this proposal is obtained in the second part, by a comparison between agmatine and its analogue alpha-methyl-agmatine, a more powerful inhibitor of the mitochondrial permeability transition. The use of this compound allows to exclude that the production of hydrogen peroxide exhibited by agmatine is one of the main causes of the mitochondrial outer membrane permeabilization observed. In the third part, the effect of the polyamines (in particular spermine and spermidine) on the permeabilization on mitochondrial membranes and on the release of pro-apoptotic factors is reported. Despite their well-known protective action against the opening of the pore, spermine and spermidine share with agmatine a similar behavior in inducing the release of some pro-apoptotic factors, even through different pathways. Thus, their mechanism of action is investigated, trying to evidence the specific membrane components involved. Finally, in the fourth part, an investigation about the mechanism of efflux of spermine is shown, focusing on the possibility that it could be linked with the efflux of ATP and phosphate, most likely in an electroneutral fashion. This observation is supported by the use of specific inhibitors of the adenine nucleotide translocator and phosphate carrier. In conclusion, the results obtained in this study first of all support the hypothesis that the permeabilization of the inner and outer membranes could be viewed as two distinct processes, even if often linked between them. Furthermore it is possible to state that the mitochondrial outer membrane permeabilization could be a contact point between intrinsic and extrinsic apoptosis, being induced both by intra- and extra-mitochondrial signals. It is already reported that the release of cytochrome c from mitochondria does not always result in apoptosis induction. In particular the redox state of the protein is crucial for the subsequent cascade of events: only oxidated cytochrome c is able to trigger apoptosis. For this reason, it is reasonable to think that the release of cytochrome c induced by biologically active amines could represent a starting point, but not a point of no return. In this context polyamines represent a useful tool to study both the mitochondrial outer membrane permeabilization and the mitochondrial permeability transition, trying to delineate structural and functional features. Moreover, in living cells polyamines are able to prevent or induce apoptosis, and the latter effect is caused by their metabolization by amine oxidases, with the production of cytotoxic metabolites. This dichotomic action depends on polyamines concentration, that is modulated, as supported by our data, also by mitochondrial polyamine cycling. More study is required to detect all structural mechanism involved in their cycling but, at least for spermine, a possible pathway of efflux is detected

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

INVOLVEMENT OF BIOGENIC ACTIVE AMINES IN MITOCHONDRIAL MEMBRANE PERMEABILIZATION AND PRO-APOPTOTIC FACTORS RELEASE

Activation of the intrinsic pathway of apoptosis is related to the permeabilization of mitochondrial membranes. This complex process is far away from being completely understood but, generally, could be divided into two steps that, depending on the experimental conditions, can be linked or independent: the permeabilization of the mitochondrial outer membrane and the opening of the mitochondrial permeability transition pore. Both processes are tightly regulated by different components that attract increasing interest as promising targets for several diseases treatment, in particular cancer. A large number of mitochondrial permeability transition pore-targeting agents, indeed, are under studies because the mitochondrial apoptotic machinery in cancer cells is structurally and functionally different from that of normal cells. Thus, mitochondrial-targeting agents are expected to have a tumor selectivity. In this scenario, a family of compounds known as biologically active amines acquires increasing importance. These amines, among which the most-known spermine, spermidine, putrescine and agmatine, are polycationic molecules at physiological pH, naturally present in almost all living species where they exert an essential role for cell growth and differentiation. It is to note that the polyamine biosynthetic pathway is very active in cancer cells and that high polyamines concentrations are often present in rapidly dividing tumor cells and growing tissues. Moreover the deregulation of polyamine metabolism may induce apoptosis. Thus, their primary role in regulating proliferation and cell death led scientists to investigate their role at a mitochondrial level. The main target of these amines seems to be the mitochondrial permeability transition: spermine, spermidine and putrescine share an inhibitory action whereas agmatine acts (in liver) as an inducer or an inhibitor, depending on the concentrations used. Furthermore in mitochondria two specific mitochondrial transporters have been detected: one for spermine, spermidine, and putrescine and one for agmatine. The aim of this work is to study the action of biologically active amines as regulators of mitochondrial membrane permeabilization and pro-apoptotic factors release in isolated rat liver mitochondria. In particular their interaction with specific membrane structures is analyzed, trying to elucidate their mechanism of action. The first part of the work evidences the agmatine double effect on the permeability transition process. In particular the attention is focused on agmatine inhibiting action. Despite its protective effect on the mitochondrial permeability transition, the amine is able to induce the release of some pro-apoptotic factors. A possible explanation relates to the induction of the mitochondrial outer membrane permeabilization. A further confirmation of this proposal is obtained in the second part, by a comparison between agmatine and its analogue alpha-methyl-agmatine, a more powerful inhibitor of the mitochondrial permeability transition. The use of this compound allows to exclude that the production of hydrogen peroxide exhibited by agmatine is one of the main causes of the mitochondrial outer membrane permeabilization observed. In the third part, the effect of the polyamines (in particular spermine and spermidine) on the permeabilization on mitochondrial membranes and on the release of pro-apoptotic factors is reported. Despite their well-known protective action against the opening of the pore, spermine and spermidine share with agmatine a similar behavior in inducing the release of some pro-apoptotic factors, even through different pathways. Thus, their mechanism of action is investigated, trying to evidence the specific membrane components involved. Finally, in the fourth part, an investigation about the mechanism of efflux of spermine is shown, focusing on the possibility that it could be linked with the efflux of ATP and phosphate, most likely in an electroneutral fashion. This observation is supported by the use of specific inhibitors of the adenine nucleotide translocator and phosphate carrier. In conclusion, the results obtained in this study first of all support the hypothesis that the permeabilization of the inner and outer membranes could be viewed as two distinct processes, even if often linked between them. Furthermore it is possible to state that the mitochondrial outer membrane permeabilization could be a contact point between intrinsic and extrinsic apoptosis, being induced both by intra- and extra-mitochondrial signals. It is already reported that the release of cytochrome c from mitochondria does not always result in apoptosis induction. In particular the redox state of the protein is crucial for the subsequent cascade of events: only oxidated cytochrome c is able to trigger apoptosis. For this reason, it is reasonable to think that the release of cytochrome c induced by biologically active amines could represent a starting point, but not a point of no return. In this context polyamines represent a useful tool to study both the mitochondrial outer membrane permeabilization and the mitochondrial permeability transition, trying to delineate structural and functional features. Moreover, in living cells polyamines are able to prevent or induce apoptosis, and the latter effect is caused by their metabolization by amine oxidases, with the production of cytotoxic metabolites. This dichotomic action depends on polyamines concentration, that is modulated, as supported by our data, also by mitochondrial polyamine cycling. More study is required to detect all structural mechanism involved in their cycling but, at least for spermine, a possible pathway of efflux is detected.L'attivazione dell’apoptosi intrinseca è correlata alla permeabilizzazione delle membrane mitocondriali. Questo complicato processo, sebbene non sia stato ancora completamente compreso, può essere generalmente suddiviso in due fasi: la permeabilizzazione della membrana mitocondriale esterna e l'apertura del poro di transizione di permeabilità mitocondriale. In base alle condizioni sperimentali tali fasi possono essere collegate o indipendenti. Diversi componenti che regolano i suddetti processi sono attualmente in fase di studio, poiché si sono rivelati bersagli promettenti per il trattamento di numerose malattie, tra le quali in particolare il cancro. Infatti, il macchinario apoptotico mitocondriale nelle cellule tumorali è strutturalmente e funzionalmente diverso da quello delle cellule normali e quindi un gran numero di composti che hanno come target la transizione di permeabilità potrebbe per tale motivo esibire una selettività tumorale. In questo contesto, una famiglia di composti noti come amine biologicamente attive acquista un'importanza crescente. Queste amine, tra cui le più note sono spermina, spermidina, putrescina e agmatina, sono molecole policationiche a pH fisiologico, naturalmente presenti in quasi tutte le specie viventi, in cui esercitano un ruolo essenziale per la crescita e la differenziazione cellulare. È da notare che la via biosintetica delle poliamine è molto attiva nelle cellule tumorali e che concentrazioni elevate di poliamine sono spesso presenti in cellule tumorali in divisione ed in tessuti in proliferazione. Inoltre, la de-regolazione del metabolismo delle poliamine può indurre l'apoptosi. Per questi motivi, il loro ruolo primario nella regolazione della proliferazione e della morte cellulare ha portato gli scienziati a studiarne il ruolo a livello mitocondriale. Il target principale delle amine biologicamente attive sembra essere la transizione di permeabilità mitocondriale: spermina, spermidina e putrescina esibiscono un'azione inibitoria mentre l’agmatina (nel fegato) si comporta da induttore o inibitore, a seconda delle concentrazioni usate. Inoltre nei mitocondri è stata dimostrata l’esistenza di due trasportatori specifici: uno per spermina, spermidina e putrescina ed uno per l’agmatina. Lo scopo di questo lavoro è quello di studiare l'azione delle amine biologicamente attive quali regolatrici della permeabilizzazione della membrana mitocondriale esterna e del rilascio dei fattori pro-apoptotici in mitocondri isolati da fegato di ratto. In particolare viene analizzata la loro interazione specifica con le strutture di membrana, allo scopo di chiarire il loro meccanismo di azione. La prima parte del lavoro evidenzia il duplice effetto dell’agmatina sul processo di transizione di permeabilità. In particolare l'attenzione è focalizzata sull’effetto inibitorio dell’amina nei confronti dell’apertura del poro di transizione mitocondriale. Infatti, nonostante il suo effetto protettivo sulla transizione di permeabilità, l'amina è in grado di indurre il rilascio di alcuni fattori pro-apoptotici. Una possibile spiegazione considera l'induzione della permeabilizzazione della membrana mitocondriale esterna. Un'ulteriore conferma di questa ipotesi è trattata nella seconda parte della tesi, per mezzo del confronto tra gli effetti mostrati dall’agmatina e dal suo analogo alfa-metil-agmatina, un più potente inibitore della transizione di permeabilità mitocondriale. L’utilizzo di tale analogo permette di escludere che la produzione di perossido di idrogeno osservata in presenza di agmatina sia una delle cause principali della permeabilizzazione della membrana esterna. Nella terza parte, è riportato l'effetto delle poliamine (in particolare della spermina e della spermidina) sulla permeabilizzazione delle membrane mitocondriali e sul rilascio dei fattori pro-apoptotici. Nonostante la ben nota azione protettiva di questi composti nei confronti dell'apertura del poro, spermina e spermidina condividono con l’agmatina un comportamento simile nell'indurre il rilascio di alcuni fattori pro-apoptotici, sebbene attraverso meccanismi diversi. Il loro meccanismo d'azione è perciò indagato, cercando di mettere in evidenza le specifiche componenti di membrana coinvolte. Infine, nella quarta parte, viene mostrato uno studio sul meccanismo di efflusso della spermina, indagandone il possibile collegamento con l'efflusso di ATP e fosfato, molto probabilmente in modo elettricamente neutro. Questa osservazione è supportata dall'uso di inibitori specifici della traslocasi degli adenin nucleotidi e del fosfato. In conclusione, i risultati ottenuti in questo studio innanzitutto sostengono l'ipotesi che la permeabilizzazione delle membrane interna ed esterna possa essere vista come due distinti processi, anche se spesso legati tra loro. Inoltre è possibile affermare che la permeabilizzazione della membrana mitocondriale esterna potrebbe essere un punto di contatto tra apoptosi intrinseca ed estrinseca, essendo indotta sia da segnali intra- che extra-mitocondriali. E’ da considerare che il rilascio di citocromo c dai mitocondri non porta in ogni caso all'induzione dell'apoptosi. In particolare lo stato redox della proteina è cruciale per la successiva cascata di eventi: solo il citocromo c ossidato, infatti, è in grado di innescare l'apoptosi. Per questo motivo, è ragionevole pensare che il rilascio di citocromo c indotto dalle amine biologicamente attive possa rappresentare un punto di partenza, ma non un punto di non ritorno. In questo contesto, le poliamine rappresentano uno strumento utile per studiare sia la permeabilizzazione della membrana esterna mitocondriale che la transizione di permeabilità mitocondriale, cercando di delinearne le caratteristiche strutturali e funzionali. Inoltre, a livello cellulare, le poliamine sono in grado di prevenire o indurre l’apoptosi e quest'ultimo effetto è causato dal loro metabolizzazione da parte delle amine ossidasi, con la produzione di composti citotossici. Questa azione dicotomica dipende dalla concentrazione delle poliamine, che è modulata, come dimostrato dai nostri dati, anche attraverso il loro ciclaggio all’interno dei mitocondri. Nonostante siano chiaramente necessari ulteriori studi per individuare tutti i meccanismi strutturali coinvolti nel loro ciclaggio, almeno per la spermina, viene individuata una possibile via di efflusso

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

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

The Americans with Disabilities Act and Equal Access to Public Spaces

Since the passage of the landmark Americans with Disabilities Act of 1990, the United States federal government, states, and localities have passed laws and created policies intended to ensure that people with disabilities had full and equal access to public spaces. Nevertheless, more than three decades after the ADA, people with disabilities continue to face architectural and other barriers to community inclusion and participation. This article describes laws, policies, and initiatives that are implemented in the United States at the federal, state, and local levels to address these barriers, examines their effectiveness, and describes the views of advocates working in furtherance of the rights of people with disabilities and the inclusiveness of public spaces. We conclude by providing brief recommendations for ways federal, state, and local governments may ensure people with disabilities have full and equal access to public spaces

Detección de huevos de toxocara sp. en lugares públicos y parasitismo en caninos de la ciudad de Salta

p. 13-15Toxocara sp., son productores de larva migrans visceralis (LMV). Las hembras eliminan huevos con la materia fecal de hospedadores habituales y en el hombre, producen el cuadro de LMV, principalmente en niños de 1 a 5 años con antecedentes de geofagia, contaminados con heces de caninos o felinos infectados. Se utilizaron las soluciones azucarada de Sheather y de Cloruro de sodio (Willis). Los métodos fueron coprológico de flotación simple y de Benbrook. De 34 muestras de materia fecal de caninos, 7 fueron positivas a Toxocara sp (21%) y 27 negativas (79%). Dos de ellas fueron sólo positivas con el método de Willis, y las otras 5 muestras positivas con los tres métodos. De 30 plazas muestreadas fueron positivas 22 (73%) correspondientes al centro y 17 (57%) correspondientes a la periferia de ellas. Coincidieron solo 15 (50%) plazas respecto a su positividad en el centro y en periferia. En estudios similares efectuados en otros países se detectó la presencia del parásito en el 53% (Paraguay), 14.6% (México), 68,3% (Cuba), 17,2 % de muestras positivas de las casas estudiadas y el 0% de los parques y «playgrounds» (Costa Rica). Es sorprendente la presencia del 73% y 57% de muestras positivas pertenecientes a plazas que son frecuentadas asiduamente por niños, por lo que es el puntapié inicial para nuevas investigaciones donde se verán beneficiados tanto la Salud Pública de los pobladores de la ciudad de Salta, los caninos, sus propietarios y los médicos veterinarios que se dediquen a pequeños animales.Fil: Martinis Mercado, Daniela. Universidad Católica de Salta. Facultad de Ciencias Agrarias y Veterinarias; Argentina.Fil: Burgos Zamudio, María José. Universidad Católica de Salta. Facultad de Ciencias Agrarias y Veterinarias; Argentina.Fil: Barrios, Pamela. Universidad Católica de Salta. Facultad de Ciencias Agrarias y Veterinarias; Argentina.Fil: Pastrana, Oscar Marcelo. Universidad Católica de Salta. Facultad de Ciencias Agrarias y Veterinarias; Argentina.Fil: Ferri, Sergio. Universidad Católica de Salta; Argentina.Fil: Binda, Javier Alejandro. Universidad Católica de Salta. Facultad de Ciencias Agrarias y Veterinarias; Argentina.Fil: Alonso, Marcelo Javier. Universidad Católica de Salta. Facultad de Ciencias Agrarias y Veterinarias; Argentina