Pamela Martinis

Mitochondrial oxidative stress induced by Ca2+ 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 so-called transition pore, that triggers bidirectional traffic of inorganic solutes and metabolites across the mitochondrial membrane. This phenomenon is caused by supraphysiological Ca2+ 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.

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 (ΔΨ). The presence of phosphate increases spermine uptake by reducing ΔpH and enhancing ΔΨ. 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 the ΔΨ value 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 in ΔΨ able 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.

Synthesis, antiproliferative and mitochondrial impairment activities of bis-alkyl-amino transplatinum complexes.

A convenient synthetic route and the characterization of complexes trans-[PtCl2(L)(PPh3)] (L=Et2NH (2), (PhCH2)2NH (3), (HOCH2CH2)2NH) (4) are reported. The antiproliferative activity was evaluated on three human tumor cell lines. The investigation on the mechanism of action highlighted for the most active complex 4 the capacity to affect mitochondrial functions. In particular, both the induction of the mitochondrial permeability transition phenomenon and an aspecific membrane damage occurred, depending on concentration.

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.

A novel enzyme with spermine oxidase properties in bovine liver mitochondria: identification and kinetic characterization.

The uptake of spermine into mammalian mitochondria indicated the need to identify its catabolic pathway in these organelles. Bovine liver mitochondria were therefore purified and their capacity for natural polyamine uptake was verified. A kinetic approach was then used to determine the presence of an MDL 72527-sensitive enzyme with spermine oxidase activity in the matrix of bovine liver mitochondria. Western blot analysis of mitochondrial fractions and immunogold electron microscopy observations of purified mitochondria unequivocally confirmed the presence of a protein recognized by anti-spermine oxidase antibodies in the mitochondrial matrix. Preliminary kinetic characterization showed that spermine is the preferred substrate of this enzyme; lower activity was detected with spermidine and acetylated polyamines. Catalytic efficiency comparable to that of spermine was also found for 1-aminododecane. The considerable effect of ionic strength on the Vmax/KM ratio suggested the presence of more than one negatively charged zone inside the active site cavity of this mitochondrial enzyme, which is probably involved in the docking of positively charged substrates. These findings indicate that the bovine liver mitochondrial matrix contains an enzyme belonging to the spermine oxidase class. Because H2O2 is generated by spermine oxidase activity, the possible involvement of the latter as an important signaling transducer under both physiological and pathological conditions should be considered.

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.

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 S1 and S2, with a negative influence of S2 on S1, has been demonstrated. The calculated binding energy is characteristic for weak interactions. S1 exhibits a lower binding capacity and higher binding affinity both of about two orders of magnitude than S2. Experiments with idazoxan, a ligand of the mitochondrial imidazoline receptor I2, demonstrate that S1 site is localized on this receptor while S2 is localized on the transport system. S1 would act as a sensor of exogenous agmatine concentration, thus modulating the transport of the amine by its binding to S2.

Agmatine and alpha-methylagmatine: permeabilizing the outer mitochondrial membrane.

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W30–11 Aktas, C.C., SW01.W5–14 Aktug, H., SW01.W5–25, SW04.S21–20 Akulich, K.A., SW01.S2–91 Akulov, A., SW04.S16–124 Akyurek, F., SW04.S16–22, SW04.S16–32, SW05.S22–17, SW06.S27–46 Akyuz, S., SW04.S16–28, SW04.S16–229 Alagoz, D., SW06.W33–21, SW06.W33–22 Al-Ajmi, N., SW03.S13–93 Alaylioglu, M., SW04.S19–51 de Alba, E., SW02.S7–106 Albano, F., SW03.S13–77 Albar, J.P., SE02-4 Albayrak, A., SW04.S16–186, SW04.S16–189 Albericio, F., SW04.S16–245, SW06.S25–50 Albino, A., SW02.W10–18 Albulescu, L., SW06.S25–12 Albulescu, R., SW06.S25–12 Alcarraz-Vizán, G., SE01-6 Aldea, I.M., SW04.S16–102 Aleksandrova, L.A., SW04.S16–281 Alekseenko, I.V., SW04.S17–3 Alekseenko, L.L., SW04.S21–33 Alekseev, A., SW04.S19–69, SW04.S19–72 Alekseev, A.V., SW03.S14–43, SW03.S14–44 Alekseev, K., SW02.S7–70 Alekseeva, A.A., SW02.W10–9, SW02.W10–10 Alekseeva, E.E., SW04.S16–287 Alekseeva, G.M., SW04.S16–190 Alekseeva, I., SW04.S16–17 Alekseeva, M., SW04.S16–25 Alekseevskaya, E., SW03.S14–23 Alelu, R., SW04.S17–61 Alenina, N., SW04.S21–27 Aleshina, G.M., SW06.S25–46 Alessio, N., SW06.S25–77 Alev, B., SW04.S16–201 Alexandre, A., SW03.S13–32 Alexandrov, A., SW04.S19–46 Alexandrova, L.A., SW04.S16–252 Alexeev, D., SW04.S16–69, SW06.S25–78, SW06.W30–30 Alezzawi, M., SW06.S27–40 Alhonen, L., SW06.W31–11, SW06.W31–17, SW06.W31–18, SW06.W31–29 Ali, M., SW03.S13–61 Aliev, T.K., SW06.W33–14 Alimova, F.K., SW04.S16–235, SW06.W29–41 Alisultanova, N., SW03.S13–84 Aliverdieva, D., SW03.S14–28 Alkalaeva, E., SW01.S2–30 Alkalaeva, E.Z., SW01.S2–75 Al-Karadaghi, S., SW02.S7–5 Allagulova, C., SW03.S13–82 Alleaume-Butaux, A., SW03.S12-14 Allegretti, M., SW04.S18–19 Allen, J.W.A., SW02.W10–25 Allen, S., SW06.W33–46 Allinne, J., SW04.S16–1 Almada, M., SW06.W30–12 Al-Maghrebi, M., SW03.S13–93 Almeida, M.R., SW04.S16–210 Almeida, R.D., SW04.S19–34 Alptekin, O., SW06.W33–21, SW06.W33–22 Alster, O., SW01.S3–40 Altan, N., SW04.S19–59 Altenfeld, A., SW01.S3–27 Altieri, F., SW02.S8–19 Altman, S., PL03 Altunoglu, E., SW04.S16–70, SW04.S16–292 Alturfan, A., SW02.S7–29 Alturfan, E., SW03.S13–121 Alva-Murillo, N., SW05.S23–29 Alvarez, M., SW04.S16–274 Alves, G., SW04.S17–31 Alves, M., SW03.S13–34, SW04.S17–21 Alves, M.G., SW03.S14–22 Alyasova, A.V., SW04.S17–57 Amado, A.M., SW04.S16–117 Amaro, M.P., SW02.S7–28 Amati, F., SW04.S16–40, SW04.S19–14, SW04.S19–18 Amato, M., SW02.W10–18 Ambicka, A., SW04.S17–66 Ambrosio, A.F., SW04.S16–39, SW04.S19–34 Amdam, G.V., SW06.W32–6 Ame, J.-C., SW01.S3–24 Ami, D., SW04.S19–10 Amico, M.D., SW06.W30–15 Aminin, D., SW06.S26–11, SW06.S26–14 Aminin, D.L., SW06.S26–3 Amoroso, M.R., SW03.S13–43 Anachkova, B., SW01.S3–15 Anannya, O., SW01.W5–18 Anatskaya, O., SW01.S1–30, SW01.S1–42, SW01.S1–59 Anatskaya, O.V., SW01.S1–32 Andersson, M., SW02.S8–9 Andican, G., SW04.S16–64, SW04.S16–70, SW04.S16–292, SW05.S22–8 Andina, S.V., SW04.S16–7 Andjelkovic, U., SW06.S28–19 Andrade, J.M., SW01.S2–37 Andrade, P.B., SW04.S16–48 Andreasson, C., SW03.S15–14 Andreea, D., SW01.S3–16 Andreev, D., SW01.S2–76, SW01.S2–80 Andreev, D.E., SW01.S2–17, SW01.S2–91 Andreeva, E., SW04.S16–226 Andrei, E., SW04.S21–13 Andriichuk, L., SW02.S7–75 Andrini, O., SW03.S12-32 Angelucci, C.B., SW02.S7–73, SW02.S7–74 Angin, Y., SW03.S12-9 Angulo, J., SE01-4 Anikanov, N., SW06.S25–13 Anikienko, K.A., SW02.W9–11 Anikov, N., SW06.S25–51 Anilanmert, B., SW06.S28–23 Anisenko, A., SW04.S16–272 Anisimova, N., SW02.W10–28 Annenkov, V.V., SW06.W31–16, SW06.W31–23 AnnMacgregor, E., SW06.W29–28 Antimonova, O.I., SE02-23 Antina, E.V., SW06.W33–66 Anton, B.M., SW01.S3–60 Anton, G., SW01.S2–58, SW04.S17–23 Antonelou, M., SW06.W30–43, SW06.W32–17 Antonenko, S., SW01.W5–20 Antonenko, Y.N., SW03.S14–16 Antonets, D., SW04.S16–115, SW04.S16–290 Antonets, D.V., SW04.S16–172 Antonny, B., SW04.S18–40 Antonov, K., SW04.S16–264 Antonov, K.V., SW04.S16–265 Antonov, V., SW04.S16–185, SW04.S16–266 Antonova, N., SW04.S16–124 Antontseva, E.V., SW05.S22–19 Antonyan, A., SW04.S16–123 Antonyan, A.A., SW04.S19–5 Antosiewicz, A., SW06.S25–19, SW06.S25–20 Anufrieva, N., SW02.W10–28 Anufrieva, N.V., SW02.W10–7, SW02.W10–20, SW02.W10–32 Anurov, M., SW02.W9–15 Ao, C.K.L., SW01.S1–52 Aono, S., SW02.S8–7 Aoun, B., SW02.S8–17 Aoyama, E., SW04.S21–29 Aparicio, R., SW02.S7–54 Appel, E., SW02.W10–4 Aradska, J.S., SW06.S25–30, SW06.S25–55 Aramini, A., SW04.S18–19 Arancia, G., SW06.W31–12 Aranda, J., SW02.S6–43 Arapi, B., SW04.S16–228 Arapidi, G., SW06.S25–13, SW06.S25–14, SW06.S25–17, SW06.S25–51 Araujo, A.P.U., SW02.S7–71 de Araujo, A.P.U., SW02.S7–82 Araz, O.S., SW04.S19–51 Arbak, S., SW06.W30–37 Arcari, P., SW02.S8–19, SW04.S16–113 Archakov, A., SW04.S16–109, SW04. S16–110, SE02-2, SE02-6, SE02-22 Archakov, A.I., SW04.S16–286, SE02-9, SE02-13, SE02-14, SE02-20, SE02-21, SE02-27 Arcone, R., SW04.S19–50 Arcovito, A., SW02.S7–48 Arcucci, A., SW03.S13–77 Arda-Pirincci, P., SW06.W30–23, SW06.W30–24 Arenas, M.I., SW04.S17–15 Arga, K.Y., SW02.W10–26, SW06.W30–18 Arias, L., SW06.S26–21 Arican, N., SW04.S19–24 Arinto, P., SW01.W5–10 Aritake, K., SW04.S16–227, SW06.S27–42 Arlt, V.M., SW04.S16–85 Arnaoutov, A., SW03.S15–20 Arnesano, F., SW03.S12-24 Arnon, R., SA04–2 Aroca, Á., SE01-4 Aronheim, A., SW01.W5–6 Aronsson, H., SW06.S27–40 Arraiano, C.M., SW01.S2–37 Arrasate, M., SW04.S19–60 Arrigo, A.P., SW03.S13–6 Arseniev, A., SW01.S3–26, SW02.S7–83, SW06.S25–64 Arseniev, A.A., SW03.S11–11 Arseniev, A.S., SW02.S7–97, SW02.S7–98 Arsenkova, O., SW04.S16–97 Arslan, C., SW04.S16–228 Arslan, S., SW03.S13–25 Artac, H., SW05.S22–17 Artamonova, T., SW02.S6–32, SW02.S7–63, SW02.S8–20 Artemov, A., SW01.S1–50 Artemyev, M.V., SW04.S16–8 Artenie, V., SW06.W33–26 Arthur, M., SW04.S16–279, SW04.S16–280, SE01-5 Arutjunyan, A., SW04.S16–218 ArzuErgen, H., SW04.S16–57 Asano, Y., SW06.W33–12 Aseev, L., SW01.S2–72 Asgeirsson, B., SW02.S8–23 Ashani, Y., SW02.W9–9, SW02.W9–10 Ashirbekov, E., SA02–11 Ashour, N., SW04.S17–61 Askar, N., SW04.S16–132 Aslan, M., SW04.S19–26 Astakhova, L., SW04.S20–8 Astakhova, L.N., SW03.S13–31