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.