**5. Conclusion**

268 Bioenergetics

A larger difference between the compounds' activities was observed in isolated rat liver mitochondria by the assessment of their ability to affect both the Ca2+-induced mitochondrial permeability transition (MPT) and lipid peroxidation. To assess the Ca2+ induced MPT, the evaluation of the drop of ∆, the increase in mitochondrial respiration associated with Ca2+ accumulation in the mitochondrial matrix, and the mitochondrial Ca2+ fluxes were carried out. Changes in these parameters help us to conclude whether the compound protects mitochondria against MPT induction and, consequently, to discern whether the compound alters mitochondrial Ca2+ homeostasis. AV-6-93, at a concentration of 10 µM, significantly protected mitochondria against MTP induction and provided complete protection at 100 µM, as revealed by its ability to prevent the depolarisation of mitochondria, the increase in mitochondrial respiration and mitochondrial Ca2+ release. These effects were comparable with that of CsA (0.75 nmol/mg protein), a specific inhibitor of the mitochondrial permeability transition pore. Diflurone was ineffective in these tests. The effectiveness of AV-6-93 can be considered to be very promising because it indicates the ability of this compound to halt mitochondrial swelling and cell death, both consequences of

A critical factor for induction of MPT is the oxidation of thiol groups of the MPT complex, creating diethyl cross-links (Costantini et al., 1996; 1998, Halestrap et al., 1997; McStay et al., 2002). Therefore, the most plausible hypothesis to explain the partial MPT protection induced by AV-6-93 is that changes in the redox-state of thiol groups of the MPT complex is provided via avoiding of diethyl cross-links. This hypothesis is supported by the observation that AV-6-93 protected mitochondria against oxidative stress. Oxidative stress was assessed by evaluating the extent of lipid peroxidation by measuring oxygen consumption and TBARs formation. Alterations of these parameters may reveal whether the compound protects mitochondria against oxidative stress, i.e., whether the compound acts as an antioxidant. AV-6-93, at concentrations up to 100 µM, protected (by about a half) mitochondria against membrane lipid peroxidation, as inferred by its ability to inhibit both oxygen consumption and TBARs formation induced by the pro-oxidant pair ADP/Fe2+. These data suggest that this compound may act as antioxidant because it can avoid both the initiation and the propagation of the oxidation of polyunsaturated fatty acid acyl chains of membrane phospholipids induced by the perferryl ion complex ADP-Fe3+-O2-, a mechanism suggested to be responsible for lipid peroxidation (Sassa et al., 1990). In contrast to AV-6-93, diflurone, under the same conditions, had no capacity to protect mitochondria against

The only common feature of both compounds was a lack of influence on mitochondrial bioenergetics, which was assessed by analysing several mitochondrial functioning parameters of the respiratory chain (respiration states 2, 3, 4, FCCP-stimulated respiration, the RCR, and the ADP/O ratio) and the oxidative phosphorylation system (∆ and phosphorylation time), using both glutamate/malate and succinate as respiratory substrates. According to the mitochondrial parameters affected, it is possible to assess how the compound interferes with mitochondrial bioenergetics: by perturbing the permeability (integrity) of the inner mitochondrial membrane (stimulation of respiration states 2 and 4), by impairing the respiratory chain (inhibition of FCCP-stimulated respiration), and/or by acting at the level of the phosphorylation system (affecting respiration state 3). Both AV-6-93 and diflurone, at concentrations of up to 100 µM, failed to significantly affect liver mitochondrial bioenergetics, as shown by the lack of effects on both glutamate/malate- and succinate-supported respiration in state 2, state 3, state 4, FCCP-stimulated respiration, RCR

the induction of the permeability transition pore.

oxidative damage induced by the pro-oxidant pair ADP/Fe2+.

and ADP/O ratios, ∆ and phosphorylation time.

The novel one-adamantane 1,4-dihydropyridine compound AV-6-93 is capable of regulating cell survival processes with regards to mitochondrial processes, such as inhibition of the induction of the permeability transition pore and prevention of oxidative stress. The effectiveness of AV-6-93 can be considered to be very promising in the treatment of neurodegenerative diseases associated with compromised mitochondrial processes, e.g., Parkinson's disease.
