**3.3 Effects of AV-6-93 and diflurone on Ca2+-induced MPT**

The effect of AV-6-93 and diflurone on Ca2+-induced MPT was studied in order to evaluate their capacity to protect mitochondria against MPT opening by measuring the decrease in ∆, the increase in oxygen consumption, and the Ca2+-induced release of mitochondrial Ca2+, which are typical phenomena that follow the induction of MPT. The amount of Ca2+ used to induce MPT was 100 nmol/mg protein.

spectrophotometrically at 530 nm. As blanks, we used control reactions performed in the absence of mitochondria and ADP/Fe2+. The amount of TBARs formed was calculated using a molar extinction coefficient of 1.56 x 105 mol-1 cm-1 and expressed as nmol TBARs/mg

The cytotoxicity data were calculated as a mean ± S.E. Statistical analysis was performed using Student's t-test or one-way analysis of variance (ANOVA), followed by a Bonferroni

The mitochondrial experiments were performed using three independent experiments with different mitochondrial preparations. The values are expressed as means ± S.E. Means were compared using one-way ANOVA for multiple comparisons, followed by Tukey's test.

In primary rat cortical neurones, AV-6-93 at concentrations of 1 and 10 µM decreased MPP+ induced cell death by 75% and 56%, respectively (Fig. 2A). Diflurone exerted the protective ability only at the highest tested concentration, 10 μM, and decreased the MPP+-induced cell death by 35% (Fig. 2B). Neither AV-6-93 nor diflurone, added without MPP+, changed cell

AV-6-93 and diflurone (up to 100 μM) were studied for their effects on mitochondrial bioenergetics by evaluating several mitochondrial respiratory chain parameters (state 2, state 3, state 4, FCCP-stimulated respiration, RCR, ADP/O ratio, ∆, and phosphorylation

The effects of AV-6-93 on glutamate/malate-supported respiratory rates (state 2, state 3, state 4 and FCCP-stimulated respiration), respiratory indices RCR and ADP/O of rat liver mitochondria were almost non-existent and insignificant at concentrations of up to 100 µM (Table 1), indicating that the compounds did not significantly affect mitochondrial

These results are demonstrated in Table 2, where AV-6-93 and diflurone, at concentrations of up to 100 µM, did not significantly affect either the ∆ induced by glutamate/malate-

As for glutamate/malate-supported respiration, the effects of AV-6-93 and diflurone on succinate-supported respiratory rates (state 2, state 3, state 4 and FCCP**)** and respiratory indices RCR and ADP/O of rat liver mitochondria were not significantly affected (results not shown), further supporting the finding that these compounds did not affect

The effect of AV-6-93 and diflurone on Ca2+-induced MPT was studied in order to evaluate their capacity to protect mitochondria against MPT opening by measuring the decrease in ∆, the increase in oxygen consumption, and the Ca2+-induced release of mitochondrial Ca2+, which are typical phenomena that follow the induction of MPT. The amount of Ca2+

**3.2 Effects of AV-6-93 and diflurone on rat liver mitochondrial bioenergetics** 

protein (Buege & Aust, 1978).

**2.10 Statistical analysis** 

multiple comparisons test.

**3. Results**

bioenergetics.

mitochondrial bioenergetics.

Statistical significance was set at *p*< 0.05.

**3.1 Protection against the cell death induced by MPP<sup>+</sup>**

viability at the highest tested concentrations (Fig. 2).

rate) using glutamate/malate as the respiratory substrate.

dependent respiration or the phosphorylation time.

used to induce MPT was 100 nmol/mg protein.

**3.3 Effects of AV-6-93 and diflurone on Ca2+-induced MPT**

Fig. 2. Influence of AV-6-93 (AV) and diflurone (D) on MPP+-induced cell death in primary rat cortical neurons (A and B, respectively). Cell death measured by Trypan blue method. Data are presented as a mean S.E. p < 0.001 vs control, t-test, \*\*\* p < 0.001 vs MPP+, one-way ANOVA followed by Bonferroni multiple comparison's test.

Targeting the Mitochondria

by Novel Adamantane-Containing 1,4-Dihydropyridine Compounds 265

Fig. 3*.* Effect of AV-6-93 on rat liver MPT induced by Ca2+.

with three different mitochondrial preparations.

Evaluation was performed by measuring succinate-supported transmembrane potential (∆) (A), oxygen consumption (B), and mitochondrial Ca2+ fluxes (C). Additions of 100 nmol calcium/mg protein (Ca2+) and 10 mM succinate (Suc); additions of AV-6-93 at the concentrations of 1, 10, and 100 M (1, 10, 100) are indicated. Assays in the absence of Ca2+ (-Ca2+); assays in the presence of Ca2+ plus CsA (0.75 nmol/mg protein (CsA); assays in the presence of Ca2+ plus CsA + 1M AV-6-93 (CsA+ 1). The traces are representative of assays

These effects were due to the entry of Ca2+ into the electronegative mitochondrial matrix (Fig. 3C), followed by the efflux of H+ for restoring the ∆. Incubation of mitochondria with


Table 1*.* Effects of AV-6-93 and diflurone on the respiratory parameters (state 2, state 3, state 4, FCCP-stimulated respiration) and respiratory indices (RCR and ADP/O ratio) of rat liver mitochondria using glutamate/malate as respiratory substrate.

The values, which are given in percentage of control (% of control), correspond to the mean ± S.E. of the respiratory parameters, evaluated in three different mitochondrial preparations, at the different indicated situations. Control values are expressed in nmol O2**.** mg-1 protein min-1: state 2 = 7.1 ± 0.7; state 3 = 39.4 ± 4.0; state 4 = 5.62 ± 0.8; FCCP-stimulated respiration = 57.14 ± 10.4.


Table 2. Effects of AV-6-93 and diflurone on glutamate/malate-dependent transmembrane potential (∆) and phosphorylation time of rat liver mitochondria.

The values correspond to the mean ± S.E. of the Δ and the phosphorylation time, evaluated in three different mitochondrial preparations, at the different indicated situations.

The results of the effect of AV-6-93 on MTP protection are depicted in Fig. 3. Under control conditions, the addition of 10 mM succinate to mitochondrial suspensions produced a ∆ of about -216 mV (negative inside mitochondria) (Fig. 3A), corresponding to respiratory state 4 (Fig. 3B). The addition of Ca2+ led to a rapid depolarisation (decrease of ∆), followed by a partial repolarisation (recover of ∆), the subsequent total depolarisation of mitochondria (Fig. 3A), and an increase in respiratory state 4 (Fig. 3B).

0.0 100.0 ± 0.0 100 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 6.8 ± 1.3 3.01 ± 0.1 1.0 100.0 ± 0.0 113.4 ± 6.7 94.4 ± 5.6 100.2 ± 8.7 6.5 ± 1.5 2.9 ± 0.1 10.0 105.0 ± 18.9 104.0 ±7.3 119.4 ± 10.0 101.9 ± 7.3 6.4 ± 1.0 2.8 ± 0.4 100.0 127.2 ± 31.9 109.9 ± 6.6 145.8 ± 44.5 91.2 ± 8.5 6.3 ± 2.7 2.8 ± 0.1

0 100.0± 0.0 100.0± 0.0 100.0± 0.0 100.0± 0.0 6.8 ± 1.3 3.01 ± 0.1 100 107.3 ± 4.8 95.9 ± 2.9 104.4 ± 5.27 94.4 ± 4.03 6.4 ± 1.2 2.9 ± 0.09

Table 1*.* Effects of AV-6-93 and diflurone on the respiratory parameters (state 2, state 3, state 4, FCCP-stimulated respiration) and respiratory indices (RCR and ADP/O ratio) of rat liver

The values, which are given in percentage of control (% of control), correspond to the mean ± S.E. of the respiratory parameters, evaluated in three different mitochondrial preparations, at the different indicated situations. Control values are expressed in nmol O2**.** mg-1 protein min-1: state 2 = 7.1 ± 0.7; state 3 = 39.4 ± 4.0; state 4 = 5.62 ± 0.8; FCCP-stimulated respiration

**ADP** 

0 -220.5 ± 5.1 21.7 ± 2.2 -216.8 ± 2.6 33.0 ± 3.0 1 -219.0 ± 4.0 21.3 ± 1.9 -217.0 ± 4.3 32.7 ± 2.9 10 -220.6 ± 4.1 23.5 ± 2.1 -218.7 ± 4.2 32.5 ± 4.5 100 -216.0 ± 5.6 23.4 ± 1.1 -212.5 ± 4.7 39.0 ± 1.7

0 -220.5 ± 5.1 21.7 ± 2.2 -216.8 ± 2.6 33.0 ± 3.0 100 -218.0 ± 1.7 21.0± 0.3 -216.0 ± 1.4 32.8 ± 2.3

Table 2. Effects of AV-6-93 and diflurone on glutamate/malate-dependent transmembrane

The values correspond to the mean ± S.E. of the Δ and the phosphorylation time, evaluated

The results of the effect of AV-6-93 on MTP protection are depicted in Fig. 3. Under control conditions, the addition of 10 mM succinate to mitochondrial suspensions produced a ∆ of about -216 mV (negative inside mitochondria) (Fig. 3A), corresponding to respiratory state 4 (Fig. 3B). The addition of Ca2+ led to a rapid depolarisation (decrease of ∆), followed by a partial repolarisation (recover of ∆), the subsequent total depolarisation of mitochondria

in three different mitochondrial preparations, at the different indicated situations.

mitochondria using glutamate/malate as respiratory substrate.

**energisation** 

potential (∆) and phosphorylation time of rat liver mitochondria.

(Fig. 3A), and an increase in respiratory state 4 (Fig. 3B).

**RCR ADP/O State 2 State 3 State 4 State** 

**FCCP** 

**<sup>Δ</sup> (mV) Phosphorylation** 

**time (s) Glu/Mal** 

**depolarisation Repolarisation**

**Oxygen consumption (% of control)** 

**Compounds (µM)** 

**AV-6-93** 

**Diflurone** 

= 57.14 ± 10.4.

**Compounds (µM)** 

**Diflurone** 

**AV-6-93**

Fig. 3*.* Effect of AV-6-93 on rat liver MPT induced by Ca2+.

Evaluation was performed by measuring succinate-supported transmembrane potential (∆) (A), oxygen consumption (B), and mitochondrial Ca2+ fluxes (C). Additions of 100 nmol calcium/mg protein (Ca2+) and 10 mM succinate (Suc); additions of AV-6-93 at the concentrations of 1, 10, and 100 M (1, 10, 100) are indicated. Assays in the absence of Ca2+ (-Ca2+); assays in the presence of Ca2+ plus CsA (0.75 nmol/mg protein (CsA); assays in the presence of Ca2+ plus CsA + 1M AV-6-93 (CsA+ 1). The traces are representative of assays with three different mitochondrial preparations.

These effects were due to the entry of Ca2+ into the electronegative mitochondrial matrix (Fig. 3C), followed by the efflux of H+ for restoring the ∆. Incubation of mitochondria with

Targeting the Mitochondria

by Novel Adamantane-Containing 1,4-Dihydropyridine Compounds 267

Fig. 4. Effect of AV-6-93 on membrane lipid peroxidation of rat liver mitochondria induced

**0 10.2 ± 1.0 1 9.6 ± 1.0 10 8.5 ± 0.5 20 7.6 ± 1.0 50 5.2 ± 2.1 100 3.1± 1.9\*** 

Table 3. Effect of AV-6-93 on membrane lipid peroxidation of rat liver mitochondria induced by the pro-oxidant pair ADP/Fe2+ evaluated by TBARs assay. The data correspond to the

Studies examining the importance of mitochondrial pathophysiology in neurodegeneration provide a target for additional treatments with agents that improve mitochondrial function, protect MPT, and/or exert antioxidant activity (Petrozzi et al., 2007). These studies lead to novel approaches in the treatment of neurodegenerative diseases, such as Parkinson's

The aim of the present study was to examine the abilities of two novel adamantanecontaining DHP analogues, AV-6-93 and diflurone, to protect against cell death induced by mitochondrial toxin MPP+ and beneficially influence mitochondrial processes in an attempt

First, we examined how both compounds acted in primary cortical cultures in response to MPP+. AV-6-93, at concentrations of 1 and 10 µM, significantly protected against MPP+ induced cell death by 75% and 56%, respectively, whereas diflurone protected against cell death by 35% at a concentration of 10 µM. Neither AV-6-93 nor diflurone, added without

**TBARs (nmol/mg protein/10 min)** 

by the pro-oxidant pair ADP/Fe2+ evaluated by oxygen consumption.

**AV-6-93 (M)** 

mean ± S.E. of three independent experiments. \**p*< 0.05 vs control (in the absence of AV-6-93).

disease, with disease-modifying drugs.

MPP+, changed cell viability.

to identify putative antiparkinsonian drugs.

**4. Discussion** 

AV-6-93 concentrations of up to 100 µM for 3 min before energisation with succinate prevented total depolarisation of mitochondria (Fig. 3A), the increase in respiratory state 4 (Fig. 3B), and the release of mitochondrial Ca2+ (Fig. 3C), suggesting that this compound has a high ability to protect mitochondria against MPT induction. Incubation of mitochondria with 0.75 nmol/mg protein, CsA, a specific inhibitor of MPT (Broekemeier et al., 1989), for 2 min before energising with succinate, either in the absence or presence of 1 μM AV-6-93, completely blocked mitochondrial depolarisation (Fig. 3A), the increase in respiratory state 4 (Fig. 3B), and the Ca2+-induced release of mitochondrial Ca2+ (Fig. 3C). These data show that these effects had been induced by MPT. In contrast to AV-6-93, diflurone, in the same concentration range, did not prevent either the depolarisation of mitochondria or the release of mitochondrial Ca2+ (results not shown), indicating that this compound did not protect mitochondria against MPT.

#### **3.4 Effects of AV-6-93 and diflurone on mitochondrial oxidative stress**

The effects of AV-6-93 and diflurone on mitochondrial oxidative damage were assessed by detecting the mitochondrial membrane lipid peroxidation induced by the pro-oxidant pair ADP/Fe2+. Lipid peroxidation was evaluated by measuring oxygen consumption (Fig. 4) and TBARs formation (Table 3). In the absence of AV-6-93 and after the addition of the prooxidant pair, it is possible to distinguish two-phase kinetics in oxygen consumption: an initial lag phase, characterized by slow oxygen consumption lasting about 2 min, is followed by a rapid oxygen consumption phase. The lag phase is probably related with the time required for the generation of a sufficient amount of the perferryl ion complex (ADP-Fe2+- O2 ADP-Fe3+-O2-), which has been suggested to be responsible for the initiation of lipid peroxidation. The rapid oxygen consumption phase is probably due to the oxidation of the polyunsaturated fatty acid acyl chain of membrane phospholipids by reactive oxygen species (ROS) and, consequently, due to the propagation phase of lipid peroxidation (Sassa et al., 1990). AV-6-93 concentrations up to 100 M enlarged the lag phase of slow oxygen consumption before the oxygen uptake burst induced by the ADP/Fe2+ complex and increased the rate of the rapid oxygen consumption phase (Fig. 4), suggesting that the compounds affected both the initiation and the propagation of lipid peroxidation of mitochondrial membranes.

These results agree with the quantitative evaluation of TBARs formation performed to confirm the protective effects of AV-6-93. The data in Table 3 show that the kinetics of TBARs formation induced by ADP/Fe2+ are similar to that observed for oxygen consumption. The same range of AV-6-93 concentrations used in the oxygen consumption assays also affected TBARs formation. TBARs formation in the absence of ADP/Fe2+ was negligible (0.44 ± 0.25 nmol/mg of protein). In contrast to AV-6-93, diflurone, in the same concentration range, did not affect oxygen consumption induced by the ADP/Fe2+ complex or TBARs formation (results not shown), indicating that this compound has no capacity to protect mitochondria against the lipid peroxidation induced by the pro-oxidant pair ADP/Fe2+.

Lipid peroxidation was evaluated by oxygen consumption and initiated by adding 1 mM ADP/0.1 mM Fe2+ to mitochondrial suspensions (Fig. 4). The traces represent typical direct oxygen consumption recordings of three experiments obtained from different mitochondrial preparations; controls in the absence of ADP/Fe2+ (–ADP/Fe2+); assays in the presence of AV-6-93 at the concentrations 1, 10, 20, 50, 100 μM (1, 10, 20, 50, 100).

Fig. 4. Effect of AV-6-93 on membrane lipid peroxidation of rat liver mitochondria induced by the pro-oxidant pair ADP/Fe2+ evaluated by oxygen consumption.


Table 3. Effect of AV-6-93 on membrane lipid peroxidation of rat liver mitochondria induced by the pro-oxidant pair ADP/Fe2+ evaluated by TBARs assay. The data correspond to the mean ± S.E. of three independent experiments.

\**p*< 0.05 vs control (in the absence of AV-6-93).
