**2.3 Primary culture of rat cortical neurons**

Primary cultures were prepared from 1-day-old Wistar rat pups, according to the method of Alho et al., 1988, with minor modifications. Briefly, cortices were dissected in ice-cold Krebs-Ringer solution (135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.4 mM KH2PO4, 15 mM glucose, 20 mM HEPES, pH 7.4, containing 0.3% bovine serum albumin) and trypsinised in 0.8% trypsin-EDTA (Invitrogen, U.K.) for 10 min at 37 °C, followed by trituration in 0.008% DNAse I solution containing 0.05% soybean trypsin inhibitor (both obtained from Surgitech AS, Estonia). Cells were resuspended in Eagle's basal medium with Earle's salts (BME, Invitrogen, U.K.), containing 10% heat-inactivated foetal bovine serum (FBS, Invitrogen, U.K.), 25 mM KCl, 2 mM GlutaMAXTM-I (Invitrogen, U.K.) and 100 g/mL gentamycin. Cells were plated onto poly-L-lysine- (Sigma Chemical Co., MO, USA) coated 48-well plates at a density of 1.8 x 105 cells/cm2. The medium was changed to NeurobasalTM-A medium containing 2 mM GlutaMAXTM-I with B-27 supplement and 100 μg/mL gentamycin 2.5 hr later. Cultures were incubated for 6 days in a 5% CO2/95% air atmosphere at 37 C, and one-fifth of the culture medium was changed on DIV 3 (day 3 *in vitro*).

Targeting the Mitochondria

response.

**2.9 Lipid peroxidation** 

by Novel Adamantane-Containing 1,4-Dihydropyridine Compounds 261

where v, V, and ∆E stand for inner mitochondrial volume, incubation medium volume, and deflection of the electrode potential from the baseline, respectively. A mitochondrial matrix volume of 1.1 µl/mg protein was assumed. No correction was made for the "passive" binding of TPP+ to the mitochondrial membranes because the purpose of the experiments was to show relative changes in potential rather than absolute values. As a consequence, we anticipate some overestimation for the ∆ values. To monitor ∆ associated with mitochondrial respiration, liver mitochondria (1 mg/ml) were incubated for 3 min in the respiratory medium described above, supplemented with 3 M TPP+, at 30 °C in the absence or presence of different concentrations of AV-6-93 or diflurone before energisation with 10 mM glutamate/5 mM malate or 10 mM succinate. When succinate was used as the respiratory substrate, the medium was supplemented with 2 µM rotenone. AV-6-93 or diflurone did not affect TPP+ binding to mitochondrial membranes or the electrode

Ca2+-induced MPT was evaluated by measuring changes in mitochondrial transmembrane potential (∆) using a TPP+ electrode, changes in oxygen consumption using a Clark-type electrode, and changes in Ca2+ fluxes using a Ca2+-selective electrode. The reactions were conducted in a medium containing 200 mM sucrose, 10 mM Mops-Tris (pH 7.4), 1 mM KH2PO4, and 10 µM EGTA, supplemented with 2 µM rotenone, as previously described (Custódio et al., 1998a, 1998b). Mitochondria (1mg/ml) that were incubated at 30 °C for 3 min (in the absence and presence of AV-6-93 or diflurone) were energised with 10 mM succinate, and the single addition of Ca2**+** (100 nmol/mg protein) was used to induce MPT. Control assays, in both the absence and presence of Ca2+ plus 0.75 nmol/mg protein

The extent of lipid peroxidation was evaluated by oxygen consumption using a Clark-type electrode at 30 ºC in an open glass chamber equipped with magnetic stirring. Mitochondria (1 mg/ml) were pre-incubated for 3 min in a medium containing 175 mM KCl, 10 mM Tris– Cl (pH 7.4), supplemented with 3 µM rotenone (in the presence or absence of tested compounds) to avoid mitochondrial respiration induced by endogenous respiratory substrates. The iron solution was prepared immediately before use and was protected from light. The changes in O2 tension were recorded in a potentiometric chart record and oxygen consumption was calculated assuming an oxygen concentration of 230 nmol/ml. Membrane lipid peroxidation was initiated by adding 1 mM ADP/0.1 mM Fe2+ as oxidizing agents.

Lipid peroxidation was also determined by measuring thiobarbituric acid reactive substances (TBARs), using the thiobarbituric acid assay (Ernster & Nordenbrand, 1967). Aliquots of mitochondrial suspensions (0.5 ml each), removed 10 min after the addition of ADP/Fe2+, were added to 0.5 ml of ice cold 40% trichloroacetic acid. Then, 2 ml of 0.67% of aqueous thiobarbituric acid containing 0.01% of 2,6-di-*tert*-butyl-*p*-cresol was added. The mixtures were heated at 90 °C for 15 min, then cooled on ice for 10 min, and centrifuged at 850 *g* for 10 min. Controls, in the absence of ADP/Fe2+, were performed under the same conditions. The supernatant fractions were collected and lipid peroxidation was estimated

**2.8 Ca2+-induced mitochondrial membrane transition pore (MPT)** 

cyclosporin A (CsA) and compound (when necessary) were also performed.

Controls, in the absence of ADP/Fe2+, were performed under the same conditions.

∆ = 59 x log (v/V) – 59 x log (10∆E/59 – 1) (1)

#### **2.4 Measurement of cell death in cytotoxicity assay**

Primary rat cortical neurons were cultured for 5 days as described above. On DIV 5, cultures were incubated with 1-methyl, 4-phenylpyridinium (MPP+, Sigma Chemical Co., MO, USA) for the following 24 hr at a concentration of 300 μM. Cells were pre-incubated with the tested compounds AV-6-93 and diflurone for 90 min followed by the addition of MPP+ and further incubation with MPP+ plus the tested compounds or a solvent (control) for the next 24 hours. Cell death was measured with a Trypan blue assay (Tymianski et al., 1993). Cells were incubated with 0.4% Trypan blue solution in phosphate buffered saline (PBS, 145 mM NaCl, 3 mM KCl, 0.42 mM Na2HPO4, 2.4 mM KH2PO4, pH = 7.4) at 37 ºC for 7 min and then washed twice with PBS and fixed with 4% paraformaldehyde in PBS. Only dead neurons were stained with Trypan blue (Tymianski et al., 1993). The fixed cultures were rinsed with PBS for microscopic observation, and approximately 150 cells per 5 fields in each well were counted to determine the number of dead cells and the total number of cells. Neuronal death was calculated as the percentage of dead cells from the total (viable plus dead) number of cells, and the obtained data were averaged for each well.

#### **2.5 Isolation of rat liver mitochondria**

Rat liver mitochondria were isolated from male Wistar rats by differential centrifugation according to conventional methods (Gazotti et al., 1979). After washing, the pellet was gently resuspended in the washing medium at a protein concentration of about 50 mg/ml. Protein content was determined by the biuret method (Gornall et al., 1949), using bovine serum albumin as a standard.

#### **2.6 Measurement of respiratory activities**

Oxygen consumption was monitored polarographically with a Clark-type electrode at 30 °C in a closed glass chamber equipped with magnetic stirring. Mitochondria (1 mg/ml) were incubated in a respiratory medium containing 130 mM sucrose, 5 mM HEPES (pH 7.2), 50 mM KCl, 2.5 mM K2HPO4, and 2.5 mM MgCl2 (in the presence and absence of AV-6-93 or diflurone) for 3 min before energisation with 10 mM glutamate/5 mM malate*.* When 10 mM succinate was used as the respiratory substrate, the reaction medium was supplemented with 2 µM rotenone. To induce state 3 respiration, adenosine diphosphate (ADP, 150 µM) was added. FCCP (p-trifluoromethoxyphenylhydrazone)-stimulated respiration was initiated by the addition of 1µM FCCP. The respiratory control ratio (RCR), which is calculated by the ratio between state 3 (consumption of oxygen in the presence of substrate and ADP) and state 4 (consumption of oxygen after ADP phosphorylation), is an indicator of mitochondrial membrane integrity. The ADP/O ratio, which is expressed by the ratio between the amounts of ADP added and the oxygen consumed during state 3 respiration, is an index of oxidative phosphorylation efficiency. Respiration rates were calculated assuming that the saturation of oxygen concentration was 250 µM at 30 ºC (Chance & Williams, 1956), and the values are expressed in percentage of control (% of control).

#### **2.7 Measurement of mitochondrial transmembrane potential**

The mitochondrial transmembrane potential (∆) was measured indirectly based on the detection of lipophilic cation tetraphenylphosphonium (TPP+) using a TPP+-selective electrode, as previously described (Kamo et al., 1979). The ∆ was estimated from the following equation (1):

$$
\Delta\psi = 59 \times \log\left(\text{v/V}\right) - 59 \times \log\left(10^{\text{AE/59}} - 1\right) \tag{1}
$$

where v, V, and ∆E stand for inner mitochondrial volume, incubation medium volume, and deflection of the electrode potential from the baseline, respectively. A mitochondrial matrix volume of 1.1 µl/mg protein was assumed. No correction was made for the "passive" binding of TPP+ to the mitochondrial membranes because the purpose of the experiments was to show relative changes in potential rather than absolute values. As a consequence, we anticipate some overestimation for the ∆ values. To monitor ∆ associated with mitochondrial respiration, liver mitochondria (1 mg/ml) were incubated for 3 min in the respiratory medium described above, supplemented with 3 M TPP+, at 30 °C in the absence or presence of different concentrations of AV-6-93 or diflurone before energisation with 10 mM glutamate/5 mM malate or 10 mM succinate. When succinate was used as the respiratory substrate, the medium was supplemented with 2 µM rotenone. AV-6-93 or diflurone did not affect TPP+ binding to mitochondrial membranes or the electrode response.

#### **2.8 Ca2+-induced mitochondrial membrane transition pore (MPT)**

Ca2+-induced MPT was evaluated by measuring changes in mitochondrial transmembrane potential (∆) using a TPP+ electrode, changes in oxygen consumption using a Clark-type electrode, and changes in Ca2+ fluxes using a Ca2+-selective electrode. The reactions were conducted in a medium containing 200 mM sucrose, 10 mM Mops-Tris (pH 7.4), 1 mM KH2PO4, and 10 µM EGTA, supplemented with 2 µM rotenone, as previously described (Custódio et al., 1998a, 1998b). Mitochondria (1mg/ml) that were incubated at 30 °C for 3 min (in the absence and presence of AV-6-93 or diflurone) were energised with 10 mM succinate, and the single addition of Ca2**+** (100 nmol/mg protein) was used to induce MPT. Control assays, in both the absence and presence of Ca2+ plus 0.75 nmol/mg protein cyclosporin A (CsA) and compound (when necessary) were also performed.

#### **2.9 Lipid peroxidation**

260 Bioenergetics

Primary rat cortical neurons were cultured for 5 days as described above. On DIV 5, cultures were incubated with 1-methyl, 4-phenylpyridinium (MPP+, Sigma Chemical Co., MO, USA) for the following 24 hr at a concentration of 300 μM. Cells were pre-incubated with the tested compounds AV-6-93 and diflurone for 90 min followed by the addition of MPP+ and further incubation with MPP+ plus the tested compounds or a solvent (control) for the next 24 hours. Cell death was measured with a Trypan blue assay (Tymianski et al., 1993). Cells were incubated with 0.4% Trypan blue solution in phosphate buffered saline (PBS, 145 mM NaCl, 3 mM KCl, 0.42 mM Na2HPO4, 2.4 mM KH2PO4, pH = 7.4) at 37 ºC for 7 min and then washed twice with PBS and fixed with 4% paraformaldehyde in PBS. Only dead neurons were stained with Trypan blue (Tymianski et al., 1993). The fixed cultures were rinsed with PBS for microscopic observation, and approximately 150 cells per 5 fields in each well were counted to determine the number of dead cells and the total number of cells. Neuronal death was calculated as the percentage of dead cells from the total (viable plus dead)

Rat liver mitochondria were isolated from male Wistar rats by differential centrifugation according to conventional methods (Gazotti et al., 1979). After washing, the pellet was gently resuspended in the washing medium at a protein concentration of about 50 mg/ml. Protein content was determined by the biuret method (Gornall et al., 1949), using bovine

Oxygen consumption was monitored polarographically with a Clark-type electrode at 30 °C in a closed glass chamber equipped with magnetic stirring. Mitochondria (1 mg/ml) were incubated in a respiratory medium containing 130 mM sucrose, 5 mM HEPES (pH 7.2), 50 mM KCl, 2.5 mM K2HPO4, and 2.5 mM MgCl2 (in the presence and absence of AV-6-93 or diflurone) for 3 min before energisation with 10 mM glutamate/5 mM malate*.* When 10 mM succinate was used as the respiratory substrate, the reaction medium was supplemented with 2 µM rotenone. To induce state 3 respiration, adenosine diphosphate (ADP, 150 µM) was added. FCCP (p-trifluoromethoxyphenylhydrazone)-stimulated respiration was initiated by the addition of 1µM FCCP. The respiratory control ratio (RCR), which is calculated by the ratio between state 3 (consumption of oxygen in the presence of substrate and ADP) and state 4 (consumption of oxygen after ADP phosphorylation), is an indicator of mitochondrial membrane integrity. The ADP/O ratio, which is expressed by the ratio between the amounts of ADP added and the oxygen consumed during state 3 respiration, is an index of oxidative phosphorylation efficiency. Respiration rates were calculated assuming that the saturation of oxygen concentration was 250 µM at 30 ºC (Chance &

Williams, 1956), and the values are expressed in percentage of control (% of control).

The mitochondrial transmembrane potential (∆) was measured indirectly based on the detection of lipophilic cation tetraphenylphosphonium (TPP+) using a TPP+-selective electrode, as previously described (Kamo et al., 1979). The ∆ was estimated from the

**2.7 Measurement of mitochondrial transmembrane potential** 

**2.4 Measurement of cell death in cytotoxicity assay**

number of cells, and the obtained data were averaged for each well.

**2.5 Isolation of rat liver mitochondria** 

**2.6 Measurement of respiratory activities** 

serum albumin as a standard.

following equation (1):

The extent of lipid peroxidation was evaluated by oxygen consumption using a Clark-type electrode at 30 ºC in an open glass chamber equipped with magnetic stirring. Mitochondria (1 mg/ml) were pre-incubated for 3 min in a medium containing 175 mM KCl, 10 mM Tris– Cl (pH 7.4), supplemented with 3 µM rotenone (in the presence or absence of tested compounds) to avoid mitochondrial respiration induced by endogenous respiratory substrates. The iron solution was prepared immediately before use and was protected from light. The changes in O2 tension were recorded in a potentiometric chart record and oxygen consumption was calculated assuming an oxygen concentration of 230 nmol/ml. Membrane lipid peroxidation was initiated by adding 1 mM ADP/0.1 mM Fe2+ as oxidizing agents. Controls, in the absence of ADP/Fe2+, were performed under the same conditions.

Lipid peroxidation was also determined by measuring thiobarbituric acid reactive substances (TBARs), using the thiobarbituric acid assay (Ernster & Nordenbrand, 1967). Aliquots of mitochondrial suspensions (0.5 ml each), removed 10 min after the addition of ADP/Fe2+, were added to 0.5 ml of ice cold 40% trichloroacetic acid. Then, 2 ml of 0.67% of aqueous thiobarbituric acid containing 0.01% of 2,6-di-*tert*-butyl-*p*-cresol was added. The mixtures were heated at 90 °C for 15 min, then cooled on ice for 10 min, and centrifuged at 850 *g* for 10 min. Controls, in the absence of ADP/Fe2+, were performed under the same conditions. The supernatant fractions were collected and lipid peroxidation was estimated

Targeting the Mitochondria

by Novel Adamantane-Containing 1,4-Dihydropyridine Compounds 263

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.

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 protein (Buege & Aust, 1978).
