The experimental protocols were approved by the Ethical Committee for the Use of Laboratory Animals of the Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Dracena. Mitochondria were isolated by standard differential centrifugation (Pedersen et al., 1978). Rats were BMS-354825 sacrificed by decapitation, and the liver was immediately removed, sliced into 50 ml of medium containing 250 mM sucrose, 1 mM EGTA, and 10 mM HEPES-KOH, pH 7.2, and homogenized three times for 15 s at 1-min intervals with a Potter-Elvehjem homogenizer. Homogenate was centrifuged at 770g for 5 min, and the resulting supernatant further

centrifuged at 9800g for 10 min. The pellet was suspended in 10 ml of medium containing 250 mM sucrose, 0.3 mM EGTA, and 10 mM HEPES-KOH, pH 7.2 and centrifuged at 4500g for 15 min. The final mitochondrial pellet was suspended in 1 ml of medium containing 250 mM sucrose and 10 mM HEPES-KOH,

pH 7.2 and was used within 3 h. The mitochondrial protein concentration was determined by a biuret assay with BSA as the standard ( Cain and Skilleter, 1987). The disrupted mitochondria were obtained by heat shock treatment after three consecutive cycles of freezing in liquid nitrogen and thawing in a water bath heated to 37 °C. The membrane fragments were kept at 4 °C and were used in the assessment of mitochondrial enzymatic activity within 3 h. Mitochondrial respiration was monitored using a Clark-type oxygen electrode (Strathkelvin Instruments Limited, Glasgow, Scotland, UK), and respiratory parameters were determined according Roxadustat to Chance and Williams (1955). One milligram of mitochondrial protein was added to 1 ml of respiration buffer containing 125 mM sucrose, 65 mM KCl, and tuclazepam 10 mM HEPES-KOH, pH 7.4, plus 0.5 mM EGTA and 10 mM K2HPO4, at 30 °C. Oxygen consumption was measured using 5 mM glutamate + 5 mM malate, 5 mM succinate (+2.5 μM rotenone) or 200 μM N,N,N,N-tetramethyl-p-phenylene diamine (TMPD) + 3 mM ascorbate as respiratory substrates in the absence (state-4

respiration) or the presence of 400 nmol ADP (state-3 respiration). The mitochondrial membrane potential (Δψ) was estimated spectrofluorimetrically using model RF-5301 PC Shimadzu fluorescence spectrophotometer (Tokyo, Japan) at the 495/586 nm excitation/emission wavelength pair. Safranine O (10 μM) was used as a probe (Zanotti and Azzone, 1980). Mitochondria (2 mg protein) energized with 5 mM glutamate + 5 mM malate were incubated in a medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES-KOH, pH 7.4, and 0.5 mM EGTA (2 ml final volume). ATP levels were determined using the firefly luciferin–luciferase assay system (Lemasters and Hackenbrock, 1976). After incubation in the presence of ABA, the mitochondrial suspension (1 mg protein/ml) was centrifuged at 9000g for 5 min at 4 °C, and the pellet was treated with 1 ml of ice-cold 1 M HClO4.