After centrifugation at 14,000 × g for 5 min at 4 °C, 100-μl aliquots of the supernatant were neutralized with 5 M KOH, suspended in 100 mM TRIS–HCl, pH 7.8 (1 mL final volume), and centrifuged at 15,000 × g for 15 min. The supernatant was tested with a Sigma/Aldrich assay kit (Catalog Number FLAA) according to the manufacturer’s instructions, and the resulting luminescence was measured using a SIRIUS Luminometer (Berthold, Pforzheim, Germany). Mitochondria (0.45 mg protein) were incubated in a medium containing 54 mM potassium acetate, 5 mM HEPES–KOH, pH 7.1, 0.1 mM EGTA, 0.2 mM EDTA, 0.1 mM sodium azide, 0.1% bovine serum albumin, 15 mM

selleck atractyloside, 1 mM antimycin A, and 0.3 mM propranolol to inhibit the inner membrane anion channel, followed by 1 mM valinomycin and juliprosopine in a final volume of 1.5 mL (Mingatto et al., 2000). The swelling was estimated from the decrease in the absorbance at 540 nm using a DU-800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). Mitochondrial hydrogen peroxide (H2O2) production was monitored spectrofluorometrically in a RF-5301 PC Shimadzu fluorescence spectrophotometer (Tokyo, Japan) using the Amplex Red assay: mitochondria were incubated with 100 mM Amplex Red and 0.025 U/mL horseradish peroxidase,

and fluorescence of the oxidized probe was measured at the 563/587 nm excitation/emission wavelength pair (Votyakova and Reynolds, 2001). Mitochondria learn more were incubated at 37 °C with 0.5 μM DPH (0.5 mg protein) or ANS (2 mg protein) plus 1 μg/mL CCCP before juliprosopine was added (2 mL final volume). The fluorescence was measured in a RF-5301 PC Shimadzu fluorescence spectrophotometer (Tokyo, Japan) at excitation

and emission wavelengths of 377 and 431 nm, respectively, for DPH (Lee et al., 1999) and 380 and 485 nm, respectively, for ANS (Slavík, 1982). The data were expressed as the means ± s.e. and significant differences were calculated by one-way analysis of variance (ANOVA) Amobarbital followed by the Dunnett’s test using GraphPad Prism software, version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). Mitochondrial oxygen consumption was monitored in the presence of varying concentrations of juliprosopine. The parameters assessed were state-3 respiration (consumption of oxygen in the presence of respiratory substrate and ADP) and state-4 respiration (consumption of oxygen after ADP has been exhausted). At the concentrations tested (5–25 μM), juliprosopine presented no effects on state-3 respiration, but it stimulated the state-4 respiration of mitochondria energized with either pyruvate plus malate, which are respiratory chain site I substrates (Fig. 2A and B), or succinate, a respiratory chain site II substrate (Fig. 2C and D), in a dose-dependent manner. This result indicated that the alkaloid acts as an uncoupler.