Neal B. Blatt, Jeffrey J. Bednarski, Roscoe E. Warner, Francesco Leonetti, Kathryn M. Johnson, Anthony Boitano, Raymond Yung, Bruce C. Richardson, Kent J. Johnson, Jonathan A. Ellman, Anthony W. Opipari Jr., Gary D. Glick
Bz-423 generates ROS but not swelling in isolated mitochondria. Measurements were conducted in duplicate as described in Methods and represent data obtained with mitochondria from four different rats. Within a given run, a larger slope corresponds to greater ROS production. For these experiments, antimycin A was added at 0.5 μM and Bz-423 at 10 μM. The r2 values for the linear regression analysis of the data in a, c, and d are greater than 0.995. (a) ROS measured in state 3 respiration buffer. The slopes of each curve after the induction phase (1,200–1,500 seconds) are 1.5 × 10–3 ± ≤15%, 2.1 × 10–3 ± ≤15%, and 1.1 × 10–3 ± ≤15% for antimycin A, Bz-423, and vehicle, respectively. P < 0.05 for vehicle vs. Bz-423 or antimycin A (Student’s t test). (b) Swelling of mitochondria in state 3 buffer. Only Ca2+ (400 μM) triggered the MPT pore. (c) ROS measured using the S15 fraction in state 3 buffer. Slopes are 0.9, 0.8, and 1.0 (× 10–3 ± ≤15%) for antimycin A, Bz-423, and vehicle, respectively. (d) ROS measured in state 4 respiration buffer. Slopes are 2.1, 1.3, and 1.3 (× 10–3 ± ≤15%) for antimycin A, Bz-423, and vehicle, respectively. P = 0.25 for vehicle vs. Bz-423; P = 0.035 for vehicle vs. antimycin A (Student’s t test). (e) Micrographs (×630) of mitochondria stained with DHE (red) to detect O2– or with DiOC6 (green) to determine ΔΨm. Each spot represents an individual mitochondrion staining with the indicated dye.