Figure 3. Openers and blockers of the putative mitoK_ATP did not affect light absorbance of the suspension of isolated brain mitochondria (A) or mitochondrial matrix volume measured with radioisotopes (B).

Mitochondria were incubated in the KCl-based incubation medium supplemented with 3 mm succinate and 3 mm glutamate. A, representative traces obtained with and without openers and blockers of the mitoK_ATP, and with valinomycin (0.4 nm). Openers (50 μm each) and blockers (0.5 mm 5-HD, 0.25 mm ATP) of putative mitoK_ATP were added as indicated to mitochondria 4–5 min after addition of mitochondria into the chamber, when the initial decrease in light absorbance was completed. The openers were added in the presence of 0.5 mm ATP. Higher and lower doses of the openers (10 and 100 μm) did not produce an effect beyond the deflection caused by a vehicle (1.5 μl DMSO, 0.5% final concentration) alone. The presence of the higher concentrations of DMSO (1–1.5% final concentration) in the medium produced a larger effect of the vehicle, but did not improve the effects of diazoxide or other DMSO-dissolved openers. Exclusion of BSA from the medium did not result in changes in the effect of the openers or blockers (not shown). Diazoxide, cromakalim and pinacidil were solubilized in DMSO. Valinomycin, minoxidil and testosterone were solubilized in ethanol. Ethanol as a vehicle (final concentration 0.5%) did not affect light absorbance measurements (not shown). B, summary of the radioisotope measurements of the mitochondrial matrix volume with and without the openers of the mitoK_ATP, and with valinomycin. Matrix volume in the control was determined in the ice-cold medium. All other matrix volume measurements were performed at 37°C. Vehicle was 0.5% DMSO. *P < 0.05 versus vehicle. Data are means ±s.e.m., n = 3.