Measuring Mitochondrial Function and BK Ca Channel

Mitochondrial respiration and ΔΨ were measured in isolated endothelial mitochondria as previously described [11 (link),64 (link)]. Oxygen uptake was determined polarographically using a Rank Bros. (Cambridge, UK) oxygen electrode in 2.8 ml of incubation medium (50 mM KCl, 70 mM sucrose, 2.5 mM KH₂PO₄, 2 mM MgCl₂, 10 mM HEPES, 10 mM Tris/HCl (pH 7.2), and 0.05% BSA) with 2 mg of mitochondrial protein (at 37 °C). Mitochondrial ∆Ψ was measured simultaneously with oxygen uptake using a tetraphenylphosphonium (TPP⁺)-specific electrode. Measurements were made in the presence of 5 mM succinate (as respiratory substrate), 4 µM rotenone (to inhibit complex I), 2 µg oligomycin (to inhibit ATP synthase), 1.7 µM carboxyatractyloside (to inhibit ATP/ADP antiporter), and 0.15 mM ATP (to activate succinate dehydrogenase). Up to 2 µM iberiotoxin (dissolved in water; Alomone Labs, Israel) or 0.3 mM paxilline (dissolved in methanol; Merck, Poznan, Poland) were used to inhibit the mitoBK_Ca channel.