Fig. 3. Characterization of mitochondrial dysfunction induced by protein thiolation.

Delineation of changes in ATP-linked oxygen consumption, proton leak, and respiratory capacity in cells treated with diamide. (A) Extracellular flux analysis was used to measure mitochondrial function in intact, adherent smooth muscle cells. To probe individual components of respiration that contributed to the consumption of oxygen, oligomycin (1 µg/ml), FCCP (1 µM), and antimycin A (10 µM) were injected sequentially. This allowed for estimation of the contribution of proton leak (Leak) and ATP demand (ATP) to mitochondrial oxygen consumption. The maximal respiratory capacity was determined using the FCCP-stimulated rate. The residual oxygen consumption that occurred after addition of antimycin A was ascribed to non-mitochondrial sources. (B) After baseline measurements of oxygen consumption rate (OCR), diamide was injected to 0 (closed squares), 0.1 (diamonds; dotted line), 0.25 (open squares), or 0.5 (triangles; dotted line) mM final concentrations. At the indicated time, oligomycin (O; 1 µg/ml) was injected and OCR was measured. FCCP (F; 1 µM) was then injected followed by another OCR measurement. Last, antimycin A (A; 10 µM) was injected and OCR was again measured. (C and D) The oligomycin-sensitive and –insensitive rates of oxygen consumption were used to calculate the amount of oxygen consumption that was linked to ATP production (panel C) and proton leak (panel D). (E) The reserve or spare respiratory capacity was calculated in control and diamide-treated cells by subtracting the maximal (FCCP-stimulated) rate of oxygen consumption from the basal OCR. N = 5 per group, *p < 0.05 vs. cells not treated with diamide.