Table 2 shows the effects
of juglone on Anticancer Compound Library chemical structure the ADP/O and respiratory control ratios (RC). As noted, juglone reduced significantly the ADP/O ratio already at the concentration of 1 μM when β-hydroxybutyrate was the substrate. At the concentration of 5 μM the ADP/O ratio could no longer be determined. The respiratory control ratio was also reduced and eventually abolished, depending on the concentration. Similar results were obtained when succinate was the substrate, but at somewhat higher concentrations. The uncoupling action of juglone was further investigated by measuring the ATPase, NADH-oxidase and succinate-oxidase activities of rat liver mitochondria. The ATPase activity was measured using mitochondria under three different conditions: intact (coupled), freeze-thawing disrupted and 2,4-dinitrophenol uncoupled. Fig. 8A shows that the ATPase activity was stimulated by juglone in the range between 1 and 10 μM, but with a maximum at 2.5 μM. The ATPase activity of disrupted and uncoupled mitochondria, however, was relatively insensitive to juglone in the range up to 2.5 or 5 μM, and inhibited at higher concentrations. The selleck chemical actions of juglone on the NADH- and succinate-oxidase activities are shown in Fig. 8B. The NADH-oxidase activity was stimulated at concentrations between 5 and 10 μM; the succinate-oxidase activity,
however, was not significantly affected. The main conclusion that can be drawn from the bulk of the data obtained in the present work is that juglone is active on liver metabolism and able to affect several metabolic routes which are linked in some way to energy oxyclozanide metabolism. In general, most observations in the perfused liver are compatible with its reported uncoupling action. The most important observations, which have also been traditionally reported for other uncouplers of oxidative phosphorylation are: a)
stimulation of oxygen consumption at low concentrations (Soboll et al., 1978 and Suzuki-Kemmelmeier and Bracht, 1989); b) diminution of the ATP content combined with diminutions in the ATP/ADP and ATP/AMP ratios (Soboll et al., 1978); c) increase in the NADH/NAD+ ratio (Soboll et al., 1978 and Suzuki-Kemmelmeier and Bracht, 1989); d) inhibition of gluconeogenesis (Kelmer-Bracht and Bracht, 1993 and Suzuki-Kemmelmeier and Bracht, 1989) from two different substrates, namely lactate and alanine; e) stimulation of glycolysis as a cytosolic compensatory phenomenon for the diminished mitochondrial ATP production (Soboll et al., 1978 and Suzuki-Kemmelmeier and Bracht, 1989); f) stimulation of glycogenolysis as a means of providing glucose 6-phosphate for the increased glycolytic flux (Lopez et al., 1998 and Soboll et al., 1978). Experiments with isolated mitochondria, based on the original observations of Makawiti et al. (1990), allowed to characterize further the actions of juglone on the organelle.