Inhibition of mitochondrial proton F0F1-ATPase/ATP synthase by polyphenolic phytochemicals

Abstract

Mitochondrial proton F0F1-ATPase/ATP synthase synthesizes ATP during oxidative phosphorylation. In this study, we examined the effects of several groups of polyphenolic phytochemicals on the activity of the enzyme. Resveratrol, a stilbene phytoalexin that is present in grapes and red wine, concentration-dependently inhibited the enzymatic activity of both rat brain and liver F0F1-ATPase/ATP synthase (IC(50) of 12 - 28 microM). Screening of other polyphenolic phytochemicals using rat brain F0F1-ATPase activity resulted in the following ranking potency (IC(50) in parenthesis): piceatannol (8 microM)>resveratrol (19 microM)=(-)epigallocatechin gallate (17 microM)>(-)epicatechin gallate, curcumin (45 microM)>genistein=biochanin A=quercetin=kaempferol=morin (55 - 65 microM)>phloretin=apigenin=daidzein (approx. 100 microM). Genistin, quercitrin, phloridzin, (+)catechin, (+)epicatechin, (-)epicatechin and (-)epigallocatechin had little effect at similar concentrations. Tannic acid, theaflavins (tea extract) and grape seed proanthocyanidin extract (GSPE) had IC(50) values of 5, 20 and 30 microg ml(-1), respectively. Several monophenolic antioxidants and non-phenolic compounds were ineffective at concentrations of 210 microM or higher. The inhibition of F0F1-ATPase by resveratrol and genistein was non-competitive in nature. The effects of polyphenolic phytochemicals were additive. Both resveratrol and genistein had little effect on the Na(+)/K(+)-ATPase activity of porcine cerebral cortex, whereas quercetin had similar inhibitory potency as for F0F1-ATPase. In conclusion, the ATP synthase is a target for dietary phytochemicals. This pharmacological property of these phytochemicals should be included in the examination of their health benefits as well as potential cytotoxicity.

Figure 1

Structures of selected phytochemicals tested for their inhibitory effects on the rat mitochondrial F0F1-ATPase/ATP synthase activity. Many have been shown to be phytoestrogens, such as stilbenes, isoflavones and flavones.

Figure 2

Effects of resveratrol on F0F1-ATPase/ATP synthase activity of rat brain mitochondrial preparations. (A) Typical examples of spectrophotometric read-out showing the absorbance change at 340 nm induced by 20 μl digitonin-solubilized brain mitochondrial preparation (51 μg protein) in the presence of ethanol vehicle, methanol vehicle, 7 μg ml⁻¹ oligomycin in methanol, 7 μM diethylstilbestrol in ethanol and 14 μM resveratrol in ethanol. Resveratrol itself and, to a lesser degree, diethylstilbestrol and oligomycin, had extinction at 340 nm, so the starting point had higher absorbance. Diethylstilbestrol was included here as a comparison. Oligomycin and efrapeptin (see below and text) were included for determination of the F0F1-ATPase activity in the preparations. (B) The concentration-dependent effect of 0.7–70 μM resveratrol on digitonin- and CHAPS-solubilized rat brain mitochondrial preparations. Data are expressed as means (s.d.) from three experiments. Also shown is the concentration-dependent effect of oligomycin and efrapeptin. For studying the effect of efrapeptin on ATPase activity, efrapeptin was pre-incubated at 26°C with the solubilized preparation for 6 min to allow for binding. The F0F1-ATPase activity in 0.7% vehicle (this and following figures) was defined as 100%. (C) Effect of resveratrol on ATP synthesis and hydrolysis catalysed by rat brain submitochondrial preparations. F0F1-ATPase activity of rat brain submitochondrial preparations was determined similarly as solubilized mitochondrial preparation except that 2 μg ml⁻¹ antimycin A was present. Data are expressed as means (s.d.) from three experiments. The F0F1-ATPase/ATP synthase activity in vehicle was defined as 100%.

Figure 3

Concentration-dependent effects of selected phytochemicals on total ATPase activity of digitonin-solubilized rat brain mitochondrial preparations. These compounds include isoflavones (A), flavones (B), catechins (C) and several others (D). Experiments were conducted as in Figure 2. Control experiments indicated that (−)epicatechin-gallate, tannic acid and several flavonoids affected ADP-induced responses, i.e. pyruvate kinase or lactate dehydrogenase; therefore, the total ATPase activity was used here. The inhibition of ADP-induced responses was from 28 to 76% at 70 μM with kaempferol<apigenin<quercetin<(−)epicatechin gallate<morin. This inhibition, however, will not significantly affect the estimation of IC₅₀ for the ATPase since ATP hydrolysis was the limiting step and the residual oligomycin-insensitive ATPase activity is low (13.4 (1.1)% of total activity). ‘NO' indicates the absence of phytochemicals.

Figure 4

Inhibition of F0F1-ATPase of digitonin-solubilized brain mitochondrial preparation by resveratrol (7 μM) and genistein (50 μM) at several concentrations of 0.1–4 mM ATP as shown by Lineweaver–Burk plots. The linear correlation coefficients for all three groups are between 0.996 and 0.999. Note the different V_max among the control, resveratrol and genistein, and the change in K_m induced by resveratrol. The control F0F1-ATPase activity of the preparation at 4 mM ATP was 0.797 (0.007) μmol ATP hydrolysed min⁻¹ (mg protein)⁻¹ from three trials performed before, during and after the experimental trials.

Figure 5

Effects of resveratrol, genistein and quercetin on porcine cerebral cortex Na⁺/K⁺-ATPase. Each point corresponds to a single trial except for control experiments, which were conducted at least three times with variation less than 3%.