Different oxidants and PKC isozymes mediate the opposite effect of inhibition of Qi and Qo site of mitochondrial complex III on calcium currents in rat cortical neurons

Biochimica et Biophysica Acta 1803 (2010) 1072–1082

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m c r

Different oxidants and PKC isozymes mediate the opposite effect of inhibition of Qi and Qo site of mitochondrial complex III on calcium currents in rat cortical neurons Pei-Ying Wu, Bin Lai, Yi Dong, Ze-Min Wang, Zi-Cheng Li, Ping Zheng ⁎ State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, China

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Article history: Received 10 March 2010 Received in revised form 29 April 2010 Accepted 3 May 2010 Available online 19 May 2010 Keywords: Cortical neurons Mitochondrial complex III Whole-cell patch-clamp Calcium currents Reactive oxygen species Protein kinase C

a b s t r a c t The inhibition of the complex III of the mitochondrial respiratory chain under hypoxia-ischemia has been observed. However, the downstream events of this inhibition remain to be studied. In this paper, we used the Qi site inhibitor antimycin A and the Qo site inhibitor myxothiazol to inhibit the Qi site and the Qo site of the complex III and studied the effect and mechanism of the inhibition of these sites on voltage-gated Ca2+ currents (ICa) in rat prefrontal neurons with whole cell patch-clamp method in slices. The results showed that antimycin A inhibited ICa, but myxothiazol increased it. Further mechanism study showed that antimycin A inhibited ICa via the H2O2-hydroxyl radicals/cPKC (mainly PKCβI) pathway, whereas myxothiazol increased ICa via the superoxide anion/nPKC (mainly the PKCδ) pathway. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Mitochondria play a central role in the survival and death of neurons. Under physiological and pathophysiological conditions, a number of changes to mitochondrial function have been observed. Among them, it has been proposed that the inhibition of mitochondrial respiration is one of the earliest events occurring in ischemic tissue [1–3]. The inhibition of the complex I, complex II, complex III and complex IV of the mitochondrial respiratory chain under hypoxiaischemia has been observed [4–11], especially the inhibition of the complex III has received considerable attention because the complex III has a special electron transfer pathway [12] and the consequence of its inhibition may be complicated. However, the downstream events of the inhibition of the complex III remain to be studied. The complex III is a complex of membrane proteins, consisting of 11 different polypeptides [13]. There are two centers in the complex III: the Qo center, where ubiquinol is oxidized; and the Qi center, where ubisemiquinone is reduced [14]. Using the Qi site inhibitor antimycin A to mimic the complex III response to hypoxia, Chandel et al studied the downstream event of the inhibition of the Qi site of the complex III and found that antimycin A could lead to the stabilization of the hypoxia-inducible factor-1α [15]. Another downstream event of the inhibition of the Qi site of the complex III from our previous

⁎ Corresponding author. State Key Laboratory of Medical Neurobiology, Fudan University Shanghai Medical College, 138 Yixueyuan Road, Shanghai, 200032, People's Republic of China. Tel.: +86 21 54237437; fax: +86 21 64174579. E-mail address: [email protected] (P. Zheng). 0167-4889/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2010.05.001

study was that it could lead to a decrease in both transient and persistent sodium currents and thus inhibit neuronal excitability [16]. In addition, our previous study found that the inhibition of the Qo site of the complex III with the Qo site inhibitor myxothiazol could increase the persistent sodium currents and the neuronal excitability [17]. However, the downstream events of the inhibition of the Qi and Qo site of the complex III may be more complicated because the products of the inhibition of these two sites are reactive oxygen species (ROS), which may have multiple potential targets. In this paper, we used antimycin A and myxothiazol to inhibit the Qi and Qo site, respectively and studied the effects and mechanism of the inhibition of these two sites on voltage-gated Ca2+ channels in rat prefrontal neurons with whole cell patch-clamp method in slices. This investigation is of significant importance because the voltage-gated Ca2+ channels have been known to play important roles in the survival of neurons under hypoxia. 2. Materials and methods 2.1. Preparation of cortical slices 14- to 20 day-old Sprague-Dawley rats were anesthetized with chloral hydrate (400 mg/kg, i.p.). All experimental procedures conformed to Fudan University as well as the international guidelines on the ethical use of animals and all efforts were made to minimize the number of the animals used and their suffering. Slices were prepared according to the procedures described previously [18]. Briefly, after the rat was decapitated, the brain was quickly excised and immersed in the ice-cold artificial cerebrospinal fluid (ACSF)

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containing 125 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, 10 mM sucrose and saturated with 95% O2/5% CO2. A block of tissue containing the medial prefrontal cortex (mPFC) was cut and placed on a layer of moistened filter paper glued to the cutting stage of a vibratome (VT1000M/E, Leica, Wetzlar, Germany). Coronal slices (380 μm) were cut and then transferred into an incubating chamber (30-32 °C) where they stayed for at least one hour before recordings began. 2.2. Visualization of pyramidal cells To visualize the cell, the slice was placed in a recording chamber and viewed with a fixed stage, upright microscope equipped with Nomarski optics and a 40X water immersion objective (Olympus, Tokyo, Japan). To increase the clarity of the image, the infrared light was used to illuminate the slice. The resultant infrared DIC (differential interference contrast) images were visualized on a black-white TV monitor through the use of a low light sensitive CCD camera. Recordings were made from pyramidal cells of the mPFC. Pyramidal cells were identified by their pyramidal shape and the presence of the apical dendrite.

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indoyl-3-yl}-3-(1-methyl-1H-indoyl-3-yl) maleimide methane sulfonate (RO318220), 5,6-bis[(4-fluorophenyl)amino]-1H-isoindole-1,3 (2H)-dione (CGP53353), rottlerin, bryostatin and thymeleatoxin were purchased from Sigma. Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) was from Calbiochem. Tetrodotoxin (TTX) was made in the Research Institute of the Aquatic Products of Hebei. Other reagents in AR grade were products of Shanghai Chemical Plant. All drugs were dissolved in dH2O, except for antimycin A, myxothiazol, PHEN, chelerythrine, CGP53353 and bryostatin, which were dissolved in DMSO. When DMSO was used as vehicle, drugs were initially dissolved in 100% DMSO and then diluted into the ACSF at a final DMSO concentration of 0.1%. In vehicle control experiments, we confirmed that the final concentration of DMSO (0.1%) in the ACSF had no detectable effects on the Ca2+ currents we observed. Antimycin A, myxothiazol, PHEN, MPG, SOD, chelerythrine, RO318220, CGP53353, rottlerin, bryostatin, thymeleatoxin, MnTBAP and TTX were applied by bath perfusion. 3. Results 3.1. Antimycin A decreases the peak amplitude of ICa, but myxothiazol increases it

2.3. Whole cell recordings The slice was continuously perfused with the ACSF. Electrodes were pulled from glass capillaries using a Narishige micropipetter puller (model PB-7, Narishige, Japan). They had a resistance of 4-6 MΩ when filled with the patch pipette solution containing 135 mM CsCl, 2 mM MgCl2·6H2O, 0.1 mM CaCl2, 1 mM EGTA, 2 mM ATP-K2, 0.1 mM GTP-Na3, 10 mM HEPES, pH adjusted to 7.4 by CsOH. To record Ca2+ currents, the external fluid was changed to the solution containing: 110 mM NaCl, 5 mM KCl, 5 mM BaCl2, 2 mM MgCl2·6H2O, 10 mM glucose, 1.25 mM NaH2PO4, 26 mM NaHCO3, 5 mM 4-AP, 10 mM TEA·Cl, pH adjusted to 7.4. Ba2+ was substituted for Ca2+ as the charge carrier in order to reduce problems associated with the voltage clamping of large Ca2+ currents [19]. Under these recording conditions, depolarizations of 500 ms in duration from -60 to 40 mV in 10 mV increments from a holding potential of -70 mV were applied to activate the voltage-gated Ca2+ currents (ICa). The current-voltage relationship of ICa recorded here was similar to those reported by other authors [20]. ICa were obvious at the potential of -30 mV, peaked at the potential of -10 mV and reversed at the potential around +30 mV (n = 6). ICa were further demonstrated by the experiment that the voltage-gated Ca2+ channel blocker cadmium (100 μM) could completely abolish these currents (n = 8). The current signals were recorded with an Axopatch 200B amplifier (Axon, Union City, USA) connected to a Digidata1200 interface (Axon, Union City, USA). The data were digitized and stored on disks using pClamp (Axon, Union City, USA). All experiments were conducted at room temperature (21–24 °C). 2.4. Data analysis Statistical significance between two groups was evaluated by Student's two-tailed t test. The significance of the differences between multiple groups at different time-points was evaluated by one-way ANOVA with a post hoc Tukey test. All values were expressed as means ± standard errors of means (SEM) and the number of cells (n) in each group was given. 2.5. Drugs ATP·K2, GTP·Na3, CsOH, CdCl2, tetraethylammonium (TEA), 4aminopyridine (4-AP), antimycin A, myxothiazol, 1,10 phenanthroline (PHEN), 2-mercaptopropionylglycine (MPG), superoxide dismutase (SOD), chelerythrine, 3-{1-[3-(amidinothio) propyl]-1H-

Addition of antimycin A and myxothiazol into the bath solution produced an opposite effect on the peak amplitude of ICa in cortical neurons. As shown in the left panels of Fig. 1, antimycin A (3 μM) significantly decreased the peak amplitude of ICa. The averaged peak amplitude of ICa was reduced from 621.7 ± 43.7 pA before antimycin A to 371.8 ± 27.8 pA at 4 min after antimycin A (n = 6, P b 0.05). The time course showed that the effect of antimycin A became apparent at 1.5 min (P b 0.05) after antimycin A and persisted throughout the rest of the experiment. We also analyzed the voltage dependence of the effect of antimycin A on ICa by constructing the current-voltage relationship of ICa before and after antimycin A. The result showed that the inhibitory effect of antimycin A was obvious when the membrane potential was depolarized to the range between -30 mV and +10 mV. The maximal inhibition percentage was 51.5% at the potential of -20 mV. However, myxothiazol (5 μM) significantly increased the peak amplitude of ICa (the right panels of Fig. 1). The averaged peak amplitude of ICa was increased from 614.1 ± 43.7 pA before myxothiazol to 802.3 ± 42.4 pA at 4 min after myxothiazol (n = 6, P b 0.05). The time course showed that the effect of myxothiazol appeared at 2 min (P b 0.05) after myxothiazol and persisted throughout the rest of the experiment. The experiment of the voltage dependence of the effect of myxothiazol showed that myxothiazol was effective when the membrane potential was within the range between -30 mV and +10 mV. The maximal potentiation percentage was 52.6 % at the membrane potential of -30 mV. 3.2. Roles of oxidants in the effects of antimycin A and myxothiazol on ICa To test the roles of oxidants in the effects of antimycin A and myxothiazol on ICa, we observed the influence of the antioxidant PHEN and MPG on the effects of antimycin A and myxothiazol. The results showed that PHEN (10 μM) or MPG (300 μM) could abolish the inhibitory effect of antimycin A on ICa (Fig. 2A and B, n = 6), but could not abolish the potentiation effect of myxothiazol on ICa (Fig. 2C and 2D, n = 6). MPG or PHEN alone had no effects on ICa (n = 6). In addition, H2O2 (100 μM) could mimic the effect of antimycin A on ICa (Fig. 3). The averaged peak amplitude of ICa was reduced from 601.5 ± 49.6 pA before H2O2 to 369.8 ± 39.4 pA at 4 min after H2O2 (n = 6, P b 0.05, compared to control). However, the superoxide scavengers SOD (10 U/ml) and MnTBAP (10 μM) could cancel the potentiation effect of myxothiazol on ICa (Fig. 4A and B, n = 6). SOD or MnTBAP alone had no effect on ICa (n = 6). We also observed the influence of MnTBAP on the effect of antimycin A. The result showed that MnTBAP

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Fig. 1. Effect of antimycin A and myxothiazol on ICa in cortical neurons. A: raw data before and 4 min after antimycin A (3 μM). Holding potential: -70 mV. B: the averaged amplitude of ICa before and 4 min after antimycin A (3 μM). n = 6, *Pb 0.05, compared to control. C: the time course of the antimycin A (3 μM) effect. n = 6, *Pb 0.05, compared to control before antimycin A. D: the averaged current-voltage relationship for the amplitude of ICa from 6 cells before and 4 min after antimycin A (3 μM). E: raw data before and 4 min after myxothiazol (5 μM). Holding pestilential: -70 mV. F: the averaged amplitude of ICa before and 4 min after myxothiazol (5 μM). n = 6, *Pb 0.05, compared to control. G: the time course of the myxothiazol (5 μM) effect. n = 6, *Pb 0.05, compared to control before myxothiazol. H: the averaged current-voltage relationship for the amplitude of ICa from 6 cells before and 4 min after myxothiazol (5 μM).

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Fig. 2. Influence of the antioxidant PHEN and MPG on the effect of antimycin A and myxothiazol on ICa in cortical neurons. A and B: influence of PHEN (10 μM) and MPG (300 μM) on the effect of antimycin A (3 μM) on ICa. n = 6, *P b 0.05, compared to PHEN or MPG before antimycin A; #P b 0.05, compared to antimycin A alone. C and D: influence of PHEN (10μM) and MPG (300 μM) on the effect of myxothiazol (5 μM) on ICa. n = 6, *P b 0.05, compared to PHEN or MPG before myxothiazol.

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(10 μM) had no significant influence on the inhibitory effect of antimycin A on ICa (Fig. 4C, n = 6).

3.3. Roles of protein kinase C (PKC) in the effects of antimycin A and myxothiazol on ICa To test the role of PKC in the effects of antimycin A and myxothiazol on ICa, we observed the influence of the PKC inhibitor chelerythrine [21] on the effects of antimycin A and myxothiazol. The results showed that in the presence of chelerythrine (2.5 μM), the effects of both antimycin A and myxothiazol disappeared (Fig. 5A and B, n = 6). Chelerythrine alone had no effects on ICa (n = 6). To examine which subtypes of PKC were responsible for the effect of antimycin A and myxothiazol on ICa, we observed the influence of

the selective PKC subtype inhibitors on the effect of antimycin A and myxothiazol. We first observed the influence of RO318220, a PKC inhibitor that was selective for the classical PKC isoforms (including α, βI, βII and γ subtypes) [22], on the effect of antimycin A and myxothiazol. As shown in Fig. 6, RO318220 (3 μM) could abolish the effect of antimycin A (Fig. 6A, n = 6), but had no influence on the effect of myxothiazol (Fig. 6B, n = 6). RO318220 alone had no effects on ICa (n = 6). Next, we examined the influence of the PKCβ-specific inhibitor CG53353 (IC50 value for PKCβII was 0.41 μM and for PKCβI was 3.8 μM) [23] on the effect of antimycin A on ICa. First, we used this compound at 4 μM to inhibit both PKCβII and PKCβI. The result showed that in the presence of CGP53353 (4 μM), the effect of antimycin A disappeared (Fig. 7A, n = 6). However, when we used 0.5 μM CGP53353, which was selective for PKCβII [24], we did not find a significant influence of CGP53353 on the effect of antimycin A (Fig. 7B, n = 6). CGP53353 ( 4 μM and 0.5 μM) alone had no effects on ICa (n = 6). When we examined the influence of rottlerin (5 μM), which was selective for PKCδ (One isoform of the novel PKC) [25], on the effect of myxothiazol, we found that rottlerin (5 μM) could abolish the effect of myxothiazol on ICa (Fig. 8A, n = 6), whereas it had no influence on the effect of antimycin A on ICa (Fig. 8B, n = 6). Rottlerin (5 μM) alone had no effects on ICa (n = 6). We also examined the effect of the classical PKC activator thymeleatoxin [26] and the PKCδ activator bryostatin [27] on ICa. The result showed that thymeleatoxin (100 nM) and bryostatin (100 nM) could mimic the effect of antimycin A and myxothiazol on ICa, respectively. The averaged peak amplitude of ICa was decreased from 644.3 ± 27.5 pA before thymeleatoxin to 407.4 ± 33.8 pA at 4 min after thymeleatoxin (Fig. 9A, n = 6, P b 0.05), whereas the averaged peak amplitude of ICa was increased from 638.7 ± 30.8 pA before bryostatin to 812.1 ± 33.9 pA at 4 min after bryostatin (Fig. 9B, n = 6, P b 0.05). 4. Discussion

Fig. 3. Effect of H2O2 on ICa in cortical neurons. A: raw data before and 4 min after H2O2 (100 μM). Holding potential: -70 mV. B: the averaged amplitude of ICa before and 4 min after H2O2 (100 μM). n = 6, *P b 0.05, compared to control. C: the time course of the H2O2 (100 μM) effect. n = 6, *P b 0.05, compared to control before H2O2.

One of the main findings of the present study is that the Qi site inhibitor antimycin A inhibits the amplitude of ICa, but the Qo inhibitor myxothiazol increases it. To our knowledge, this is the first report about the opposite effect of the inhibition of these two sites on ICa. We further explored the signaling pathways that connected the inhibition of these two sites to the changes in ICa. Both antimycin A and myxothiazol are highly selective inhibitors of the mitochondrial complex III. In our previous study, using the method described by Balijepalli et al [28], we confirmed that antimycin A and myxothiazol indeed could inhibit the activity of the complex III. The activity of complex III was reduced by 20.0 ± 4.6% and 17.0 ± 2.8% (n = 5) in response to 3 μM antimycin A and 5 μM myxothiazol, respectively. These results are consistent with those reported for the antimycin A-induced inhibition (32.2 ± 6.8%) [29] of the complex III and the ischemia-induced inhibition (22%) [30] of the complex III. Moreover, the binding site of antimycin A and myxothiazol in the complex III has been studied by structure-activity analysis using either synthetic analogues of the inhibitors or resistant mutants of the protein [31–36] and by X-ray crystallography [37,38]. These studies clearly demonstrated that antimycin A targeted specifically the Qi site of the complex III, whereas myxothiazol targeted specifically the Qo site of the complex III. However, as analyzed by the following discussion, the signaling pathways that connect the inhibition of the Qi and Qo site to ICa are different and thus the influence of the inhibition of these two sites on ICa is opposite. It has been known that the action of antimycin A is to block specifically the electron transfer from heme bL to ubisemiquinone in the Qi site [39] and increases the formation of ubisemiquinone radical,

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Fig. 4. Influence of superoxide scavengers on the effect of myxothiazol and antimycin A on ICa in cortical neurons. A and B: influence of the superoxide scavengers SOD (10 U/ml) and MnTBAP (100 μM) on the effect of myxothiazol (5 μM) on ICa. n = 6, #P b 0.05, compared to myxothiazol alone. C: influence of MnTBAP (100 μM) on the effect of antimycin A (3 μM) on ICa. n = 6, *P b 0.05, compared to MnTBAP before antimycin A.

which can result in ROS generation. Therefore, it is most likely that antimycin A produces its effect on ICa by ROS. This hypothesis is confirmed by the present results that the antioxidant MPG or PHEN can completely abolish the effect of antimycin A on ICa. Moreover, we propose that among different ROS, H2O2 may play an important role in the antimycin A-induced inhibition of ICa. The evidence supporting this statement is (1) since the Qi site is oriented toward the mitochondrial matrix [40,41] where there are high levels of Mnsuperoxide dismutase that can transfer the entered superoxide anion into H2O2 [42], H2O2 should be one of the major products after the inhibition of the Qi site by antimycin A. This statement has been demonstrated by the study from Chen et al [43]; (2) exogenous application of H2O2 can mimic the effect of antimycin A on ICa (the present result). However, it appears that H2O2 needs to have access to

heme Fe2+ to be converted to hydroxyl radicals in the Fenton reaction. The evidence supporting this statement is (1) PHEN, as a metal chelator, can chelate iron effectively and thus act as a powerful antioxidant due to its inhibition of iron-catalyzed Fenton reaction [44]. So the present result that PHEN can cancel the effect of antimycin A on IC suggests that the produced H2O2 needs heme Fe2+ to be converted to hydroxyl radicals; (2) although MPG, as a thiol group–containing compound, can react with all reactive oxygen species, it has a preference for hydroxyl radicals in its scavenging action [45,46]. Thus, the present result that MPG can cancel the effect of antimycin A on ICa suggests that hydroxyl radicals may play an important role in the effect of antimycin A on ICa. However, it appears that the Qo inhibitor myxothiazol produces the increasing effect on ICa via another ROS pathway. In the Qo site,

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Fig. 5. Influence of the wide spectrum PKC inhibitor chelerythrine on the effect of antimycin A and myxothiazol on ICa in cortical neurons. A: influence of chelerythrine (2.5 μM) on the effect of antimycin A (3 μM) on ICa. n = 6, #P b 0.05, compared to antimycin A alone. B: influence of chelerythrine (2.5 μM) on the effect of myxothiazol (5 μM) on ICa. n = 6, #P b 0.05, compared to myxothiazol alone.

Fig. 6. Influence of the cPKC inhibitor RO318220 on the effect of antimycin A and myxothiazol on ICa in cortical neurons. A: influence of RO318220 (3 μM) on the effect of antimycin A (3 μM) on ICa. n=6, #Pb 0.05, compared to antimycin A alone. B: influence of RO318220 (3 μM) on the effect of myxothiazol (5 μM) on ICa. n=6, *Pb 0.05, compared to RO318220 before myxothiazol.

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Fig. 7. Influence of the cPKCβ inhibitor CG53353 (4 μM) and the cPKCβII inhibitor CG53353 (0.5 μM) on the effect of antimycin A on ICa in cortical neurons. A: influence of CG53353 (4 μM) on the effect of antimycin A (3 μM) on ICa. n = 6, #P b 0.05, compared to antimycin A alone. B: influence of CG53353 (0.5 μM) on the effect of antimycin A (3 μM) on ICa. n = 6, *P b 0.05, compared to CG53353 before antimycin A.

Fig. 8. Influence of the PKCδ inhibitor rottlerin on the effect of antimycin A and myxothiazol on ICa in cortical neurons. A: influence of rottlerin (5 μM) on the effect of myxothiazol on ICa. n = 6, #P b 0.05, compared to myxothiazol alone. B: influence of rottlerin (5 μM) on the effect of antimycin A ( 3μM) on ICa. n = 6, *Pb 0.05, compared to rottlerin before antimycin A.

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Fig. 9. Effect of the cPKC activator thymeleatoxin (100 nM) and the nPKCδ activator bryostatin (100 nM) on ICa in cortical neurons. n = 6, *P b 0.05, compared to control.

the oxidation of ubiquinol (QH2) delivers two electrons divergently to the Rieske iron–sulfur cluster (ISP) and the bL heme (Fig. 10). The binding site of myxothiazol in the Qo site has been examined by a number of studies. Brasseur et al proposed that the myxothiazol binding pocket was underneath the cd helices of cytochrome b [47]. Using X-ray crystallography analysis Zhang et al also proposed that the myxothiazol binding site was at the site from which the electron transfer from the ubisemiquinone to the bL heme occurred [48]. With the analysis of the absorption spectrum of bL heme and ISP, it was shown that myxothiazol caused solely a red-shift of the bL heme's spectrum [49]. Thus, for the bifurcated electron transfer path in the Q0 site, myxothiazol blocks the electron transfer from the ubisemiquinone to the bL heme. Thus, after the binding of myxothiazol, one electron still moves to ISP, but the pathway of another electron from the ubisemiquinone to the bL heme is blocked, so resulting in the formation of ubisemiquinone radicals, which then leave the Qo site and generate superoxide anion. Moreover, since the x-ray structure of the complex III reveals that the Qo site is oriented toward the intermembrane space [40,41], the superoxide anion generated at this site can move easily into the cytoplasm [50]. This statement has been confirmed by the result that myxothiazol can promote the release of the superoxide anion from the mitochondria into the cytoplasm [51–53]. Therefore, it is likely that myxothiazol produces the increasing effect on I Ca by the superoxide anion. This hypothesis is confirmed by our results that the superoxide scavenger MnTBAP and SOD can completely abolish the effect of myxothiazol. Moreover, our results suggested that it was the superoxide anion itself rather than the H2O2 and hydroxyl radicals that mediated the effect of myxothiazol on ICa because MPG and PHEN had no influence on the effect of myxothiazol.

Although myxothiazol is a potent inhibitor of the complex III, it has also been found to inhibit the activity of the complex I [54]. This ability of myxothiazol can potentially complicate interpretation of results as the complex I [55] is also a source of ROS production. However, since the NADH dehydrogenase site of electron leak from the complex I [56] is located in the matrix side of the inner mitochondrial membrane [57], the superoxide anion production from the complex I is directed into the mitochondrial matrix, where the produced superoxide anion is transferred into H2O2 by Mnsuperoxide dismutase. So, even though some ROS was produced due to an inhibition of the complex I by myxothiazol, the ROS entering from the mitochondria into the cytoplasm was H2O2, rather than superoxide anion. It has been known that ROS can activate PKC [58–62]. Therefore, it is possible that the activation of PKC is involved in the effect of antimycin A and myxothiazol on ICa. To test this hypothesis, we observed the influence of the wide spectrum PKC inhibitor chelerythrine on the effect of antimycin A and myxothiazol. The result showed that in the presence of chelerythrine the effect of both antimycin A and myxothiazol disappeared, suggesting that the activation of PKC played a key role in the effects of antimycin A and myxothiazol on ICa. However, since the effects of antimycin A and myxothiazol on ICa are opposite, we hypothesize that antimycin A and myxothiazol may produce an opposite effect on ICa via activating different PKC isozymes. PKC family includes at least 12 isozymes, which are classified into three major groups [63]. The first group is the conventional PKC (cPKC), which includes α, βI, βII and γ types. The second group, called the novel PKC (nPKC), includes δ, ε, η and θ type. The third group is the atypical PKC (aPKC), including ζ and λ type. To test the role of different PKC isozymes in the effect of antimycin A and myxothiazol on ICa, we observed the influence of the selective PKC isozyme inhibitors on the effect of antimycin A and myxothiazol. The results showed that the cPKC inhibitor could abolish the effect of antimycin A, but had no influence on the effect of myxothiazol; the cPKCβ inhibitor could abolish the effect of antimycin A, but the cPKCβII inhibitor had no influence; the nPKCδ inhibitor could abolish the effect of myxothiazol, but had no influence on the effect of antimycin A. These results suggest that the cPKC (mainly the cPKCβI) plays a key role in the inhibitory effect of antimycin A on ICa, whereas the nPKC (mainly the nPKCδ) plays a key role in the increasing effect of myxothiazol on ICa. This statement was further supported by our results that the cPKC activator could mimic the inhibitory effect of antimycin A on ICa, whereas the nPKCδ activator could mimic the increasing effect of myxothiazol on ICa. In conclusion, the Qi site inhibitor antimycin A inhibited ICa, but the Qo site inhibitor myxothiazol increased it; antimycin A inhibited ICa via the H2O2-hydroxyl radicals/cPKC (mainly cPKCβI) pathway, whereas myxothiazol increased ICa via the superoxide anion/nPKC (mainly the nPKCδ) pathway (Fig. 10). Significance – The present findings are of important significance for revealing the signal transduction pathways that connect the inhibition of the Qo and Qi site to voltage-gated Ca2+ channels. This finding may also provide some evidence for the possible functional significance of the inhibition of the complex III under hypoxiaischemia.

Acknowledgments This work was supported by Project 30900424, 30670653 and 30821002 of Foundation of National Natural Science of China, the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2009CB52201) and Project B119 of Shanghai Leading Academic Discipline.

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Fig. 10. Schematic representation of the signaling pathways that connect the inhibition of the Qi site by antimycin A and the inhibition of the Qo site by myxothiazol to ICa in cortical neurons. QH2: ubiquinol; ISP: Rieske iron–sulfur cluster; MYX: myxothiazol; ANT: antimycin A.

References [1] K.R. Wagner, M. Kleinholz, R.E. Myers, Delayed onset of neurologic deterioration following anoxia/ischemia coincides with appearance of impaired brain mitochondrial respiration and decreased cytochrome oxidase activity, J Cereb Blood Flow Metab 10 (1990) 417. [2] N. Bouaziz, M. Redon, L. Quere, J. Remacle, C. Michiels, Mitochondrial respiratory chain as a new target for anti-ischemic molecules, Eur J Pharmacol 441 (2002) 35. [3] P. Lipton, Ischemic cell death in brain neurons, Physiol Rev 79 (1999) 1431. [4] J.P. Bolanos, A. Almeida, J.M. Medina, Nitric oxide mediates brain mitochondrial damage during perinatal anoxia, Brain Res 787 (1998) 117. [5] F. Dagani, D. Curti, F. Marzatico, Effect of Ca2+-homopantothenate and mild hypoxia on some enzyme activities evaluated in subcellular fractions from different rat brain regions, Mol Chem Neuropathol 10 (1989) 157. [6] A. Almeida, M. gado-Esteban, J.P. Bolanos, J.M. Medina, Oxygen and glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones but not in astrocytes in primary culture, J Neurochem 81 (2002) 207. [7] J.C. Chavez, P. Pichiule, J. Boero, A. Arregui, Reduced mitochondrial respiration in mouse cerebral cortex during chronic hypoxia, Neurosci Lett 193 (1995) 169. [8] K.R. Wagner, M. Kleinholz, R.E. Myers, Delayed onset of neurologic deterioration following anoxia/ischemia coincides with appearance of impaired brain mitochondrial respiration and decreased cytochrome oxidase activity, J Cereb Blood Flow Metab 10 (1990) 417. [9] K.L. Allen, A. Almeida, T.E. Bates, J.B. Clark, Changes of respiratory chain activity in mitochondrial and synaptosomal fractions isolated from the gerbil brain after graded ischaemia, J Neurochem 64 (1995) 2222. [10] K.J. Brooks, I.P. Hargreaves, T.E. Bates, Nitric-oxide-induced inhibition of mitochondrial complexes following aglycaemic hypoxia in neonatal cortical rat brain slices, Dev Neurosci 22 (2000) 359. [11] K.J. Brooks, I.P. Hargreaves, T.E. Bates, Protection of respiratory chain enzymes from ischaemic damage in adult rat brain slices, Neurochem Res 33 (2008) 1711. [12] A. Matsuno-Yagi, Y. Hatefi, Ubiquinol:cytochrome c oxidoreductase. The redox reactions of the bis-heme cytochrome b in unenergized and energized submitochondrial particles, J Biol Chem 272 (1997) 16928. [13] Z. Zhang, L. Huang, V.M. Shulmeister, Y.I. Chi, K.K. Kim, L.W. Hung, A.R. Crofts, E.A. Berry, S.H. Kim, Electron transfer by domain movement in cytochrome bc1, Nature 392 (1998) 677.

[14] S. Junemann, P. Heathcote, P.R. Rich, On the mechanism of quinol oxidation in the bc1 complex, J Biol Chem 273 (1998) 21603. [15] N.S. Chandel, D.S. McClintock, C.E. Feliciano, T.M. Wood, J.A. Melendez, A.M. Rodriguez, P.T. Schumacker, Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing, J Biol Chem 275 (2000) 25130. [16] B. Lai, L. Zhang, L.Y. Dong, Y.H. Zhu, F.Y. Sun, P. Zheng, Inhibition of Qi site of mitochondrial complex III with antimycin A decreases persistent and transient sodium currents via reactive oxygen species and protein kinase C in rat hippocampal CA1 cells, Exp Neurol 194 (2005) 484. [17] B. Lai, L. Zhang, L.Y. Dong, Y.H. Zhu, F.Y. Sun, P. Zheng, Impact of inhibition of Qo site of mitochondrial complex III with myxothiazol on persistent sodium currents via superoxide and protein kinase C in rat hippocampal CA1 cells, Neurobiol Dis 21 (2006) 206. [18] Z. Wang, P. Zheng, Characterization of spontaneous excitatory synaptic currents in pyramidal cells of rat prelimbic cortex, Brain Res 901 (2001) 303. [19] J.M. Doughty, M. Barnes-Davies, Z. Rusznak, C. Harasztosi, I.D. Forsythe, Contrasting Ca2+ channel subtypes at cell bodies and synaptic terminals of rat anterioventral cochlear bushy neurones, J Physiol 512 (Pt 2) (1998) 365. [20] X.M. Li, J.M. Yang, D.H. Hu, F.Q. Hou, M. Zhao, X.H. Zhu, Y. Wang, J.G. Li, P. Hu, L. Chen, L.N. Qin, T.M. Gao, Contribution of downregulation of L-type calcium currents to delayed neuronal death in rat hippocampus after global cerebral ischemia and reperfusion, J Neurosci 27 (2007) 5249. [21] S.J. Chmura, E. Nodzenski, R.R. Weichselbaum, J. Quintans, Protein kinase C inhibition induces apoptosis and ceramide production through activation of a neutral sphingomyelinase, Cancer Res 56 (1996) 2711. [22] T. Leppanen, U. Jalonen, H. Kankaanranta, R. Tuominen, E. Moilanen, Inhibition of protein kinase C beta II downregulates tristetraprolin expression in activated macrophages, Inflamm Res 57 (2008) 230. [23] S.W. Lee, H.B. Kwak, W.J. Chung, H. Cheong, H.H. Kim, Z.H. Lee, Participation of protein kinase C beta in osteoclast differentiation and function, Bone 32 (2003) 217. [24] S.W. Lee, H.B. Kwak, W.J. Chung, H. Cheong, H.H. Kim, Z.H. Lee, Participation of protein kinase C beta in osteoclast differentiation and function, Bone 32 (2003) 217. [25] M. Gschwendt, H.J. Muller, K. Kielbassa, R. Zang, W. Kittstein, G. Rincke, F. Marks, Rottlerin, a novel protein kinase inhibitor, Biochem Biophys Res Commun 199 (1994) 93.

1082

P.-Y. Wu et al. / Biochimica et Biophysica Acta 1803 (2010) 1072–1082

[26] I.E. Baldeiras, R.M. Santos, L.M. Rosario, Protein kinase C isoform specificity of cholinergic potentiation of glucose-induced pulsatile 5-HT/ insulin release from mouse pancreatic islets, Biol Res 39 (2006) 531. [27] A.C. Carpenter, J.S. Alexander, Endothelial PKC delta activation attenuates neutrophil transendothelial migration, Inflamm Res 57 (2008) 216. [28] S. Balijepalli, M.R. Boyd, V. Ravindranath, Inhibition of mitochondrial complex I by haloperidol: the role of thiol oxidation, Neuropharmacology 38 (1999) 567. [29] D. Janssens, E. Delaive, J. Remacle, C. Michiels, Protection by bilobalide of the ischaemia-induced alterations of the mitochondrial respiratory activity, Fundam Clin Pharmacol 14 (2000) 193. [30] G. Petrosillo, F.M. Ruggiero, V.N. Di, G. Paradies, Decreased complex III activity in mitochondria isolated from rat heart subjected to ischemia and reperfusion: role of reactive oxygen species and cardiolipin, FASEB J 17 (2003) 714. [31] D.E. Robertson, F. Daldal, P.L. Dutton, Mutants of ubiquinol-cytochrome c2 oxidoreductase resistant to Qo site inhibitors: consequences for ubiquinone and ubiquinol affinity and catalysis, Biochemistry 29 (1990) 11249. [32] T. Tron, M. Crimi, A.M. Colson, E.M. Degli, Structure/function relationships in mitochondrial cytochrome b revealed by the kinetic and circular dichroic properties of two yeast inhibitor-resistant mutants, Eur J Biochem 199 (1991) 753. [33] N. Tokutake, H. Miyoshi, H. Nakazato, H. Iwamura, Inhibition of electron transport of rat-liver mitochondria by synthesized antimycin A analogs, Biochim Biophys Acta 1142 (1993) 262. [34] J.P. di Rago, A.M. Colson, Molecular basis for resistance to antimycin and diuron, Q-cycle inhibitors acting at the Qi site in the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae, J Biol Chem 263 (1988) 12564. [35] H. Kim, D. Xia, C.A. Yu, J.Z. Xia, A.M. Kachurin, L. Zhang, L. Yu, J. Deisenhofer, Inhibitor binding changes domain mobility in the iron-sulfur protein of the mitochondrial bc1 complex from bovine heart, Proc Natl Acad Sci U S A 95 (1998) 8026. [36] B.M. Geier, H. Schagger, U. Brandt, A.M. Colson, J.G. Von, Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit, Eur J Biochem 208 (1992) 375. [37] D. Xia, C.A. Yu, H. Kim, J.Z. Xia, A.M. Kachurin, L. Zhang, L. Yu, J. Deisenhofer, Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria, Science 277 (1997) 60. [38] Z. Zhang, L. Huang, V.M. Shulmeister, Y.I. Chi, K.K. Kim, L.W. Hung, A.R. Crofts, E.A. Berry, S.H. Kim, Electron transfer by domain movement in cytochrome bc1, Nature 392 (1998) 677. [39] H. Kim, D. Xia, C.A. Yu, J.Z. Xia, A.M. Kachurin, L. Zhang, L. Yu, J. Deisenhofer, Inhibitor binding changes domain mobility in the iron-sulfur protein of the mitochondrial bc1 complex from bovine heart, Proc Natl Acad Sci U S A 95 (1998) 8026. [40] S. Iwata, J.W. Lee, K. Okada, J.K. Lee, M. Iwata, B. Rasmussen, T.A. Link, S. Ramaswamy, B.K. Jap, Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex, Science 281 (1998) 64. [41] J.L. Smith, Secret life of cytochrome bc1, Science 281 (1998) 58. [42] D. Han, E. Williams, E. Cadenas, Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space, Biochem J 353 (2001) 411. [43] Q. Chen, E.J. Vazquez, S. Moghaddas, C.L. Hoppel, E.J. Lesnefsky, Production of reactive oxygen species by mitochondria: central role of complex III, J Biol Chem 278 (2003) 36027. [44] T.L. Vanden Hoek, Z. Shao, C. Li, P.T. Schumacker, L.B. Becker, Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes, J Mol Cell Cardiol 29 (1997) 2441.

[45] S. Sekili, P.B. McCay, X.Y. Li, M. Zughaib, J.Z. Sun, L. Tang, J.I. Thornby, R. Bolli, Direct evidence that the hydroxyl radical plays a pathogenetic role in myocardial "stunning" in the conscious dog and demonstration that stunning can be markedly attenuated without subsequent adverse effects, Circ Res 73 (1993) 705. [46] R. Bolli, M.O. Jeroudi, B.S. Patel, O.I. Aruoma, B. Halliwell, E.K. Lai, P.B. McCay, Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion. Evidence that myocardial "stunning" is a manifestation of reperfusion injury, Circ Res 65 (1989) 607. [47] G. Brasseur, A.S. Saribas, F. Daldal, A compilation of mutations located in the cytochrome b subunit of the bacterial and mitochondrial bc1 complex, Biochim Biophys Acta 1275 (1996) 61. [48] Z. Zhang, L. Huang, V.M. Shulmeister, Y.I. Chi, K.K. Kim, L.W. Hung, A.R. Crofts, E.A. Berry, S.H. Kim, Electron transfer by domain movement in cytochrome bc1, Nature 392 (1998) 677. [49] U. Brandt, H. Schagger, and J.G.von, Characterisation of binding of the methoxyacrylate inhibitors to mitochondrial cytochrome c reductase, Eur J Biochem 173 (1988) 499. [50] J. St-Pierre, J.A. Buckingham, S.J. Roebuck, M.D. Brand, Topology of superoxide production from different sites in the mitochondrial electron transport chain, J Biol Chem 277 (2002) 44784. [51] J. Sun, B.L. Trumpower, Superoxide anion generation by the cytochrome bc1 complex, Arch Biochem Biophys 419 (2003) 198. [52] T.A. Young, C.C. Cunningham, S.M. Bailey, Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol, Arch Biochem Biophys 405 (2002) 65. [53] F.L. Muller, A.G. Roberts, M.K. Bowman, D.M. Kramer, Architecture of the Qo site of the cytochrome bc1 complex probed by superoxide production, Biochemistry 42 (2003) 6493. [54] E.M. Degli, A. Ghelli, M. Crimi, E. Estornell, R. Fato, G. Lenaz, Complex I and complex III of mitochondria have common inhibitors acting as ubiquinone antagonists, Biochem Biophys Res Commun 190 (1993) 1090. [55] E.M. Degli, A. Ghelli, M. Crimi, E. Estornell, R. Fato, G. Lenaz, Complex I and complex III of mitochondria have common inhibitors acting as ubiquinone antagonists, Biochem Biophys Res Commun 190 (1993) 1090. [56] E.M. Degli, A. Ghelli, M. Crimi, E. Estornell, R. Fato, G. Lenaz, Complex I and complex III of mitochondria have common inhibitors acting as ubiquinone antagonists, Biochem Biophys Res Commun 190 (1993) 1090. [57] N. Grigorieff, Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I), Curr Opin Struct Biol 9 (1999) 476. [58] E. Klann, E.D. Roberson, L.T. Knapp, J.D. Sweatt, A role for superoxide in protein kinase C activation and induction of long-term potentiation, J Biol Chem 273 (1998) 4516. [59] L.T. Knapp, E. Klann, Potentiation of hippocampal synaptic transmission by superoxide requires the oxidative activation of protein kinase C, J Neurosci 22 (2002) 674. [60] L.T. Knapp, E. Klann, Superoxide-induced stimulation of protein kinase C via thiol modification and modulation of zinc content, J Biol Chem 275 (2000) 24136. [61] Y. Banno, Y. Nozawa, Hydrogen peroxide-induced phospholipase D activation and its PKC dependence are modulated by pH changes in PC12 cells, Biochem Biophys Res Commun 312 (2003) 1087. [62] D. Polosukhina, K. Singaravelu, B.J. Padanilam, Activation of protein kinase C isozymes protects LLCPK1 cells from H2O2 induced necrotic cell death, Am J Nephrol 23 (2003) 380. [63] K.J. Way, E. Chou, G.L. King, Identification of PKC-isoform-specific biological actions using pharmacological approaches, Trends Pharmacol Sci 21 (2000) 181.