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Am J Physiol Heart Circ Physiol 291: H2067-H2074, 2006. First published June 23, 2006; doi:10.1152/ajpheart.00272.2006
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Opening mitoKATP increases superoxide generation from complex I of the electron transport chain

Anastasia Andrukhiv, Alexandre D. Costa, Ian C. West, and Keith D. Garlid

Department of Biology, Portland State University, Portland, Oregon

Submitted 15 March 2006 ; accepted in final form 12 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Opening the mitochondrial ATP-sensitive K+ channel (mitoKATP) increases levels of reactive oxygen species (ROS) in cardiomyocytes. This increase in ROS is necessary for cardioprotection against ischemia-reperfusion injury; however, the mechanism of mitoKATP-dependent stimulation of ROS production is unknown. We examined ROS production in suspensions of isolated rat heart and liver mitochondria, using fluorescent probes that are sensitive to hydrogen peroxide. When mitochondria were treated with the KATP channel openers diazoxide or cromakalim, their ROS production increased by 40–50%, and this effect was blocked by 5-hydroxydecanoate. ROS production exhibited a biphasic dependence on valinomycin concentration, with peak production occurring at valinomycin concentrations that catalyze about the same K+ influx as KATP channel openers. ROS production decreased with higher concentrations of valinomycin and with all concentrations of a classical protonophoretic uncoupler. Our studies show that the increase in ROS is due specifically to K+ influx into the matrix and is mediated by the attendant matrix alkalinization. Myxothiazol stimulated mitoKATP-dependent ROS production, whereas rotenone had no effect. This indicates that the superoxide originates in complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain.

reactive oxygen species; mitochondrial ATP-sensitive potassium channel; signaling; protein kinase C

ISCHEMIA-REPERFUSION injury in the heart can be reduced by ischemic preconditioning (IPC) (36) or by administration of an ATP-sensitive K+ (KATP) channel opener before ischemia (19, 31). Both modes of protection depend on mitochondrial KATP (mitoKATP) opening (17) and on an increase in reactive oxygen species (ROS) (38), which act as second messengers of cardioprotection (11). We have hypothesized that ROS arise from mitochondria as a consequence of mitoKATP opening (14), a suggestion that is supported by studies in cells (24, 38, 44). Still lacking, however, is a direct demonstration that mitoKATP opening leads to increased mitochondrial ROS production. Moreover, the mechanism and site of ROS production are unknown.

Electrophoretic K+ influx into respiring mitochondria causes 1) lowered mitochondrial membrane potential ({Delta}{Psi}m), 2) matrix alkalinization as K+ replaces expelled protons, and 3) matrix swelling as weak acid anions replace OH– (10). One of these effects must be responsible for the increased ROS production. Lowering {Delta}{Psi}m is known to decrease the rate of ROS production, so this effect can be excluded. Matrix swelling is believed to be protective of the energy status of cells in certain circumstances (15, 16, 23) but is not believed to increase ROS production. We therefore hypothesized that net K+ influx will cause matrix alkalinization, which will, in turn, cause an increase in mitochondrial ROS production (15). These hypotheses are tested and confirmed in this study.

Here, we demonstrate for the first time that mitoKATP opening increases ROS production in isolated heart and liver mitochondria. We show, by comparison with valinomycin-induced K+ influx, that this effect can be completely explained in terms of K+ influx into the mitochondrial matrix. The K+-induced increase in ROS production is maximal at the K+ flux mediated by mitoKATP and is depressed at higher K+ influx rates. We show that mitoKATP opening causes matrix alkalinization and that ROS production increases strongly with increasing matrix pH. Finally, studies with inhibitors of the electron transport chain indicate that the site of mitoKATP-dependent ROS production is complex I (NADH:ubiquinone oxidoreductase).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (Cbx-H2DCFDA), and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) were purchased from Molecular Probes (Eugene, OR). Protein kinase C (PKC) isozyme-specific peptide antagonists {epsilon}V1–2 (EAVSLKPT) and {delta}V1–1 (SFNSYELGSL) were synthesized with a purity of >98% by EZBiolab (Westfield, IN). All other chemicals were from Sigma-Aldrich Chemical (St. Louis, MO).

Mitochondrial isolation. Mitochondria were prepared from liver and heart of male 220- to 240-g Sprague-Dawley rats exactly as described previously (9). Isolated mitochondria were suspended in "sucrose buffer" containing 250 mM sucrose, 10 mM HEPES, and 2 mM K-EGTA. The stock suspensions were stored on ice and kept aerobic with gentle stirring. Mitochondrial protein was estimated with the biuret method by using BSA as standard (18).

Measurements of Cbx-DCF and Amplex Red fluorescence. We found with heart mitochondria that we could only detect ROS reproducibly with an internal (matrix) probe, whereas with liver mitochondria we could detect ROS by using an external probe. A possible reason for this difference is the relatively greater amount of matrix catalase in the matrix of heart mitochondria (1, 41). Isolated heart mitochondria were diluted to ~5 mg protein/ml in assay medium and incubated with 500 µM Cbx-H2DCFDA for 10 min at room temperature with stirring and oxygen access. During this period, the Cbx-H2DCFDA is hydrolyzed by endogenous esterases to Cbx-H2DCF. The suspension was then diluted 10-fold with mitochondrial isolation buffer and centrifuged at 9,000 g for 5 min to remove external probe, and the final pellet was resuspended in sucrose buffer. ROS production by 0.25 mg mitochondrial protein/ml was measured in assay medium at wavelengths of 503 nm (excitation) and 530 nm (emission) (32).

Isolated liver mitochondria were added directly to the assay medium at 0.25 mg mitochondrial protein/ml. ROS production was measured in assay medium using Amplex Red fluorogenic probe at wavelengths of 550 nm (excitation) and 590 nm (emission) (53). Amplex Red (2 µM) and horseradish peroxidase (4.5 U/ml) were added to assay medium.

Assay medium contained 10 mM glutamate, 2 mM malate, 0.1 mM Pi, 90 mM K-MES, 20 mM imidazole, 12 mM K-TES, 10 mM K-EGTA, 1.38 mM MgCl2, 7.0 mM CaCl2, 200 µM ATP, and 1 µg/ml oligomycin, pH 7.4. Osmolality lay between 270 and 280 mosmol/kgH2O, except where noted, as determined by freezing-point depression. All experiments were performed at 30°C unless otherwise noted.

Measurements of BCECF fluorescence. Matrix pH was measured in isolated rat heart mitochondria as described by Jung et al. (21). Briefly, isolated rat heart mitochondria were incubated with 8 µM BCECF-AM fluorescent probe in the mitochondrial isolation buffer for 10 min at room temperature and aerated by gentle stirring. The mitochondrial suspension was then diluted 10-fold with mitochondrial isolation buffer and spun at 9,000 g for 5 min to remove excess probe. Mitochondria were then resuspended in mitochondrial isolation buffer at ~25 mg protein/ml and stored as described previously. The pH-dependent fluorescence was then measured in the assay medium described above with protein concentration of 0.25 mg/ml by using 509 nm (excitation) and 535 nm (emission) wavelengths. Assays were carried out at 30°C.

Data analysis. Data are given as means ± SE. P values of < 0.05 using paired Student's t-test were considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
KATP channel openers and valinomycin increase ROS production in heart mitochondria. The representative fluorescence traces in Fig. 1A were obtained from heart mitochondria containing Cbx-H2DCF and respiring in K+ medium. Increasing fluorescence indicates a continuous oxidation of Cbx-H2DCF to Cbx-DCF, predominantly by ROS in the matrix (43). We have previously shown that mitoKATP is open in our mitochondrial preparations; that it is closed on adding 200 µM ATP, and opened again on the further addition of channel openers such as diazoxide (7-chloro-3-methyl-2H-1,2,4-benzothiadiazine 1,1-dioxide) or cromakalim [3-hydroxy-2,2-dimethyl-4-(2-oxopyrrolidin-1-yl)-chroman-6-carbonitrile]; and that the pharmacologically opened channel can be closed with agents such as glibenclamide or 5-hydroxydecanoate (5-HD) (10). As shown in Fig. 1A, addition of ATP (200 µM) reduced the rate of Cbx-DCF fluorescence increase, presumably by inhibiting mitoKATP. Additions of 30 µM diazoxide (or 50 µM cromakalim; not shown) increased ROS production in the presence of ATP, whereas 5-HD (300 µM) reversed the increase in observed ROS production induced by diazoxide and cromakalim. Addition of diazoxide or cromakalim in the absence of ATP or the addition of 5-HD in the absence of a KATP channel opener had no effect on Cbx-DCF fluorescence.


Figure 1
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  		Fig. 1. Mitochondrial reactive oxygen species (ROS) production increases with K+ uptake. A: relative fluorescence of Cbx-DCF is plotted against time. Probe-loaded mitochondria (0.25 mg mitochondrial protein/ml) were suspended in assay medium described in METHODS. ATP (200 µM) was present in all experiments except for the trace marked "none." ATP, no further additions; Val, valinomycin (1 pmol/mg protein); Dzx, diazoxide (30 µM); 5-HD, 5-hydroxydecanoate (300 µM). B: rates of H2O2 production by rat heart mitochondria were obtained from the initial slopes of traces such as those shown in A and are plotted as percentage of ATP-inhibited control rate obtained in the absence of drug. Addition of ROS scavenger N-(2-mercaptopropyonyl)glycine (MPG) (1 mM) decreased ROS production in the presence of diazoxide (30 µM) and valinomycin (1 pmol/mg protein) about 10-fold. Results are means and SE of 4 experiments.

 
Figure 1B contains a summary of three to four similar experiments on the effects of diazoxide and valinomycin on ROS production. Note that valinomycin (1 pmol/mg mitochondrial protein) also increased the ATP-inhibited rate of ROS production, but, as previously shown in whole cells (24), 5-HD had no effect on the valinomycin-stimulated ROS production because the K+-specific ionophore valinomycin acts independently of mitoKATP. Addition of 1 mM ROS scavenger N-(2-mercaptopropyonyl)glycine (MPG) decreased both diazoxide- and valinomycin-induced ROS production rates below the ATP-inhibited level (Fig. 1B). Same experiments with 0.3 mM MPG were performed with similar results. On the basis of these results, we conclude that the mitoKATP-dependent increase in ROS is the result of increased K+ influx into the matrix.

Biphasic dependence of ROS production on K+ influx. The finding in Fig. 1 that increased K+ flux leads to increased mitochondrial ROS production is surprising because increased K+ cycling causes increased electron transport (10), which should suppress ROS production (22, 30, 48). We therefore examined the effect of the magnitude of K+ influx on ROS production, with the results shown in Fig. 2. Note that there is a biphasic dependence on valinomycin concentration in both rat heart (Fig. 2A) and rat liver (Fig. 2B) mitochondria. At low concentrations, valinomycin caused a dose-dependent increase in ROS production, and at higher concentrations, valinomycin caused a dose-dependent decrease in ROS production. The latter effect is expected, whereas the former effect is not. The peak ROS increase detected with valinomycin was ~40% over baseline in both heart and liver mitochondria, and similar stimulation was observed by diazoxide and cromakalim. In heart mitochondria, the peak occurred at a titer of ~1 pmol valinomycin/mg mitochondrial protein. In liver mitochondria, the peak of ROS production occurred at a titer of ~0.6 pmol valinomycin/mg mitochondrial protein. As reported in Table 1, these valinomycin concentrations yield a K+ influx for the respective mitochondria that closely matches that caused by KATP channel openers. It is remarkable that the increased K+ influx induced by KATP channel openers is matched so precisely to K+ influx levels where ROS production is maximally enhanced.


Figure 2
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  		Fig. 2. Mitochondrial ROS production exhibits biphasic dependence on K+ influx. A: heart mitochondria. Rates of H2O2 production (obtained from traces as in Fig. 1A) expressed as percentage of ATP-inhibited control rate are plotted against concentrations of valinomycin ({blacksquare}) or carbonylcyanide p-chlorophenylhydrazone (CCCP; bullet). Dashed line represents the mean H2O2 production rate in the presence of 30 µM diazoxide (see Table 1). The curves are representative of 4 independent experiments. The peak increase in response to valinomycin was 43 ± 4% (n = 4) and occurred at 1.1 ± 0.1 pmol valinomycin per mg of mitochondrial protein. B: liver mitochondria. Rates of H2O2 production (obtained from plots as in Fig. 1A) expressed as percentage of ATP-inhibited control rate are plotted against concentrations of valinomycin ({blacksquare}). Dashed line represents the mean H2O2 production rate in the presence of 30 µM diazoxide (see Table 1). Curves are representative of 5 independent experiments. The peak increase in response to valinomycin was 44 ± 5% (n = 4) and occurred at 0.6 ± 0.1 pmol valinomycin per mg of mitochondrial protein. Crom, cromakalim; A-Red, Amplex Red.

 

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  		Table 1. K+ flux-dependent ROS production in rat heart and liver mitochondria

 
Higher concentrations of valinomycin progressively diminish ROS production (Fig. 2, A and B). This is, of course, expected, for valinomycin will progressively increase K+ cycling, causing apparent uncoupling and accelerated respiration, as we have recently demonstrated elsewhere (10). Indeed, uncoupling by a conventional proton-conducting ionophore, over a concentration range giving the same stimulation of respiration (10), shows no stimulation of ROS but simply a monotonic decline (Fig. 2A), as has been reported many times before (22, 30, 48). From these results, we conclude that the biology of mitoKATP is attuned to causing a maximal, but nevertheless modest, stimulation of ROS production. It seems clear that the biphasicity is the result of two ongoing processes with opposite consequences. We see that increased K+ cycling leads to a decline in ROS; it remains to be determined how increasing K+ influx at levels below the peak values leads to an increase in ROS.

Which effect of increased K+ influx causes increased ROS production? Net K+ influx into the matrix has three direct effects on mitochondria: mild uncoupling and lowering of mitochondrial membrane potential ({Delta}{Psi}), matrix swelling, and matrix alkalinization (10). We exclude an effect of mild uncoupling, because we observe a monotonic decrease in ROS production at all concentrations of uncoupler (Fig. 2A; and data not shown). The swelling caused by diazoxide-induced mitoKATP opening corresponds to an increase of matrix water of <20% (10, 16). We tested the effect of matrix swelling on the basal (ATP inhibited) rate of ROS production by lowering the osmolality of the assay medium from 270 to 160 mosmol/KgH2O (causing a 50% increase in matrix volume). There was no significant difference in ROS production (Fig. 3). We then suspended mitochondria in media both more acidic and more alkaline than our standard pH 7.4 medium. Alkaline medium strongly increased ROS production, whereas acidic medium lowered it (Fig. 3).


Figure 3
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  		Fig. 3. Mitochondrial ROS production is increased by matrix alkalinization. Rat heart mitochondria preloaded with 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein (Cbx-H2DCF) probe, were incubated in the presence of 200 µM ATP. H2O2 production was measured at different osmotic strengths (270 vs. 160 mosmol/KgH2O) and different medium pH values (pH 6.9 vs. pH 7.9). The rightmost two columns addressed the effect of matrix acidification on the valinomycin-induced increase in H2O2 production (medium pH = 7.4). The matrix was acidified by substituting 25 mM K-acetate for 25 mM K-MES. Data are presented as means and SE of 3 independent experiments.

 
To distinguish between effects of extramitochondrial and matrix pH, we replaced the MES in the assay medium with 25 mM acetate and reexamined the effect of 1 pmol valinomycin/mg mitochondrial protein. As shown in Fig. 3, acetate prevented the increase in ROS production caused by valinomycin. From these results, we infer that the immediate cause of increased ROS production is matrix alkalinization secondary to net K+ influx.

Alkalinization of the matrix, as protons ejected by respiration are replaced by K+ ions, will be partly although not completely offset by the acidifying effect of the electroneutral uptake of phosphate and other anions. Matrix alkalinization as a result of mitoKATP channel opening has recently been confirmed experimentally by loading the matrix with the membrane-permeant acetoxymethyl ester of the pH-sensitive fluorescent probe BCECF (10). The same technique has been used here to confirm the comparable effects of valinomycin and pharmacological channel openers. Representative traces are contained in Fig. 4A, and the results of a series of experiments are summarized in Fig. 4B. The mitoKATP openers cromakalim (50 µM) and diazoxide (not shown) caused an increase in matrix pH. A similar effect was observed with valinomycin (1 pmol/mg protein) and 4-phorbol-12-beta-myristate-13-acetate (PMA), which has recently been shown to cause mitoKATP opening by activating an endogenous mitochondrial PKC{epsilon} (9). As expected, 5-HD abolished the effects on matrix pH of each of these agents, except for valinomycin. These experiments confirm that net K+ influx causes matrix alkalinization and show once again that the effect of maximum channel opening by diazoxide or cromakalim is quantitatively matched by 1 pmol/mg valinomycin.


Figure 4
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  		Fig. 4. Effects of increasing K+ influx on matrix pH. A: experimental traces. Fluorescence from matrix-loaded 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) is plotted vs. time. BCECF (pKa 6.98) indicates matrix pH because it is 6 times more fluorescent at pH 9 than at pH 5. Probe-loaded mitochondria were suspended in assay medium as described in METHODS. 200 µM ATP was present in all experiments. None, no further additions. Further additions in the indicated traces were made after 25-s incubation: Val, valinomycin (1 pmol/mg protein); crom, cromakalim (50 µM); 5-HD, 5-hydroxydecanoate (300 µM). B: average rates of pH change. Rates of matrix pH change were obtained from traces such as those shown in A and are represented as percentage of ATP-inhibited control. Valinomycin (1 pmol/mg protein), cromakalin (50 µM), and the PKC activator 4-phorbol-12-beta-myristate-13-acetate (PMA; 0.2 µM) each increased d(pHi)/dt about 3-fold, where pHi is pH in the mitochondrial matrix. Effects of all these agents, except valinomycin, were blocked by 5-HD (300 µM). Data are presented as means and SE of 3 independent experiments.

 
Source of ROS. We may expect ROS arising from complex III to be inhibited by myxothiazol, whereas ROS arising from complex I will be stimulated by the downstream block imposed by myxothiazol (37) (and see DISCUSSION). In Fig. 5 we show the effects of myxothiazol and rotenone on ROS production in mitochondria incubated with glutamate plus malate, as detected by the matrix-loaded Cbx-H2DCF probe. Myxothiazol increased probe oxidation while rotenone had no significant effect, suggesting that basal ROS is formed at a site upstream of complex III. Myxothiazol also increased probe oxidation in the presence of valinomycin, suggesting that the ROS occurring as a result of increased K+ influx also is formed at a site upstream of complex III.


Figure 5
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  		Fig. 5. Effects of myxothiazol (Myx) and rotenone (Rot) on mitochondrial ROS production. Data were obtained from plots as in Fig. 1A and are expressed as percentage of ATP-inhibited control rate. Myxothiazol and rotenone were each present at 1 µM, and valinomycin was at 1 pmol/mg mitochondrial protein. Data are means and SE of 3 independent experiments.

 
ROS increased by protein kinase G + cGMP and by phorbol ester. Diazoxide and valinomycin are equally able to increase K+ influx into the matrix (10) and to increase mitochondrial ROS production (Fig. 1). However, these compounds may not be relevant to the in vivo situation, in which we have hypothesized that mitoKATP opening causes increased ROS production when opened during IPC (14). Protein kinase G (PKG) is a known mediator in the cardioprotective signaling pathway (39), and PKG + cGMP have recently been shown to induce opening of mitoKATP in isolated rat heart mitochondria via a mitochondrial PKC{epsilon} (9). In Fig. 6A we show that exogenous PKG + cGMP also induced increased ROS production in isolated mitochondria, to an extent comparable to that observed with valinomycin and diazoxide (cf. Fig. 1). This effect was blocked not only by 5-HD but also by chelerythrine, an inhibitor of PKC. In control experiments (not shown), PKG or cGMP alone had no effect on ROS production.


Figure 6
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  		Fig. 6. Protein kinase G (PKG) + cGMP increases mitochondrial ROS via PKC{epsilon} and mitochondrial ATP-sensitive K+ channel (mitoKATP). Rates of H2O2 production by rat heart mitochondria were obtained from the initial slopes of traces such as those shown in Fig. 1A and are plotted as percentage of ATP-inhibited control rate. A: PKG + cGMP. 0.25 U/ml PKG + 20 µM cGMP increased mitochondrial ROS production by 45%, similar to the increase observed with valinomycin or diazoxide (Table 1). The effect of PKG + cGMP was inhibited by 300 µM 5-HD, a mitoKATP inhibitor; by 50 nM chelerythrine (Chel), a general PKC inhibitor; and by the PKC{epsilon}-specific inhibitor peptide {epsilon}V1–2 (0.5 µM); but not by the PKC{delta}-specific inhibitor peptide {delta}V1–1 (0.2 µM). Data are means and SE of 3 independent experiments. B: PMA. PMA (0.2 µM ), a general PKC activator, increased ROS production by 50%. This effect was inhibited by 300 µM 5-HD, by 50 nM chelerythrine, by 50 nM Ro-318220 (Ro), and by 0.5 µM {epsilon}V1–2. The PKC{delta} inhibitor Gö-6983 (50 nM; Go) and the PKC{delta}-specific inhibitor peptide {delta}V1–1 (0.2 µM) failed to block this PMA effect. Data are means and SE of 3 independent experiments.

 
To determine which PKC isoform is responsible for the observed effects, we used the isoform-specific peptides described by Chen et al. (6). We found that PKG-induced ROS production was inhibited by the PKC{epsilon}-specific inhibitor peptide {epsilon}V1–2 but was not inhibited by the PKC{delta}-specific inhibitor peptide {delta}V1–1 (Fig. 6A). In control experiments (not shown), the peptides alone had no effect on ROS production. Together, these results demonstrate that the increased mitochondrial ROS production caused by exogenous PKG + cGMP requires participation of endogenous PKC{epsilon} and mitoKATP. They also show that isolated mitochondria constitutively contain sufficient endogenous PKC{epsilon} to transmit the cytosolic signal to mitoKATP, supporting the conclusion of Baines et al. (4).

Not surprisingly, a similar response was observed when the phorbol ester PMA was added to isolated mitochondria, for PMA activates PKC directly (45). In Fig. 6B we show that PMA increased ROS production in isolated mitochondria by 50 ± 7%, and this effect was blocked by 5-HD and chelerythrine. The isoform-specific inhibitor peptides used in Fig. 6 confirmed that PKC{epsilon}, and not PKC{delta}, is the isoform that mediates PMA-induced ROS production. We also studied the effects of the PKC inhibitors Ro-318220 and Gö-6983. The former is thought to inhibit PKC{epsilon} (51), while Gö-6983 is known to inhibit the {delta}-isoform (20). In control experiments (not shown) these compounds alone had no effect on ROS production. As shown in Fig. 6B, Ro-318220 (50 nM) but not Gö-6983 (50 nM) blocked PMA-induced ROS increase, again excluding the participation of PKC{delta} in this process.


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The proposal that mitoKATP opening leads to increased mitochondrial ROS production (14) is widely accepted, based in part on studies in intact cells (24, 38, 44) and in part on the effects of free radical scavengers on cardioprotection in isolated intact hearts (3, 8, 12, 40, 47, 49). Nevertheless, there has been no direct demonstration that mitoKATP opening causes increased ROS production, nor has any mechanism been put forward for this effect. This study addresses these issues, beginning with a demonstration that mitoKATP opening in isolated rat heart mitochondria leads to increased ROS production (Fig. 1). Several reports have indicated that Cbx-H2DCF is oxidized to the fluorescent fluorescein derivative by H2O2, particularly in the presence of redox active iron (e.g., in heme), but that it will also be oxidized by ONOO– or ·OH radicals (32, 43). We used the carboxy-derivative of 2',7'-dichlorodihydrofluorescein here as it is reported to be better retained in the compartment in which it is generated (2, 43). We believe that our matrix-loaded probe is probably reporting the generation of H2O2 formed in the matrix by the dismutation of O2–· radicals released to the matrix side of the inner mitochondrial membrane. Matrix superoxide dismutase converts superoxide to H2O2 (1), which presumably has a sufficient lifetime to play a signaling role as a second messenger (11).

Valinomycin, at a dose causing the same K+ influx as a KATP channel opener, quantitatively reproduces the increased ROS production caused by mitoKATP openers (Fig. 1). This fact strongly suggests that K+ influx is directly and completely responsible for the increased ROS production that follows channel activation. We next sought to determine the mechanism by which K+ influx increases ROS in mitochondria. It should be understood that mitoKATP opening will lead only briefly to net K+ inflow but will establish a persistent steady-state characterized by lowered {Delta}{Psi}, increased matrix volume, and increased matrix pH (10). The data in Fig. 2 show that even very small amounts of a classical proton-cycling uncoupler reduces ROS production, as has been shown by others (22, 30, 46, 48). Thus the proton-cycling induced by the K+ cycle cannot itself enhance ROS production. Likewise, the data in Fig. 3 show that simply increasing matrix volume by itself does not increase ROS production. On the other hand, ROS production is markedly increased by alkaline pH (Fig. 3). Furthermore, when acetic acid inflow largely counteracts the matrix alkalinizing effect of K+ inflow, the stimulatory effect of valinomycin on ROS production is blocked (Fig. 3). We conclude, therefore, that matrix alkalinization is the direct cause of increased ROS production.

The interrelated equilibria involved in matrix pH regulation in the presence of phosphate, glutamate, malate, and acetate are complex (15, 34, 50). Therefore, direct confirmation of matrix alkalinization under the conditions that provoke increased matrix ROS production is important confirmation of the argument put forward here. The data in Fig. 4 confirm that increasing K+ conductance causes matrix alkalinization, whether the increased K+ inflow is caused by valinomycin, pharmacological channel openers, or the physiological mechanisms that use endogenous PKC{epsilon}.

Superoxide is formed when a single electron from a redox center in the electron transport chain transfers directly to molecular oxygen (46). The mechanisms of ROS production in mitochondria have been thoroughly discussed by a number of authors (5, 7, 13, 25, 28–30, 33, 35, 37, 46, 48, 52). Two factors permit identification of the complex from which the ROS originate. First, inhibition of any complex in the electron transport chain causes the upstream redox centers to become highly reduced. Second, at constant PO2, superoxide formation is pseudo-first order with the concentration of the reduced site. Superoxide of mitochondrial origin arise from three major sites (mechanisms): 1) the o-site of complex III, 2) redox centers in complex I that have been reduced by reverse electron transport from the Q-pool, and 3) redox centers in complex I that have been reduced by NADH.

Apropos of the fact that we use a matrix-loaded probe, mechanisms 2 and 3 produce ROS largely or exclusively to the matrix (7, 25, 27, 35). Mechanism 1 is largely blocked by myxothiazol (42), mechanism 2 is inhibited by rotenone (30, 48), and mechanism 3 is enhanced by myxothiazol and rotenone (7, 28, 35, 37, 48). In our studies, ROS detection was stimulated by myxothiazol, indicating little or no contribution by mechanism 1. Lack of inhibition by rotenone indicates little or no contribution by mechanism 2. The sensitivity to uncoupler, stimulation by myxothiazol, and relative lack of effect of rotenone shown by the data in Fig. 5 indicate mechanism 3 as a major source of ROS in these experiments.

Complex I contains numerous redox centers that may be capable of partial reduction of O2, and identifying the primary site has proved elusive. Some authors favor components at or near the weakly-reducing N2 non-heme-iron made strongly reducing by back pressure from the transmembrane protonmotive force (5, 29, 37). Other authors consider the flavin or one of the strongly reducing non-heme-iron centers to be more likely (13, 25–28, 48). Recently, Galkin and Brandt (13) found that a mutant complex I lacking detectable iron-sulfur cluster N2 exhibited the same rate of ROS production as wild-type, which appears to exclude the N2 site as the source of ROS. These authors conclude that single electrons are delivered to oxygen from the FMNH2 or FMN semiquinone either directly or via hydrophilic ubiquinone derivatives.

The effect of matrix pH on mechanism 3 has been studied previously. Importantly, our results in Fig. 3 are in agreement with the finding that O2–· production toward the matrix by the highly reducing site in complex I is increased strongly as the pH is increased from 6.8 to 8.6 (13, 25). This result permits a simple explanation for the biphasic dependence of ROS production on valinomycin concentration (Fig. 2B). As K+ influx is increased in the low range, the effect of matrix alkalinization on ROS production exceeds the negative effect of increased electron transport rates. ROS production progressively increases until the increasing rate due to alkalinization equals the declining rate due to increased electron transport. At this point, increased uncoupling due to K+ cycling causes the same effect on ROS production as does increased uncoupling due to a classical uncoupler, and higher values of K+ influx cause ROS to decline.

Our mechanistic conclusions are summarized in the diagrams of Fig. 7, which focuses on the mechanism by which increasing matrix pH causes increased ROS production from complex I, and Fig. 8, which deals with the overall physiology of mitoKATP-dependent ROS production. Bradykinin mimics IPC by generating ROS, and it does so via cGMP activation of PKG (39). We have shown further in isolated mitochondria that PKG + cGMP induce mitoKATP opening via an endogenous PKC{epsilon} (9). The data in Fig. 6 complete this pathway by showing that PKG + cGMP and PMA stimulate mitochondrial ROS production in a PKC{epsilon}-dependent and mitoKATP-dependent manner. Although our studies have the limitation of being performed in vitro on isolated mitochondria, the results are in concordance with observations in cells. We therefore propose that mitoKATP opening in vivo, mediated either through cell signaling or by addition of a KATP channel opener, causes increased ROS production by the mechanisms described in Figs. 7 and 8.


Figure 7
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  		Fig. 7. Effect of matrix pH on ROS production from complex I. Electrons are passed to ubiquinone (Q) from complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain. Although the site of superoxide production has been controversial, recent data suggest that the single electrons are delivered to oxygen from the FMNH2 or FMN semiquinone (13). It can be seen in the diagram that the reduction of ubiquinone requires two matrix protons. Electron flow will therefore be retarded at these points as matrix pH increases, causing increased reduction at the flavin site and consequent increase in steady-state superoxide production. IMS, intermembrane space.

 

Figure 8
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  		Fig. 8. MitoKATP-dependent ROS production. The cytosolic steps of the cardioprotective signaling pathway end with phosphorylation of an unknown outer mitochondrial membrane protein by PKG. This is followed by unknown steps that lead to activation of a mitochondrial PKC{epsilon} (step 1), which in turn phosphorylates and opens mitoKATP (step 2a). It is possible to bypass PKG by activating PKC{epsilon} directly, for example, by using a phorbol ester. It is possible to bypass both PKG and PKC{epsilon} by adding a KATP channel opener such as diazoxide (step 2b). MitoKATP opening causes net K+ uptake into the matrix (step 3), which increases matrix pH (step 4). Increased pH causes a relative retardation of electron flow (step 5) and consequent increase in superoxide production (step 6), as described in the diagram of Fig. 7. Matrix superoxide dismutase converts the superoxide to H2O2 (step 7), which is now available by diffusion to act as a second messenger to activate other kinases (step 8). IM, inner membrane; OM, outer membrane.

 

   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grants HL-67842 and HL-36573 (to K. D. Garlid).


   ACKNOWLEDGMENTS
 
We are grateful to Craig Semrad, Brian Corry, and Heather Barnes for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. D. Garlid, Dept. of Biology, Portland State Univ., PO Box 751, Portland, OR 97207 (e-mail: garlid{at}pdx.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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