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SPECIAL SECTION ON SYSTEMS BIOLOGY OF THE MITOCHONDRION
Anesthesiology Research Laboratories, Departments of 1Anesthesiology and 2Physiology, 3Cardiovascular Research Center, Medical College of Wisconsin, 4Veterans Affairs Medical Center Research Service, and 5Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin
Submitted 28 April 2006 ; accepted in final form 25 July 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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m). To test this hypothesis, we measured effects of NS-1619, a putative mtKCa channel opener, and valinomycin, a K+ ionophore, on mitochondrial respiration, m, and ROS generation in guinea pig heart mitochondria. NS-1619 (30 µM) increased state 2 and 4 respiration by 5.2 ± 0.9 and 7.3 ± 0.9 nmol O2·min–1·mg protein–1, respectively, with the NADH-linked substrate pyruvate and by 7.5 ± 1.4 and 11.6 ± 2.9 nmol O2·min–1·mg protein–1, respectively, with the FADH2-linked substrate succinate (+ rotenone); these effects were abolished by the mtKCa channel blocker paxilline. m was not decreased by 10–30 µM NS-1619 with either substrate, but H2O2 release was increased by 44.8% (65.9 ± 2.7% by 30 µM NS-1619 vs. 21.1 ± 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H2O2 release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained m, despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K+ influx during states 2 and 4 increases mitochondrial respiration while maintaining m; this allows singlet electron uptake by O2 and ROS generation. mitochondrial bioenergetics; heart mitochondria
m), and matrix alkalinization (22). On the other hand, Holmuhamedov et al. (17) demonstrated in isolated rat heart mitochondria that mtKATP channel opening markedly depolarized m, accelerated respiration, slowed ATP production, and increased matrix volume. Liu et al. (25) further supported the hypothesis that matrix K+ influx stimulates mitochondrial respiration by reporting an increase in flavoprotein fluorescence in substrate-depleted medium after treatment with the mtKATP channel opener diazoxide. Matrix K+ influx may play an essential role in cardiac pharmacological preconditioning. In addition to the mtKATP channel, there is increasing evidence for a role of mitochondrial Ca2+-sensitive K+ (mtKCa) channel opening in cardioprotection. Xu et al. (42) found large-conductance KCa (mtBKCa) channels in the IMM of guinea pig ventricular cells and demonstrated their protective potency against ischemia-reperfusion injury. Recently, Cao et al. (5) demonstrated that pharmacological preconditioning was initiated by the mtBKCa channel activator 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619) in isolated rat hearts. Blockade of mtBKCa channels abolished the reduction of infarct size caused by ischemic preconditioning, which suggested a key role of mtBKCa channel opening in ischemic preconditioning (35). Opening of mtBKCa channels by NS-1619 also triggered delayed preconditioning against ischemia-reperfusion injury in mice (41). Sato et al. (34) confirmed the protective effect of NS-1619 in isolated cardiac myocytes, where pretreatment with NS-1619 reduced mitochondrial Ca2+ overload-induced cell death and increased flavoprotein fluorescence. These authors suggested that this increase in flavoprotein fluorescence, which indicates a more oxidized redox state, is caused by enhanced mitochondrial respiration as a consequence of K+ influx into the mitochondrial matrix.
We reported recently (37) that preconditioning with NS-1619 aids in preserving the mitochondrial redox state, lowers reactive oxygen species (ROS) production, and reduces mitochondrial Ca2+ overload during ischemia and reperfusion in isolated guinea pig hearts. This protection was blocked by bracketing NS-1619 treatment with the BKCa channel blocker paxilline or the SOD mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride. This finding supported the hypothesis that ROS play a key role in triggering cardiac preconditioning and that this effect is modulated by matrix K+ influx and subsequent changes in mitochondrial bioenergetics. Indeed, a role for ROS in initiating ischemic and pharmacological preconditioning by mtKATP channel opening is strongly supported by individual studies (12, 19, 31, 40) and is summarized in recent reviews (16, 38, 43).
It is unknown how mtBKCa channel opening alters mitochondrial function and what impact such alterations have on mitochondrial ROS generation. Because a key initiating trigger in cardiac preconditioning may be K+ influx-related ROS generation, we investigated effects of drug-induced mtBKCa channel opening and valinomycin-induced K+ influx on respiration, m, and H2O2 release rate in cardiac isolated mitochondria. Our aim was to determine the respiratory conditions by which matrix K+ influx, specifically via the mtBKCa channel, can induce an increase in mitochondrial ROS generation, an essential condition required for initiation of cardiac preconditioning.
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MATERIALS AND METHODS |
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Mitochondrial isolation. Heart mitochondria were isolated from ketamine-anesthetized guinea pigs (250–300 g) of either sex by differential centrifugation as described previously (33) with moderate modifications. Briefly, ventricles were excised, placed in an isolation buffer (200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 5 mM MOPS, 1 mM EGTA, and 0.1% BSA, with pH adjusted to 7.15 with KOH), and minced into 1-mm3 pieces. The suspension was homogenized for 15 s in 2.5 ml of isolation buffer containing 5 U/ml protease (Bacillus licheniformis) and for another 15 s after addition of 17 ml of isolation buffer. The suspension was centrifuged at 8,000 g for 10 min, the pellet was resuspended in 25 ml of isolation buffer and centrifuged at 750 g for 10 min, the supernatant was centrifuged at 8,000 g for 10 min, and the final pellet was suspended in 0.5 ml of isolation buffer and kept on ice. The protein content was determined by the Bradford method (2). All isolation procedures were conducted at 4°C. All experiments were conducted at a maintained temperature of 27°C, rather than 37°C, via a circulatory water system to allow time to assess drug effects at a lower respiratory rate.
Mitochondrial O2 consumption. O2 consumption was measured polarographically using a respirometric system (model S 200A, Strathkelvin Instruments, Glasgow, Scotland). Mitochondria (0.25 mg protein/ml) were suspended in respiration buffer containing 130 mM KCl, 5 mM K2HPO4, 20 mM MOPS, 2.5 mM EGTA, 1 µM Na4P2O7, and 0.1% BSA, with pH adjusted to 7.15 with KOH. Buffer Ca2+ concentration was <100 nM as assessed by the fluorescent dye indo 1. Respiration was initiated by administration of 10 mM pyruvate or 10 mM succinate + 10 µM rotenone. State 3 respiration was determined after addition of 125 nmol (or 5 µl of 25 mM) of ADP. The respiratory control index (RCI) was calculated as the state 3-to-state 4 ratio. Results are expressed as absolute changes from control in nanomoles of O2 per milligram per minute or as percentage of control.
Mitochondrial m.
Mitochondrial m was monitored during state 2–4 respiration with the substrates pyruvate and succinate + rotenone in a cuvette-based spectrophotometer (model QM-8, Photon Technology International) operating at excitation and emission wavelengths of 503 and 527 nm, respectively, in the presence of the fluorescent dye rhodamine 123 (50 nM). Mitochondria (0.5 mg/ml) were suspended in respiration buffer. m is expressed as the percentage of rhodamine 123 fluorescence in the presence of fully coupled mitochondria relative to the fluorescence after addition of 4 µM carbonyl cyanide-m-chlorophenylhydrazenone, a mitochondrial uncoupler.
Mitochondrial H2O2 release. Rates of mitochondrial H2O2 release were measured spectrophotometrically (model QM-8, Photon Technology International) with pyruvate or succinate + rotenone during state 2–4 respiration using the fluorescent dye Amplex red (25 µM) in the presence of 0.1 U/ml horseradish peroxidase. Excitation and emission wavelengths were set to 530 and 583 nm, respectively. Mitochondria (0.5 mg/ml) were suspended in respiration buffer, and rates of H2O2 release are expressed as percentage of baseline H2O2 release (after substrate addition). Baseline H2O2 levels were calibrated from a mean of three standard curves of photon counts over a range of 10–200 nM H2O2 (added to assay medium in the presence of reactants, Amplex red, and horseradish peroxidase); each regression was linear (R > 0.99). Time controls received 0.3% DMSO, the dye vehicle.
Chemicals and reagents. Rhodamine 123, Amplex red, and indo 1 were purchased from Molecular Probes (Eugene, OR) and KCl from EMD Chemicals (Gibbstown, NJ); all other chemicals were purchased from Sigma Chemical. NS-1619, paxilline, and Amplex red were dissolved in DMSO before they were added to the experimental buffer.
Statistical analyses. All data were analyzed using customized software developed in Matlab (MathWorks, Natick, MA). Group data were compared by analysis of variance. If F values (P < 0.05) were significant, post hoc comparisons of means tests (Student-Newman-Keuls) were considered statistically significant at P < 0.05 (2-tailed). Values are means ± SE.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To test whether these effects of NS-1619 were due to mtBKCa channel opening, we added 5 µM paxilline in the absence or presence of 30 µM NS-1619 (Figs. 2 and 3). Paxilline alone had no effect on state 2 respiration, indicating that mtBKCa channels were closed under the experimental conditions. Preadministration of paxilline (Fig. 1) attenuated the 30 µM NS-1619-induced increase in state 2 (14.6 ± 4.3% vs. 46.9 ± 7.2%) and state 4 (9.8 ± 1.7% vs. 31.3 ± 5.7%) respiration with pyruvate as substrate (Fig. 2) and abolished the NS-1619-induced increase in state 2 (5.3 ± 2.0% vs. 21.4 ± 4.2%) and state 4 (1.5 ± 1.6% vs. 13.5 ± 1.9%) respiration with succinate + rotenone as substrate (Fig. 3). State 3 respiration was not affected by 30 µM NS-1619, but administration of paxilline before NS-1619 decreased state 3 respiration by 17.4 ± 3.1% vs. control with pyruvate as substrate (Fig. 2). With succinate + rotenone as substrate, paxilline had no effect on the NS-1619-induced decrease in state 3 respiration. The decrease in RCI with pyruvate was not affected by prior administration of paxilline but was blunted (19.6 ± 3.4% vs. 29.0 ± 3.9%) with succinate + rotenone. These data demonstrate that NS-1619 increases mitochondrial state 2 and 4, but not state 3, respiration by activating mtBKCa channels.
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m.
We measured fluorescence of the membrane potential-sensitive dye rhodamine 123 to examine the effects of NS-1619 and valinomycin on m. Data are shown as raw photon counts per second (Table 2) and as percentage of maximal depolarization by carbonyl cyanide-m-chlorophenylhydrazenone (Fig. 4). Administration of 30 µM NS-1619 had no effect on m with the complex I substrate pyruvate (0.7 ± 0.2% vs. 0% baseline) or the complex II substrate succinate + rotenone (–0.4 ± 0.3% vs. 0% baseline; Fig. 4, A and B). A significant depolarization of m was detected after administration of 50 µM NS-1619, and this effect was substrate independent: 2.7 ± 0.4% vs. baseline for pyruvate and 2.7 ± 0.6% vs. baseline for succinate + rotenone. State 2 and 4 respiratory rates were approximately threefold higher with succinate + rotenone than with pyruvate (Table 1). The large ADP-induced (state 3) peak depolarization was unaffected by 30 or 50 µM NS-1619. During state 4 respiration with pyruvate, effects on m were similar to those of state 2, i.e., depolarization only at 50 µM NS-1619. With succinate + rotenone, 30 µM NS-1619 promoted a hyperpolarization and 50 µM NS-1619 caused no change in m relative to the controls. These data indicate that, during states 2 and 4, NS-1619 maintains (or increases) m, which favors generation of O2
–. The extent and duration of ADP-induced depolarization were slightly enhanced in the 50 µM NS-1619-treated group compared with the time controls; however, these effects did not reach statistical significance (data not shown).
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–, by direct electron transfer to O2 (electron leak), which is converted to H2O2 by SOD (39). In the presence of the complex I substrate pyruvate, activation of mtBKCa channels by 30 µM NS-1619 had no effect on the state 2 mitochondrial H2O2 release rate compared with the DMSO-treated time controls: –2.2 ± 1.9% vs. 3.5 ± 5.6% (Fig. 5B). In contrast, with succinate + rotenone, which stimulated at least a threefold faster rate of respiration and potential for electron leak, 30 µM NS-1619 increased the rate of H2O2 release by 44.8%: 65.9 ± 2.7% for NS-1619 vs. 21.1 ± 3.8% for time controls. Furthermore, the large 30 µM NS-1619-induced increase in H2O2 release rate was sensitive to mtBKCa channel blockade; pretreatment with 5 µM paxilline (n = 5) blunted the effect of NS-1619 to 29.4 ± 4.9% vs. 65.9 ± 2.7% for NS-1619 alone (Fig. 5B).
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m under both substrate conditions. During state 4 respiration, there was no change in H2O2 release rates by 30 or 50 µM NS-1619 with pyruvate (data not displayed); with succinate + rotenone during state 4, 30 µM NS-1619 significantly increased the H2O2 release rate (24.3 ± 2.5%) over the no-drug control, whereas 50 µM NS-1619 had no effect (–3.5 ± 3.3%, n = 4, P < 0.05; data not plotted).
Effect of valinomycin on respiration, m, and ROS release rate.
To support the hypothesis that matrix K+ influx causes ROS generation when respiration is increased but m is maintained, we conducted a series of experiments using the K+ ionophore valinomycin with succinate + rotenone in normal-K+ buffer. The experimental protocol was the same as for NS-1619 experiments. The low concentration (0.25 nM) of valinomycin increased the state 2 respiration rate by 10.3 ± 2.8% over the control rate, had no significant effect on m, and increased mitochondrial H2O2 release rate by 19.6% over the control rate (Fig. 6). In comparison, 1 nM valinomycin increased the state 2 respiration rate by 16 ± 3.9%, depolarized m significantly over control, and did not alter the H2O2 release rate. Similar to the results for lower concentrations of NS-1619, these data show that, at a low concentration, valinomycin can also enhance ROS release when respiration is increased but m remains fully polarized.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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m only at high concentrations (50 µM). 4) Valinomycin (0.25 nM) and 30 µM NS-1619 markedly increased mitochondrial ROS production in the presence of succinate + rotenone, because a high resting m is maintained in the face of markedly enhanced respiration. Our data offer a possibility by which ROS can be generated by submaximal K+-induced H+ leak into the mitochondrial matrix. A schematic representation of the putative pathway from mtBKCa channel opening to ROS generation is shown in Fig. 7. As matrix K+ influx increases through the mtBKCa channel, an equivalent K+ efflux is balanced by inward proton leak, which would accelerate respiration while maintaining m and, thus, allow greater electron leak and O2– generation. Greater K+ influx would saturate the K+/H+ exchanger, causing a decline in m and O2– generation.
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µH)]-dissipating channels by expending µH, in part to eject the K+ that enters the matrix via activated K+ channels via an electroneutral K+/H+ exchanger. This decrease in µH would stimulate respiration to compensate for a net proton leak and, consequently, maintain m. Our finding that 10–30 µM NS-1619 stimulated respiration but did not depolarize m validates this theory. Furthermore, we detected an inhibition of respiration under ADP-stimulated state 3 conditions. The insensitivity of this inhibitory effect to paxilline indicates an mtBKCa channel-independent effect of NS-1619. A similar effect of NS-1619 was described by Debska et al. (7), who showed that NS-1619 reduced the O2 consumption rate after maximal stimulation of electron flow by the mitochondrial uncoupler dinitrophenol in glioma cells. In addition, Kowaltowski et al. (22) demonstrated a decrease in maximal respiratory rates in uncoupled mitochondria by the KATP channel openers diazoxide and pinacidil, but this effect was apparently unrelated to mitochondrial KATP channel activity, because the decrease in respiration was also observed in K+-free medium.
Most recently, Costa et al. (6) examined the effects of matrix K+ influx in isolated rat heart mitochondria respiring on succinate + rotenone with oligomycin added to inhibit ATP synthesis at complex V and, thus, matrix proton influx, which prevents a depolarization of m. Under these conditions, they demonstrated that matrix K+ uptake is increased by mtKATP channel opening or by valinomycin and that this increase results in matrix swelling, mild uncoupling, and matrix alkalinization; these effects were blocked by ATP, glibenclamide, and 5-hydroxydecanoate. They proposed that the increase in matrix volume stimulates K+-H+ antiport activity, which tends to mitigate the matrix expansion indirectly by extrusion of K+ for H+. Finally, our group has reported that, similar to NS-1619, the KATP channel openers diazoxide and pinacidil decreased ADP-stimulated (state 3) respiration but increased respiration in the presence of oligomycin (state 4) [preliminary observations (32)]; the latter, but not the former, state condition was blocked by the KATP channel inhibitors 5-hydroxydecanoate and glibenclamide.
Subthreshold mtBKCa channel opening does not alter m.
In these experiments, we have demonstrated that mtBKCa channel activation by 10–30 µM NS-1619 had no effect on m. We observed a slight depolarization only at 50 µM NS-1619, a concentration higher than that used in our previous study to examine effects of mtBKCa channel opening on ex situ mitochondrial function or to initiate preconditioning (37). However, Sato et al. (34) observed that 30 µM NS-1619 reduced m by 11% in ventricular myocytes; the difference between their study and ours may be due to their use of substrate-depleted intact myocytes.
The effect of matrix K+ influx through mtKATP channels on m remains controversial. This controversy may be due in part to the concentrations of drugs used to open these channels. Although a depolarizing effect of m by activation of mtKATP channels has been reported (17, 18, 28), Kowaltowski et al. (22) argued against those findings, because they believed that the concentrations of KATP channel opener were too high and that the depolarization was not due to a KATP channel-specific effect. Our data for the mtBKCa channel support their observations on the mtKATP channel. They estimated that pharmacological concentrations of an mtKATP channel opener would reduce m by only 1–2 mV. Our data also support the general hypotheses by O'Rourke (29) and Kowaltowski et al. that matrix K+ influx stimulates respiration by a reduced µH due to exchange of K+ for H+ via the K+/H+ exchanger. We propose that mtBKCa channel opening and matrix K+ influx leads to H+ influx (leak) by K+/H+ exchange, which in turn enhances matrix H+ pumping and increases electron flow and, in resting states, prevents depolarization of m, because protons cannot enter the matrix via complex V ATP synthase. The bioenergetic consequence of mtKCa channel opening would be accelerated cycling of K+ between the matrix and the intermembrane space (i.e., matrix K+ inflow through mtKCa channel and K+ extrusion via K+/H+ exchange) and an increase in mitochondrial respiration. Pronounced depolarization of m would occur only when the compensatory mechanism is exhausted, e.g., if matrix K+ influx exceeds K+/H+ exchange capability, or under state 3 conditions.
Mitochondrial BKCa channel opening enhances mitochondrial ROS generation. Our data demonstrate that opening of mtBKCa channels by 10–30 µM NS-1619 enables increased ROS generation in isolated heart mitochondria during resting state conditions with the complex II substrate succinate + rotenone. This increase in ROS generation was sensitive to paxilline, which indicates a mtKCa channel opening-induced effect. The consequence of matrix K+ influx by mtKATP channel opening in terms of mitochondrial ROS generation has been frequently discussed and remains quite controversial. Several laboratories demonstrated that activation of KATP channels causes increased ROS production (12, 13), whereas Ferranti et al. (11) reported that KATP channel opening decreases ROS generation. The interrelation between mitochondrial ROS generation and basal and inducible H+ leak by uncoupling proteins and the AMP-adenine nucleotide translocator pathway has been expertly reviewed recently by Brookes (3).
The larger increase in H2O2 release rate in our study was induced by NS-1619 at 30 µM, a concentration that also accelerated mitochondrial respiration but had no effect on m. It appears unlikely that accelerated respiration (i.e., electron flow) alone can increase ROS generation, because 50 µM NS-1619, which further stimulated respiration, had no effect on the rate of mitochondrial H2O2 release. It is well accepted that mitochondrial ROS production is strongly dependent on m; ROS generation can decrease rapidly with even a slight depolarization of m (20, 21, 36). We suggest that 50 µM NS-1619 leads to net K+ influx that is not replaced by H+ because of saturated K+/H+ exchange; this would cause a slight m depolarizing effect and more effective electron transfer with less chance for electron leak and ROS generation. Our finding that a low concentration (0.25 nM) of the K+ ionophore valinomycin also increased ROS generation while m was not depolarized supports the hypothesis that submaximal matrix K+ influx can increase ROS production. The higher concentration (1 nM) of valinomycin, which depolarized m in a manner similar to that of 50 µM NS-1619, not only did not enhance, but actually decreased, the rate of H2O2 release. Brookes et al. (4) observed that 0.01–1 nM valinomycin was sufficient to fully establish m, whereas 3 nM valinomycin caused secondary proton flux with depolarization of m, a condition that is also not expected to increase ROS release.
Despite our plausible mechanism for increased ROS generation by induced matrix K+ influx, it is difficult to explain why no increase in ROS generation was detected by 30 µM NS-1619 with 10 mM pyruvate. The net effect of 30 µM NS-1619 on enhancement of state 2 respiration was greater with succinate + rotenone than with pyruvate: 11.6 ± 2.9 and 7.3 ± 0.9 nmol O2·mg–1·min–1, respectively. It is possible that the smaller increase in respiration and electron flow with pyruvate induces less ROS release, which may not be detectable by our fluorescence methods. There are several lines of evidence that diazoxide, a putative KATP-sensitive channel opener, can inhibit complex II and retard succinate-supported, but not NADH-supported, respiration (10, 16, 24, 30). In our study, it is unlikely that NS-1619 had a similar indirect role to inhibit complex II as a source for ROS generation, because this effect was sensitive to paxilline (Fig. 5B) and respiration was not slower with succinate + rotenone. Pyruvate has also been reported to decrease formation of H2O2 by changing the redox state (1) and to scavenge OH· (9). In trial runs, we observed that 30 µM NS-1619 enhanced ROS release with 
-ketoglutarate and glutamate as an NADH-linked substrate. In our isolated heart model, the presence of pyruvate in the perfusate did not effectively scavenge the upstream reactant O2–, which is generated during ischemia and reperfusion (19, 37). Another possibility is that the increase in ROS generation is elicited only by FADH2-linked substrates. Reports by several laboratories indicate that, in addition to complexes I and III, complex II is a site of O2– generation in mitochondria (23, 26, 44). In future studies, it will be important to answer the following questions. 1) Does mtKCa channel activation induce ROS generation with NADH-linked substrates? 2) Where are the sites of ROS generation induced by matrix K+ influx and concomitant K+/H+ exchange? 3) Which reactant (O2–, H2O2, OH·, or others) leads to preconditioning?
Possible limitations.
Mitochondria isolated from their cells are not in a physiological environment, inasmuch as there is cross talk between these compartments and ATP is consumed due to cellular energy requirements. Respiration was decreased during state 3 by NS-1619, and this was not blocked by paxilline, suggesting that this effect is independent of matrix K+ influx or that these channels cannot be opened during state 3 respiration. This may indicate that these channels are normally open only during initial cell stress when mitochondria are in a reduced state. Another concern is that ROS were generated only under succinate + rotenone conditions. This may relate to the smaller baseline release of ROS with pyruvate than with succinate as the substrate. As with the putative mtKATP channel openers, there is a concern that the prominent protective effect of mtBKCa channels openers is not actually mediated by increasing matrix K+ influx, but by another mechanism. There is limited evidence for these channels in cardiac IMM. However, we too have found good coverage of protein fragments of BKCa - and 
-subunits in several trypsinated purified preparations of guinea pig heart IMM using mass spectrometry (nanoprobe liquid chromatography-electrospray ionization-mass spectrometry) to the second profile (unpublished observations). Finally, exogenous opening of this channel may give erroneous results compared with blockade of endogenously opened channels, because the agonist may have biphasic or unrelated effects at higher concentrations.
Summary and conclusions.
Our results support the hypothesis that matrix K+ influx through activated mtBKCa channels modulates specific changes in mitochondrial bioenergetics, i.e., an imbalance between m and electron flow. Opening of mtBKCa channels by NS-1619 accelerates mitochondrial respiration during the resting states but depolarizes m only at higher concentrations. Moreover, these conditions of accelerated respiration coupled with maintained m are suitable for allowing singlet electron leak capable of enhancing O2– production. Thus it is possible that the increase in respiration compensates for a reduced µH due to mtBKCa channel activation and the K+/H+ exchange that maintains m. This mitochondrial mechanism may underlie, at least in part, the preconditioning effect of mtKCa channel opening as a consequence of enhanced ROS generation.
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ACKNOWLEDGMENTS |
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This work has been published, in part, in abstract form (FASEB J 19: 346.13, 2005; FASEB J 20: 213.1, 2006; Biophys J 88: 440a, 2005; Anesthesiology 103: A477, 2005).
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FOOTNOTES |
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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.
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120 s (treatment effects on state 2 were measured beginning at 120 s) in the presence or absence of 5 µM paxilline.
