Anesth Analg 2004;99:1308-1315
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000134804.09484.5D
CARDIOVASCULAR ANESTHESIA
Preconditioning by Isoflurane Is Mediated by Reactive Oxygen Species Generated from Mitochondrial Electron Transport Chain Complex III
Lynda M. Ludwig, BS,
Katsuya Tanaka, MD,
Janis T. Eells, PhD,
Dorothee Weihrauch, DVM PhD,
Paul S. Pagel, MD PhD,
Judy R. Kersten, MD, and
David C. Warltier, MD PhD
Departments of Anesthesiology, Pharmacology and Toxicology, and Medicine (Division of Cardiovascular Diseases), the Medical College of Wisconsin and the Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin and the Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin.
Address correspondence and reprint requests to David C. Warltier, MD, PhD, Medical College of Wisconsin, MEB-M4280, 8701 Watertown Plank Road, Milwaukee, WI 53226. Address email to warltier{at}mcw.edu
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Abstract
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Reactive oxygen species (ROS) mediate volatile anesthetic preconditioning. We tested the hypothesis that isoflurane (ISO) generates ROS from electron transport chain complexes I and III. Rabbits (n = 55) underwent 30 min coronary artery occlusion followed by 3 h reperfusion and received 0.9% saline, the complex I inhibitor diphenyleneiodonium (DPI; 1.5 mg/kg bolus followed by 1.5 mg/kg over 1 h), or the complex III inhibitor myxothiazol (MYX; 0.1 mg/kg bolus followed by 0.3 mg/kg over 1 h) in the absence and presence of 1.0 minimum alveolar concentration ISO. ISO was administered for 30 min and discontinued 15 min before coronary occlusion. Infarct size and ROS production (n = 32) were determined using triphenyltetrazolium staining and ethidium-DNA fluorescence, respectively. Adenosine triphosphate (ATP) synthesis in mitochondria obtained from rabbit hearts (n = 24) subjected to drug interventions was measured by luciferin-luciferase luminometry. ISO significantly (P < 0.05) reduced infarct size (19% ± 4%) as compared with control (39% ± 4%). MYX (35% ± 4%), but not DPI (24% ± 2%), abolished this protection. ISO increased ethidium-DNA fluorescence (83 ± 11 U) as compared with control (40 ± 12 U). MYX (35 ± 3 U), but not DPI (78 ± 9 U), abolished ROS generation. DPI and MYX selectively reduced complex I- and complex III-mediated ATP synthesis, respectively. ROS generated from electron transport chain complex III mediate ISO-induced cardioprotection.
IMPLICATIONS: The electron transport chain complex III inhibitor myxothiazol, but not the complex I inhibitor diphenyleneiodonium, abolished isoflurane-induced protection against ischemia-reperfusion injury and reactive oxygen species production in rabbits. Reactive oxygen species generated from electron transport chain complex III act in the signal transduction process mediating preconditioning by isoflurane in vivo.
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Introduction
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Small bursts of reactive oxygen species (ROS) have been strongly implicated in the initiation of signal transduction pathways during myocardial preconditioning. ROS scavengers abolish volatile anesthetic-induced reductions in myocardial infarct size (1,2). Pretreatment with small concentrations of ROS also mimics these antiischemic effects in isolated cardiac myocytes (3). We previously demonstrated that isoflurane directly stimulates ROS production that subsequently contributes to cardioprotection against irreversible ischemic injury in vivo (2). However, the source of these ROS has yet to be identified.
The antiischemic actions of volatile anesthetics have been shown to be mediated by adenosine type 1 (A1) receptors, inhibitory guanine nucleotide binding proteins (Gi), protein kinase C (PKC), ROS, and sarcolemmal and mitochondrial adenosine triphosphate (ATP)-sensitive potassium (KATP) channels. Opening of mitochondrial KATP channels by isoflurane triggers preconditioning by generating ROS (4). Mitochondrial KATP channel opening also depolarizes the inner mitochondrial membrane and alters the oxidation-reduction state (5). These results suggest that the mitochondrial electron transport chain may represent a source of ROS production. Previous findings indicate that complexes I (nicotinamide adenine dinucleotide [NADH] dehydrogenase) and III (ubisemiquinone) are the primary ROS-forming sites of the mitochondrial electron transport chain (6). We tested the hypothesis that isoflurane protects against myocardial infarction by generating ROS from mitochondrial electron transport chain complexes I and III.
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Methods
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All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin. Furthermore, all conformed to the Guiding Principles in the Care and Use of Animals (7) of the American Physiological Society and were in accordance with the Guide for the Care and Use of Laboratory Animals (8).
Male New Zealand white rabbits weighing between 2.5 and 3.0 kg were anesthetized with IV sodium pentobarbital (30 mg/kg) as previously described (2). Additional doses of pentobarbital were titrated as required to assure that pedal and palpebral reflexes were absent throughout the experiment. A tracheostomy was performed through a ventral midline incision, and the trachea was cannulated. The rabbits were ventilated with positive pressure using an air-oxygen mixture (FIO2 = 0.33). Arterial blood gas tensions and acid-base status were maintained within normal physiological ranges (pH 7.35–7.45, PaCO2 25–40 mm Hg, and PaO2 90–150 mm Hg) by adjusting the respiratory rate or tidal volume throughout the experiment. Body temperature was maintained using a heating blanket. Heparin-filled catheters were inserted into the right carotid artery and the left jugular vein for measurement of arterial blood pressure and fluid or drug administration, respectively. IV fluids (0.9% saline) were administered at 15 mL · kg–1 · h–1 for the duration of the experiment. A left thoracotomy was performed at the fourth intercostal space, and the heart was suspended in a pericardial cradle. A prominent branch of the left anterior descending coronary artery (LAD) was selected, and a silk ligature was placed around this artery approximately halfway between the base and apex for the production of coronary artery occlusion and reperfusion. Each rabbit was anticoagulated with 500 U of heparin immediately before LAD occlusion. Coronary artery occlusion was verified by the presence of epicardial cyanosis in the ischemic zone, and reperfusion was confirmed by observing an epicardial hyperemic response. A heparin-filled catheter was inserted into the left atrium for the administration of dihydroethidium in experiments designed to detect ROS production. Hemodynamics were continuously recorded on a polygraph throughout experimentation. Representative measurements of heart rate and mean arterial blood pressure were obtained at one point in time during each particular segment of the experimental protocol. Averages for distinct time intervals were calculated for each rabbit group.
The experimental design is illustrated in Figure 1. Baseline systemic hemodynamics were recorded 30 min after instrumentation was completed. Rabbits were randomly assigned to one of six experimental groups using a partial Latin square design. All rabbits underwent a 30 min LAD occlusion followed by 3 h reperfusion. Rabbits received IV vehicle (0.9% saline), diphenyleneiodonium (DPI) (Sigma-RBI, Natick, MA; 1.5 mg/kg bolus followed by 1.5 mg/kg over 1 h), or myxothiazol (MYX) (Sigma-RBI, Natick, MA; 0.1 mg/kg bolus followed by 0.3 mg/kg over 1 h) in the absence and presence of isoflurane (1.0 minimum alveolar concentration [MAC]). Isoflurane was administered for 30 min and discontinued 15 min before coronary artery occlusion. This served to create an acute memory period characteristic of preconditioning. End-tidal concentrations of isoflurane were measured at the tip of the tracheostomy tube with an infrared anesthetic analyzer that was calibrated with known standards before and during experimentation.
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Figure 1. Schematic illustration of the protocol used in infarct size experiments. DPI = diphenyleneiodonium; MYX = myxothiazol.
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Myocardial infarct size was measured as previously described (2). Briefly, at the end of each experiment, the LAD was reoccluded and 3 mL of patent blue dye was injected IV. The left ventricular area-at-risk (LVAAR) for infarction was separated from surrounding normal areas (stained blue), and the 2 regions were incubated at 37°C for 30 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M phosphate buffer adjusted to pH 7.4. After overnight storage in 10% formaldehyde, infarcted and noninfarcted myocardium within the AAR were carefully separated and weighed. Infarct size was expressed as a percentage of the AAR. Rabbits that developed intractable ventricular fibrillation and those with an AAR <15% of LV mass were excluded from subsequent analysis.
ROS were detected using dihydroethidium in a parallel series of six additional groups using the same interventions described above for the infarct size experiments. The ROS probe dihydroethidium (2.2 mg) was rapidly injected into the left atrium 5 min before the administration of isoflurane or at the corresponding time point in control rabbits not exposed to the volatile anesthetic. Isoflurane was discontinued after 30 min, and the rabbits were killed after 1 h with an overdose of pentobarbital. The heart was rapidly excised, and the LV was isolated, divided into 4 sections of equal size, and frozen in liquid nitrogen for subsequent analysis.
ROS were detected using dihydroethidium fluorescence as previously described (2). Briefly, cryostat sections (20 µm) of the LV were mounted on standard microscope slides. Using a laser fluorescence imaging system mounted on a confocal microscope, images were recorded and stored for subsequent off-line analysis on a computer workstation equipped with image analysis software. Use of the 40x objective yielded a 400x end magnification on a 292 x 195 µm2 digital image (768 x 512 pixels). Excitation was produced using a Krypton-Argon laser at a wavelength of 488 nm, and emitted fluorescence was measured at 550 nm after long pass filtering. The pixel intensity of each myocyte nucleus was determined. Background was identified as an area without cells or with minimal cytosolic fluorescence. In each rabbit, 20 images were obtained and approximately 6 to 8 dihydroethidium-stained myocardial cells were analyzed in each image by subtraction of background fluorescence from the pixel intensity of the myocardial nuclei.
In three separate groups, rabbits received vehicle, DPI, or MYX in the absence of isoflurane as described for infarct size experiments. Cardiac mitochondria were isolated using differential centrifugation. Briefly, the LV was isolated and minced into 1-mm pieces in cold isolation buffer containing 200 mM mannitol, 50 mM sucrose, 1 mM EGTA, 5 mM MOPS, and 0.1% bovine serum albumin (BSA). The pH was adjusted to 7.15 using KOH. The tissue was rinsed clear of blood with isolation buffer and transferred to a glass Potter-Elvehjem homogenizing vessel. Immediately after the addition of 2.5 mg protease (P-5459, Sigma) diluted in 2.5 mL isolation buffer, the tissue was gently homogenized on ice with a Teflon pestle (Dupont, Wilmington, DE). Fifteen mL of cold isolation buffer was added to dilute the protease after 30 s, and the tissue was further homogenized for an additional 60 s. Exposure to concentrated protease was limited to 30 s to maintain mitochondrial integrity and increase yield. The tissue suspension was centrifuged at 8000 g for 10 min to remove the protease. The pellet was resuspended in 3.5 mL isolation buffer, and an additional 25 mL isolation buffer was added before the suspension was centrifuged a second time at 700g for 10 min to remove cellular debris. The supernatant containing the mitochondrial fraction was further centrifuged at 8000g for 10 min. The pellet was washed by resuspension in 3.5 mL isolation buffer without EGTA and centrifuged a final time at 8000g for 10 min. The final mitochondrial pellet was resuspended to 1 g original tissue weight in cold isolation buffer without EGTA. All procedures were performed at 4°C, and protein concentration was determined by the Bradford method (BIO-RAD, Hercules, CA).
ATP synthesis was determined in isolated cardiac mitochondria using luciferin-luciferase luminometry. Briefly, mitochondria (10 µg/mL) were preincubated for 10 min in buffer containing 180 mM sucrose, 45 mM KH2PO4, 10 mM Mg-acetate, 1 mM EDTA, 1 mM pyrophosphate, 1 g/L BSA, and 150 µM adenosine diphosphate (ADP) in a 25°C water bath. Mitochondrial ATP production was initiated by addition of respiratory chain substrates that drive electron flow through complex I (1 mM pyruvate + 1 mM malate), complex II (20 mM succinate), complex I and II (1 mM pyruvate + 1 mM malate + 20 mM succinate), or complex IV (5 mM ascorbate + 50 mM N,N,N',N'-tetramethyl-p-phenylenediamine). Three 50-mL aliquots were removed from each incubation at 4-min intervals. The reaction was terminated using 500 µL of 2.5% (v/v) trichloracetic acid. All reactions were performed in triplicate. Samples were neutralized with 100 µL of 1 M Tris base and assayed for ATP by luciferin-luciferase luminescence using a bioluminescent somatic cell assay kit (FL-AA, Sigma). The rate of ATP synthesis was expressed as micromoles of ATP synthesized per min per milligram of mitochondrial protein.
Statistical analysis of data within and between groups was performed with analysis of variance for repeated measures followed by Student-Newman-Keuls test. Statistical significance was defined as P < 0.05. All data are expressed as mean ± SEM
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Results
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Fifty-five rabbits were instrumented to obtain 45 successful myocardial infarct size experiments. Three rabbits were excluded because the AAR was <15% of LV mass (1 control, 1 isoflurane alone, and 1 isoflurane + MYX). Seven rabbits were excluded because of intractable ventricular fibrillation (1 control, 2 isoflurane alone, 2 MYX alone, and 2 DPI alone).
No differences in baseline systemic hemodynamics were observed among groups (Table 1). Isoflurane significantly (P < 0.05) decreased mean arterial blood pressure and rate-pressure product in the presence or absence of DPI or MYX. Hemodynamics returned to baseline values 15 min after isoflurane had been discontinued. Coronary artery occlusion and reperfusion produced similar decreases in mean arterial blood pressure and rate-pressure product in each experimental group.
Body weight, LV weight, AAR weight, and LVAAR mass were similar among groups (Table 2). Isoflurane significantly reduced infarct size (19% ± 4% of the LVAAR) as compared with control experiments (39% ± 4%; Fig. 2). MYX and DPI alone had no effect on infarct size (37% ± 4% and 42% ± 2%, respectively). The protective effect of isoflurane was abolished by MYX (35% ± 4%) but not DPI (24% ± 2%).
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Figure 2. Myocardial infarct size expressed as a percentage of the left ventricular area at risk (AAR) in rabbits receiving saline (CON), diphenyleneiodonium (DPI) alone, myxothiazol (MYX) alone, and 1.0 MAC isoflurane (ISO) in the absence and presence of DPI (ISO+DPI) or myxothiazol (ISO+MYX). *Significantly (P < 0.05) different from CON;  significantly (P < 0.05) different from ISO.
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Thirty-two rabbits were instrumented for measurement of ROS production. Ethidium-DNA fluorescence was detected in all images examined (Fig. 3). Isoflurane increased ethidium-DNA fluorescence as compared with control experiments (83 ± 11 versus 40 ± 12 U, respectively; Fig. 4). MYX (35 ± 3 U), but not DPI (78 ± 9), abolished isoflurane-induced increases in fluorescence. MYX and DPI alone had no effect on fluorescence intensity (37 ± 3 and 44 ± 3 U, respectively). Mean arterial blood pressure and rate-pressure product were lower in isoflurane-treated rabbits (Table 3) before administration of dihydroethidium.
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Figure 3. Representative photomicrographs demonstrating enhanced production of superoxide anion by the expression of fluorescent ethidium bound to nuclear DNA. The fluorescence in myocardial nuclei in rabbits treated with 1.0 MAC isoflurane (ISO) in the absence and presence of DPI (ISO+DPI) was more intense than that observed in rabbits that were treated with the other interventions. ISO = isoflurane; MYX = myxothiazol; DPI = diphenyleneiodonium.
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Figure 4. Histogram depicting dihydroethidium fluorescence in rabbits receiving saline (CON), diphenyleneiodonium (DPI) alone, myxothiazol (MYX) alone, and 1.0 MAC isoflurane (ISO) in the absence and presence of DPI (ISO+DPI) or myxothiazol (ISO+MYX). *Significantly (P < 0.05) different from CON; significantly (P < 0.05) different from ISO.
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Twenty-four rabbits were instrumented for measurement of the rate of ATP synthesis in isolated cardiac mitochondria exposed to drug interventions in vivo (Fig. 5). DPI (1.8 ± 0.3 µmoles ATP · min–1 · mg–1), but not MYX (3.1 ± 0.4 µmoles ATP · min–1 · mg–1), decreased the rate of complex I-induced ATP synthesis as compared with control experiments (3.7 ± 0.4 µmoles ATP · min–1 · mg–1). Both DPI and MYX decreased the rate of complex I and II-induced ATP synthesis (3.2 ± 0.4 and 3.2 ± 0.5 µmoles ATP · min–1 · mg–1, respectively) as compared with control experiments (5.2 ± 0.6 µmoles ATP · min–1 · mg–1). DPI (1.9 ± 0.3 and 0.8 ± 0.2 µmoles ATP · min–1 · mg–1, respectively) and MYX (2.3 ± 0.2 and 1.1 ± 0.3 µmoles ATP · min–1 · mg–1, respectively) had no effect on complex II- and complex IV-induced ATP synthesis as compared to control experiments (2.1 ± 0.2 and 1.1 ± 0.1 µmoles ATP · min–1 · mg–1, respectively).
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Figure 5. Rates of adenosine triphosphate (ATP) synthesis in isolated mitochondria after previous in vivo administration of saline (CON), diphenyleneiodonium (DPI), or myxothiazol (MYX). Mitochondrial ATP production was initiated by addition of respiratory chain substrates that drive electron flow through complex I (1 mM pyruvate + 1 mM malate), complex II (20 mM succinate), complex I and II (1 mM pyruvate + 1 mM malate + 20 mM succinate), or complex IV (5 mM ascorbate + 50 mM N,N,N',N'-tetramethyl-p-phenylenediamine). *Significantly (P < 0.05) different from CON.
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Discussion
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The mitochondrial electron transport chain generates large amounts of ROS that have been shown to be deleterious during ischemia and reperfusion injury (9), but smaller quantities of ROS produced by mitochondria paradoxically trigger myocardial protection (10). Mitochondrial KATP channel activation by a variety of pharmacological agonists results in a protective effect that is associated with ROS generation in isolated ventricular myocytes (11) and intact hearts (12). For example, the mitochondrial KATP channel agonist diazoxide increased the fluorescence intensity of a mitochondrial-specific ROS probe in a human atrial-derived cell line (11). Morphine and the selective 
1-opioid agonist BW373U86 enhanced ventricular myocyte viability and oxidation of the hydrogen peroxide-sensitive probe 2'7'-dichlorofluorescin. This latter action was abolished by pretreatment with MYX (13). Acetylcholine-induced myocardial protection occurred concomitant with increased ROS formation that was also inhibited by MYX (14). These data suggest that small bursts of ROS that are produced in response to preconditioning stimuli may be generated by the mitochondrial electron transport chain complex III.
Our laboratory demonstrated that isoflurane directly causes superoxide anion production in myocardium in vivo independent of ischemia and reperfusion (2). This process was shown to be dependent on opening of mitochondrial KATP channels (4). The results of the present investigation confirm and extend our previous findings and demonstrate that the complex III inhibitor MYX, but not the complex-I inhibitor DPI, abolished reductions in myocardial infarct size produced by isoflurane. The results further demonstrate that MYX inhibits isoflurane-induced production of superoxide anion as detected by ethidium-DNA fluorescence. MYX inhibits electron transfer between reduced coenzyme Q and the iron sulfur protein of complex III and has been previously shown to reduce ROS production (15,16). DPI has also been shown to inhibit ROS generation in isolated mitochondria (17), but this complex I inhibitor (18) had no effect on isoflurane-induced superoxide anion generation.
Measurement of the rate of ATP synthesis in isolated mitochondria after in vivo administration of MYX and DPI was used to validate the specificity of the mitochondrial respiratory chain inhibitors. The results demonstrate that DPI selectively inhibited complex I of the electron transport chain because DPI depressed the rates of complex I- and complex I+II-driven ATP synthesis, but did not affect ATP synthesis through complexes II or IV. In addition, MYX was indirectly shown to antagonize complex III of the electron transport chain, as this drug selectively reduced the rate of complex I+II-driven ATP synthesis without altering the activities of complex I or II alone. These data suggest that electron flow through complex III was prevented by MYX. Nevertheless, a proton gradient may have been maintained across the mitochondrial membrane that was capable of driving ATP synthesis when substrates for either complex I or complex II alone were used to initiate electron transport activity. However, the combined presence of substrates for both complexes I and II most likely stimulated an increased transfer of electrons to complex III, and therefore, pretreatment with MYX produced a selective reduction in the rate of ATP synthesis under these conditions. Thus, the present results indicate that complex III of the mitochondrial electron transport chain is the source of superoxide anion produced by volatile anesthetics and the ROS are an element of the signal transduction pathway responsible for preconditioning.
The relative balance between the generation of ROS and their neutralization by endogenous antioxidants appears to be a critical regulator of signal transduction and mitochondrial homeostasis. Inhibition of the electron transport chain may increase (16) or decrease (15–17) ROS formation. Antimycin A, a complex III inhibitor that prevents electron transfer between cytochrome b and ubisemiquinone, stimulates superoxide production that is attenuated by myxothiazol (16). Thus, the correlation between complex III inhibition and ROS production is dependent on site-specific inhibition of the Q-cycle (15). The present findings support the contention that isoflurane may alter complex III activity and stimulate ROS formation by acting at a site within the Q-cycle distinct from MYX. In contrast, one investigation indicated that volatile anesthetics inhibit complex I of the electron transport chain in cardiac submitochondrial particles (19). However, the present investigation did not specifically determine if isoflurane somehow selectively inhibited electron transport chain activity at one or more complexes to stimulate ROS production. Moreover, the possibility that volatile anesthetics modulate other intracellular pathways to increase the intracellular concentration of ROS cannot be entirely excluded. These objectives will require further investigation to clarify.
The downstream signal transduction pathway(s) that may be modulated by ROS during myocardial preconditioning remain unclear. ROS activate PKC, restore contractility, and limit the extent of myocardial infarction (3). Hydrogen peroxide also selectively activates G
i and Go proteins (20). Mitochondrial-derived ROS have been shown to activate p38 mitogen-activated protein kinase (21). Additionally, ROS may modulate KATP channel activity (22). Therefore, it appears highly likely that production of ROS by volatile anesthetics may have multifactorial actions through several of these intracellular mediators to cause protection against ischemic injury. This tantalizing hypothesis will require further investigation to confirm, however.
ROS generation from the electron transport chain may preserve mitochondrial bioenergetic function that is vital for cytoprotection against myocardial ischemia. Moderate disturbances of mitochondrial homeostasis produced by brief ischemic stimuli or pharmacological agents, including volatile anesthetics, may promote myocardial tolerance to ischemic stress by reducing Ca2+ overload, preventing the activation of necrotic or apoptotic pathways, or attenuating oxidant stress (23). Sevoflurane improved cardiac function in isolated hearts subjected to global ischemia by attenuating mitochondrial Ca2+ overload (24). Thus, the present and previous results (24) suggest that volatile anesthetics may modulate the activity of the electron transport chain to generate signaling amounts of ROS that preserve mitochondrial function and ultimately contribute to myocardial protection.
The present findings should be interpreted within the constraints of several potential limitations. The LVAAR for infarction and coronary collateral blood flow represent major determinants of myocardial infarct size. The AAR was similar among experimental groups, and rabbits exhibited minimal coronary collateral blood flow. Thus, it appears unlikely that the present results were substantially affected by these variables. Isoflurane caused similar hemodynamic effects in the presence or absence of MYX or DPI, and there were no differences in hemodynamics among groups after the volatile anesthetic had been discontinued to create the acute memory period of anesthetic preconditioning. Therefore, the present results occurred independent of many of the hemodynamic determinants of myocardial oxygen consumption during administration of isoflurane or the mitochondrial electron transport chain inhibitors. The rate-pressure product, an indirect index of myocardial oxygen consumption, was also similar among experimental groups. DPI may also inhibit NADPH oxidase, a major source of ROS in neutrophils and vascular cells. Our findings demonstrate that isoflurane stimulates ROS production in cardiac myocytes and that DPI did not inhibit isoflurane-induced reductions in infarct size or increases in ROS generation. Thus, it is unlikely that NADPH oxidase plays a critical role in producing the signaling levels of ROS that are required for triggering myocardial protection by isoflurane. Experiments using dihydroethidium as an indicator of superoxide anion production may underestimate the rate of superoxide anion generation because this ROS probe may catalyze the dismutation of superoxide anion (25). Cytochrome c may oxidize dihydroethidium (25), but it is very unlikely that any substantial cytochrome c release occurred during the dihydroethidium experiments because these rabbits were not subjected to ischemia and reperfusion. It is also possible that generation of an oxygen radical species other than superoxide is stimulated by isoflurane administration to initiate the signaling events involved in myocardial protection.
In summary, the present findings indicated that the electron transport chain complex III inhibitor MYX, but not the complex I inhibitor DPI, abolishes isoflurane-induced reductions in myocardial infarct size and increases in ROS. These results indicate that ROS generated from mitochondrial electron transport chain complex III are crucial intracellular mediators of isoflurane-induced preconditioning in vivo.
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Acknowledgments
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Supported, in part, by the American Heart Association (Dallas, TX) Northland Affiliate grant 0151487Z (JTE), National Institutes of Health grants HL 03690 (JRK), HL 63705 (JRK), HL 54820 (DCW), GM 066730 (DCW) and GM 08377 (DCW) from the United States Public Health Service (Bethesda, MD).
The authors thank David A. Schwabe, BSEE (Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin) and Michele M. Henry, BS (Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin) for technical assistance, and Mary Lorence-Hanke, AA (Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin) for assistance in preparation of the manuscript.
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Accepted for publication May 20, 2004.
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A. S. Midzak, J. Liu, B. R. Zirkin, and H. Chen
Effect of Myxothiazol on Leydig Cell Steroidogenesis: Inhibition of Luteinizing Hormone-Mediated Testosterone Synthesis but Stimulation of Basal Steroidogenesis
Endocrinology,
June 1, 2007;
148(6):
2583 - 2590.
[Abstract]
[Full Text]
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Abstract
Introduction
