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Anesth Analg 2005;100:46-53
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000139346.76784.72

CARDIOVASCULAR ANESTHESIA

Anesthetic Preconditioning: The Role of Free Radicals in Sevoflurane-Induced Attenuation of Mitochondrial Electron Transport in Guinea Pig Isolated Hearts

Matthias L. Riess, MD, PhD*,{dagger},§, Leo G. Kevin, FCARCSI*, Joseph McCormick, BS{ddagger}, Ming T. Jiang, PhD*, Samhita S. Rhodes, PhD*, and David F. Stowe, MD, PhD*,§,||,¶,#

*Anesthesiology Research Laboratories, Departments of Anesthesiology and §Physiology and ||Cardiovascular Research Center, {ddagger}Medical College of Wisconsin, Milwaukee, Wisconsin; {dagger}Westfälische-Wilhelms-Universität, Münster, Germany; and ¶Veterans Affairs Medical Center Research Service and #Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin

Address correspondence and reprint requests to David F. Stowe, MD, PhD, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Address e-mail to dfstowe{at}mcw.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Cardioprotection by anesthetic preconditioning (APC) can be abolished by nitric oxide (NO·) synthase inhibitors or by reactive oxygen species (ROS) scavengers. We previously reported attenuated mitochondrial electron transport (ET) and increased ROS generation during preconditioning sevoflurane exposure as part of the triggering mechanism of APC. We hypothesized that NO· and other ROS mediate anesthetic-induced ET attenuation. Cardiac function and reduced nicotinamide adenine dinucleotide (NADH) fluorescence, an index of mitochondrial ET, were measured online in 68 Langendorff-prepared guinea pig hearts. Hearts underwent 30 min of global ischemia and 120 min of reperfusion. Before ischemia, hearts were temporarily perfused with superoxide dismutase, catalase, and glutathione to scavenge ROS or NG-nitro-L-arginine-methyl-ester (L-NAME) to inhibit NO· synthase in the presence or absence of 1.3 mM sevoflurane (APC). APC temporarily increased NADH before ischemia, i.e., it attenuated mitochondrial ET. Both this NADH increase and the cardioprotection by APC on reperfusion were prevented by superoxide dismutase, catalase, and glutathione and by NG-nitro-L-arginine-methyl-ester. Thus, ROS and NO·, or reaction products including peroxynitrite, mediate sevoflurane-induced ET attenuation. This may lead to a positive feedback mechanism with augmented ROS generation to trigger APC secondary to altered mitochondrial function.

IMPLICATIONS: Nitric oxide and other reactive oxygen species mediate sevoflurane-induced attenuation of mitochondrial electron transport in Langendorff-prepared hearts. This may lead to a positive feedback mechanism that initiates cardiac anesthetic preconditioning and attenuates ischemia/reperfusion injury.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Enhanced formation of reactive oxygen species (ROS) has been implicated as an essential component of the triggering mechanism of cardiac anesthetic preconditioning (APC): different scavengers of ROS abolish or attenuate the otherwise protective effects of temporary exposure to a volatile anesthetic against cardiac ischemia/reperfusion (I/R) injury (1–4). We recently reported temporary increases in the reduced form of nicotinamide adenine dinucleotide (NADH) (5,6) and in ROS (4) during preconditioning exposure to sevoflurane. An increase in NADH suggests attenuated mitochondrial (m) electron transport (ET) (Fig. 1). This could explain the mildly increased ROS generation as a signal during anesthetic exposure. Superoxide (O2· –) could be formed along one or more sites of the ET system, and O2· – itself or its reactants could act as a signal on further downstream effectors of APC, such as tyrosine kinase or protein kinase C cascades (7,8). This latter action is postulated to cause a preconditioning memory effect and to lead to opening of mitochondrial and/or sarcolemmal adenosine triphosphate (ATP)-sensitive K+ (KATP) channels and cardioprotection (9,10). However, the exact signaling cascade from the anesthetic exposure to attenuated I/R injury is not well elucidated, and it remains unclear how and where ROS are formed during the anesthetic exposure. Because various radicals and reactants, including nitric oxide (NO·) and peroxynitrite (ONOO–), have been described to attenuate mET at different sites (11,12), it is possible that O2· – or reactants formed initially during the anesthetic exposure further attenuate mET and so exert a positive feedback effect on the ET system (10).



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  		Figure 1. Mitochondrial electron transport and reactive oxygen species formation. Reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2) are reoxidized to nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD) at Complex I and II of the electron transport system in the inner mitochondrial membrane, which provides the electrons that reduce oxygen to H2O at Complex IV. Electron transport along the electron transport system from Complex I or II via ubiquinone (UQ), Complex III, cytochrome c (cyt c), and Complex IV provides the energy to actively pump protons from the mitochondrial matrix to the intermembrane space and establish a proton gradient and a membrane potential across an otherwise impermeable inner mitochondrial membrane. This membrane potential is the driving force for protons to reenter the mitochondrial matrix through Complex V and drive adenosine triphosphate (ATP) synthesis. Complexes I and III are potential sources of electron leakage and superoxide (O2· –) production. Attenuated electron transport by a volatile anesthetic, e.g., at Complex III, would lead to increased levels of NADH and/or FADH2 and decreased levels of NAD and/or FAD. In contrast, accelerated electron transport, as hypothesized with mitochondrial ATP-sensitive K+(KATP) channel opening, would lead to decreased levels of NADH and FADH2 and increased levels of NAD and FAD. O2· –, possibly after reacting with nitric oxide (NO·) to form peroxynitrite (ONOO–), could further attenuate electron transport at Complex I and possibly lead to increased O2· – formation. ADP = adenosine diphosphate.

 
The purpose of this study was to investigate whether the previously reported attenuation of mET by sevoflurane is mediated in part by ROS or reactants. To do so, we infused ROS scavengers and an NO· synthase (NOS) inhibitor during sevoflurane exposure and assessed changes in mET by online measurements of NADH fluorescence in Langendorff-prepared guinea pig hearts.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The investigation conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health publication No. 85-23, revised 1996) and was approved by the Institutional Animal Care and Use Committee (Medical College of Wisconsin, Milwaukee, WI). Our methods have been described in detail previously (1,4–6). In short, left ventricular pressures (LVP) of 68 Langendorff-prepared guinea pig hearts perfused with Krebs-Ringer solution at 55 mm Hg and 37°C were measured isovolumetrically with a saline-filled latex balloon inserted into the left ventricle. At the beginning of each experiment, the balloon volume was adjusted to achieve a diastolic (dia) LVP of 0 mm Hg, so that any subsequent increase in diaLVP reflected an increase in left ventricular wall stiffness, or diastolic contracture. Characteristic data derived from LVP measurements were systolic (sys) and diaLVP and the maximal and minimal first derivatives of LVP as indices of contractility and relaxation, respectively. Spontaneous heart rate (HR) was monitored with bipolar electrodes placed in the right atrial and ventricular walls. Coronary flow (CF) was measured at constant temperature and perfusion pressure by an ultrasonic flowmeter (T106X; Transonic, Ithaca, NY) placed directly into the aortic (a) inflow line. Outflow (v) PO2 tension was measured continuously online with a Clark-type oxygen electrode (Model 203B; Instech, Plymouth Meeting, PA), and myocardial oxygen consumption (MVO2) was calculated as (PO2a –PO2v) · 24 µL of oxygen per milliliter (at 760 mm Hg and 37°C) · CF · heart wet weight–1. All analog signals were digitized (PowerLab/16 SP; ADInstruments, Castle Hills, Australia) and recorded at 200 Hz (Chart & Scope Version 3.6.3; ADInstruments) on a Power Macintosh computer (Apple, Cupertino, CA) for later analysis with MATLAB (The MathWorks, Natick, MA) and Microsoft Excel (Microsoft Corp., Redmond, WA) software. All variables were averaged over a sampling period of 2.5 s.

Sevoflurane (Abbott Laboratories, North Chicago, IL) was bubbled into the Krebs-Ringer perfusate with an agent-specific vaporizer (Vapor 2000; Dräger Medizintechnik GmbH, Lübeck, Germany) placed in the oxygen/CO2 gas mixture line. Samples of coronary perfusate were collected to measure sevoflurane concentrations by gas chromatography. The measured inflow concentration was 1.3 ± 0.1 mM, equivalent to 8.9 ± 0.7 vol% at 37°C. This large sevoflurane concentration was chosen to achieve better cardioprotection on reperfusion (5) and, therefore, a better discrimination of preconditioned versus nonpreconditioned hearts (6). Although this concentration is too large to maintain long-term general anesthesia, the inspired concentration can be used temporarily during mask induction to speed the onset of anesthesia (13). As previously reported (1), we used a combination of O2· – dismutase (SOD, 50 U/mL; Sigma, St. Louis, MO), catalase (50 U/mL; Sigma), and glutathione (0.5 mM; Sigma) (SCG) to scavenge ROS, i.e., to rapidly convert released O2· – to H2O, and 100 µM NG-nitro-L-arginine-methyl-ester (L-NAME; Sigma) to inhibit NOS.

Autofluorescence is widely used to measure mET in myocardial tissue (5,6,9,14–17). To assess the fluorescence of reduced NADH, each experiment was conducted in a light-blocking Faraday cage. The distal end of a fiberoptic cable was placed gently against the left ventricular anterior wall; the proximal ends were connected to a modified spectrophotometer (Photon Technology International, London, Canada). NADH fluorescence was excited at 350 nm. The shutter was opened only for 2.5-s recording intervals to prevent photobleaching. Fluorescence emissions were filtered at 405 ± 15 nm and 460 ± 10 nm (Chroma Technology Corp., Brattleboro, VT), and intensities were measured by photomultipliers (Photon Technology International).

Although autofluorescence at 460 nm could also arise from unknown intracellular constituents or cytosolic NADH, most is derived from mNADH (15). This is supported by preliminary experiments (n = 3) showing that 100 µM 2,4-dinitrophenol, an uncoupler of mET, leads to a 65% ± 13% decrease in NADH fluorescence in our model, whereas inhibition of mET by 100 µM antimycin A, a Complex III blocker, results in a 30% ± 10% increase in NADH fluorescence. This indicates that the measured NADH signal is indeed primarily of mitochondrial origin. Motion artifacts in the NADH fluorescence at 460 nm are diminished by using 405 nm as a reference wavelength that is less sensitive to changes in NADH; thus, the ratio of the intensities at 460 and at 405 nm is interpreted as a measure of NADH (16). The use of these two wavelengths also accounts for possible alterations in myoglobin light absorption, e.g., by hypoxia (16). NADH is quantitated in arbitrary fluorescence units.

Hearts were assigned to one of seven experimental groups as displayed in Figure 2. After stabilization, each experiment lasted 200 min. The 6 ischemic groups underwent 30 min of global no-flow ischemia by clamping the aortic inflow and 120 min of reperfusion. If ventricular fibrillation occurred, a bolus of 250 µg of lidocaine was immediately injected into the aortic cannula. All data were collected from hearts in sinus rhythm. At the end of each experiment, hearts were removed, and ventricular infarct size was determined by staining with 0.1% 2,3,5-triphenyltetrazolium chloride followed by cumulative planimetry, as described previously (6).



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  		Figure 2. Experimental protocol. Hearts were randomly assigned to one of seven groups. 1) Nonischemic time control hearts (TC; n = 12) were perfused with Krebs-Ringer (KR) solution for 200 min. 2) Untreated ischemic control hearts (CON; n = 12) were not exposed to sevoflurane or given an antagonist for 50 min before ischemia. 3) Anesthetic preconditioning (APC) hearts (n = 12) were exposed to 1.3 mM sevoflurane for 15 min, followed by 30 min of washout before ischemia. 4) APC + SCG hearts (n = 8) were perfused with the combination of reactive oxygen species scavengers (SCG comprises 50 U/mL superoxide dismutase, 50 U/mL catalase, and 0.5 mM glutathione) from 5 min before to 15 min after a 15-min exposure to 1.3 mM sevoflurane, followed by 15 min of washout before ischemia. 5) SCG hearts (n = 8) were perfused with SCG alone for 35 min, followed by 15 min of washout before ischemia. 6) APC + L-NAME hearts (n = 8) were perfused with 100 µM of the nitric oxide synthase inhibitor NG-nitro-L-arginine-methyl-ester (L-NAME) from 5 min before to 15 min after a 15-min exposure to 1.3 mM sevoflurane, followed by 15 min of washout before ischemia. 7) L-NAME hearts (n = 8) were perfused with L-NAME alone for 35 min, followed by 15 min of washout before ischemia. There were no differences in sevoflurane concentrations among exposed hearts, and sevoflurane was undetectable in the effluent 30 min after its washout before ischemia. All ischemic hearts underwent 30 min of global no-flow ischemia followed by 120 min of reperfusion.

 
All data are mean ± SEM. Among groups, data for a given variable and time were compared by analysis of variance (Super ANOVA 1.11 software for Macintosh; Abacus Concepts, Inc., Berkeley, CA). If F values (P < 0.05) were significant, Student-Newman-Keuls post hoc tests were used to compare the groups. Differences among means were considered significant when P < 0.05 (two tailed).


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
No differences in baseline values (time t = 0 min) for any given cardiac variable (data not shown) or NADH (Fig. 3) were observed among groups. At 120 min of reperfusion, cardioprotection in APC hearts was evidenced by improved return of LVP and its peak derivatives as indices of contractility and relaxation, CF, and MVO2, by normalized mitochondrial redox state as measured by NADH fluorescence (Table 1) and by attenuated infarct size (Fig. 4).



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  		Figure 3. Time course of reduced nicotinamide adenine dinucleotide (NADH) autofluorescence before, during, and after exposure to 1.3 mM sevoflurane for 15 min (anesthetic preconditioning [APC]; n = 12) with and without perfusion of the reactive oxygen species scavenger combination SCG (superoxide dismutase, catalase, and glutathione; n = 8) (A) or the nitric oxide synthase inhibitor NG-nitro-L-arginine-methyl-ester (L-NAME) (n = 8) (B) from 5 min before to 15 min after sevoflurane exposure. Control experiments were no treatment (CON; n = 12), or SCG (n = 8) (A) or L-NAME (n = 8) (B) alone. Data are separated for better display; however, CON and APC data in panels A and B are identical, and statistical tests included all groups. Data are mean ± SEM. {dagger}P < 0.05 versus CON; §P < 0.05 versus APC + SCG; #P < 0.05 versus SCG; ¶P < 0.05 versus APC + L-NAME; {int}P < 0.05 versus L-NAME.

 

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  		Table 1. Cardiac Variables at 120 Minutes of Reperfusion After 30 Minutes of Ischemia
 


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  		Figure 4. Ventricular infarct size after 120 min of reperfusion. Data are mean ± SEM. *P < 0.05 versus TC; {dagger}P < 0.05 versus CON; {ddagger}P < 0.05 versus APC. TC = time control; CON = control; APC = anesthetic preconditioning; SCG = superoxide dismutase, catalase, and glutathione; L-NAME = NG-nitro-L-arginine-methyl-ester.

 
When perfused alone, neither the ROS scavenger combination SCG nor the NOS inhibitor L-NAME affected any of these variables at 120 min of reperfusion. However, both SCG and L-NAME abolished the cardioprotective effects of preconditioning by sevoflurane (Table 1, Fig. 4). HR was not different among groups at 120 min of reperfusion (Table 1).

Exposure to 1.3 mM sevoflurane for 15 min before ischemia led to decreased sysLVP, contractility and relaxation, HR, and MVO2 and to increased CF (t = 20 min; Table 2). These depressant effects were not abolished by concurrent perfusion with SCG or L-NAME. SCG alone had no effect on these cardiac variables, whereas perfusion with L-NAME temporarily decreased CF and MVO2 (Table 2). All sevoflurane-induced changes were completely reversible after washout of sevoflurane immediately before ischemia (t = 50 min; data not shown).


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  		Table 2. Cardiac Variables at 15 Minutes of Sevoflurane Exposure 30 Minutes Before Ischemia
 
Exposure to 1.3 mM sevoflurane led to an increase in NADH fluorescence (Fig. 3) of approximately 30% of that observed during ischemia (data not shown). Although perfusion of SCG (Fig. 3A) or L-NAME (Fig. 3B) alone had no significant effect on NADH, either one abolished the sevoflurane-induced increase in NADH. The sevoflurane-induced NADH changes were completely reversible after washout of sevoflurane immediately before ischemia (t = 50 min; Fig. 3).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our study shows that ROS scavengers and the NOS inhibitor L-NAME not only abolished APC-induced cardioprotection, but also prevented the anesthetic-induced attenuation of mET before ischemia, as assessed by NADH fluorescence in isolated hearts. This study, and our previous studies, suggests that anesthetic-induced attenuation of mET with enhanced generation of O2· –, as well as available NO·, is required to trigger APC.

The role of ROS in effecting cardiac injury on aerobic reperfusion after ischemia is now well known (4,18). Sources of ROS on reperfusion are believed to be mitochondrial oxidoreductases, primarily Complexes I and III (Fig. 1) (19,20), as well as nicotinamide adenine dinucleotide (phosphate)/NADH oxidases, xanthine oxidase, cyclooxygenase/lipoxygenase, cytochrome P450, and uncoupled NOS (21). Excess ROS levels cause cell damage by oxidizing DNA, proteins, carbohydrates, and membrane phospholipids.

However, a mild generation of ROS has been implicated in playing a central role in triggering cardiac preconditioning, including APC. For example, concurrent administration of the ROS scavengers Mn(III)tetrakis(4-benzoic acid)porphyrine chloride (MnTBAP, a SOD mimetic), N-(2-mercaptoproprionyl)glycine, or N-acetyl-cysteine during a preconditioning isoflurane exposure abolished the attenuation of myocardial infarction in in vivo rabbit models (2,3). In addition, functional protection by sevoflurane-induced preconditioning was also abolished in the guinea pig in vitro heart by MnTBAP (4) or by the scavenger combination used in this study, SCG (1). This was reflected not only by a better functional return on reperfusion, but also by attenuated formation of O2· – (4) and other downstream reactants, including ONOO– (1). Moreover, in one of these studies we also showed, during the anesthetic exposure, a reversible increase in O2· – generation measured by increased dihydroethidium fluorescence that was prevented by MnTBAP (4). The fact that MnTBAP abolished APC suggests that O2· – plays a more important role in the triggering mechanism than just being a precursor for hydrogen peroxide (H2O2), which has also been found to precondition (22): if H2O2 or other downstream radicals were the only ROS necessary to trigger APC, then MnTBAP would be expected to lead to enhanced rather than to abolished preconditioning. The anesthetic-induced enhancement in formation of O2· – and possibly downstream radicals such as H2O2 is thought to then trigger tyrosine kinase and protein kinase C cascades (7,8) and mediate a memory effect that lasts beyond the elimination of the volatile anesthetic and stimulates KATP channel opening.

Little is known about the exact source and mechanism of O2· – formation during the preconditioning anesthetic exposure and its relation to the opening of mKATP channels. Their opening has been reported as both the cause (23) and effect (22) of ROS formation in triggering preconditioning. Probable mitochondrial sources of O2· – are Complex I and Complex III (19,20,24). Because, even under physiological conditions, a small fraction of all electrons are transferred to oxygen to form O2· –, any condition causing accelerated ET, e.g., exercise, may cause an increase in the absolute rate of ROS formation (25). Uncoupling, i.e., the dissociation of mET and phosphorylation, is one way to accelerate ET and therefore increase [O2· –]. The opening of mKATP channels could be another: according to one school of thought, mitochondrial membrane potential would be slightly dissipated by K+ influx from the cytosol; in reaction, ET and proton pumping would accelerate to maintain the membrane potential as the driving force for ATP synthesis (17). If this is true, then mKATP channel opening may cause increased [O2· –] (26) by mildly uncoupling oxidative phosphorylation and therefore may act as a trigger of APC.

However, it is not clear whether mKATP channels actually open under physiological conditions during the anesthetic exposure before ischemia. Although several studies reported increased autofluorescence of oxidized flavin adenine dinucleotide as a sign of accelerated ET due to mKATP channel opening (9,17,27), this was typically done in resting myocytes starved overnight or perfused with substrate-free solution; thus, these studies do not allow any conclusion as to the effect of volatile anesthetics on mKATP channel opening under more physiological conditions (28). Moreover, most reports on the triggering role of mKATP channels in APC rely solely on the putative specificity of 5-hydroxydecanoic acid as an mKATP channel antagonist, and this has recently been challenged (29). It is possible that mKATP channels act mainly as effectors of APC: their opening with subsequent K+ influx and concomitant water uptake is believed to prevent a decrease in mitochondrial matrix volume that would otherwise occur during ischemia and, in this manner, protect mitochondrial function during ischemia and upon reperfusion (28). Prior anesthetic exposure would merely sensitize them to open earlier and to a greater extent during ischemia (9).

Another possibility to increase [O2· –] as a trigger of APC could be partial inhibition of the mET system. When ET is attenuated while the components of the ET system are still functional, electrons are more likely than under physiological circumstances to leak and form O2· – (19,24). Several volatile anesthetics were long ago found to increase NADH (30,31). We have recently reported a reversible increase in reduced NADH during the preconditioning exposure to sevoflurane in intact hearts (5,6). Increased NADH means relatively less mitochondrial oxidation of reducing equivalents than are being produced, i.e., a relative attenuation of ET (Fig. 1). Thus, anesthetic-induced attenuation of mET could result in increased [O2· –] and downstream reactants and could in this way trigger APC.

ROS have been described not only to result from attenuated mET, but also to cause attenuated mET (11). Indeed, this study shows that the increase in NADH, i.e., attenuated ET, during sevoflurane exposure is abolished by the ROS scavenger SCG. In another study with isolated cardiac mitochondria, we found that the O2· – scavengers MnTBAP and N-tert-butyl-a-(2-sulfophenyl)nitrone sodium prevented the sevoflurane-induced attenuation of mitochondrial respiration for Complex I substrates (pyruvate with malate), but not for Complex II substrates (succinate with rotenone to block Complex I) (32). Taken together, our results suggest that attenuation of mET by sevoflurane at Complex I is mediated by O2· – itself or O2· – reactants, whereas the sevoflurane-induced attenuation at other sites is likely caused by different mechanisms. This offers the interesting possibility of a positive feedback mechanism and amplification between O2· –, likely formed at Complex III (33), and an attenuated ET at Complex I that may then lead to more formation of O2· – and downstream reactants to trigger APC (Fig. 1).

Most NO· is released from endothelial cells. Therefore, studies in intact hearts provide a more physiological balance between NO· and other ROS than studies in isolated myocytes or subcellular preparations. Because NO· is a free radical produced constitutively and because O2· – reacts faster with NO· to produce ONOO– than it does with SOD, NO· is an important factor to be considered when APC is studied in intact hearts. Interestingly, endothelial NOS can produce either NO· or O2· – (21).

NO· and ONOO– have been found to attenuate mET (12), and the NOS inhibitor L-NAME reversed APC in a previous study (1). Therefore, we tested the hypothesis that NO·, in addition to other ROS such as O2· –, is directly or indirectly involved in the sevoflurane-induced attenuation of ET. Although inhibiting NOS alone did not have a significant effect on mET, it prevented the sevoflurane-induced attenuation of mET; this suggests that NO· (from a nonmyocyte or possibly an m source) contributes to the modulation of mET by sevoflurane (Fig. 1).

Because of our approach with two different APC antagonists, SCG and L-NAME, we are limited in understanding the exact mechanism. Although O2· – and NO· appear to be involved in APC, the actual ROS or reactants remain unknown. Neither SCG nor L-NAME has an untoward effect in the absence of ischemia, except that CF and subsequently MVO2 are slightly decreased by L-NAME, which suggests that there is continuous formation of NO· by the vascular endothelium. Thus, the modulating role of NO· could be permissive in that its presence allows the formation of ONOO– generated from O2· – and NO·, with ONOO– as the ET-attenuating molecule. If the formation of either precursor is reduced, ONOO– cannot be produced. Another possibility is that sevoflurane triggers endothelial NOS to produce O2· –, which then attenuates mET. In this case also, either inhibiting NOS or scavenging oxygen would result in the observed restoration of mET. Of course, ROS and NO· may have other roles in APC in addition to the attenuation of ET. Like ROS, NO· may also play a role downstream of attenuated ET and thereby mediate preconditioning (22).

In summary, our study shows that the role of NO· and other ROS in the preconditioning cascade triggered by temporary anesthetic exposure is more complex than previously thought. Both the NOS inhibitor L-NAME and the ROS scavenger combination SCG not only abolished the cardioprotection by APC, but also prevented the attenuation of mET during the preconditioning exposure to sevoflurane, as assessed by NADH fluorescence. This study supports the role of temporarily altered mitochondrial function as a trigger for APC and contributes to our understanding of the phenomenon of preconditioning to attenuate myocardial I/R injury.


   Acknowledgments
 
Supported in part by Grants Ri 1132/1-1 (MLR) from the German Research Foundation (Bonn, Germany), 0355608Z (DFS) from the American Heart Association (Dallas, TX), and HL58691 (DFS) from the National Institutes of Health (Bethesda, MD).

The authors thank Amadou K. S. Camara, PhD, James Heisner, BS, Srinivasan G. Varadarajan, MD (all Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI), and Janis T. Eells, PhD (College of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI) for their valuable contributions to this study.


   Footnotes
 
Presented in part at the annual meeting of the American Society of Anesthesiologists, San Francisco, CA, October 11–15, 2003.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication June 22, 2004.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press