Anesthetic preconditioning: triggering role of reactive oxygen and nitrogen species in isolated hearts

Departments of ¹ Anesthesiology and ² Physiology, ³ Biophysics Research Institute, and ⁴ Cardiovascular Research Center, Medical College of Wisconsin 53226, and ⁵ Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We postulated that anesthetic preconditioning (APC) is triggered by reactive oxygen/nitrogen species (ROS/RNS). We used the isolated guinea pig heart perfused with L-tyrosine, which reacts with ROS and RNS to form strong oxidants, principally peroxynitrite (ONOO), and then forms fluorescent dityrosine. ROS scavengers superoxide dismutase, catalase, and glutathione (SCG) and NO· synthesis inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) were given 5 min before and after sevoflurane preconditioning stimuli. Drugs were washed out before 30 min of ischemia and 120 min of reperfusion. Groups were control (nontreated ischemia control), APC (two, 2-min periods of perfusion with 0.32 ± 0.02 mM of sevoflurane; separated by a 6-min period of perfusion without sevoflurane), SCG, APC + SCG, L-NAME, and APC + L-NAME. Effluent dityrosine at 1 min reperfusion was 56 ± 6 (SE), 15 ± 5, 40 ± 5, 39 ± 4, 35 ± 4, and 33 ± 5 units (P < 0.05 vs. APC), respectively; left ventricular pressure (%baseline) at 60 min of reperfusion was 30 ± 5, 60 ± 4, 35 ± 5, 37 ± 5, 44 ± 4, and 47 ± 4; and infarct size (%total heart weight) was 50 ± 5, 19 ± 2, 48 ± 3, 46 ± 4, 42 ± 4, and 45 ± 2. Thus APC is initiated by ROS as shown by improved function, reduced infarct size, and reduced dityrosine on reperfusion; protective and ROS/RNS-reducing effect of APC were attenuated when bracketed by ROS scavengers or NO· inhibition.

guinea pig; experimental; pathophysiology; contractile function; infarct size

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CARDIAC ISCHEMIC PRECONDITIONING (IPC), first described in 1986 (21), is most often assessed by observations of reduced infarct size, attenuated mechanical dysfunction, or limited ultrastructural abnormality on reperfusion after prolonged ischemia (3, 5, 16, 25, 27, 28, 43). Anesthetics can also precondition hearts against ischemic-reperfusion (IR) injury (3, 7, 14, 25, 28). For example, Novalija and Stowe (28) reported that anesthetic preconditioning (APC) with sevoflurane mimics IPC by improving vascular, mechanical, and metabolic function in isolated hearts.

The role of reactive oxygen species (ROS) in effecting cardiac injury on aerobic reperfusion after ischemia is now well known (11, 32). The seemingly paradoxical role of ROS and reactive nitrogen species (RNS) in triggering or mediating IPC has gained increasing importance (23, 33, 37). Studies using inhibitors of nitric oxide (NO·) synthase during IR injury suggest the specific role of NO· in cardioprotection is unclear (12, 16, 22, 23, 33, 42-44). However, because NO· is a free radical produced constitutively and because superoxide (O·) reacts faster with NO· than it does with superoxide dismutase (SOD), NO· is a factor that must be considered in IR injury. We showed that NO· release is improved along with vascular and mechanical function after IPC and APC with sevoflurane and that these effects are blocked by ATP-sensitive K (K_ATP) channel inhibition (25, 28). Peroxynitrite (ONOO), the product of NO· and O·, is released on reperfusion after ischemia (45) and may be more protective than its precursor free radicals, because with its short half-life, it can react with other molecules to produce a long-lasting NO· carrier (24). Several studies indirectly support the notion that volatile anesthetics cause release of free radicals by mitochondrial or other oxidases in cardiac and vascular tissue (8, 34), but the stimulus for this release is not known. Sevoflurane (46) and isoflurane (29) have been proposed to cause formation of ROS directly in cardiac tissues. It may be possible that an anesthetic can cause free radical release from itself or the tissue with which it interacts.

The objective of this study was to explore a possible causal relationship between APC and the generation of ROS/RNS. We tested whether anesthetic-induced cardioprotection causes reduced ROS/RNS formation during IR injury, and whether anesthetic preconditioning is triggered in part by ROS/RNS formation during anesthetic exposure as evidenced by inhibited APC-induced cardioprotection by ROS scavengers and a NO· inhibitor given during anesthetic exposure and by restored production of ROS/RNS on reperfusion.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Langendorff heart preparation. The investigation conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health No. 85-23, Revised 1996). Prior approval was obtained from the Medical College of Wisconsin animal studies committee. The preparation has been described in detail previously (2, 3, 25, 28, 39). Guinea pigs (n = 56) were prepared by the Langendorff method and perfused via the aortic root at 55 mmHg with a 37°C oxygenated, modified Krebs-Ringer (KR) solution as described previously (2, 3, 32, 35, 52).

Left ventricular pressure (LVP), spontaneous heart rate (HR), atrioventricular conduction time, and coronary flow (CF) were measured continuously. Coronary sinus effluent was collected by placing a small catheter into the right ventricle through the pulmonary artery after the ligation of both venae cavae. Coronary sinus venous PO₂ tension (Pv_O2) was also measured continuously on-line with an O₂ Clark-type electrode (model 203B, Instech; Plymouth Meeting, PA). Percent O₂ extraction was calculated as 100 · (Pa_O2  Pv_O2/Pa_O2), where Pa_O2 is arterial PO₂; myocardial O₂ consumption (MO₂) was calculated as CF/g · (Pa_O2  Pv_O2) · 24 µl O₂/ml at 760 mmHg; and cardiac efficiency was calculated as systolic-diastolic LVP · HR/MO₂.

Effluent was spot collected during reperfusion and frozen for later analysis of creatine kinase (Creatine Kinase Flex reagent cartridge, Dade Behring Dimension; Newark, DE; sensitivity > 10 U/l). If ventricular fibrillation (VF) occurred, a 0.25-ml bolus of lidocaine (250 µg) was administered immediately via the aortic cannula. Data were collected only from hearts naturally in, or converted, to sinus rhythm. Infarct size was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) staining method and expressed as a percentage of the total heart weight (2).

Dityrosine fluorescent indicator of peroxynitrite formation in coronary effluent. ONOO is the product of free radicals O· (superoxide) and NO·. This reaction proceeds with a rate constant comparable to the reaction between O· with SOD (k = 10⁹ M¹ · s¹). We used the method of Yasmin et al. (45) in isolated hearts to estimate ROS and RNS production by the reaction of these species with L-tyrosine in phosphate buffer at pH 6.0 to form the fluorescent product dityrosine (DTY) (1). Both authentic ONOO and decomposed ONOO were prepared by the method described by Villa et al. (41). The sensitivity and linearity of this reaction were tested in KR solution containing 0.3 mM L-tyrosine and authentic ONOO (0.1-10 µmol/l) or the equivalent volume of decomposed ONOO for 15 min at 37°C as shown previously (19). Formation of DTY was analyzed in the incubation solution by measuring fluorescence spectra, excitation (_ex 320 nm) and emission (_em 410 nm), at room temperature using a spectrophotofluorometer (Perkin Elmer model LS 50B, Beaconsfield, Buckinghamshire, UK). DTY concentration measured by HPLC is linearly related to fluorescence intensity (r² > 0.99); the detection limit for DTY is reported as 0.05 µmol/l (45). Reaction of L-tyrosine with authentic ONOO to produce DTY in KR buffer occurs completely within 1 min, and the product is stable in room air for over 1 h (45). Collected effluent samples were kept at 3°C until measured for DTY concentration within 15 min at 25°C.

Protocol. Figure 1 shows the experimental design. There were six L-tyrosine-treated groups subjected to IR and one untreated time control group (data not shown); each group comprised eight hearts. Each experiment lasted 200 min beginning 30 min after equilibration. Hearts were assigned randomly into six 30-min global ischemia groups. In three groups, hearts were exposed to two, 2-min periods of perfusion with sevoflurane delivered by vaporizer (2.5 vol%); these periods were separated by a 6-min period of perfusion without sevoflurane (APC). Sevoflurane was detected by gas chromatography (GC-8AIF, Shimadzu; Kyoto, Japan) as described previously (25). Aortic inflow concentration was 0.32 ± 0.02 mmol/l, which is equivalent to 2.27 ± 0.14 vol% for a minimal alveolar concentration of ~1.03 ± 0.06. Sevoflurane was not detectable in the effluent at the end of the 15-min washout period before ischemia. To determine whether anesthetic-induced preconditioning is triggered by ROS/RNS, scavengers of ROS or the NO· synthesis inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) were given beginning 5 min before sevoflurane, during sevoflurane exposures, and for 5 min after the second sevoflurane exposure. ROS scavengers used to block triggering were SOD (S, 50 µmol/l), catalase (C, 50 µmol/l), and glutathione (G, 0.5 mmol/l) (or called SCG); L-NAME concentration was 100 µmol/l. Two additional groups of hearts were pretreated with the combination of the ROS scavengers or with L-NAME alone to rule out residual effects of these drugs.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Schema for protocols used in six randomized groups of guinea pig hearts. Anesthetic preconditioning (APC) pulses were elicited by exposing hearts to 2.5% sevoflurane at normal cardiac perfusion pressure for two 2-min periods. Combination of reactive oxygen species (ROS) scavengers, superoxide dismutase (SOD, 50 U/ml), catalase (50 U/ml), and glutathione (0.5 mmol/l) (SCG), or NG-nitro-L-arginine methyl ester (L-NAME, 100 µmol/l) were infused for 5 min before, during, and 5 min after APC.

CF responses to bolus adenosine (0.2 ml of 200 mM stock), 100 µmol/l nitroprusside, and 10 nmol/l bradykinin were tested at the end of reperfusion (at 200 min). For DTY concentration measurements, coronary effluent samples (2.7 ml) were collected at baseline (15 min), 1 min after each exposure to sevoflurane, 1 min before global ischemia, each minute during the first 5 min of reperfusion, and at 10 min of reperfusion. For creatine kinase measurements, effluent was collected 1 min before ischemia and at 1, 5, and 15 min of reperfusion.

Statistical analysis. All data were expressed as means ± SE. Within-group data (time effect) for a given variable were compared with a baseline control period (at 15 min) by Duncan's comparison of means test whenever univariate analysis of variance for repeated measures were significant (Super ANOVA 1.11 software for Macintosh from Abacus Concepts; Berkeley, CA). Among-group data (treatment effect) at specific time points (at 15, 23, 30, 81, 110, 140, and 200 min) were analyzed using multivariate analysis for repeated measures. If F values (P < 0.05) were significant, post hoc comparisons of means compared with the baseline control period (at 15 min) (Student's t-test with Duncan's adjustment for multiplicity) were used to differentiate treatment groups. The incidence of VF versus sinus rhythm was determined by ²-analysis, and differences in VF duration were determined by unpaired t-tests. Differences among means were considered statistically significant when P  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Electrical and mechanical effects. For all groups before ischemia (at 50 min) and after reperfusion (at 200 min), there were no differences in HR, respectively, 255 ± 4 and 258 ± 3 beats/min, or atrioventricular conduction time, 75 ± 2 and 74 ± 2 ms. These values were averaged for all groups (P > 0.1). The only dysrhythmia observed on reperfusion was VF, which occurred in all ischemic groups. The incidence of VF for each group, including repeat VF in a given heart, was control, 99%; APC, 38%; SCG, 98%; APC + SCG; 92%, L-NAME, 87%; and APC + L-NAME, 80% (P < 0.05). When VF occurred, its onset occurred within 1 min of reperfusion in control, SCG, APC + SCG, and L-NAME groups at 1.7 ± 0.2 min in the APC + L-NAME group, and much later, at 4.7 ± 0.4 min, in the APC group (P < 0.05).

No differences in baseline values (at 15 min) for a given cardiac variable were observed among the seven groups (nonischemia time control data not shown). Figures 2-4 display changes in mechanical and metabolic variables in the six IR groups. Systolic-diastolic (developed) LVP (Fig. 2) was not different among groups before ischemia (at 15, 23, and 30 min) but was reduced in all groups after ischemia. Developed LVP was markedly greater in the APC group than in the control group throughout reperfusion, whereas it was different from the control group in the SCG group at 110 min and in the L-NAME group at 110 and 140 min, but neither were different from the control group at 200 min. In the APC + SCG group, developed LVP was not different from the control group on reperfusion except at 80 min. In the APC + L-NAME group, developed LVP was different from the control group on reperfusion at 80 and 140 min but not at 200 min. End-diastolic LVP (not displayed) for each group at time 80 and 200 min, respectively, was 23 ± 4 to 12 ± 3 for control, 12 ± 3 to 2 ± 1 mmHg for APC, 15 ± 3 to 14 ± 4 mmHg for SCG, 36 ± 5 to 20 ± 5 mmHg for APC + SCG, 19 ± 4 to 13 ± 4 mmHg for L-NAME, and 22 ± 4 to 14 ± 3 mmHg for APC + L-NAME. End-diastolic LVP was lower (P < 0.05) for the APC group at both times except at 80 min for SCG.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Time course for developed left ventricular (LV) pressure before, during, and after global ischemia in control and sevoflurane preconditioned groups (APC), with and without the combination of ROS scavengers (SCG) or L-NAME. SCG or L-NAME slightly reduced baseline developed LV pressure. Developed LV pressure was least reduced in APC-treated groups on reperfusion. Other details in text. sys, Systolic; dia, diastolic.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Time course for myocardial O2 consumption (MO2, in µl O2 · g1 · min1) before, during, and after global ischemia in control and sevoflurane preconditioned groups (APC), with and without combination of ROS SCG or L-NAME. Note that MO2 was nearly restored during reperfusion after APC compared with other groups.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Time course for efficiency index (in mmHg · beat · g1 · 0.1 µl O21) before, during, and after global ischemia in control and sevoflurane preconditioned groups (APC) with and without combination of ROS scavengers (SCG) or L-NAME. Note that cardiac efficiency was markedly improved during reperfusion after APC.

Metabolic effects, infarct size, and formation of ROS/RNS. Percent O₂ extraction (data not displayed) was 65.7 ± 4.3% for the APC group at 30 min of reperfusion (at 110 min), which was not significantly different from 71.9 ± 4.9% for control, 69.6 ± 5.1% for SCG, 72.5 ± 4.4% for APC + SCG, 69.7 ± 4.5% for L-NAME, and 67.8 ± 3.1% for APC + L-NAME group. MO₂ (in µl · g¹ · min¹; Fig. 3) was below baseline in each group but highest in the APC group; in other treated groups, MO₂ was no different from that of the control group during reperfusion (at 140 and 200 min). Cardiac efficiency index (Fig. 4; in mmHg · beat/g¹ · µl O₂¹ min¹) was depressed after global ischemia in all groups immediately after ischemia and increased most in the APC group during later reperfusion. In the other treated groups, cardiac efficiency was generally lower than in the APC group but higher than in the control group. Table 1 shows that CF was higher throughout reperfusion in the APC group than in all other groups and returned to the baseline level only in the APC group. Flow was lowest at 1 min of reperfusion (at 80 min) in the control group and was similarly lower in all but the APC group at 120 min of reperfusion (at 200 min).

View this table: [in this window] [in a new window]   Table 1.   Effects of APC on coronary flow in control, APC, SCG, APC + SCG, L-NAME, and APC + L-NAME groups before, during, and after sevoflurane exposure and on reperfusion after ischemia

Myocardial infarct size (Fig. 5) was significantly reduced in the APC group compared with all other treated groups and the control group; there were no differences among the other treated groups. Table 2 summarizes effects of APC on creatine kinase in control, APC, SCG, APC + SCG, L-NAME, and APC + L-NAME groups on reperfusion after ischemia. Creatine kinase was not detected at 15 and 50 min of perfusion in any group.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Myocardial infarct size expressed as a percentage of total heart weight in guinea pig hearts receiving combination of ROS scavengers (SCG) or L-NAME in the presence or absence of preconditioning by sevoflurane. Note the large protection against infarction by APC compared with other groups.

View this table: [in this window] [in a new window]   Table 2.   Effects of APC on creatine kinase in control, APC, SCG, APC + SCG, L-NAME, and APC + L-NAME groups before, during, and after sevoflurane exposure and on reperfusion after ischemia

Dityrosine fluorescence (Fig. 6), a marker of ROS/RNS production, was not detected in the effluent before ischemia in any group with or without sevoflurane treatment. After global ischemia, dityrosine fluorescence was markedly increased in the control group; dityrosine fluorescence was reduced by 73 ± 4, 32 ± 3, 30 ± 3, 37 ± 4, and 41 ± 4% in APC, SCG, APC + SCG, L-NAME, and APC + L-NAME groups, respectively, compared with the control group at 1 min of reperfusion. Release of dityrosine (relative fluorescence units/l)(ml · g¹ · min¹) was significantly lower (P < 0.05) after APC (114 ± 2) compared with control (263 ± 6) at 1 min reperfusion.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Release of fluorescent dityrosine into the coronary effluent during and after sevoflurane exposure and after global ischemia in CON and sevoflurane preconditioned groups (APC), with and without combination of ROS scavengers (SCG) or L-NAME. Values are expressed as the change in relative units of fluorescence from the baseline value measured in effluent before treatments. Note the change in the time scale. Dityrosine concentration was reduced by 73% after APC and less so in other groups.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We found that APC induced by temporary exposure to sevoflurane was characterized by significantly improved postischemic contractility, basal flow, metabolism, and cardiac work efficiency throughout reperfusion. Moreover, APC was exhibited by a significant reduction in infarct size after 120 min of reperfusion and markedly reduced creatine kinase production. Dityrosine is an indirect marker of ROS/RNS production; it reflects formation of strong oxidants of which ONOO is likely the principal oxidant. Accompanying the cardioprotective effects of prior anesthetic exposure was less formation of dityrosine. It was clear that the protective effects of APC were initiated before the onset of ischemia ("memory phase") because the anesthetic was washed out before ischemia. Importantly, APC-induced protective effects on function and infarct size were abolished, or nearly so, by ROS scavengers and attenuated by NO· inhibition.

Anesthetic preconditioning. It is now well known that APC, i.e., exposure of the heart to a volatile anesthetic followed by its washout, protects the heart against subsequent ischemia-reperfusion injury (3,6, 14, 18, 28, 35). APC is as protective as IPC in many models and has the distinct advantage that ischemia is not required to initiate the preconditioning phenomena. It has been difficult to understand how these divergent stimuli, anesthetic exposure and brief ischemia, can both lead to preconditioning. Common mechanisms between IPC and APC are K_ATP channel opening and activation of protein kinase cascades (14, 18, 27, 35). Using a protocol identical to the present study, we reported that APC is as effective as IPC on improving basal and NO·-mediated CF as well as cardiac rhythm, perfusion, mechanical, and metabolic function (25). Moreover, the protective effects of APC and IPC were antagonized by glibenclamide, suggesting a common final mechanism via activation of K_ATP channels.

We propose that volatile anesthetics trigger preconditioning in part by inducing formation of ROS/RNS because APC was nearly abolished by bracketing anesthetic exposure with perfusion of ROS scavengers. The triggering effect could involve induction by NO·, O·, H₂O₂, ·OH, or other reactive species. The initial bursts of ROS could have a direct action to stimulate intracellular pathways that elicit cardioprotection, or ROS formation could be modulated by sarcolemmal or mitochondrial K_ATP channels, as postulated for ROS in IPC (5). A caveat of this proposal is that we could not detect dityrosine in the effluent during anesthetic exposure before ischemia. We have observed, however, that two, 2-min pulses of ischemia before a longer index ischemia caused a significant formation of dityrosine (26). It is possible that our technique for assessing ROS was not sensitive enough to detect a small release of strong oxidants such as ONOO into extracellular fluid during anesthetic exposure.

Another finding was that blocking endothelial NO synthase with L-NAME during anesthetic exposure reduced dityrosine formation on reperfusion, with a partial inhibitory effect on function compared with APC alone, but did not reduce infarct size. Moreover, compared with nonpreconditioned controls, L-NAME alone slightly improved function during early reperfusion while reducing initial dityrosine release. The ROS scavengers alone had little effect on reperfusion injury when given before ischemia. We have reported that volatile anesthetics did not alter bradykinin-induced NO· release (10), so we suggest that exposure to sevoflurane before ischemia induces formation of ROS rather than inhibiting formation of RNS.

Role of ROS and RNS in anesthetic preconditioning. The role of ROS in effecting cardiac injury on aerobic reperfusion after ischemia is now well known (11, 32). ROS have long been regarded as toxic byproducts of anaerobic metabolism. ROS include the free radicals O·, HO· (hydroxyl), NO·, and LOO· (lipid peroxyl); non-free radicals include H₂O₂, ONOO, and HOCl. Major sources of ROS are believed to be NAD(P)H oxidases, other mitochondrial oxidases (primarily complexes I and III), xanthine oxidase, cyclooxygenase-lipoxygenase, cytochrome P-450, and uncoupled NO synthase (9, 13, 36, 38, 40). Excess ROS cause cell damage by oxidizing DNA, protein, carbohydrates, and membrane phospholipids.

A limitation of our method is that it does not allow determination of the exact nitrogen or oxygen reactive species that are involved in anesthetic preconditioning. We also cannot know the relative contributions of these reactive species by the dityrosine detection method. We speculate that normally the O·/NO· ratio is low in vascular endothelium. Inhibiting NO· production before ischemia might deplete the pool of NO· to react with O·, thus mediating APC, if index ischemia occurs. Increasing the O·/NO· ratio by L-NAME could favor prooxidant (via O·) rather than antioxidant effects of NO· so that APC is induced by the higher resting formation of O·. On reperfusion, excess NO· might inhibit cytochrome oxidase so that electron backup in the electron transport chain leads to more O· formation by leakage of electrons from the electron carriers (30).

The combination of ROS scavengers SCG completely abolished all cardioprotective effect of APC, but L-NAME also attenuated the protective effects of APC. We speculate that APC is mediated primarily by an increase in O· rather than by a decrease in NO· because: 1) in the same model, volatile anesthetics neither stimulated NO· release nor enhanced bradykinin-induced NO· release (10); 2) L-NAME may exert long-lasting effects that extend into the reperfusion period thus blocking the potentially beneficial scavenging effect of NO· to destroy O· on reperfusion; 3) in the same model brief pulses of ischemia induce dityrosine formation that is completely blocked by the same ROS scavengers (26); and 4) sevoflurane has been reported to generate free radicals (46). It is clear that the precise role of NO· in APC remains to be determined.

It may appear paradoxical that volatile anesthetics trigger formation of ROS. It is not believed that anesthetics directly cause cardiac ischemia; in contrast, they reduce metabolism and contractility. Because they suppress metabolism, anesthetics may stimulate formation of a small quantity of ROS to confer protection in a manner similar to IPC. It has long been known that under certain conditions, anesthetics can react with cell constituents to undergo reductive metabolism with release of ROS (20). Several anecedotal reports suggest that anesthetics do (8, 15, 34) or do not (17) alter release of reactive oxidants, including NO· (31), after ischemia or oxidative stress-induced injury. Anesthetics could cause release of free radicals by mitochondrial or other oxidases in cardiac and vascular tissue or they could modify the activity of released reactive oxidants. Tanguay et al. (34) reported that pretreatment of hearts with an anesthetic attenuated the effect of free radicals produced by electrolysis on reducing coronary flow and LVP. Sevoflurane (46) and isoflurane (29) have also been proposed to cause formation of ROS in cardiac tissues. Using the electron-spin resonance spin-trap 5,5'-dimethyl-pyrroline-N-oxide (DMPO), ·OH and/or O· was detected in the bath of isolated canine mesenteric artery rings exposed to sevoflurane; the ·OH radical was eliminated in the presence of SOD (46). Park et al. (29) demonstrated that isoflurane-induced endothelium-dependent constriction of coronary arteries in vitro was inhibited in the presence of ROS scavengers SOD or mannitol. These studies do not prove, but strongly suggest, that an anesthetic can cause free radical release from itself or from the tissue with which it interacts.

The role of RNS in APC is even less clear than the role of ROS. Because most NO· is released from endothelial cells, studies in intact heart models are useful because the balance of NO· and O· is likely more physiological than in isolated myocytes. Involvement of O· radicals in cardiac IR injury necessarily involves NO· radicals because these molecules rapidly interact. It is also possible that anesthetics increase the sensitivity of cells to ROS or cause endothelial NO synthase to produce O· radicals (40) rather than trigger their release directly. Volatile anesthetics have been reported to interfere with NO· to reduce its vasorelaxation effects (4); this could arise from anesthetic-induced O· formation and its reaction with NO· to form ONOO, thus deactivating NO·. In the same model, we (3) reported that APC improves myocardial function and perfusion, reduces dysrhythmias and improves responses to vasodilators with restored NO· production in isolated guinea pig hearts (25); these effects were blocked by the K_ATP channel blocker glibenclamide.

The present study clearly points to a relationship between APC and ROS/RNS and a commonality between IPC and APC via ROS. There is reduced dityrosine formation suggestive of ONOO on reperfusion after IPC. The protection afforded by APC is markedly attenuated by ROS scavengers and by inhibited NO· formation. It remains unclear whether the presence of NO· affords protection before ischemia but loses that protective role on reperfusion. This may appear contradictory because it is thought that NO· might help to scavenge O· and to reduce damage to ROS during ischemia and reperfusion. However, it is possible that the presence of O·, but not NO·, is important in triggering APC, whereas continued NO· release on reperfusion is important to preserve cardiac function.

Volatile anesthetics are often selected for patients with coronary artery disease at risk for ischemia and infarction during cardiac and noncardiac surgery. Temporary ischemia is often induced during cardiac surgery and angioplasty. Prior administration of a volatile anesthetic may be a more practical, but just as efficacious, method to protect the heart. Further research will determine whether the improved cardiac function and reduced damage by APC results from improved Ca²⁺ homeostasis coupled to reduced ROS/RNS formation during ischemia-reperfusion and whether anesthetic exposure alters mitochondrial function and activates certain intracellular signaling pathways that lead to K_ATP channel opening.

	ACKNOWLEDGEMENTS

The authors thank Cathleen Berglund, Mary Lorence-Hanke, James Heisner, Sarah Laabs, Anita Tredeau, and Mary Ziebell for valuable contributions to this study.

	FOOTNOTES

This research was supported in part by National Heart Lung and Blood Institute Grants R01-HL-58691 and R01-5T32GM-08377, and by American Heart Association Grant 0020503Z. Portions of this work have appeared in abstract form: Novalija E, An JZ, Camara AK, Varadarajan SG, and Stowe DF. Anesthesiology 93: A679, 2000; Novalija E, Varadarajan SG, Heisner JS, Chen Q, Camara AK, An JZ, and Stowe DF. FASEB J 15: A1133, 2001; Novalija E, Heisner JS, Camara A, Varadarajan SG, An JZ, and Stowe DF. Anesth Analg 92: S35, 2001; Novalija E, Heisner JS, Camara AK, Varadarajan SG, An JZ, Chen Q, and Stowe DF. Biophys J: 80: 581A, 2001; and Novalija E, Hogg N, Camara AK, Varadarajan SG, and Stowe DF. Anesthesiology 95: A104, 2001.

Address for reprint requests and other correspondence: E. Novalija, Medical College of Wisconsin, Depts. of Anesthesiology and Physiology, M4280, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: novalija{at}mcw.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.

First published February 28, 2002;10.1152/ajpheart.01056.2001

Received 3 December 2001; accepted in final form 26 February 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1.   Amado, R, Aeschbach R, and Neukom H. Dityrosine: in vitro production and characterization. Methods Enzymol 107: 377-388, 1984[Web of Science][Medline].

2.   An, JZ, Varadarajan SG, Camara A, Chen Q, Novalija E, Gross GJ, and Stowe DF. Blocking Na⁺/H⁺ exchange reduces [Na⁺]_i and [Ca²⁺]_i load after ischemia and improves function in intact hearts. Am J Physiol Heart Circ Physiol 281: H2398-H2409, 2001[Abstract/Free Full Text].

3.   An, JZ, Varadarajan SG, Novalija E, and Stowe DF. Ischemic and anesthetic preconditioning reduces [Ca²⁺] and improves Ca²⁺ responses in intact hearts. Am J Physiol Heart Circ Physiol 281: H1508-H1523, 2001[Abstract/Free Full Text].

4.   Blaise, G, To Q, Parent M, Lagarde B, Asenjo F, and Sauve R. Does halothane interfere with the release, action, or stability of endothelium-derived relaxing factor/nitric oxide? Anesthesiology 80: 417-426, 1994[Web of Science][Medline].

5.   Cohen, MV, Baines CP, and Downey JM. Ischemic preconditioning: from adenosine receptor to K_ATP channel (Review). Annu Rev Physiol 62: 79-109, 2000[Web of Science][Medline].

6.   Cohen, PJ. Effect of anesthetics on mitochondrial function (Review). Anesthesiology 39: 153-164, 1973[Web of Science][Medline].

7.   Cope, DK, Impastato WK, Cohen MV, and Downey JM. Volatile anesthetics protect the ischemic rabbit myocardium from infarction. Anesthesiology 86: 699-709, 1997[Web of Science][Medline].

8.   Durak, I, Kurtipek O, Ozturk HS, Birey M, Guven T, Kavutcu M, Kacmaz M, Dikmen B, Yel M, and Canbolat O. Impaired antioxidant defence in guinea pig heart tissues treated with halothane. Can J Anaesth 44: 1014-1020, 1997[Web of Science][Medline].

9.   Duranteau, J, Chandel NS, Kulisz A, Shao Z, and Schumacker PT. Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes. J Biol Chem 273: 11619-11624, 1998[Abstract/Free Full Text].

10.   Fujita, S, Roerig DL, Chung WW, Bosnjak ZJ, and Stowe DF. Volatile anesthetics do not alter bradykinin-induced release of nitric oxide or L-citrulline in crystalloid perfused guinea pig hearts. Anesthesiology 89: 421-433, 1998[Web of Science][Medline].

11.   Henry, TD, Archer SL, Nelson D, Weir EK, and From AH. Enhanced chemiluminescence as a measure of oxygen-derived free radical generation during ischemia and reperfusion. Circ Res 67: 1453-1461, 1990[Abstract/Free Full Text].

12.   Hotta, Y, Otsuka-Murakami H, Fujita M, Nakagawa J, Yajima M, Liu W, Ishikawa N, Kawai N, Masumizu T, and Kohno M. Protective role of nitric oxide synthase against ischemia-reperfusion injury in guinea pig myocardial mitochondria. Eur J Pharmacol 380: 37-48, 1999[Web of Science][Medline].

13.   Ide, T, Tsutsui H, Kinugawa S, Utsumi H, Kang D, Hattori N, Uchida K, Arimura K, Egashira K, and Takeshita A. Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing myocardium. Circ Res 85: 357-363, 1999[Abstract/Free Full Text].

14.   Ismaeil, MS, Tkachenko I, Gamperl AK, Hickey RF, and Cason BA. Mechanisms of isoflurane-induced myocardial preconditioning in rabbits. Anesthesiology 90: 812-821, 1999[Web of Science][Medline].

15.   Johnson, ME, Sill JC, Uhl CB, Halsey TJ, and Gores GJ. Effect of volatile anesthetics on hydrogen peroxide-induced injury in aortic and pulmonary arterial endothelial cells. Anesthesiology 84: 103-116, 1996[Web of Science][Medline].

16.   Jones, SP, Girod WG, Palazzo AJ, Granger DN, Grisham MB, Jourd'Heuil D, Huang PL, and Lefer DJ. Myocardial ischemia-reperfusion injury is exacerbated in absence of endothelial cell nitric oxide synthase. Am J Physiol Heart Circ Physiol 276: H1567-H1573, 1999[Abstract/Free Full Text].

17.   Kashimoto, S, Kume M, Ikeya K, and Kumazawa T. Effects of sevoflurane and isoflurane on free radical formation in the post-ischaemic reperfused heart. Eur J Anaesthesiol 15: 553-558, 1998[Web of Science][Medline].

18.   Kersten, JR, Schmeling TJ, Pagel PS, Gross GJ, and Warltier DC. Isoflurane mimics ischemic preconditioning via activation of K_ATP channels: reduction of myocardial infarct size with an acute memory phase. Anesthesiology 87: 361-370, 1997[Web of Science][Medline].

19.   Malencik, DA, Sprouse JF, Swanson CA, and Anderson SR. Dityrosine: preparation, isolation, and analysis. Anal Biochem 242: 202-213, 1996[Web of Science][Medline].

20.   Morio, M, Yuge O, and Fujii K. Biotransformation and toxicity of inhalational anaesthetics (Review). Can J Anaesth 37: S116-S123, 1990.

21.   Murry, CE, Jennings RB, and Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124-1136, 1986[Abstract/Free Full Text].

22.   Nakamura, M, Thourani VH, Ronson RS, Velez DA, Ma XL, Katzmark S, Robinson J, Schmarkey LS, Zhao ZQ, Wang NP, Guyton RA, and Vinten-Johansen J. Glutathione reverses endothelial damage from peroxynitrite, the byproduct of nitric oxide degradation, in crystalloid cardioplegia. Circulation 102: III332-III338, 2000.

23.   Nakano, A, Liu GS, Heusch G, Downey JM, and Cohen MV. Exogenous nitric oxide can trigger a preconditioned state through a free radical mechanism, but endogenous nitric oxide is not a trigger of classical ischemic preconditioning. J Mol Cell Cardiol 32: 1159-1167, 2000[Web of Science][Medline].

24.   Nossuli, TO, Hayward R, Jensen D, Scalia R, and Lefer AM. Mechanisms of cardioprotection by peroxynitrite in myocardial ischemia and reperfusion injury. Am J Physiol Heart Circ Physiol 275: H509-H519, 1998[Abstract/Free Full Text].

25.   Novalija, E, Fujita S, Kampine JP, and Stowe DF. Sevoflurane mimics ischemic preconditioning effects on coronary flow and nitric oxide release in isolated hearts. Anesthesiology 91: 701-712, 1999[Web of Science][Medline].

26.   Novalija, E, Heisner JS, Camara AK, Varadarajan SG, An JZ, Chen Q, and Stowe DF. Ischemic preconditioning (IPC) reduces peroxynitrite release and improves function on reperfusion after global ischemia in isolated hearts (Abstract). Biophys J 80: 581A, 2001.

27.   Novalija, E, Hogg N, Camara AK, Varadarajan SG, and Stowe DF. Reactive oxygen species (ROS) are possible triggers for sevoflurane preconditioning (SPC) in isolated hearts (Abstract). Anesthesiology 95: A104, 2001.

28.   Novalija, E, and Stowe DF. Prior preconditioning by ischemia or sevoflurane improves cardiac work per oxygen use in isolated guinea pig hearts after global ischemia. Adv Exp Med Biol 454: 533-542, 1998[Web of Science][Medline].

29.   Park, KW, Dai HB, Lowenstein E, Darvish A, and Sellke FW. Oxygen-derived free radicals mediate isoflurane-induced vasoconstriction of rabbit coronary resistance arteries. Anesth Analg 80: 1163-1167, 1995[Abstract].

30.   Poderoso, JJ, Carreras MC, Lisdero C, Riobo N, Schopfer F, and Boveris A. Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch Biochem Biophys 328: 85-92, 1996[Web of Science][Medline].

31.   Sigmon, DH, Florentino-Pineda I, Van Dyke RA, and Beierwaltes WH. Halothane impairs the hemodynamic influence of endothelium-derived nitric oxide. Anesthesiology 82: 135-143, 1995[Web of Science][Medline].

32.   Simpson, PJ, and Lucchesi BR. Free radicals and myocardial ischemia and reperfusion injury (Review). J Lab Clin Med 110: 13-30, 1987[Web of Science][Medline].

33.   Takano, H, Tang XL, Qiu Y, Guo Y, French BA, and Bolli R. Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res 83: 73-84, 1998[Abstract/Free Full Text].

34.   Tanguay, M, Blaise G, Dumont L, Beique G, and Hollmann C. Beneficial effects of volatile anesthetics on decrease in coronary flow and myocardial contractility induced by oxygen-derived free radicals in isolated rabbit hearts. J Cardiovasc Pharmacol 18: 863-870, 1991[Web of Science][Medline].

35.   Toller, WG, Gross ER, Kersten JR, Pagel PS, Gross GJ, and Warltier DC. Sarcolemmal and mitochondrial adenosine triphosphate- dependent potassium channels: mechanism of desflurane-induced cardioprotection. Anesthesiology 92: 1731-1739, 2000[Web of Science][Medline].

36.   Turrens, JF, and Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J 191: 421-427, 1980[Web of Science][Medline].

37.   Vanden Hoek, TL, Becker LB, Shao ZH, Li CQ, and Schumacker PT. Preconditioning in cardiomyocytes protects by attenuating oxidant stress at reperfusion. Circ Res 86: 541-548, 2000[Abstract/Free Full Text].

38.   Vanden Hoek, TL, Shao Z, Li C, Schumacker PT, and Becker LB. Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes. J Mol Cell Cardiol 29: 2441-2450, 1997[Web of Science][Medline].

39.   Varadarajan, SG, An JZ, Novalija E, Smart SC, and Stowe DF. Changes in [Na⁺]_i, compartmental [Ca²⁺], and NADH with dysfunction after global ischemia in intact hearts. Am J Physiol Heart Circ Physiol 280: H280-H293, 2001[Abstract/Free Full Text].

40.   Vasquez-Vivar, J, Martasek P, Hogg N, Karoui H, Masters BS, Pritchard KA, Jr, and Kalyanaraman B. Electron spin resonance spin-trapping detection of superoxide generated by neuronal nitric oxide synthase (Review). Methods Enzymol 301: 169-177, 1999[Web of Science][Medline].

41.   Villa, LM, Salas E, Darley-Usmar VM, Radomski MW, and Moncada S. Peroxynitrite induces both vasodilatation and impaired vascular relaxation in the isolated perfused rat heart. Proc Natl Acad Sci USA 91: 12383-12387, 1994[Abstract/Free Full Text].

42.   Weselcouch, EO, Baird AJ, Sleph P, and Grover GJ. Inhibition of nitric oxide synthesis does not affect ischemic preconditioning in isolated perfused rat hearts. Am J Physiol Heart Circ Physiol 268: H242-H249, 1995[Abstract/Free Full Text].

43.   Xuan, YT, Tang XL, Qiu Y, Banerjee S, Takano H, Han H, and Bolli R. Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits. Am J Physiol Heart Circ Physiol 279: H2360-H2371, 2000[Abstract/Free Full Text].

44.   Yang, BC, and Mehta JL. Inhibition of nitric oxide does not affect reperfusion-induced myocardial injury, but it prevents lipid peroxidation in the isolated rat heart. Life Sci 61: 229-236, 1997[Web of Science][Medline].

45.   Yasmin, W, Strynadka KD, and Schulz R. Generation of peroxynitrite contributes to ischemia-reperfusion injury in isolated rat hearts. Cardiovasc Res 33: 422-432, 1997[Abstract/Free Full Text].

46.   Yoshida, K, and Okabe E. Selective impairment of endothelium-dependent relaxation by sevoflurane: oxygen free radicals participation. Anesthesiology 76: 440-447, 1992[Web of Science][Medline].