AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 279: H2431-H2438, 2000;
0363-6135/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (46)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bassenge, E.
Right arrow Articles by Bünger, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bassenge, E.
Right arrow Articles by Bünger, R.
Vol. 279, Issue 5, H2431-H2438, November 2000

Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state

Eberhard Bassenge1, Olaf Sommer1, Michael Schwemmer1, and Rolf Bünger2

1 Institute for Applied Physiology, University of Freiburg, D-79104 Freiburg/Breisgau, Germany; and 2 Department of Physiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals (O2-·). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical source of O2-·, implying the NADH concentration-to-NAD+ concentration ratio ([NADH]/[NAD+]) as a determinant of ROS. To test this hypothesis, cardiac NADHox and ROS formation were measured as influenced by pyruvate or L-lactate. Pre- and postischemic Langendorff guinea pig hearts were perfused at different pyruvate/L-lactate concentrations to alter cytosolic [NADH]/[NAD+]. NADHox and ROS were measured with the use of lucigenin chemiluminescence and electron spin resonance, respectively. In myocardial homogenates, pyruvate (0.05, 0.5 mM) and the NADHox blocker hydralazine markedly inhibited NADHox (16 ± 2%, 58 ± 9%). In postischemic hearts, pyruvate (0.1-5.0 mM) dose dependently inhibited ROS up to 80%. However, L-lactate (1.0-15.0 mM) stimulated both basal and postischemic ROS severalfold. Furthermore, L-lactate-induced basal ROS was dose dependently inhibited by pyruvate (0.1-5.0 mM) and not the xanthine oxidase inhibitor oxypurinol. Pyruvate did not inhibit ROS from xanthine oxidase. The data suggest a substantial influence of cytosolic NADH on cardiac O2-· formation that can be inhibited by submillimolar pyruvate. Thus cytotoxicities due to cardiac ischemia-reperfusion ROS may be alleviated by redox reactants such as pyruvate.

reduced nicotinamide adenine dinucleotide oxidase; heart; ischemia-reperfusion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MANY DISEASE STATES (atherosclerosis, hypercholesterolemia, hypertension, diabetes, ischemia-reperfusion injury, inflammation, congestive heart failure) are known to be associated with enhanced production of reactive oxygen species (ROS) and plasma lactate levels. Increased ROS formation may also reduce levels of vasodilator nitric oxide and enhance formation of the cytotoxic peroxynitrite (12). Oxidoreductases such as the membrane-associated NADH oxidase contribute to ROS formation in isolated cardiomyocytes (19). These NADH-dependent enzymes appear to be controlled, in part, by the redox state of NADH, i.e., the ratio of NAD+ concentration ([NAD+]) to NADH concentration ([NADH]) times H+ concentration ([H+]): [NAD+]/([NADH] · [H+]) (2, 5). The redox state of cytoplasmic NADH closely correlates with the tissue pyruvate concentration-to-L-lactate concentration ratio ([pyruvate]/[L-lactate]). Sustained changes in redox reactants such as pyruvate or L-lactate may be associated with altered activity or expression of lactate dehydrogenase (LDH) and glutathione peroxidase (7) and can also affect the antioxidant status of the glutathione system, i.e., the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) (25).

In the present study, we imposed cytosolic redox changes using exogenous pyruvate or L-lactate. We tested the hypothesis that ROS formation, if mediated via extramitochondrial tissue NADH oxidase activity, would be inhibited by pyruvate and stimulated by L-lactate, consistent with the concept of redox control of the NADH oxidase by the cytosolic NADH/NAD+ system. The dose-response relations between NADH oxidase activity and exogenous pyruvate were quantitated in crude homogenates of heart, liver, and aorta and compared with those of the known oxidase inhibitor hydralazine (8). We also established the full dose-response effects of pyruvate versus L-lactate on ROS production in intact perfused hearts that were subjected to a brief 5-min ischemia-reperfusion protocol. We observed striking dose-dependent inhibitions of cardiac NADH oxidase and postischemic ROS formation by pyruvate, even when applied in submillimolar near-physiological concentrations.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Heart preparation. The hearts of ketamine (100 mg/kg im)/xylazine (4 mg/kg im)-anesthetized guinea pigs were quickly isolated and perfused at constant flow with a Krebs-Henseleit solution containing (in mM) 5.5 glucose, 1.25 CaCl2, 120 NaCl, 25 NaHCO3, 1.2 MgSO4, 1.2 NaH2PO4, and 4 KCl, equilibrated with 95% O2-5% CO2 (pH 7.4) at 37°C. Initial perfusion rate was set so that perfusion pressure was 60 mmHg. During the course of the experiments, perfusion rate was held constant at 11 ml/min, and pressures varied between 55 and 65 mmHg, well within the coronary flow autoregulatory range of the guinea pig heart (11). After an initial stabilization period of 20 min, hearts were electrically paced at 250 beats/min. The protocol conformed to the National Research Council's Guide for the Care and Use of Laboratory Animals.

Five-minute ischemia-reperfusion protocol. After the initial stabilization period of 20 min, the paced hearts were subjected to a control ischemia-reperfusion period composed of 5 min of zero-flow ischemia followed by 25 min of reperfusion (Fig. 1, period I). During the last 15 min of the first reperfusion, pyruvate, L-lactate, or no additional substrate was added to the perfusion medium followed by a second ischemia-reperfusion period (Fig. 1, period II) in which redox parameters were altered as indicated. Coronary effluent samples for determination of ROS (see below) were collected every 10 s during the first minute of reperfusion; thereafter, samples were collected at 1-min intervals. Time controls (n = 3) showed no appreciable differences between the two periods in terms of the postischemic ROS release (data not shown); this indicated that the first 5-min ischemia-reperfusion period I did not precondition the myocardium against ROS production in period II.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1.   Sequential ischemia-reperfusion protocol. Solid bars, 5-min ischemic periods; open bars, preischemic/postischemic periods; periods I and II, first and second, respectively, ischemia-reperfusion periods. Pyruvate, lactate, or no additional substrate was administered, starting at 10 min of the first reperfusion period, i.e., from min 35 to 80 of the protocol.

ROS release in perfused hearts measured by electron spin resonance. Cardiac ROS formation was determined in the presence or absence of different concentrations of the redox reactants L-lactate and pyruvate. The new spin trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H) was used as detailed previously; this technique records oxyradicals, O2-· (superoxide radical), ONOO- (peroxynitrite), and ·OH (hydroxyl radical), with a rate constant of 3.2 × 103 M-1 · s-1 when O2-· reacts with the spin trap CP-H (8). CP-H concentration in the perfusate was 0.8 mM; 50 µM desferoxamine was added to minimize formation of radicals due to trace amounts of transition metals of the perfusion buffer. The stable paramagnetic reaction product, 3-carboxy-proxyl, was identified and quantitated by electron spin resonance (ESR) spectroscopy with the use of an EMX-A spectrometer (Bruker, Karlsruhe, Germany). The ESR settings were as follows: modulation frequency, 30 Hz; receiver gain, 105; microwave power, 20 mW; microwave frequency, 9.72 GHz; modulation amplitude, 1 G; conversion time, 327 ms; detector time constant, 655 ms; field sweep, 60 G; and center field, 3,474 G. Calibration of the ESR spectrometer was carried out with the use of standards supplied by the manufacturer.

Potential underestimation of ROS using CP-H spin traps in presence of pyruvate versus L-lactate. O2-· was generated by xanthine (200 µM)/xanthine oxidase (2-10 mU/ml) (X/XO) in potassium phosphate buffer (10 mM, pH 7.4, 37°C) containing 50 µM desferoxamine. The system was free from NADH and LDH. ESR quantification of ROS was done with the use of 0.7 mM CP-H as scavenger in the presence or absence of pyruvate (5-10 mM) or lactate (5 mM). Activity of the O2-· generator was ascertained by use of 1 mM oxypurinol, which completely inhibited the reaction (data not presented).

Cytosolic versus mitochondrial sites of the antioxidant feature of pyruvate. To differentiate between mitochondrial and cytosolic antioxidant effects of pyruvate under conditions of the increased oxidant stress of reperfusion, we used 0.5 mM alpha -cyano-3-hydroxycinnamate (n = 2) to selectively inhibit mitochondrial pyruvate uptake in the guinea pig heart (3) without affecting plasmalemmal monocarboxylate transport or cytosolic pyruvate metabolism (17).

Contribution of XO to cardiac ROS formation. Guinea pig coronary endothelium has XO, the activity of which could contribute to ROS release from the whole heart. To assess the possible contribution of xanthine-xanthine dehydrogenase/oxidase (X/XO) to total cardiac ROS formation, the effects of the X/XO inhibitor oxypurinol (0.1-0.5 mM) were measured in hearts the ROS production of which was stimulated by 10 mM L-lactate. Separate experiments demonstrated that 0.1 mM oxypurinol inhibited the activity of commercially available xanthine dehydrogenase (XOD; 0.05 U) by 80%.

NADH oxidase activities in crude homogenates of myocardium, aorta, and liver. The activities of NADH oxidase (NADHox) were measured in crude homogenates prepared by pulverization of frozen tissue with a dismembranator (Braun, Melsungen, Germany). ROS formation in 10-mg homogenate samples was measured with the use of 0.25 M lucigenin according to Allen (1) in 10 mM HEPES buffer, incubated with 100 µM NADH. A microplate luminometer (Berthold, Pforzheim, Germany) was used to measure the chemiluminescence intensity. For calibration, X/XO (80 mM/0.05 U) was used.

All chemicals except CP-H (Alexis, San Diego, CA) were purchased from Sigma (Deisenhofen, Germany).

Statistics and fitting nonlinear kinetics. Data are expressed as means ± SE. Student's t-tests for unpaired samples with P < 0.05 were considered significant.

Nonlinear data sets (Figs. 2 and 3) were fitted iteratively by use of the maximum likelihood method (11). In the homogenate analyses (Fig. 2), regression coefficients were estimated assuming rectangular hyperbolic kinetics
%inhibition<IT>=</IT>[pyruvate]<IT>/</IT>(<IT>&agr;·</IT>[pyruvate]<IT>+&bgr;</IT>)
where 1/alpha is maximum inhibition, beta  is rate constant, and beta /alpha is the half-maximal effective concentration. Unlike the classical double-reciprocal approach, hyperbolic models are defined for zero agonist concentration, which improves precision of fits. Regression coefficients ± SE values were estimated by use of the Newton-Raphson algorithm. Confidence in estimated SE values was obtained by residual analyses, as detailed elsewhere (11).


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2.   Semilogarithmic plot of pyruvate-induced inhibition of NADH oxidase (NADHox) activity in crude homogenates of guinea pig hearts, aortas, and livers. Zero percent NADHox inhibition, homogenate NADHox activity in the absence of exogenously added pyruvate. Already subphysiological (50 µM) pyruvate produced statistically significant (P < 0.01), albeit modest, inhibitions of measured NADHox activities. But even excess (50 mM) pyruvate failed to completely inhibit NADHox. Values are means ± SE; n = 4.



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 3.   Dose-dependent inhibition by pyruvate of postischemic reactive oxygen species (ROS) production in constant-flow (11 ml/min) perfused guinea pig hearts. A value of 100% represents basal release before pyruvate infusion and the 5-min zero-flow ischemia. A significant (P < 0.05) suppression of postischemic ROS formation was obvious within the first 10 s of reperfusion (near peak ROS release) with physiological pyruvate concentrations near 100 µM. Inset: pyruvate inhibition of total release during 60 s of reperfusion; 100 mM pyruvate inhibited total postischemic ROS release by ~25%, and 5 mM pyruvate inhibited ~80% of total ROS release. Data are single values from 4 different experiments.

Postischemic ROS release (Fig. 3) was fit by use of a lognormal function
rate of ROS release (<IT>% </IT>of basal rate)

<IT>=1+a<SUB>1</SUB>·</IT>exp{−<IT>0.5</IT>[ln (<IT>x/a<SUB>2</SUB></IT>)<IT>/a<SUB>3</SUB></IT>]<SUP><IT>2</IT></SUP>}
where a1, a2, and a3 are the coefficients to be estimated and x is the time elapsed during reperfusion in seconds. Total extra ROS released during postischemia was obtained by integration of the function over 60 s of reperfusion by use of Simpson routines (Gauss 3.5, Aptech Systems) and subtraction of the basal release as extrapolated from the preischemic data.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of pyruvate on homogenate ROS formation. Figure 2 shows that exogenously added pyruvate produced a concentration-dependent inhibition (0.05-50 mM) of ROS formation. The sigmoid inhibition patterns demonstrated that 0.05 mM pyruvate, a subphysiological concentration, modestly but significantly attenuated ROS formation (15 ± 1.5%). However, even at the extreme dose of 50 mM, pyruvate did not completely inhibit ROS formation of the homogenates. From the fits, it was estimated that the maximum ROS inhibition by pyruvate was 58.3 ± 7%, significantly less than 100%. At cardioprotective concentrations between 1 and 5 mM (9, 13, 24), pyruvate inhibited ROS formation in homogenates by ~55%. Furthermore, Fig. 2 shows that pyruvate was also an effective inhibitor of ROS formation in homogenates from aorta and liver.

In a separate series of experiments, we analyzed dose-response relations between hydralazine, an inhibitor of NADHox in rabbit aorta (21), and NADHox in heart homogenates. Hydralazine also caused dose-dependent decreases in oxidase activities in guinea pig heart homogenates. Threshold concentrations for ROS inhibition were near 0.08 mM; maximum inhibitions were below 100% and occurred near 8 mM (78 ± 7.8%, n = 6; data not shown). Thus the pyruvate inhibition kinetics of Fig. 2 compared well with those obtained with hydralazine: with both agents, threshold inhibitory doses were around 0.1 mM, and maximum inhibitions were approached at concentrations between 5 and 10 mM. Neither agent was able to completely inhibit ROS formation. Thus the NADHox inhibition kinetics due to pyruvate compared well with those due to hydralazine. Pyruvate was nearly as powerful an inhibitor of homogenate ROS formation (and hence inferred NADHox activity) as hydralazine in the guinea pig cardiac homogenates.

Effects of pyruvate versus L-lactate on pre- and postischemic cardiac ROS formation. Pyruvate markedly inhibited ROS formation in postischemic hearts. Quantitative data are depicted in Fig. 3. In the absence of pyruvate, peak ROS release after a brief 5-min period of zero-flow ischemia occurred ~12 s after start of reperfusion. With increased perfusate pyruvate concentrations, time to peak ROS release appeared to increase, whereas peak height decreased. Within 60 s, postischemic ROS formation reached preischemic control levels. A smooth lognormal function fits the data readily and with high precision (Fig. 3 and see METHODS).

After integration of the postischemic ROS release functions, pyruvate inhibition of the reperfusion burst in ROS formation was expressed as 1 - (total measured release/total uninhibited ROS release) and plotted as a percentage against the perfusate pyruvate level (Fig. 3). A 25% decrease of total postischemic ROS occurred at 0.11 ± 0.001 mM pyruvate, and a 50% decrease occurred at 0.81 ± 0.001 mM pyruvate. At 6.15 ± 0.0024 mM, pyruvate inhibited postischemic ROS by 75% (not 100%), which compared with the 58% maximum inhibition of homogenate ROS formation due to NADHox (Fig. 2).

Figure 4 and Table 1 show that postischemic ROS release was markedly stimulated by perfusate L-lactate, in sharp contrast to the inhibition by pyruvate. With physiological levels (1-2 mM) of L-lactate, peak postischemic ROS release increased by ~100%, or twofold, relative to preischemic release (Fig. 4); with 5 mM L-lactate, peak postischemic ROS release increased further, about threefold. For comparison, an equimolar 5 mM pyruvate reduced peak ROS release to ~10% according to Fig. 3, a nearly 30-fold difference compared with the stimulation by L-lactate.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 4.   Effects of 1 or 5 mM L-lactate on basal and postischemic ROS release in constant-flow (11 ml/min) perfused guinea pig hearts. Data are from a representative experiment (all other relevant experiments are in Table 1). Arrows indicate start of reperfusion after 5 min of zero-flow ischemia.


                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Formation of ROS in guinea pig hearts is enhanced by lactate

Figure 5 shows that L-lactate also markedly and dose dependently stimulated basal and preischemic ROS release. ROS release more than doubled at >= 10 mM L-lactate. Figure 6 shows that perfusate pyruvate effectively and dose dependently inhibited the lactate (10 mM)-stimulated cardiac ROS formation: at the low physiological concentration of 0.3 mM, pyruvate inhibited ROS formation by ~30%. At cardioprotective levels (1-5 mM), pyruvate inhibited the lactate-induced ROS formation by ~40%.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of L-lactate on basal ROS formation in nonischemic constant-flow perfused guinea pig hearts. At concentrations between 1 and 10 mM, L-lactate enhanced dose dependently and more than 2-fold the basal release of ROS. L-Lactate enhancement of basal ROS formation was similar in magnitude to the peak postischemic ROS release in the control of Fig. 3. Values are means ± SE; n = 5. Significantly different from controls without lactate: *P < 0.05 and **P < 0.01.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6.   Pyruvate inhibition of the lactate (10 mM)-stimulated basal ROS release in constant-flow perfused guinea pig heart. Inhibition of ROS release expressed as percentage of the lactate (10 mM)-stimulated level. Values are means ± SE; n = 5.

To test whether endothelial X/XO contributed to guinea pig cardiac ROS release, we added the X/XO inhibitor oxypurinol (0.1-0.5 mM) to the perfusate. Oxypurinol inhibited >80% activity of isolated XO (see METHODS) but did not measurably affect the lactate (10 mM)-stimulated formation of ROS from the perfused hearts (Fig. 7).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 7.   Effects of the xanthine/xanthine oxidase (X/XO) inhibitor oxypurinol on lactate (10 mM)-stimulated formation of ROS in isolated constant-flow perfused guinea pig hearts. Values are means ± SE; n = 5. *P < 0.05 vs. basal.

Pyruvate has the intramolecular configuration of an alpha -ketomonocarboxylate. This is a reactive keto-enol configuration that has the potential to scavenge H2O2 and possibly other ROS nonenzymatically. This cell-free mechanism is effective with millimolar pyruvate and is independent of cellular redox systems (e.g., Ref. 6). To determine whether millimolar pyruvate directly scavenged ROS as measured by our ESR technique, we used the in vitro X/XO system as a O2-· generator in the absence and presence of 5-10 mM pyruvate or L-lactate. Table 2 shows that pyruvate or L-lactate had no measurable effects on ROS formation by the NADH- and LDH-free O2-· generator, but 0.1 and 1 mM oxypurinol inhibited measured ROS formation by 80 and 100%, respectively (data not shown).

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Lack of nonenzymatic scavenging of superoxide radicals

Effects of blocking mitochondrial pyruvate uptake by alpha -cyano-3-hydroxycinnamate. Hearts were perfused with 2 mM pyruvate (in the presence of 5 mM glucose) in the presence and absence of 0.5 mM alpha -cyano-3-hydroxycinnamate to inhibit mitochondrial pyruvate uptake (3). Under these conditions, alpha -cyano-3-hydroxycinnamate reduces mitochondrial pyruvate oxidation by approx 80% without inhibiting pyruvate sarcolemmal transport or cytosolic pyruvate metabolism (3, 17, 25). In terms of ROS formation, we observed that alpha -cyano-3-hydroxycinnamate did not appreciably alter preischemic ROS release [2.42 (control) vs. 2.17 µmol/min (alpha -cyano-3-hydroxycinnamate), n = 2]. In agreement with Fig. 3, peak postischemic ROS release was markedly inhibited by 2 mM pyruvate, increasing only slightly (19%) in the 2 mM pyruvate-perfused precinnamate controls. A quantitatively similar inhibition was found in the presence of alpha -cyano-3-hydroxycinnamate, where postischemic ROS release remained similarly attenuated, increasing by only 22% (mean of n = 2). Thus disabling mitochondrial pyruvate uptake in the intact heart did not appreciably change the potency of pyruvate as an inhibitor of postischemic ROS production. This is consistent with a predominantly extramitochondrial mechanism of pyruvate as an inhibitor of superoxide formation in the heart under the present conditions.

In a separate series of experiments, we demonstrated the lack of effects of millimolar pyruvate on superoxide formation by X/XO as detected by ESR (see Table 2).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first report delineating the full dose-response relations between antioxidant pyruvate and cardiac NADHox activity or postischemic ROS production, respectively, in guinea pig hearts. The main subcellular site for this antioxidant effect of pyruvate appeared to be cytoplasmic. This was indicated by the inability of alpha -cyano-3-hydroxycinnamate, a selective inhibitor of mitochondrial pyruvate uptake (3), to disable pyruvate as an antioxidant with regard to cardiac ROS formation. Such antioxidant effects differ mechanistically from the energetic, inotropic, anaplerotic, and allosteric enhancements at the pyruvate dehydrogenase, all of which are thought to require mitochondrial uptake of pyruvate (4, 15, 17, 18, 24). Furthermore, our new ROS inhibition data extend previous reports showing that pyruvate can raise the level of cardiac GSH (25), the natural cellular antioxidant. Also, on the basis of the intramolecular alpha -keto-configuration of pyruvate, we reported earlier that millimolar pyruvate could protect thiol-containing ATPases in cell-free systems against H2O2-induced inactivation (6). However, such direct nonenzymatic scavenger effects did not appear to play a role under the present conditions, because 5-10 mM pyruvate did not measurably inhibit ROS formation by a O2-· generator based on the NADH- and LDH-free X/XO system (Table 2).

Consistent with a significant extramitochondrial NADH-linked mechanism of ROS formation in the guinea pig heart, L-lactate, the physiological redox antagonist of pyruvate, markedly stimulated both basal and postischemic ROS production. Threshold doses for lactate stimulation of ROS were near its physiological level of 1 mM. Thus, all in all, our results are in harmony with the concept that the extracellular pyruvate/L-lactate system can substantially modulate cardiovascular NADHox activity and hence ROS production via a mechanism(s) involving cytosolic NADH (19).

Pyruvate as a cytosolic antioxidant. It may seem puzzling that pyruvate, known as the chief oxidant in the LDH reaction, functions as an antioxidant in the cytoplasm. Recalling, however, that in accordance with mass-action principles, pyruvate shifts the LDH equilibrium toward L-lactate at the expense of cytoplasmic [NADH] + [H+], it is clear that pyruvate reduces available free cytosolic NADH. Extramitochondrial NADH is substrate and electron donor in the membrane-associated NADHox activity, which suggests the mechanism for the observed pyruvate-induced reduction of ROS via the LDH-linked NADH. Thus redox control of the tissue NADHox activity is intimately linked to the LDH equilibrium and hence the pyruvate-L-lactate redox couple.

ROS formation in crude homogenates, mainly representing NADHox activity, proved strikingly sensitive to exogenous pyruvate (Fig. 2). Even at the very small dose of 50 µM, which is below the normal plasma pyruvate range of 100-200 µM, pyruvate significantly inhibited NADHox activity by 10% in cardiac homogenates and up to 30% in aorta or liver homogenates. Kinetic analyses of these data revealed that the half-maximal inhibition of NADHox activity occurred with physiological pyruvate concentrations near 100 µM. With respect to ROS release in postischemic hearts (reflecting metabolic flux through the intact in situ NADHox system), pyruvate was somewhat less effective. Whereas 100 µM pyruvate inhibited postischemic ROS release by ~25% (Fig. 3), the estimate for the half-maximal inhibitory dose was 0.81 ± 0.001 mM, about four- to eightfold the physiological levels between 0.1 and 0.2 mM. Nevertheless, our findings suggest that pyruvate could markedly and effectively inhibit NADHox-linked ROS (superoxide) production in the cardiovascular system even at low physiological concentrations. Similarly, in inflamed knee joint synovial and other biofluids, physiological doses of pyruvate protect against H2O2 and hydroxyl radicals (10, 22).

On the other hand, our estimates predict that exogenous pyruvate, even when used at extremely high concentrations of >= 10 mM, fails to fully inhibit cardiac, aortic, or hepatic tissue NADHox activities (Fig. 2). This result is in certain contrast to earlier observations demonstrating that 5-10 mM pyruvate fully prevented H2O2-induced thymocyte and endothelial apoptosis (Ref. 23 and unpublished observations), completely normalized postischemic cardiac energetics and left ventricular pressure development (4), fully prevented metabolic acidosis and delayed cerebral death in porcine hemorrhagic shock (20), and strongly improved cardiac performance in patients with chronic heart failure (9). Whether these differences in analytical NADHox/ROS parameters versus physiological and clinical beneficial outcomes reflect different sites and mechanisms of action remains to be elucidated.

Disabling pyruvate entry into the mitochondria did not affect the antioxidant potential of pyruvate in terms of the NADHox and ROS parameters (Figs. 2 and 3); this could be inferred from the lack of effect of alpha -cyano-3-hydroxycinnamate in a dose that is known to selectively inhibit ~80% of mitochondrial pyruvate uptake in the guinea pig heart. In contrast, the same or comparable experimental conditions have been shown to blunt the pyruvate-induced enhancement of myocardial energetics and the metabolic inotropy of this compound as well (15-18). Metabolic strategies targeting mitochondrial energetics and cellular function seem to require 1-5 mM pyruvate for maximum effectiveness (4, 9, 13, 17), doses that are 10- to 50-fold the normal pyruvate plasma levels. Because such high pyruvate concentrations also oxidize the cytosolic NADH system, they would combine salutary redox and antioxidant effects in the cytoplasm with the energetic and inotropic improvements mediated by the mitochondria.

Potential role of monocarboxylate transport. Hearts utilizing physiological (0.3 mM) pyruvate (in presence of 5 mM glucose) exhibited a moderate three- to fourfold increase in the intracellular [pyruvate]/[L-lactate] ratio, implying a similar increase in the free cytosolic [NAD+]/[NADH] ratio (2). Whether such or an even smaller increase in [NAD+]/[NADH] might explain the 15-25% inhibition of homogenate NADHox by pyruvate in doses of 50 µM (Fig. 2) and/or the 25% attenuation of postischemic ROS production by 100 µM pyruvate (Fig. 3) was not directly proven nor was it excluded. Any pyruvate-induced [NADH] + [H+] consumption in the cytoplasm would reduce the availability of NADH for NADHox, which would inhibit flux through the oxidase system and reduce superoxide formation. Alternatively, low doses of pyruvate could reduce superoxide formation via the monocarboxylate transporter on the cell membrane, a mechanism independent from metabolic redox effects. Pyruvate and L-lactate are known to compete for myocardial uptake and release via the monocarboxylate transport protein, but pyruvate has an ~5- to 10-fold higher affinity to the transporter than L-lactate. This renders pyruvate an effective and natural inhibitor of myocardial L-lactate uptake; preliminary data place the inhibitor constant (Ki) for pyruvate at approx 100 µM (unpublished observations), i.e., into the range of concentrations where we obtained measurable inhibitions of NADHox and ROS formation. Such transport competition may be particularly useful for the heart in vivo, because it is permanently perfused with ~0.2 mM pyruvate at 1-10 mM L-lactate.

Only supraphysiological doses of pyruvate (>= 5 mM) oxidize the cytosolic [NAD+]/[NADH] ratio by orders of magnitude (2, 5, 17). Such massive cytosolic oxidation greatly reduces the reductive potential of NADH at the catalytic sites of (lactate) dehydrogenases and hence at those of competing NADHox systems. Conversely, a substantially lower [NAD+]/[NADH] ratio due to excess L-lactate (>= 10 mM) would predict large increases in steady-state levels of ROS due to NADHox activity; these predicted opposite effects of high pyruvate versus high lactate are consistent with the ROS data in Figs. 3-6.

No role for GSH? High pyruvate can strengthen antioxidant defenses of the myocardium by raising the GSH/GSSG ratio (25). This reductive pyruvate effect on the GSH system is mechanistically unrelated to the oxidation of [NAD+]/[NADH] in the cytosol. It is thought that the GSH/GSSG concentration ratio rises in response to a pyruvate-induced accumulation of a citrate (3, 25) that is an allosteric inhibitor of phosphofructokinase (PFK). The resulting glucose 6-phosphate accumulation upstream of PFK provides substrate pressure at the glucose-6-phosphate dehydrogenase; this enzyme is rate limiting for metabolic flux through the pentose phosphate shunt that provides NADPH for reductive syntheses as well as the reduction of GSSG to GSH. The pyruvate-induced myocardial citrate accumulation is greatly dependent on mitochondrially mediated anaplerosis, as alpha -cyano-3-hydroxycinnamate strongly inhibits this pyruvate effect (25). In contrast, alpha -cyano-3-hydroxycinnamate did not disable pyruvate as an inhibitor of postischemic cardiac ROS formation, suggesting that the GSH system was not the first line of defense against superoxide formation by the NADHox under the present conditions.


    ACKNOWLEDGEMENTS

The technical assistance of A. Hoinkes is gratefully acknowledged. We thank L. Böhm for help in preparing the manuscript.


    FOOTNOTES

This work was supported by grants from the German Heart Foundation, Frankfurt/Main, Germany, and from the Uniformed Services University (RO 76HB) and the National Institute of Allergy and Infectious Diseases (RO 76HD), Bethesda, MD.

Address for reprint requests and other correspondence: E. Bassenge, Institute for Applied Physiology, Univ. of Freiburg, Herrmann Herder Str. 7, D-79104 Freiburg/Breisgau, Germany (E-mail: angphys{at}uni-freiburg.de).

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.

Received 11 April 2000; accepted in final form 16 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allen, RC. Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. Methods Enzymol 133: 449-493, 1986[ISI][Medline].

2.   Bünger, R. Compartmented pyruvate in perfused working heart. Am J Physiol Heart Circ Physiol 249: H439-H449, 1985[Abstract/Free Full Text].

3.   Bünger, R, and Mallet RT. Mitochondrial pyruvate transport in working guinea pig heart. Work-related vs. carrier-mediated control of pyruvate oxidation. Biochim Biophys Acta 1151: 223-236, 1993[Medline].

4.   Bünger, R, Mallet RT, and Hartman D. Pyruvate-enhanced phosphorylation potential and inotropism in normoxic and postischemic isolated working heart. Near-complete prevention of reperfusion contractile failure. Eur J Biochem 180: 221-223, 1989[ISI][Medline].

5.   Bünger, R, Mallet RT, and Hartman D. Redox manipulation of free cardiac adenylates, and purine nucleoside release. In: Myocardial Energy Metabolism, edited by de Jong JW.. Boston, MA: Martinus Nijhoff, 1987, p. 67-81.

6.   Clough, D, and Bünger R. Protection by pyruvate against inhibition of Na+,K+-ATPase by a free radical generating system containing t-butylhydroperoxide. Life Sci 57: 931-943, 1995[ISI][Medline].

7.   De Groot, MJ, van Helden MA, de Jong YF, Coumans WA, and van der Vusse GJ. The influence of lactate, pyruvate, and glucose as exogenous substrates on free radical defense mechanisms in isolated rat hearts during ischaemia and reperfusion. Mol Cell Biochem 146: 147-155, 1995[ISI][Medline].

8.   Dikalov, S, Skatchov M, and Bassenge E. Spin trapping of superoxide radicals and peroxynitrite by 1-hydroxy-3-carboxy-pyrrolidine and 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine and the stability of corresponding nitroxyl radicals towards biological reactants. Biochem Biophys Res Commun 231: 701-704, 1997[ISI][Medline].

9.   Hermann, HP, Pieske B, Schwarzmüller E, Keul J, Just H, and Hasenfuss G. Haemodynamic effects of intracoronary pyruvate in patients with congestive heart failure: an open study. Lancet 353: 1321-1323, 1999[ISI][Medline].

10.   Herz, H, Blake R, and Grootveld M. Multicomponent investigations of the hydrogen peroxide- and hydroxyl radical-scavenging antioxidant capacities of biofluids: the roles of endogenous pyruvate and lactate. Free Radic Res 26: 19-35, 1997[ISI][Medline].

11.   Kang, YH, Mallet RT, and Bünger R. Coronary autoregulation and purine release in normoxic heart at various cytoplasmic phosphorylation potentials: disparate effects of adenosine. Pflügers Arch 421: 188-199, 1992[ISI][Medline].

12.   Kurose, I, Wolf RE, Grisham MB, and Granger DN. Hypercholesterolemia enhances oxidant production in mesenteric venules exposed to ischemia/reperfusion. Arterioscler Thromb Vasc Biol 18: 1583-1588, 1998[Abstract/Free Full Text].

13.   Lasley, RD, Bünger R, Zhou Z, and Mentzer RM. Metabolically based treatment of stunned myocardium. J Card Surg 9: 469-473, 1994[ISI][Medline].

14.   Liedtke, AJ, Nellis SH, Neely JR, and Hughes HC. Effects of treatment with pyruvate and tromethamine in experimental myocardial ischemia. Circ Res 39: 378-387, 1976[Abstract/Free Full Text].

15.   Mallet, RT, and Bünger R. Energetic modulation of cardiac inotropism and sarcoplasmic reticular calcium uptake. Biochim Biophys Acta 1224: 22-32, 1994[Medline].

16.   Mallet, RT, and Bünger R. Metabolic protection of post-ischemic phosphorylation potential and ventricular performance. Adv Exp Med Biol 346: 233-241, 1993[Medline].

17.   Mallet, RT, and Sun J. Mitochondrial metabolism of pyruvate is required for its enhancement of cardiac function and energetics. Cardiovasc Res 42: 149-161, 1999[Abstract/Free Full Text].

18.   Martin, BJ, Valdivia HH, Bünger R, Lasley RD, and Mentzer RMJ Pyruvate augments calcium transients and cell shortening in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 274: H8-H17, 1998[Abstract/Free Full Text].

19.   Mohazzab-H, KM, Kaminski PM, and Wolin MS. Lactate and PO2 modulate superoxide anion production in bovine cardiac myocytes: potential role of NADH oxidase. Circulation 96: 614-620, 1997[Abstract/Free Full Text].

20.   Mongan, PD, Fontana JL, Chen R, and Bünger R. Intravenous pyruvate prolongs survival during hemorrhagic shock in swine. Am J Physiol Heart Circ Physiol 277: H2253-H2263, 1999[Abstract/Free Full Text].

21.   Münzel, T, Kurtz S, Rajagopalan S, Thoenes M, Berrington WR, Thompson JA, Freeman BA, and Harrison DG. Hydralazine prevents nitroglycerin tolerance by inhibiting activation of a membrane-bound NADH oxidase. A new action for an old drug. J Clin Invest 98: 1465-1470, 1996[ISI][Medline].

22.   O'Donnell-Tormey, J, Nathan CF, Lanks K, DeBoer CJ, and de la Harpe J. Secretion of pyruvate. An antioxidant defense of mammalian cells. J Exp Med 165: 500-514, 1987[Abstract/Free Full Text].

23.   Ramakrishnan, N, Chen R, McLain DE, and Bünger R. Pyruvate prevents hydrogen peroxide-induced apoptosis. Free Radic Res 29: 283-295, 1998[ISI][Medline].

24.   Scholz, TD, Laughlin MR, Balaban RS, Kupriyanov VV, and Heineman FW. Effect of substrate on mitochondrial NADH, cytosolic redox state, and phosphorylated compounds in isolate hearts. Am J Physiol Heart Circ Physiol 268: H82-H91, 1995[Abstract/Free Full Text].

25.   Tejero-Taldo, MS, Caffrey JL, Sun J, and Mallet RT. Antioxidant properties of pyruvate mediate its potentiation of beta -adrenergic inotropism in stunned myocardium. J Mol Cell Cardiol 31: 1862-1872, 1999.

Am J Physiol Heart Circ Physiol 279(5):H2431-H2438
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 Hum Exp ToxicolHome page
J. Lee, W. Kwon, Y. Jo, G. Suh, and Y. Youn
Protective effects of ethyl pyruvate treatment on paraquat-intoxicated rats
Human and Experimental Toxicology, January 1, 2008; 27(1): 49 - 54.
[Abstract] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. Carvajal, E. Zarrinpashneh, O. Szarszoi, F. Joubert, Y. Athea, P. Mateo, B. Gillet, S. Vaulont, B. Viollet, X. Bigard, et al.
Dual cardiac contractile effects of the {alpha}2-AMPK deletion in low-flow ischemia and reperfusion
Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H3136 - H3147.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
K. Schulze, C. Duschek, R. D. Lasley, and R. Bunger
Adenosine enhances cytosolic phosphorylation potential and ventricular contractility in stunned guinea pig heart: receptor-mediated and metabolic protection
J Appl Physiol, March 1, 2007; 102(3): 1202 - 1213.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Heinen, A. K. S. Camara, M. Aldakkak, S. S. Rhodes, M. L. Riess, and D. F. Stowe
Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential
Am J Physiol Cell Physiol, January 1, 2007; 292(1): C148 - C156.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
W. J. Lee, I. K. Lee, H. S. Kim, Y. M. Kim, E. H. Koh, J. C. Won, S. M. Han, M.-S. Kim, I. Jo, G. T. Oh, et al.
{alpha}-Lipoic Acid Prevents Endothelial Dysfunction in Obese Rats via Activation of AMP-Activated Protein Kinase
Arterioscler. Thromb. Vasc. Biol., December 1, 2005; 25(12): 2488 - 2494.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
E. M. Knott, M.-G. Ryou, J. Sun, A. Heymann, A. B. Sharma, Y. Lei, M. Baig, R. T. Mallet, and A. H. Olivencia-Yurvati
Pyruvate-fortified cardioplegia suppresses oxidative stress and enhances phosphorylation potential of arrested myocardium
Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1123 - H1130.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
R. T. Mallet, J. Sun, E. M. Knott, A. B. Sharma, and A. H. Olivencia-Yurvati
Metabolic Cardioprotection by Pyruvate: Recent Progress
Experimental Biology and Medicine, July 1, 2005; 230(7): 435 - 443.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
Y. J. Woo, M. D. Taylor, J. E. Cohen, V. Jayasankar, L. T. Bish, J. Burdick, T. J. Pirolli, M. F. Berry, V. Hsu, and T. Grand
Ethyl pyruvate preserves cardiac function and attenuates oxidative injury after prolonged myocardial ischemia
J. Thorac. Cardiovasc. Surg., May 1, 2004; 127(5): 1262 - 1269.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
Y.-J. Lee, I.-J. Kang, R. Bunger, and Y.-H. Kang
Enhanced survival effect of pyruvate correlates MAPK and NF-{kappa}B activation in hydrogen peroxide-treated human endothelial cells
J Appl Physiol, February 1, 2004; 96(2): 793 - 801.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G. Kristo, Y. Yoshimura, J. Niu, B. J. Keith, R. M. Mentzer Jr., R. Bunger, and R. D. Lasley
The intermediary metabolite pyruvate attenuates stunning and reduces infarct size in in vivo porcine myocardium
Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H517 - H524.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
U. M. Fischer, C. S. Cox Jr, S. J. Allen, R. H. Stewart, U. Mehlhorn, and G. A. Laine
The antioxidant N-acetylcysteine preserves myocardial function and diminishes oxidative stress after cardioplegic arrest
J. Thorac. Cardiovasc. Surg., November 1, 2003; 126(5): 1483 - 1488.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. Ochiai, J. Zhang, G. Gong, Y. Zhang, J. Liu, Y. Ye, X. Wu, H. Liu, Y. Murakami, R. J. Bache, et al.
Effects of augmented delivery of pyruvate on myocardial high-energy phosphate metabolism at high workstate
Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1823 - H1832.
[Abstract] [Full Text] [PDF]

Home page
 Eur. J. Cardiothorac. Surg.Home page
R. T. Smolenski, M. Amrani, J. Jayakumar, P. Jagodzinski, C. C. Gray, A. T. Goodwin, I. A. Sammut, and M. H. Yacoub
Pyruvate/dichloroacetate supply during reperfusion accelerates recovery of cardiac energetics and improves mechanical function following cardioplegic arrest
Eur. J. Cardiothorac. Surg., June 1, 2001; 19(6): 865 - 872.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (46)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bassenge, E.
Right arrow Articles by Bünger, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bassenge, E.
Right arrow Articles by Bünger, R.