(Circulation. 1996;94:2580-2586.)
© 1996 American Heart Association, Inc.
Articles |
Role of Nitric Oxide and Its Interaction With Superoxide in the Suppression of Cardiac Muscle Mitochondrial Respiration
Involvement in Response to Hypoxia/Reoxygenation
the Department of Physiology, New York Medical College, Valhalla.
Correspondence to Michael S. Wolin, PhD, Professor, Department of Physiology, New York Medical College, Valhalla, NY 10595.
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Abstract |
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Background Nitric oxide (NO); superoxide anion (O2.d-); the reaction product of NO with O2.d-, peroxynitrite (ONOO-); and ischemia/reperfusion have all been reported to inhibit respiration in isolated mitochondria. However, the specific species involved in the inhibition of respiration in intact tissues are poorly understood.
Methods and Results O2 consumption in isolated cardiac muscle from bovine calf hearts was quantified by use of a Clark-type electrode. Exogenous and endogenous sources of NO, from S-nitroso-N-acetylpenicillamine (SNAP) and bradykinin or carbachol, reversibly inhibited respiration, whereas the O2.- releasing agent, pyrogallol (PG), inhibited respiration in a manner that was only partially reversed when examined 15 minutes after the removal of PG. The generation of ONOO- with SNAP+PG caused a potentiation of the O2.--elicited inhibition of respiration when examined 15 minutes after the removal of the ONOO- generating system. Tiron (a scavenger of O2.-) did not alter the actions of SNAP, but it attenuated the direct inhibitory effects of PG±SNAP and essentially eliminated the suppression of respiration observed 15 minutes after removal of the O2.- or ONOO- generating system. Urate (a scavenger of ONOO-) antagonized only the actions of PG+SNAP. After exposure of muscle slices to a model of hypoxia (15 minutes) and reoxygenation (10 minutes), respiratory inhibition was observed. This reoxygenation-induced inhibition was potentiated by L-arginine, the substrate for NO biosynthesis, and was markedly blocked by nitro-L-arginine (an NO synthase inhibitor), Tiron, or urate.
Conclusions The potentially physiological reversible regulation of respiration in cardiac muscle by NO is converted to an effect that does not show rapid reversibility under conditions in which ONOO- forms, and this could contribute to cardiac dysfunction in situations such as hypoxia/reoxygenation.
Key Words: hypoxia • endothelium-dependent factors • oxygen
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Before the discovery of a role for NO in physiological processes, an undefined substance released by activated macrophages suppressed mitochondrial respiration in bacteria and cultured mammalian cells.1 2 Subsequently, it was suggested that this substance is NO derived from the metabolism of L-Arg,3 4 and NO was shown to have mitochondrial inhibitory actions similar to the macrophage-derived factor.5 However, recent studies indicate that activated macrophages appear to produce appreciable amounts of ONOO- as a result of the simultaneous generation of both NO and O2.-.6 ONOO- has also been shown to inhibit cellular respiration.7 8 9 Thus, it is unclear whether the suppression of respiration by inflammatory-related processes is due to NO or to the endogenous formation of ONOO-.
Recent in vivo studies in dogs from our institution10 11 and by others12 have shown that endogenous NO production may regulate tissue O2 consumption, suggesting an important physiological role for NO. Studies of tissue respiration by isolated slices of canine skeletal and cardiac muscle have established that stimulation of the vascular endothelium by receptor mechanisms can inhibit mitochondrial O2 consumption through the formation of NO from L-Arg in a manner that is independent of stimulation of cGMP signaling.11 13 Studies on subcellular fractions of tissues enriched in mitochondria and synaptosomes and on astrocytes have provided evidence that the suppression of mitochondrial respiration by NO is rapidly reversible.14 15 16 17 18 It has been assumed that NO mediates the potentially physiological type of regulation of respiration in these systems. Because ONOO- readily forms under conditions of endothelial stimulation,19 the actual species mediating respiratory inhibition needs to be better characterized.
Reperfusion of ischemic tissues is necessary to restore its normal function; however, it paradoxically initiates poorly understood processes that lead to tissue injury and organ dysfunction. Impaired mitochondrial function and increased production of O2.- are very common reperfusion-associated events. The activities of components of mitochondrial respiratory chain have been documented to be markedly reduced during postischemic reperfusion or posthypoxic reoxygenation.20 21 22 Previous studies have suggested that mitochondrial dysfunction results in increased production of O2.- by this organelle after exposure of cardiac muscle to ischemia/reperfusion. O2.- has also been reported to be a potent inhibitor of mitochondrial aconitase.23 24 However, potential roles for species including NO, O2.-, and ONOO- in the process contributing to mitochondrial dysfunction in hypoxia/reoxygenation and ischemia/reperfusion remain to be defined.
The species of NO responsible for its regulation of respiration in intact tissues in physiological and pathophysiological states are in general poorly understood. In addition, the properties of reversibility of respiratory inhibition may depend on the reactive species involved. The present study was designed to investigate the species of NO that could regulate tissue respiration under physiological conditions in a manner that permits comparisons of the sensitivity and reversibility of respiratory inhibition under conditions of elevated NO, O2.-, and NO together with O2.-. Moreover, the present study is looking into species that mediate respiratory suppression induced by a model of short-term hypoxia/reoxygenation.
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Materials
Catalase, HEPES, uric acid, bradykinin, carbachol, L-Arg, DNP, luminol, lucigenin, superoxide dismutase from bovine blood, and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) were obtained from Sigma Chemical Co. Pyrogallol was purchased from Baker Chemical Co. L-NNA was obtained from Aldrich Chemical Co. SNAP was synthesized as described.25 DNP was dissolved in dimethyl sulfoxide. All other chemicals were dissolved in Krebs' solution containing (mmol/L) NaCl 118, KCl 4.7, CaCl2 1.5, NaHCO3 25, KH2PO4 1.2, MgSO4 1.1, and glucose 5.6, pH 7.4.
Tissue Preparation
Bovine hearts were obtained from a slaughterhouse as described.26 Myocardial muscle from the left ventricle of these bovine hearts was cut into thin 
50-mg slices, which were initially incubated in Krebs' solution (at 37°C) gassed continuously with 21% O2/5% CO2 (balance N2) before use.
Measurement of Respiration Rates
Oxygen uptake by cardiac muscle slices was quantified by a Clark-type O2 electrode (Yellow Springs Instruments) in 3 mL Krebs' solution buffered with 10 mmol/L HEPES-NaOH, pH 7.4, at 37°C as previously described.11 Tissue respiration was calculated as the decrease rate of O2 concentration after the addition of muscle segments, assuming an initial [O2] of 224 nmol/mL. SNAP, pyrogallol, and the combination of these two probes were examined for their effects on cardiac muscle O2 consumption. The effects of these agents were also studied in the presence of Tiron, SOD, or uric acid, scavengers of O2.- and ONOO-.27 The measured rate of O2 consumption by pyrogallol in the absence of tissue was subtracted from all measurements of tissue respiration made in the presence of this agent. All experiments with pyrogallol were performed in the presence of 420 U/mL catalase to destroy hydrogen peroxide formed from the dismutation of O2.-. Effects of NO biosynthesis stimulators, bradykinin and carbachol, on cardiac muscle O2 consumption were also studied. The typical observation time for respiration measurements in the presence or absence of these agents was 6 to 8 minutes. Tissue respiration was also recorded 15 minutes after the washout of the probes examined. The mitochondrial uncoupler DNP was added 15 minutes after the washout of SNAP, pyrogallol, or the combination of these two probes to examine effects of these probes on the DNP-potentiated rate of tissue respiration. At the end of each measurement, 1 mmol/L NaCN was added, which eliminated all increases in O2 consumption. Data were analyzed as the percent change of the rate of respiration observed in the absence of NO or O2.- generating systems (60 nmol·min-1·g-1) for each muscle slice studied.
Detection of ONOO- and O2
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Cardiac muscle slices (150 mg) were prepared as indicated above for respiration studies, with each segment of muscle placed in plastic scintillation minivials equilibrated at 37°C containing 0.25 mmol/L luminol or lucigenin for the detection of ONOO-19 or O2.-,26 respectively, and other additions in a final volume of 1 mL of air-equilibrated Krebs' solution buffered with 10 mmol/L HEPES-NaOH, pH 7.4. As previously described,26 the chemiluminescence was measured in a liquid scintillation counter with single active photomultiplier tube positioned in out-of-coincidence mode. All manipulations were performed in the darkroom with minimal light. After 5 minutes of dark adaptation, vials containing all components except for SNAP and/or pyrogallol (blanks) were counted twice for 0.1 minute and then recounted twice after SNAP and/or pyrogallol were placed in each vial. Averaged counts from blanks were then subtracted from the average of the relatively constant levels of chemiluminescence produced under each condition to obtain the data reported as counts per minute.
Studies of a Model of Hypoxia/Reoxygenation
The duration of hypoxia in a model of hypoxia/reoxygenation was designed to resemble conditions under which cardiac stunning occurs. In this model, cardiac muscle slices were exposed to an atmosphere of 95%N2/5%CO2 (PO2 <10 mm Hg) for 15 minutes in a 10-mL tissue bath thermostated at 37°C. This treatment was subsequently followed by exposure of the muscle slice to 10 minutes of gassing with 21%O2/5%CO2 (balance N2). Tissue respiration was examined before and after hypoxia/reoxygenation treatment with the methods described in the previous section. L-Arg, Tiron, and uric acid were added before reoxygenation and L-NNA was added before hypoxia to examine the effects of these agents on tissue O2 consumption in the model of hypoxia/reoxygenation (these agents were not present during respiration measurements). O2 consumption of muscle slices after hypoxia/reoxygenation treatment was also determined in the presence of DNP.
Statistical Analysis
Data are reported as mean±SE. Differences in the mean values were analyzed with unpaired and, when appropriate, paired Student's t test. Values of P<.05 were considered statistically significant. A Bonferroni correction was made when multiple groups were compared.
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Effects of Exogenous NO on Cardiac Muscle O2 Consumption
SNAP was used as an exogenous NO releasing agent because it is well characterized for its effects on inhibiting mitochondrial respiration, stimulating the soluble form of guanylate cyclase and other NO-mediated processes.11 13 25 28 Administration of SNAP in cumulative, increasing concentrations over the range of 10-7 to 10-4 mol/L significantly and concentration dependently inhibited cardiac muscle O2 consumption by 11±2% to 35±2% (Fig 1
). This concentration-dependent effect of SNAP remained unaltered in the presence of 10 mmol/L Tiron, an intracellular O2.- scavenger. Tiron itself caused a small increase in respiration, which reached statistical significance (Fig 1). Tissue respiration was reduced by 17±2% or 28±3% at an individual dose of 10-5 or 10-4 mol/L SNAP, respectively (Fig 2). This inhibition of O2 consumption was reversible because respiration was not significantly reduced when examined 15 minutes after SNAP was washed out of the tissue (Fig 2). The inhibitory effect of the higher dose of SNAP was not altered in the presence of 10 mmol/L Tiron or a scavenger of ONOO-, 100 µmol/L uric acid (Fig 3A). As Fig 4 shows, 1 mmol/L DNP, a mitochondrial uncoupler, enhanced tissue respiration by 71±13%; similarly, O2 consumption by muscle slices during the postwashout period after SNAP treatment was elevated by 64±10% on exposure to DNP, consistent with the concept of reversible inhibition by NO.
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Effects of Endogenous NO on Cardiac Muscle O2 Consumption
Similar to previous studies in isolated canine skeletal11 and cardiac13 muscle, a stimulus of endogenous NO generation, carbachol, dose dependently suppressed cardiac muscle respiration by 11±2% to 31±2% over the range of 10-7 to 10-4 mol/L (n=4, not shown), which was abolished by pretreatment with 100 µmol/L L-NNA, a specific NO synthase inhibitor (n=4, not shown). Tissue respiration was also reduced by individual doses (0.1 mmol/L) of carbachol or bradykinin, another stimulus of NO biosynthesis, by 18±1% or 19±1%, respectively (Fig 5). This respiratory inhibition by bradykinin was not detectable 15 minutes after the washout of this peptide, whereas a markedly reduced inhibitory effect of carbachol was observed under these washout conditions (Fig 5).
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Effects of O2.- on Cardiac Muscle O2 Consumption
Pyrogallol, a probe compatible with physiological preparations that generates O2.- through autoxidation,29 reduced cardiac muscle respiration by 20±2% and 46±6% at doses of 10-5 and 10-4 mol/L, respectively (Fig 2
). In contrast to the reversible effects of SNAP, 14±2% or 24±3% inhibition, respectively, remained after a 15-minute recovery period after pyrogallol was washed out of the tissue (Fig 2). Tiron antagonized the initial (Fig 3A) and postwashout (Fig 3B) respiratory inhibitory effects of pyrogallol, whereas 100 µmol/L urate did not alter the actions of pyrogallol. As Fig 4 shows, DNP elevated tissue respiration in the postwashout period of pyrogallol treatment by 37±6%, which is significantly smaller than the control response to DNP, suggesting that the effects of O2.- are not rapidly reversible.
Effects of ONOO- on Cardiac Muscle O2 Consumption
When SNAP and pyrogallol were simultaneously present in the buffer, ONOO- was likely to readily form through the reaction between NO and O2.- as a result of the rate constant for this reaction of 6.7x109 (mol/L)-1·s-1.9 The combination of SNAP plus pyrogallol, at 10-5 mol/L each, increased the degree of inhibition of tissue respiration above the effects of each individual agent to 31±3% (Fig 2). However, after washout of these probes and a 15-minute recovery period, a 27±2% inhibition of tissue respiration was observed (Fig 2). Thus, the conditions used to generate ONOO- caused respiration to be inhibited in a manner that did not appear to be reversed during the 15-minute washout period. Both the acute and prolonged inhibitory effects of conditions promoting ONOO- formation were markedly attenuated by either 10 mmol/L Tiron or 100 µmol/L uric acid (Fig 3A and 3B). As Fig 4 shows, DNP enhanced tissue respiration in the postwashout period after treatment with both SNAP and pyrogallol by 33±4%, which is also significantly smaller than the control response to DNP, indicating that effects of ONOO- are persistent under conditions in which respiration is uncoupled from oxidative phosphorylation.
Comparison of the Concentration-Dependent Actions of Scavengers on the Inhibition of Respiration With the Detection of ONOO- and O2.-
Experiments were conducted to examine relationships between the concentration-dependent effects of scavengers on the attenuation of the respiratory inhibitory actions of ONOO- and/or O2.- with the detection of these species. Because mitochondria are an intracellular organelle, some of these studies were also conducted in the presence of a concentration of SOD (0.3 µmol/L) that eliminated the detection of O2.- and ONOO- in buffer to evaluate relationships between the intracellular generation and the action of these reactive species. Fig 6A shows the concentration-dependent effects of Tiron on the actions and detection of ONOO-. The data in this figure suggest that the concentration dependence of the effects of Tiron on preventing the inhibition of respiration observed 15 minutes after exposure to ONOO- generation was closely associated with the scavenging of ONOO- detected by luminol chemiluminescence observed from cardiac muscle in the presence of SOD. As shown by the data in Fig 6A, Tiron was observed to be a markedly more potent scavenger of ONOO- in buffer compared with levels detected from cardiac muscle in the presence of SOD. Fig 6B shows the concentration-dependent effects of urate on the actions and detection of ONOO-. The data in this figure suggest that the concentration dependence of the effects of urate on preventing the inhibition of respiration observed 15 minutes after exposure to ONOO- generation was also closely associated with the scavenging of ONOO- detected by luminol chemiluminescence observed from cardiac muscle in the presence of SOD. The data in this figure also indicate that urate was observed to be a more potent scavenger of ONOO- in buffer compared with levels detected from cardiac muscle in the presence of SOD. Fig 6C shows the concentration-dependent effects of Tiron on the actions and detection of O2.-. The data in this figure suggest that the concentration dependence of the effects of Tiron on preventing the inhibition of respiration observed 15 minutes after exposure to O2.- generation was closely associated with the scavenging of O2.- detected by lucigenin chemiluminescence. In these experiments, there does not appear to be a statistically significant difference in detection of the potency of the scavenging effects of Tiron on O2.- between conditions of buffer and cardiac muscle in the presence of SOD. Because urate did not significantly alter the inhibition of respiration by O2.- generation (Fig 3) or the detection of O2.- produced by 0.1 mmol/L pyrogallol (not shown), the concentration-dependent effects of urate on the respiratory actions and detection of O2.- were not further studied. As a result of the data in Fig 6A and 6B suggesting that the formation of intracellular ONOO- may be the primary source of this species involved in the inhibition of respiration, the effects of 0.3 µmol/L SOD on the inhibition of respiration 15 minutes after exposure to ONOO- were evaluated. As shown by the data in Fig 6D, the presence of SOD did not alter the inhibitory effects of ONOO- on respiration. In contrast, similar studies shown in Fig 6D examining the effects of 0.3 µmol/L SOD on the inhibition of respiration 15 minutes after exposure to O2.- indicated that the addition of SOD was observed to partially alter the inhibitory effects of O2.- on respiration.
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Effects of Hypoxia/Reoxygenation on Cardiac Muscle O2 Consumption
Cardiac muscle respiration was reduced by 25±2% when measured after short-term hypoxia/reoxygenation (Fig 7). This respiratory inhibition was potentiated to 30±1% by addition of 1 mmol/L L-Arg before reoxygenation, whereas this inhibition was reduced to 13±1% by addition of 0.1 mmol/L L-NNA before hypoxia (Fig 7), suggesting an involvement of NO in the inhibition of respiration induced by hypoxia/reoxygenation. The observed degree of respiratory inhibition induced by hypoxia/reoxygenation was also suppressed by either 10 mmol/L Tiron or 100 µmol/L uric acid (Fig 7), suggesting an involvement of O2.- and ONOO- in the inhibition of respiration elicited by hypoxia/reoxygenation. Addition of 1 mmol/L DNP after hypoxia/reoxygenation elevated cardiac muscle respiration by 27±1% (Fig 4), which is significantly less than the control response to DNP, indicating that the impairment of mitochondrial respiration in this model of hypoxia/reoxygenation is also observed under conditions of uncoupled respiration.
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Discussion |
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Our results demonstrate that an NO donor, SNAP, inhibits O2 consumption in isolated bovine cardiac muscle in a manner that is reversed when the muscle is washed free of SNAP. Similarly, release of endogenous NO through the activation of bradykinin receptors or muscarinic cholinergic receptors by the acetylcholine analog carbachol also reversibly suppresses tissue respiration. Effects of SNAP remain unaltered in the presence of Tiron, an intracellular scavenger of O2.-, or uric acid, a scavenger of ONOO-, suggesting that NO directly suppresses mitochondrial respiration and that endogenous ONOO- formation is not necessary for the action of NO. Recently, cytochrome oxidase has been shown to be inhibited by nanomolar levels of NO in a manner that appears to be rapidly reversible,14 15 16 17 which may be due to the ability of this enzyme to metabolize NO.30 31
In the present study, the O2.- releasing agent pyrogallol also inhibits O2 consumption in isolated bovine cardiac muscle, and a large portion of the inhibition remained 15 minutes after washout of pyrogallol. Both effects of pyrogallol were significantly prevented in the presence of Tiron but not by uric acid, indicating that O2.- also inhibits tissue respiration. Mitochondrial aconitase is a possible site of action of O2.- because O2.- is a potent inhibitor of this enzyme.8 23 24 It has been proposed that the inactivation of aconitase occurs through the oxidation of its Fe-S center by O2.-, resulting in the release of Fe2+, and the addition of Fe2+ has been reported to promote restoration of aconitase activity.23 24 32 In the present study, much of the inhibition of respiration by pyrogallol remained after the 15-minute recovery period, consistent with previous studies on aconitase and the inhibition of respiration in intact tissues through processes thought to involve O2.-.24
The generation of ONOO- from the simultaneous presence of SNAP and pyrogallol was observed in the present study to cause a greater suppression on O2 consumption in isolated bovine cardiac muscle than seen with the generation of NO or O2.- alone. Whereas the effects of the presence of SNAP and pyrogallol on respiration appeared to be additive with each other, respiration did not show a significant recovery after washout. It has recently been reported that exposure of cultured macrophages to 100-fold larger doses of SNAP and pyrogallol for 24 hours resulted in a similar potentiation of the inhibition of respiration associated with a corresponding loss of cell viability.33 Thus, the formation of ONOO- appears to potentiate a mechanism of inhibition of respiration that does not readily recover with time. The attenuation of the inhibitory effect of SNAP plus pyrogallol by Tiron and uric acid strongly supports a role for ONOO- in the suppression of respiration by the combination of these agents. A potential site of action of ONOO- is aconitase because this enzyme is much more sensitive to the actions of ONOO- compared with NO.8 9 In addition, complexes I and II of the mitochondrial electron transport chain,7 cytochrome c reductase and cytochrome oxidase,34 and mitochondrial ATPase7 are additional sites that have been observed to be inhibited by ONOO-. Observations in the present study of an inhibitory effect on DNP-stimulated respiration after washout of the ONOO- and O2.- generating systems are consistent with a direct effect of these reactive species on mitochondrial metabolism or electron transport, independent of possible changes in energy consumption that alter oxidative phosphorylation. Thus, in contrast to the attenuating effects of O2.- on NO-elicited stimulation of tissue guanylate cyclase activity and cGMP-mediated function,26 the interaction of NO with this O2 species enhances the potency and duration of its inhibitory effects on mitochondrial respiration.
Studies developed for comparison of the concentration dependence of the actions of Tiron and urate provided evidence suggesting the importance of intracellular ONOO- formation in the inhibition of respiration and confirmed their O2.- and ONOO- scavenging activity. The very close association between the concentration-dependent attenuation of respiratory inhibition after pretreatment with O2.- and NO generating systems by Tiron and urate and the detection of tissue-derived ONOO- (compared with extracellular detection of ONOO- in buffer, shown in Fig 6A and 6B) are consistent with a mechanism involving intracellular ONOO-. Experiments shown in Fig 6D examining the effects of ONOO- on respiration in the presence of a concentration of SOD that essentially eliminates the detection of extracellular ONOO- were observed not to alter the actions of ONOO- generation, which further supports the primary role of intracellular ONOO- formation in the inhibition of respiration. Similar experiments examining the effects of Tiron and SOD on the attenuation of respiratory inhibition remaining at 15 minutes after washout of the O2.- generating systems with the detection of tissue-derived O2.- appeared to uncover evidence that extracellular sources of O2.- also seem to participate in the actions of this species. Consistent with this concept, the studies reported in Fig 6C and 6D show the close association between the concentration-dependent effect of Tiron on the attenuation of respiratory inhibition after pretreatment with the O2.- generating system and the detection of both tissue-derived and extracellular (buffer) O2.-. In addition, exogenous SOD significantly suppressed the respiratory inhibitory effects of pyrogallol. Thus, an analysis of the concentration dependence of scavenging actions of Tiron and urate suggests that intracellular ONOO- generation is the primary source of this species that participates in the inhibition of respiration.
The similarities in the concentration dependence of the actions of Tiron and urate on attenuation of the inhibition of respiration and inhibition of the detection of O2.- and ONOO- are also consistent with known actions of these scavenger probes. This is mentioned here because both Tiron and urate are known to react with additional reactive species that potentially form during oxidative tissue injury.35 36 The lack of an observed effect of urate on the inhibition of respiration by O2.- generation is consistent with an absence of a role for O2.--derived reactive O2 species and free radicals that are scavenged by urate in the actions on pyrogallol. Based on known mechanisms of actions of these probes and their concentration-dependent effects of the detection of intracellular ONOO- shown in Fig 6A and 6B, it is likely that Tiron prevented the formation of ONOO- by scavenging O2.-, and Tiron may have also directly reacted with ONOO-, whereas urate appears to have functioned through directly reacting with ONOO-.19 27 Thus, on the basis of the known actions of the scavengers used, the effects of these scavengers on the detection of O2.- and ONOO- and the reactive O2 and N2 species that inhibit respiration, it appears that Tiron attenuated the effects of pyrogallol and that Tiron and urate suppressed the actions of pyrogallol+SNAP primarily through reducing the tissue levels of O2.- and ONOO-, respectively.
Cardiac muscle respiration is depressed after exposure to 15 minutes of hypoxia and 10 minutes of reoxygenation in the presence or absence of the mitochordrial uncoupler DNP, consistent with a direct effect of this treatment on mitochondrial function. The inhibition of respiration under these conditions is potentiated by L-Arg and suppressed by blockade of NO synthase activity with L-NNA, suggesting a considerable role for NO biosynthesis in the effect of hypoxia/reoxygenation. In addition, this reoxygenation-associated respiratory inhibition is also attenuated by Tiron or urate, suggesting an involvement of O2.- and ONOO-, respectively, in this process. An overproduction of O2.- is well documented in multiple postischemic reperfused heart models in studies that used spin-trap reagents37 38 and chemiluminescence detection techniques. 39 Because the L-Arg–NO pathway is oxygen dependent, the reintroduction of O2 should favor increased NO production, and nitrite measurements40 41 and spin-trapping studies42 provided evidence for overproduction of NO during reoxygenation. In addition, inhibition of NO synthase protects hearts from ischemia and reperfusion injury,43 44 and administration of substrate for NO synthesis promotes detrimental effects on reperfusion injury.45 46 Therefore, ONOO- is very likely to be formed endogenously during posthypoxic reoxygenation when large amounts of both NO and O2.- are generated simultaneously. Indeed, dityrosine, an oxidation product generated by ONOO-, is detected in isolated hearts after ischemia/reperfusion.47 Impairment of complex I and II and cytochrome c reductase in the mitochondrial respiratory chain is observed during reperfusion,20 21 22 which resembles the dysfunction elicited by ONOO-. Because in the present study the concentrations of tissue metabolites that accumulate during ischemia were likely to have been reduced as a result of diffusion into the Krebs' solution–bicarbonate buffer used, our model of hypoxia/reoxygenation does not reproduce certain metabolic conditions that occur during ischemia/reperfusion. However, the data in our study are consistent with ONOO- forming during hypoxia/reoxygenation in amounts sufficient to inhibit tissue respiration. It is interesting that urate attenuates respiratory suppression by hypoxia/reoxygenation in this study. This effect of urate suggests a very important role for this endogenous antioxidant in protecting humans and primates from injury caused by hypoxia/reoxygenation because the plasma level of urate increases to concentrations close to its solubility limit during primate evolution.36
The results of the present study demonstrate in intact cardiac muscle that NO itself has a completely reversible inhibitory effect on respiration, whereas the formation of ONOO- from its interaction with O2.- promotes enhanced suppression of respiration that is no longer rapidly reversed in the absence of NO production. Thus, NO itself, through a reversible inhibition of mitochondrial cytochrome oxidase, may participate in the physiological control of tissue O2 consumption and related aspects of mitochondrial function. However, the ONOO- mechanism examined in this study is probably an important contributor to the inactivation of respiration in situations in which it is formed such as inflammatory processes or cardiac hypoxia/reoxygenation and ischemia/reperfusion.6 40 48 Interestingly, the respiratory-inhibiting effect of ONOO- is prevented by urate at concentrations of this free radical scavenger that have been retained in blood during the evolution of primates and man.
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Acknowledgments |
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This work was supported by US Public Health Service grants HL-31069 and HL-43023.
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Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 14-17, 1995 (Circulation. 1995;92:I-45. Abstract.), and the Experimental Biology '96 Meeting, Washington DC, April 14-17, 1996 (FASEB J. 1996;10:A582. Abstract.).
Received May 2, 1996; revision received June 25, 1996; accepted July 8, 1996.
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