Lactate and Po2 Modulate Superoxide Anion Production in Bovine Cardiac Myocytes
Potential Role of NADH Oxidase
Background Lactate increases lucigenin chemiluminescence (CL)–detectable superoxide anion (O2•−) generation in bovine vascular smooth muscle and endothelium, and a microsomal flavoprotein-containing NADH oxidase whose activity is regulated by Po2 and cytosolic NAD(H) redox appears to be the detected source of O2•− production. Little is known about the importance of this O2•−-producing system in cardiac myocytes.
Methods and Results In isolated bovine cardiac myocytes, lactate (10 mmol/L) increased lucigenin-detectable O2•− levels to ≈1.8 times baseline, whereas pyruvate (10 mmol/L) and mitochondrial probes did not increase the detection of O2•−. A nonmitochondrial NADH oxidase activity, found in microsomes containing a cytochrome b558, was a major source of O2•− production in the homogenate of myocytes, because NADH (0.1 mmol/L) increased basal lucigenin CL >100-fold. NADPH oxidases, mitochondria, and xanthine oxidase were minor sources of detectable O2•− production. However, mitochondria released H2O2 in the presence of 5 mmol/L succinate and 30 μmol/L antimycin, based on its detection as catalase-inhibitable luminol (+horseradish peroxidase)–elicited CL. Diphenyliodonium (DPI), an inhibitor of flavoprotein-containing oxidases, significantly attenuated basal, lactate, and NADH-elicited lucigenin CL. Hypoxia eliminated myocyte lucigenin CL, and posthypoxic reoxygenation caused an 8.6-fold increase in the detection of O2•− that was potentiated by lactate and inhibited by DPI.
Conclusions NADH oxidase activity linked to cytosolic NAD(H) redox appears to be a key source of O2•− production in cardiac myocytes that could contribute to oxidant signaling mechanisms and injury upon exposure to changes in Po2 and metabolites produced under hypoxia, such as lactate. These processes could contribute to the previously observed potentiation of injury caused by lactate in cardiac ischemia/reperfusion.
Reactive oxygen species seem to play an important role in the pathophysiology of H/R and I/R injury in various organs, including the myocardium.1 2 3 4 Although examination of the significance of ROS in injury to cardiac muscle has been a major area of investigation, the importance of certain potential sources of production of these species has not been systematically studied. Mitochondria and xanthine oxidase have often been implicated in this increase in ROS after H/R or I/R in myocardium.1 2 3 4 5 6 7 Our group has previously investigated in isolated bovine microvessels and myocytes the effect of a 30-minute hypoxia followed by reoxygenation treatment on the steady-state level of an endogenous basal source of O2•− that was detected by lucigenin-elicited CL.7 In this previous study, the basal level of O2•− was not altered by H/R in either the microvessels or myocytes. However, the addition of substrate for xanthine oxidase caused an increase in lucigenin-detectable O2•− in microvessels but not myocytes exposed to H/R, consistent with an increased expression of xanthine oxidase activity in the microvessels. In general, only minimal consideration has been given to the possibility that there might be sources of ROS generation in cardiac muscle other than xanthine oxidase and mitochondria.
We have previously reported evidence in isolated endothelium-removed bovine calf pulmonary and coronary artery and coronary artery endothelial cells that O2•− production by an NADH-oxidoreductase(s) with NADH oxidase activity is the major source of O2•− detected in these tissues by lucigenin-elicited CL.8 9 10 In these tissues, NADH oxidase activity seems to be linked to the redox state of cytosolic NADH, because lactate but not pyruvate increases the observed level of O2•−.10 11 12 13 Previous studies examining the role of NADH oxidase as a source of production of O2•− (or H2O2) in cardiac muscle have focused on developing the concept that it originates from NADH dehydrogenase of the mitochondrial electron transport chain14 15 or a mitochondrial system that has been claimed to have the rather unusual property of being able to directly utilize extramitochondrial NADH.16 17 The O2•−-producing oxidase activity our group has characterized in vascular tissue utilizes predominantly NADH, not NADPH, and its rate of O2•− production is sensitive to physiological changes in Po2. Thus, the requirements of this system for NADH and oxygen as substrates permit it to potentially function as a cellular sensor of metabolic states that alter cytosolic NAD(H) redox and Po2.9 11 12 13 18 19 Our previous studies in both pulmonary and coronary arterial smooth muscle suggest that many of the responses of these vascular segments to changes in Po2 are activated as a result of ROS produced by this oxidase, and in both of these vascular segments, posthypoxic reoxygenation is associated with a transient overproduction of O2•−.9 19 We have also shown that NADH-dependent O2•− production appeared to originate from a microsomal electron transport chain containing a DPI-inhibitable flavoprotein site and spectrally detectable cytochrome b558.18 19 The objective of the present study was to examine whether an NADH oxidase linked to cytosolic NAD(H) redox was an important source of O2•− production in isolated and homogenized calf cardiac myocytes.
Lucigenin (bis-N-methylacridinium nitrate), luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), HEPES, MOPS, NADH, NAD, NADPH, lactic acid, sodium pyruvate, sodium succinate, antimycin A, rotenone, sodium arachidonate, hypoxanthine, catalase (bovine liver), horseradish peroxidase (type VI), SOD (bovine blood), and Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid) were purchased from Sigma Chemical Co. DPI chloride was purchased from Aldrich Chemical Co. Other chemicals were analyzed reagent grade from Baker Chemical Co. Lactate solutions (1 mol/L) were prepared by dissolving lactic acid in water followed by adjustment of pH with NaOH (1 mol/L) to ≈7.4.
Preparation of Calf Heart Myocytes
Calf heart myocytes were prepared as previously described.7 Ventricular muscle obtained from slaughterhouse-derived bovine calf hearts was finely ground with a commercial meat processor at 4°C. The resulting myocardial tissue was suspended in a 1:3 dilution of Krebs-HEPES buffer at 4°C containing the following (mmol/L): NaCl 118, KCl 4.7, CaCl2 1.5, MgSO4 1.1, KH2PO4 1.2, glucose 5.6, and HEPES 10, adjusted to pH 7.4 with NaOH. The muscle suspension was then homogenized at the maximum speed for five periods of 5 seconds in a Sorvall Omnimixer (Du Pont Instruments, Sorvall Biomedical Division). The homogenized tissue was poured over a 95-μm nylon mesh sieve (Small Parts Inc) to remove the microvessel fraction and other larger tissue components. The brownish filtrate containing cardiac myocytes was then examined by light microscopy. If the preparation appeared to contain <90% cardiac myocytes and/or if fibrous connective tissue, microvessel segments, or nonmyocyte tissue components were observed, the myocyte fraction was briefly rehomogenized and sieved through 75-μm mesh. The final filtrate was centrifuged at 650g (4°C) for separation of intact myocytes from broken myocytes and other tissue elements. The resulting pellet was then extensively washed with cooled Krebs-HEPES solution over 25-μm mesh. When examined by light contrast microscopy, the myocyte fraction obtained from this procedure contained >90% cardiac myocytes, generally in the form of rod-shaped single myofibrils or as cells aligned in short chains or small clusters free of detectable vascular elements. Isolated myocytes were then incubated for 30 to 45 minutes in Krebs bicarbonate (containing 25 mmol/L NaHCO3) in a tissue bath gassed with 21% air/5% CO2 before their immediate use in the experiments described. When intact myocytes were studied, their viability was assessed as previously described.7
Preparation of the Homogenate Fraction and Microsomes From Calf Heart Myocytes
The calf heart myocyte homogenate was prepared by adaptation of methods previously used with vascular myocytes and endothelium.9 10 11 Briefly, after isolation, myocytes (1 g blotted wet wt/30 mL buffer) were homogenized in 20 mmol/L MOPS-KOH buffer (pH 7.4) containing 250 mmol/L sucrose at 0°C to 5°C in an Eberbach homogenizer at maximum speed with five treatments of 20-second duration. A microsomal fraction was prepared from the homogenate by differential centrifugation by adapting methods previously used with vascular myocytes.20 Briefly, the homogenate was centrifuged at 29 000g for 15 minutes, and the supernatant obtained was centrifuged at 100 000g for 1 hour. The pelleted material was resuspended in MOPS-sucrose buffer for studies on cardiac myocyte microsomes, and the post–100 000g supernatant fraction was saved for use as a control in studies on microsomes.
CL Measurement of O2•− and H2O2 Production
By methods previously described,7 intact calf heart myocytes were placed in plastic scintillation minivials containing 250 μmol/L lucigenin and other additions in a final volume of 1 mL air-equilibrated Krebs solution buffered with 10 mmol/L HEPES-NaOH (pH 7.4). Experiments on the homogenate fraction used 0.05 to 0.1 mg/mL protein and were conducted in air-equilibrated MOPS-sucrose buffer, pH 7.4, containing 50 μmol/L lucigenin.8 9 10 The CL elicited by O2•− in the presence of lucigenin was measured in a liquid scintillation counter (Mark V, TmAnalytic) with a single active photomultiplier tube positioned in out-of-coincidence mode. All manipulations were performed in the darkroom with minimal light. Samples were initially at 37°C, but the temperatures subsequently equilibrated with the ambient temperature of ≈34°C. After a 5-minute dark adaptation, vials containing all components, with the exception of intact myocytes (blanks), were counted once for 0.1 minute and then recounted three times after myocytes (≈150 mg) were placed in each vial. Blanks of ≈103 counts were then subtracted from the average of the relatively constant levels of CL produced under each condition by the myocytes to obtain the data reported as cpm/g myocytes in the “Results.” In studies on tissue homogenate samples, after quantification of the vial blank, samples were counted immediately within 2 minutes of addition of substrates for O2•− production to the myocyte homogenate fraction to minimize the effects of time-dependent changes in metabolite levels (eg, NADH). The sensitivity and chemical specificity of the lucigenin-elicited light-yielding reaction for O2•− has been reported previously.21 For peroxide detection, 10 μmol/L luminol plus 1 μmol/L horseradish peroxidase were substituted for lucigenin.22
Experiments in which intact myocytes were studied for the effects of changes in Po2 on CL measurement of O2•− were conducted in a single photon counting apparatus constructed in a light-tight box similar to that previously described.9 19 In these experiments, intact myocytes (≈0.8 to 1.0 g/mL) were incubated in Krebs bicarbonate buffer containing 0.25 mmol/L lucigenin in a continuously gassed 1-cm2 spectrophotometer cuvette mounted in a thermostatted (37°C) cell holder on the surface of a Lucite light guide (with a shutter cover) directed into a cooled Thorn EMI photomultiplier tube (model 9235B). A Thorn EMI amplifier-discriminator (model C604) and photon counter (model C660) were used to quantify CL. The counts were integrated over 5-second periods by the photon counter, and an analog signal of the integrated counts was continuously recorded on a Grass model 7 polygraph recorder. Data are reported as counts·5 s−1·g myocytes−1 after subtraction of the background of ≈250 counts/5 s.
Spectral Detection of Microsomal Cytochromes
Spectral studies were conducted as previously described18 in 50 mmol/L sodium phosphate buffer, pH 7.4, with ≈0.5 mg/mL microsomal protein in both the sample and reference cuvettes. After the baseline or oxidized cytochrome spectra were re-corded over the 500- to 620-nm range, a few grains (to avoid sample dilution) of sodium dithionite were added to the sample cuvette to generate reduced cytochromes in an anaerobic environment, and these spectra were subsequently recorded immediately after dithionite was added.
Data were evaluated by a one-way ANOVA with Scheffé’s post hoc test to determine statistical significance between groups. The acceptable level of significance was P<.05. The number of experimental determinations (n) in all cases is equal to the number of animals from which myocytes were obtained. Data in the figures are depicted as mean±SEM.
Effects of Lactate, Mitochondrial Inhibitors, and DPI on Lucigenin-Detectable O2•− in Isolated Calf Heart Myocytes
Experiments in our previous study on isolated bovine cardiac myocytes using lucigenin for the detection of O2•− indicated that inhibition of Cu,Zn-SOD increased the level of CL measured by the scintillation counter method.7 In that previous study, it was also observed that the tissue-permeable scavenger of O2•− Tiron but not added SOD decreased the amount of CL detected. Thus, basal levels of myocyte-derived lucigenin CL appear to originate from an intracellular source of O2•− production. As shown by the data in Fig 1⇓, 10 mmol/L lactate increased the amount of CL detected to ≈1.8 times baseline. To evaluate whether mitochondrial NADH oxidase activity14 15 participates in the sources of O2•− detected by lucigenin, the effects of two inhibitors of mitochondrial electron transport, rotenone and antimycin, were evaluated. As shown by the data in Fig 1⇓, these inhibitors of mitochondrial electron transport did not significantly alter the amount of basal or lactate-elicited CL detected. The mitochondrial probes may actually have a small stimulating effect on basal CL, because the increase in lactate-elicited CL did not reach statistical significance in the presence of these probes. As shown in Fig 1⇓, the flavoprotein inhibitor DPI (10 μmol/L) reduced basal lucigenin CL by 27%, and this probe completely eliminated the lactate-elicited increase in the detection of O2•−.
Conditions for the Detection of Lactate-Stimulated O2•− Production in Homogenized Calf Heart Myocytes
In the homogenate fraction of calf heart myocytes, lactate was able to increase CL only in the presence of added NAD (Fig 2A⇓), presumably as a result of NADH formation by lactate dehydrogenase. As shown by the data in Fig 2A⇓, lactate, pyruvate, NAD, or pyruvate+NAD did not elicit detectable increases in CL over the background level of the myocyte homogenate. As shown by the data in Fig 2B⇓, NADH (0.1 mmol/L), a product of the lactate dehydrogenase reaction, caused a prominent increase in lucigenin CL. The increase in CL caused by NADH was significantly decreased by the scavengers of O2•− SOD (3 μmol/L) and Tiron (10 mmol/L), further implicating O2•− as the source of CL. Preincubation of the homogenate of calf heart myocytes for 15 minutes with the flavoprotein inhibitor DPI (1 μmol/L) also resulted in a significant decrease in the observed level of NADH-derived CL (Fig 2B⇓).
Evidence That NADH Oxidase Is a Major Source of Lucigenin-Detectable O2•− Production in the Homogenate of Calf Heart Myocytes
Like our studies in calf pulmonary and coronary arteries,8 9 experiments were conducted with lucigenin for the detection of sources of O2•− production potentially present in the homogenate of calf heart myocytes. O2•− production from xanthine oxidase was determined by examining the increase in lucigenin-elicited CL caused by 100 μmol/L hypoxanthine. NAD(P)H oxidoreductase was examined by determining the effect of 100 μmol/L NADH or NADPH, and arachidonic acid–metabolizing enzymes were determined by quantifying the increase in CL produced by 10 μmol/L arachidonic acid over the levels observed in the presence of NADPH. The data in Fig 3A⇓ indicate that NADH oxidase activity was the major source of O2•− production detected by lucigenin. Xanthine oxidase, arachidonic acid–metabolizing enzymes, and NADPH oxidases were observed to be minor sources of O2•− production by this method. Therefore, NADH oxidase appears to be a major source of O2•− detected by lucigenin in the homogenate of calf heart myocytes.
Mitochondria as a Source of O2•− Production in Calf Heart Myocytes
Because lucigenin is a poor detector of mitochondrial electron transport chain–derived O2•− production,8 we examined the intramitochondrial production of O2•− indirectly by measuring catalase-inhibitable luminol (10 μmol/L)+horseradish peroxidase (1 μmol/L)–dependent CL for detection of H2O2 released from mitochondria in the presence of combinations of 5 mmol/L succinate and 30 μmol/L antimycin. Mitochondrial O2•− production is known to be maximized by the presence of the combination of these two agents, and the H2O2 released is thought to originate from intramitochondrial sources of O2•− production.15 As shown in Fig 3B⇑, the combination of succinate and antimycin caused the largest observed increase in luminol CL, and this increase was inhibited back to basal levels by 1 μmol/L catalase. Under similar conditions, lucigenin did not detect an increase in O2•− in the presence of succinate and antimycin (see Fig 3A⇑), which is consistent with lucigenin being a poor detector of mitochondrial O2•− production. The increase in lucigenin-detectable O2•− production produced by NADH (5.56±0.55×107 cpm/mg, n=9) in the myocyte homogenate also appeared to be independent of mitochondrial function, because its rate of O2•− production was not significantly altered by the presence of 30 μmol/L rotenone (5.11±0.57×107 cpm/mg, n=9).
Detection of a Cytochrome b558 in Microsomes Containing NADH Oxidase Activity
In preliminary experiments, conducted in a manner similar to our studies on NADH oxidase in vascular tissue,20 it was observed that microsomal membranes obtained by differential centrifugation of the homogenate contained the NADH-dependent O2•−-producing activity. In addition, an NADH-dependent SOD-inhibitable source of lucigenin CL could not be detected in the supernatant fraction obtained after removal of the microsomes by centrifugation at 100 000g. When this microsomal fraction was examined for the presence of a cytochrome b558 by spectral methods under anoxic reducing conditions produced by dithionite, a prominent absorbance was observed at 558 nm (see Fig 4⇓).
Effects of H/R in the Absence and Presence of Lactate, Pyruvate, or DPI on the Detection of O2•− by Lucigenin in Isolated Calf Heart Myocytes
Time-dependent changes in lucigenin CL from isolated calf heart myocytes as a function of exposure to H/R were measured in the single photon counting CL detection apparatus described in “Methods.” Fig 5⇓ contains summary data showing the steady-state level of CL above the lucigenin-containing buffer background elicited by the presence of myocytes under an atmosphere of 21% O2/5% CO2 (balance N2; Po2≈150 mm Hg). This basal level of production of O2•− by myocytes was essentially eliminated upon exposure to a hypoxic environment (Po2≈8 to 10 mm Hg) produced by a subsequent gassing of the myocytes with 95% N2/5% CO2. As shown in Fig 5⇓, reoxygenation with 21% O2 after a 30-minute exposure to hypoxia produced a transient 8.6-fold increase in CL over the initial levels observed with 21% O2, followed by a subsequent decay over 20 minutes back to the levels initially observed under 21% O2.
In intact calf heart myocytes, 10 mmol/L lactate produced a significant increase in the steady-state level of myocyte lucigenin CL observed under 21% O2 atmosphere, whereas the presence of 10 mmol/L pyruvate or 10 μmol/L DPI depressed the detected level of O2•− (see Fig 5⇑). None of these probes altered the essentially undetectable level of CL observed under hypoxia. The presence of lactate but not pyruvate caused a marked potentiation of detection of the transient increase in lucigenin CL observed during posthypoxic reoxygenation. The presence of DPI markedly reduced but did not eliminate the transient increase in the level of lucigenin CL observed during posthypoxic reoxygenation.
The results of the present study are consistent with bovine calf cardiac myocytes having a lucigenin-detectable basal source of O2•− production that is increased by lactate. This basal source of O2•− production in intact myocytes was also shown to be regulated by changes in Po2 and to be dependent on a flavoprotein-containing oxidase. The major source of O2•− production detected by lucigenin in the homogenate of calf heart myocytes is derived from flavoprotein-dependent NADH oxidase activity, which appears to originate from a microsomal electron transport chain containing a cytochrome b558. As might be anticipated, O2•−-producing systems associated with other tissues, such as xanthine oxidase and NADPH oxidases or arachidonic acid–metabolizing enzymes, were not major sources of O2•− generation in calf heart myocytes. Although mitochondria seem to be a significant site of generation of H2O2, lucigenin does not appear to detect the intramitochondrial pool of O2•− that is likely to be the source of H2O2 observed to be released from mitochondria. Thus, a flavoprotein-containing oxidase whose activity is controlled by cytosolic NAD(H) redox and Po2 appears to be an important source of O2•− production in bovine cardiac myocytes.
Bovine calf cardiac myocytes have previously been shown to express a basal intracellular source of production of O2•− that is detected by lucigenin-elicited CL.7 This basal production of O2•− is shown in the present study to be modulated by changes in Po2, increased in the presence of 10 mmol/L lactate, and inhibited slightly by 10 mmol/L pyruvate potentially through the hypothesized mechanisms included in the model in Fig 6⇓. Since the major tissue metabolites of lactate are pyruvate and NADH, lactate deydrogenase-derived NADH is likely to be the key metabolite of lactate that contributes to the generation of O2•−. Consistent with this role for cytosolic NADH in the production of O2•− is the observation in this study in the homogenate of cardiac myocytes that NADH oxidase is a major source of O2•− production. Previous studies have reported the detection of an O2•−-producing NADH oxidase activity in cardiac muscle, which has been claimed to have the unusual property that it is a mitochondrial system that uses extramitochondrial NADH.16 We suspect that this previously detected O2•−-producing NADH oxidase activity is similar to the NADH oxidase identified in the present study. However, since O2•− production by both of these NADH oxidase systems is not altered by rotenone, it is likely that these oxidases are not linked to the mitochondrial NADH dehydrogenase, which is thought to utilize extramitochondrial NADH. This interpretation is based on more recent studies from the same group17 indicating that the NADH dehydrogenase activity linked to extramitochondrial NADH is inhibited by rotenone. The inhibitory effects of DPI on O2•− production in intact and homogenized cardiac myocytes are consistent with a flavoprotein component in the detected O2•−-producing oxidase. Our previous studies on the properties and subcellular localization of O2•− production by a lactate and cytosolic NADH redox–regulated NADH oxidase in bovine pulmonary arterial smooth muscle are consistent with this activity originating from a flavoprotein and b558-type cytochrome containing microsomal electron transport chain.18 19 On the basis of the results of the present study, cardiac myocytes appear to have a similar microsomal NADH-dependent O2•−-producing electron transport chain containing a cytochrome b558 whose activity is regulated by cytosolic NAD(H) redox.
In the present study, attempts were made to detect several other potential sources of O2•− production in the homogenate of bovine cardiac myocytes. It is well established that exposure of cardiac muscle to H/R or I/R can lead to conversion of xanthine dehydrogenase to xanthine oxidase.1 2 23 Since hypoxia and ischemia also seem to generate the substrates hypoxanthine and xanthine that are required for the expression of xanthine oxidase activity, xanthine oxidase appears to become a major source of O2•− in the myocardium upon reoxygenation or reperfusion.23 24 However, the results of our previous study in intact bovine cardiac myocytes did not detect a hypoxanthine-elicited increase in O2•− production either before or after H/R.7 Consistent with these previous observations and the known specific localization of xanthine dehydrogenase/oxidase to endothelium,25 the present study did not detect evidence for xanthine oxidase activity in homogenized myocytes. Since NADPH oxidases and arachidonic acid–metabolizing enzymes have also been shown to be potential sources of O2•− production,26 27 28 experiments in the present study also examined the importance of these systems as myocyte sources of O2•− generation. In the calf myocyte homogenate, the presence of 100 μmol/L NADPH was observed to elicit a small but statistically significant increase in lucigenin-dependent CL. Arachidonic acid plus NADPH was used to detect O2•− generation by enzymes such as cyclooxygenase and lipoxygenase.28 Under these conditions, arachidonic acid did not cause a further increase in lucigenin CL. Although the origin of the minor NADPH oxidase activity that was detected was not further examined, it is possible that this activity originates from utilization by NADH oxidases of NADPH as a substrate or from NADPH oxidases that remain to be identified. Observations made in the present study are consistent with an absence of evidence in the literature for xanthine oxidase, arachidonic acid–metabolizing enzymes, and NADPH oxidases as major sources of production of O2•− in cardiac myocytes.
Mitochondria are considered to be an important source of generation of O2•− in cardiac myocytes.4 5 6 14 This concept is supported by observations in the present study on the detection of H2O2 release from mitochondria under conditions that maximize intramitochondrial generation of O2•− (succinate+antimycin15 ). These conditions are thought to maximize O2•− production as a result of blocking by antimycin of electron flow in the mitochondrial electron transport chain in a manner that permits succinate dehydrogenase to transfer electrons in a forward and reverse direction to the previously identified key sites of O2•− production associated with NADH dehydrogenase (complex I) and ubiquinone–cytochrome b.14 15 It should be noted that our method of detection of mitochondrial H2O2 release is expected to markedly underestimate the actual rates of mitochondrial O2•− and H2O2 production, because it does account for O2 metabolites that are metabolized within the mitochondria. Previous studies on mitochondrial NADH–dependent O2•− production indicate that inhibitors that block electron transport at sites proximal (rotenone) and distal (antimycin) to the location of electron transfer to O2 cause decreased and increased production of O2•−, respectively.14 15 Thus, O2•− production by the rotenone-inhibitable NADH dehydrogenase suggested to be an inner mitochondrial membrane enzyme utilizing extramitochondrial (cytosolic) NADH16 17 would be expected to be altered by the presence of rotenone. In the present study, rotenone was found not to significantly alter the level of lucigenin-detectable O2•− observed in the presence of lactate or NADH in intact or homogenized myocytes, respectively. The observed minor stimulating effects of rotenone and antimycin on basal myocyte CL and the apparent suppression of the increase caused by lactate in the presence of these inhibitors may originate from potential effects of these electron transport inhibitors on the control of cytosolic NAD(H) redox by mitochondrial function, as has been observed in our previous studies on calf pulmonary arterial smooth muscle.12 We have previously suggested that lucigenin appears to be a poor detector of intramitochondrial O2•−.8 Data in the present study indicate that lucigenin CL was not increased by the intramitochondrial source of O2•− that results in the mitochondrial release of H2O2 that is detectable as catalase-inhibitable increases in luminol CL. Thus, the major source of lucigenin-detectable O2•− appears to originate from an NADH oxidase activity that is not linked to intramitochondrial O2•− production.
The lucigenin-detectable source of production of O2•− observed in intact cardiac myocytes is suppressed by hypoxia, and posthypoxic reoxygenation elicits a transient overproduction of O2•−. A key role for NADH oxidase in these Po2-elicited changes in O2•− detection is supported by the inhibition by DPI and the enhancement by lactate of the measured levels of lucigenin CL under these conditions. Although 10 mmol/L pyruvate was observed to cause a small decrease in lucigenin CL under basal conditions, it did not significantly alter the transient overproduction of O2•− observed during posthypoxic reoxygenation. Our studies on the Po2 dependence of O2•− production at constant levels of NADH by a microsomal preparation of NADH oxidase isolated from bovine pulmonary arterial smooth muscle determined that it is suppressed by hypoxia and that posthypoxic reoxygenation elicits a transient overproduction of O2•−.18 Thus, O2•− production by the microsomal NADH oxidase is suppressed by decreases in physiological levels of Po2, and a transient overproduction of O2•− during posthypoxic reoxygenation is a property of the effects of hypoxia on this electron transport chain. Isolated bovine pulmonary19 and coronary9 arterial smooth muscle show Po2-elicited changes in lucigenin-detectable O2•− that appear to originate from NADH oxidase upon exposure to H/R and are similar to the responses observed in cardiac myocytes and pulmonary arterial microsomes containing NADH oxidase activity. Interestingly, a comparison with O2•− production by previously studied bovine vascular smooth muscle preparations8 9 19 suggests that bovine cardiac myocytes have an ≈10-fold greater level of NADH oxidase activity and a markedly increased spectral absorbance of microsomal cytochrome b558 and that the myocytes generally show a greater basal level of lucigenin CL, particularly after exposure to H/R. On the basis of studies in a variety of tissue preparations, including cardiac muscle, H/R and I/R also appear to cause the accumulation of tissue lactate and an increase in the levels of cytosolic NADH.29 30 Thus, increased levels of lactate and cytosol-ic NADH that accumulate during the hypoxic exposure are also likely to contribute to the transient overproduction of O2•− observed in bovine cardiac myocytes during posthypoxic reoxygenation.
Experiments in the present study have identified an NADH oxidase whose activity appears to be controlled by cytosolic NADH as an important novel source of O2•− production in bovine cardiac myocytes. As hypothesized by interactions shown in the model in Fig 6⇑, the activity of this system seems to be controlled in myocytes by changes in Po2 and lactate in a manner suggesting that O2•− production by this NADH oxidase may be of importance in signaling processes involving reactive O2 species in cardiac myocytes. NADH oxidase–derived O2•− production may also have an important role in the injury caused by exposure of cardiac muscle to I/R, because the accumulation of tissue lactate and the associated increase in cytosolic NADH have previously been suggested to actively participate in metabolic processes that contribute to tissue injury under these conditions.29 30 Heart failure is an additional pathophysiological state in which NADH oxidase may participate in increased myocardial O2•− production, because we have observed elevated levels of lucigenin CL and NADH oxidase activity in myocytes isolated from failing human and canine hearts.31 32
Selected Abbreviations and Acronyms
|ROS||=||reactive oxygen species|
This work was supported by grants HL-31069 and HL-43023 from the National Heart, Lung, and Blood Institute.
Presented in part at the Experimental Biology ’95 Conference, Atlanta, Ga, April 9-13, 1995, and published in abstract form (FASEB J. 1995;9:A12).
- Received August 6, 1996.
- Revision received December 16, 1996.
- Accepted January 20, 1997.
- Copyright © 1997 by American Heart Association
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