Laboratory of Oxygen Metabolism, University Hospital, School of Medicine and Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, 1120 Buenos Aires, Argentina; and Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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Isolated rat heart perfused with 1.5-7.5
µM NO solutions or bradykinin, which activates endothelial NO
synthase, showed a dose-dependent decrease in myocardial O2
uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,
P < 0.05) and to 1.2 ± 0.1 µM O2 · min1 · g
tissue
1 (10 µM bradykinin, n = 10,
P < 0.05). Perfused NO concentrations correlated with an
induced release of hydrogen peroxide (H2O2) in
the effluent (r = 0.99, P < 0.01). NO markedly
decreased the O2 uptake of isolated rat heart mitochondria
(50% inhibition at 0.4 µM NO, r = 0.99,
P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b and
cytochrome c, which accounts for the effects in O2
uptake and H2O2 release. Most NO was bound to
myoglobin; this fact is consistent with NO steady-state concentrations
of 0.1-0.3 µM, which affect mitochondria. In the intact heart,
finely adjusted NO concentrations regulate mitochondrial O2
uptake and superoxide anion production (reflected by
H2O2), which in turn contributes to the
physiological clearance of NO through peroxynitrite formation.
Langendorff preparation; regulation of myocardial oxidative metabolism; nitrosylmyoglobin; role of oxygen free radicals; peroxynitrite
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INTRODUCTION |
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THE NITROGEN RADICAL nitric oxide (NO) is a physiological vasodilator in arterial vascular beds such as vertebral, pulmonary, and coronary arteries (17). The effects of NO are mediated by its binding to the heme group of guanylyl cyclase to produce guanosine 3',5'-cyclic monophosphate, which relaxes vascular smooth muscle by lowering cytoplasmic Ca2+ levels (12).
The pharmacological use of nitrites, nitrates, and derived compounds to alleviate symptoms of coronary insufficiency is well known by physicians. It is now accepted that the pharmacological actions of nitrates and nitrites are mediated by the release of NO (12). In the last years, solutions of NO or NO donors have been reported to produce coronary vasodilation, to protect ischemic myocardium, and to prevent the deleterious effects of reperfusion in the heart (16).
Recently, interest has been focused on the actions of NO on
mitochondrial function and on oxidative metabolism of mammalian cells.
NO reversibly inhibits cytochrome oxidase and O2 uptake in
skeletal muscle and heart mitochondria (5, 20) and in heart
submitochondrial particles prepared from rat heart (20). Previously,
inhibition of mitochondrial enzymes by NO was described as a toxic and
nonreversible effect rather than as a physiological and regulatory
action (3). Toxic effects are understood to be primarily produced by
peroxynitrite (ONOO), the product of the reaction of NO
with superoxide anion (
; see Ref. 1).
The effects of ONOO
appear to be selectively exerted on
matrix mitochondrial enzymes such as aconitase and on membrane-bound
enzymes such as succinate dehydrogenase (3).
On the other hand, the overproduction of superoxide radicals and
derived reactive O2 species has been recognized as the
initial molecular phenomenon in experimental and clinical myocardial
ischemia-reperfusion (30). Mitochondria are the main source of cellular
O2 free radicals; and its
dismutation product H2O2 are by-products of
mitochondrial electron transfer from a deviation of electrons that
reduce O2 in collisional noncatalyzed reactions (25).
Irreversible inhibition of electron transfer and increase in
production are obtained in the presence
of the nonphysiological drug antimycin. We have reported that
0.5-1 µM NO is able to inhibit mitochondrial electron transfer
at the ubiquinol-cytochrome b-cytochrome c region of
the respiratory chain and to produce an increase in
and H2O2
production in submitochondrial particles and in rat heart mitochondria
(20). The mitochondrial production of
seemed to revert NO inhibitory effects on cytochrome oxidase. On this
basis, it is interesting to consider the hypothesis that the heart,
under physiological conditions, is subjected to a NO regulation of
O2 uptake and mitochondrial electron transfer with an
effect in
production in a much more
causal way than previously recognized.
The aim of the present study was to determine whether changes in O2 uptake and in H2O2 release could be observed in the beating heart of the Langendorff preparation after the infusion of a NO gas solution, the NO donor S-nitrosoglutathione (GSNO), or autacoids that promote NO release, like bradykinin. The experimental conditions of the Langendorff preparation do not rule out some degree of myocardial ischemia, but they allow approximation of the physiological setting in which O2 concentration can be considered to be at the edge of effectively regulating respiration (8).
The results suggest that NO released by endothelium not only dilates
the vessels but also plays a regulatory role increasing the generation
of and lowering heart O2
uptake.
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MATERIAL AND METHODS |
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General experimental design. The effects of different doses of NO, GSNO, and bradykinin were tested in different heart preparations. In each experiment, cardiac physiological functions such as mean coronary perfusion pressure, left ventricular developed pressure, heart O2 uptake, and release of NO and H2O2 were assessed simultaneously. To determine NO and H2O2 concentrations, 10-ml samples of the effluent perfusate of the isolated rat hearts were collected every 5 min. The samples were taken before NO or bradykinin infusion to set the basal values, during NO and bradykinin infusion, and up to 30 min after the treatments were discontinued. Control hearts perfused with saline solution during a similar period (50 min, n = 4) neither exhibited changes in O2 uptake, aortic coronary pressure, and developed left ventricular pressure nor released detectable NO or H2O2 during the experiments. The direct effect of NO on mitochondrial respiration was determined by measuring the O2 uptake of isolated rat heart mitochondria supplemented with NO. To obtain enough mitochondrial protein concentration and considering the weight of the rat hearts (1-1.3 g), two animals were used in each experiment, and four experiments were done to assess the NO effects on mitochondrial functions.
Animals. Eighty female Sprague-Dawley rats weighing 250-300 g fasted overnight with ad libitum access to water were used. Rats were anesthetized with ether. To remove blood cells, 60 units of sodium heparin were administered by the tail vein before the heart was excised. The protocol followed international ethics guidelines for laboratory animals.
Heart perfusion and pressure determinations.
Hearts were perfused by the Langendorff technique (21) at 37°C with a
perfusion medium consisting of 128 mM NaCl, 4.7 mM KCl, 1 mM
MgCl2, 0.4 mM NaH2PO4, 20.2 mM
NaHCO3, 1.3 mM CaCl2, 5 mM glucose, and 2 mM
pyruvic acid (pH 7.4) that was oxygenated with 95% O2-5%
CO2. Hearts were paced at 5 Hz with a pacemaker. The
aortic-coronary flow was kept constant at a rate of 6-7 ml/min with a peristaltic pump (model 7554-20; Cole Parmer, Chicago IL). Mean
aortic-coronary perfusion pressure was obtained from the perfusion
line. Developed left ventricular pressure (peak systolic left
ventricular pressure end diastolic left ventricular pressure) was
determined at a fixed preload (5-7 mmHg) with a balloon-tipped catheter directly inserted in the left ventricle. The lines were connected to a pressure transducer (78342A monitor; Hewlett-Packard, Boeblingen, Germany), and pressure curves were recorded in Dyne programmable multichannel equipment (Dyne, Buenos Aires, Argentina).
Infusion of NO. After 15 min of perfusion and under stable beating and perfusion conditions, NO or GSNO was added in parallel to the perfusion medium with a Harvard pump at a rate of 0.2 ml/min to achieve concentrations of 1-8 µM NO and of 0.1-1.5 mM GSNO in the perfusate. At these slow rates, the degassed NO solution did not diminish PO2 of the perfusion fluid by >2.8%. The electrochemical detection of NO in the effluent during the perfusion with true NO solutions was ~3-10% of the added NO (0.14, 0.17, and 0.27 µM for 2, 5, and 7.5 µM, respectively). The NO concentration in the effluent amounted to ~0.6% of added GSNO (20). The NO levels in the effluent decreased promptly to be undetectable a few minutes after NO infusion was stopped.
Measurement of myocardial O2 uptake. The O2 uptake was calculated from the difference between the O2 content in the input minus the content in the output multiplied by the perfusate flow according to the Fick principle. The difference in O2 content was determined with an on-line Clark type electrode and recorded continuously.
Detection of NO. The NO concentration in the perfused solutions and in the effluent was measured with a sensitive NO electrode (ISO-NO; World Precision Instruments, Sarasota, FL) connected to a recorder. The electrode was calibrated daily with standard nitrite solutions in acid medium.
Measurement of H2O2 in the effluent. The concentration of H2O2 in the effluent samples was determined fluorometrically at 315 nm (excitation) and 425 nm (emission) in an F-2000 Hitachi spectrofluorometer (Hitachi, Tokyo, Japan), using an assay medium containing 12 U/ml horseradish peroxidase and 500 µM p-hydroxyphenilacetic acid in 2-ml cuvettes at 37°C under soft magnetic stirring (2, 20). Determinations were performed in duplicates with and without 0.1 µM catalase, and the difference between both fluorescence measurements was calculated as H2O2 concentrations (11).
Mitochondrial respiration. Rat heart mitochondria were isolated in 0.23 M mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.3; see Ref. 20). Mitochondrial O2 uptake was determined polarographically with a Clark type O2 electrode in a reaction medium consisting of 0.23 M mannitol, 70 mM sucrose, 0.2 mM EDTA, 5 mM Na2HPO4-KH2PO4, and 20 mM Tris · HCl buffer (pH 7.4) at 30°C. O2 uptake rates were measured with 8 mM succinate as substrate in the presence (state 3 O2 uptake) or absence (state 4 O2 uptake) of 0.2 mM ADP as phosphate acceptor. Cytochrome differential spectra of rat heart submitochondrial particles obtained by sonication of rat heart mitochondria as previously described (20) were recorded with a model 356 Perkin-Elmer-Hitachi double beam-double wavelength spectrophotometer. Mitochondria and submitochondrial particles were exposed to different GSNO concentrations by 8 min to provide a known rate of NO release (d[NO]/dt) and a known effective NO concentration (20). Cytochrome redox state was controlled by adding succinate and, to achieve a complete cytohrome reduction, with sodium dithionite. The supplementation with NO did not modify the final reduction level produced by sodium dithionite.
Myoglobin spectra in heart slices.
Rat hearts were placed in liquid nitrogen immediately after NO
infusion, and 6- to 7-µm slices from the left ventricles were obtained with an electronic microtome at 20°C (4). The slices were
placed on thin glass covers that were vertically adhered to one of the
transparent walls of quartz cuvettes. The wavelength scan spectra were
recorded between 560 and 600 nm in a Hitachi U-3000 spectrophotometer.
The absorbance difference between 581 and 592 nm was proportional to
oxyMb concentrations with an extinction coefficient
(
581-592) = 11 mM/cm (2).
Infusion of bradykinin. Bradykinin was infused at 1-10 µM. As reported by Zhang et al. (29) in isolated myocardial slices, the effects of bradykinin were potentiated by inhibition of kininase II with enalapril (10 mg/kg), which was administered to the rats in the tail vein 30 min before the start of the experiments. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) was added to the perfusion medium at a concentration of 100 µM. In other experiments, indomethacin (100 µM) was added to bradykinin to rule out prostacyclin-dependent effects.
Chemicals and drugs. GSNO was synthesized at 25°C by reacting equimolar concentrations (200 mM) of reduced glutathione in 100 mM phosphate buffer (pH 7.4) with sodium nitrite in 100 mM HCl. NO solutions (1-1.5 mM) were obtained freshly on the day of the experiment or on the day before by bubbling 99% pure NO gas in N2-degassed water for 30 min at room temperature and afterward were kept at 4°C. Other chemicals were purchased from Sigma Chemical (St. Louis, MO).
Statistics. The statistical analysis included paired Student's t-test, one-way analysis of variance, Dunnett's test, and regression studies.
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RESULTS |
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Effects of NO infusion on myocardial O2 uptake and coronary
perfusion.
The infusion of either NO solutions or GSNO to the isolated and
perfused rat heart clearly induced dose-dependent decreases in
myocardial O2 uptake and coronary perfusion pressure and a slight nonsignificant change in developed left ventricular pressure. A
comparison between the percentage of variation with respect to control
values induced by 1.5-7.5 µM NO infusion on heart functions is
shown in Fig. 1. O2 uptake
decreased progressively from 3.2 ± 0.3 (basal value) up to 1.6 ± 0.1 µmol
O2 · min1 · g
tissue
1 (at 7.5 µM NO, P < 0.05; controls
n = 6, each NO concentration subgroup n = 4, total
n = 18). Once infusion was stopped, O2 uptake recovered to the basal condition after 10-15 min of perfusion. In
the same conditions, mean coronary perfusion pressure decreased from 80 ± 3 to 50 ± 5 mmHg (total n = 18, P < 0.05)
and returned back to the basal values within 10 min after stopping NO
infusion. The left ventricular developed pressure did not significantly change by effects of NO infusion (from 100 ± 10 to 85 ± 2
mmHg). The hearts did not exhibit any functional impairment up to 60 min after NO infusion was stopped.
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Effects of NO on mitochondrial O2 uptake.
The O2 uptake of isolated rat heart mitochondria was 42 ± 6 ng atoms O2 min1 · mg
protein
1 in state 4 (without ADP) and 165 ± 17 ng
atoms O2 · min
1 · mg
protein
1 in state 3 (with ADP), with succinate as
substrate. The exposition to NO inhibited 50% state 3 O2 uptake at ~0.4 µM and reached maximal 80%
inhibition at ~0.9 µM NO (33 ± 3 ng atoms O2
min
1 · mg protein
1, total
n = 16, P < 0.05). The negative relationship
between NO concentration and mitochondrial state 3 O2
uptake is shown in Fig. 2.
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Kinetics of H2O2 release after NO infusion. The infusion of either NO solutions or the NO donor GSNO induced the appearance of H2O2 in the effluent. The release of H2O2 was detected ~5-10 min after the end of NO perfusion as a transient phenomenon, increasing to maximal levels in ~10-15 min and decreasing afterward (Fig. 3). Both the concentration of H2O2 in the perfusate and the amount of H2O2 released (determined as the area under the curve of H2O2 release as a function of time) depended linearly on the NO concentration in the perfusion fluid between 1.5 and 7.5 µM NO (Fig. 3, inset). Similarly, maximal H2O2 concentrations in the effluent fluid also correlated with the NO concentrations recovered in the perfusion fluid during GSNO infusion (Fig. 4).
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Cytochrome reduction. The cytochrome differential spectra of rat heart submitochondrial particles supplemented with NO showed that addition of 0.8 mM succinate, at relatively low substrate concentrations, produced a complete reduction of cytochrome c and cytochrome a-a3 with no further reduction after addition of 8 mM succinate and sodium dithionite; by contrast, cytochrome b remained only partially reduced (Fig. 5).
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Spectra of myoglobin in myocardium slices. The NO-dependent changes in the tissue spectrum in the 560- to 580-nm region were similar to the NO-dependent changes in the spectrum of pure oxymyoglobin solutions (2). The infusion of 2-7 µM NO produced a decrease in the absorption peak of tissue oxymyoglobin at 581 nm that was linearly related to NO concentrations (Fig. 6). The conservation of the absorption at 592 nm, at the isosbestic point, indicates the lack of myoglobin loss and the stoichiometric change of oxymyoglobin to metmyoglobin (Fig. 6).
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The action of bradykinin. The infusion of 1-10 µM bradykinin induced a decrease of ~30-40% in coronary perfusion pressure and O2 uptake. The changes in perfusion pressure and O2 uptake were associated with an increase in NO concentration in the effluent. Bradykinin infusion also promoted an increase in H2O2 concentrations in the effluent in which the time course was similar to that obtained after perfusion with NO (Fig. 7).
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DISCUSSION |
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Coronary infusion of NO, either as an NO solution or released by GSNO or by bradykinin-activated endothelium, transiently and reversibly decreased aortic perfusion coronary pressure and myocardial O2 uptake and elicited the release of H2O2 in the effluent fluid of the isolated and perfused beating rat heart.
The decrease in myocardial O2 uptake was not the result of a diminished energy demand secondary to a decreased heart performance. Actually, the effects of NO on heart contractility parameters are still controversial, since they probably depend on the doses, i.e., very high doses of NO are required to achieve detrimental effects on the contractility of rat cardiac muscle (28). Hence, the decrease in myocardial O2 uptake is understood here as derived from a direct NO effect on mitochondrial electron transfer and O2 uptake, similar to those previously reported in mitochondria isolated from skeletal muscle (5) and heart (20). In this study, half-inhibition of heart mitochondria state 3 respiration was achieved at ~0.4 µM NO. The decrease in mitochondrial O2 uptake was mostly due to the inhibition of cytochrome oxidase, the final step of the electron transfer chain. In agreement, Cleeter et al. (5) and our group (20) reported that NO half-inhibition of rat heart mitochondrial cytochrome oxidase occurs at even lower levels in the range of 0.05-0.1 µM NO.
In addition, this study shows a significant correlation between NO and
the amounts of H2O2 released by the heart.
Mitochondrial production of and
H2O2 requires an inhibition of mitochondrial
electron transfer as is the case for ADP-controlled state 4 respiration or after the addition of nonphysiological inhibitors like
antimycin or in the presence of NO. In this way, NO is the unique
physiological substance that blocks more than one site in the
mitochondrial respiratory chain. In addition to cytochrome oxidase, NO
reversibly inhibits the segment cytochrome b-cytochrome
c in which electron transfer capacity is adequately measured by
succinate-cytochrome c reductase activity (20). In accord, a
blockade between cytochrome b-cytochrome c in the presence of NO and succinate was shown by absorption spectral studies
(Fig. 5).
In a previous work, we reported that heart mitochondria exposed to 0.4 µM NO produced ~0.07 nmol
H2O2 · min1 · mg
protein
1 (20). Thus, considering that 1 g of cardiac
tissue provides 54 mg of mitochondrial protein (7), it is apparent that
the H2O2 production by the whole heart in
conditions of 50% inhibition of O2 uptake should be ~3.8
nmol · min
1 · g
tissue
1. The concentration of
H2O2 detected in the effluent (0.04 µM) accounts for the recovery of 0.28 nmol · min
1 · g
tissue
1 and thus ~7.4% of the produced
H2O2. The relatively low amount of
H2O2 recovered in the effluent appears to
indicate the effective operation of tissue detoxification pathways.
Mitochondrial and cytosolic catalase and glutathione peroxidase are
certainly using myocardial H2O2. Moreover,
underestimation of H2O2 in the effluent perfusate may be caused by the remaining red blood cells and by the
release, during heart perfusion, of hydrogen donors for the peroxidase
reaction used in the detection of H2O2.
The NO concentrations in the perfusion fluid that were able to inhibit
O2 uptake were 10-100 times higher than the ones that produce cellular effects through the guanylyl cyclase activation. Distribution of NO in the perfused myocardium seemed homogeneous and
not limited by NO diffusion; the diffusion distance was calculated as
130 µm, considering a diffusion constant of 4 × 106
cm2 · s (13, 27). The heart
steady-state intracellular NO concentration reached during infusion
will depend on NO concentration in the infusion fluid, on the perfusate
flow, and on NO myocardial uptake, including NO binding to cellular and
mitochondrial proteins like myoglobin and cytochrome oxidase.
Particularly these two hemoproteins react with NO with great affinity,
with diffusion-limited rate constants and, in the case of myoglobin,
the binding is followed by immediate release of metmyoglobin (2).
Consequently, myoglobin markedly shortens NO half-life and decreases NO
concentration in the perfused heart (13). The tissue metmyoglobin
content was found inversely related to NO concentrations, as shown by absorption spectrophotometry (Fig. 6) and recently by electron spin
resonance (15).
According to Fick's law, the myocardial uptake of NO is expressed by the product of the difference between NO concentrations in the influent ([NO]inf) and in the effluent ([NO]eff) by the coronary perfusion flow
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(1) |
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It is thought that a better O2 myocardial distribution is developed when maximal mitochondrial state 3 O2 uptake is lowered by endothelial NO release in response to hypoxia or ischemia (20). In this way, NO increases O2 supply through coronary vasodilation and allows O2 to diffuse further along its gradient, reaching more cells and mitochondria and lowering the steepness of the PO2 gradient in the anoxic-normoxic transition (20). The same regulatory action has been proposed for skeletal muscle (22). These effects of NO could afford a generalized adaptive response; an increase in whole body O2 uptake has been reported in the conscious dog after the administration of NO synthase inhibitors (23).
The role of NO in the regulation of O2 uptake in the
perfused heart is depicted in Fig. 8. The
NO-induced production of H2O2 was not found to
be deleterious for the heart performance as proposed by other studies
(26). An increased NO concentration in the cell and mitochondria will
sequentially inhibit cytochrome oxidase and electron transfer between
cytochrome b and cytochrome c; the production at the second site of
inhibition sets a feedback mechanism in which an increased steady-state
concentration of
will remove cytochrome
oxidase inhibition by clearing NO to form ONOO
(NO +
ONOO; see Ref. 20). At an
intracellular concentration of ~0.1 µM NO, O2 could
only insignificantly remove NO in mitochondria, by oxidation, in
several hours (9). In contrast, according to Beckman (1), the very fast
diffusion-controlled NO/
reaction
assures a prompt regulatory NO clearance. It is apparent that NO and
are also able to interact in vivo;
tolerance to NO donors, like nitrites, has been proposed to be based on
an excessive
release by myocardium
(18), and a protective myocardial action of NO or of bradykinin has
been suggested in ischemia-reperfusion (19, 29).
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The interplay of NO and reactive O2 species seems to
constitute a suitable mechanism for physiological regulation of
O2 uptake in the heart and other tissues. The molecular
details in terms of involved reactive species and the whole concept of
oxidative stress have to be reexamined in terms of the interdependence
of and NO steady-state concentrations
and of ONOO
formation.
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ACKNOWLEDGEMENTS |
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This work was supported by a research grant from Fundación Perez Companc and by grant ME001 from the University of Buenos Aires.
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FOOTNOTES |
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Address for reprint requests: J. J. Poderoso, Laboratory of Oxygen Metabolism, Hospital de Clínicas "José de San Martín," Cordoba 2351, 1120 Buenos Aires, Argentina.
Received 2 May 1997; accepted in final form 10 September 1997.
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