High Glucose Impairs Voltage-Gated K+ Channel Current in Rat Small Coronary Arteries
Hyperglycemia is associated with impaired endothelium-dependent dilation that is due to quenching of NO by superoxide (O2· −). In small coronary arteries (CAs), dilation depends more on smooth muscle hyperpolarization, such as that mediated by voltage-gated K+ (Kv) channels. We determined whether high glucose enhances O2·− production and reduces microvascular Kv channel current and functional responses. CAs from Sprague-Dawley rats were incubated 24 hours in medium containing either normal glucose (NG, 5.5 mmol/L d-glucose), high glucose (HG, 23 mmol/L d-glucose), or l-glucose (LG, 5.5 mmol/L d-glucose and 17 mmol/L l-glucose). O2·− production was increased in HG arteries. Whole-cell patch clamping showed a reduction of 4-aminopyridine (4-AP)–sensitive current (Kv current) from smooth muscle cells of HG CAs versus NG CAs or versus LG CAs (peak density was 9.95±5.3 pA/pF for HG versus 27.8±6.8 pA/pF for NG and 28.5±5.2 pA/pF for LG; P<0.05). O2·− generation (xanthine+xanthine oxidase) decreased K+ current density, with no further reduction by 4-AP. Partial restoration was observed with superoxide dismutase and catalase. Constriction to 3 mmol/L 4-AP was reduced in vessels exposed to HG (13±5%, P<0.05) versus NG (30±7%) or LG (34±4%). Responses to KCl and nifedipine were not different among groups. Superoxide dismutase and catalase increased contraction to 4-AP in HG CAs. This is the first direct evidence that exposure of CAs to HG impairs Kv channel activity. We speculate that this O2·−-induced impairment may reduce vasodilator responsiveness in the coronary circulation of subjects with coronary disease or its risk factors.
Cardiovascular disease is the leading cause of death in the Western world, and diabetes mellitus (DM) is a major factor underlying its development. Many detrimental effects of diabetes are linked to elevations in serum glucose that is punctuated by elevations in superoxide (O2·−). Hyperglycemia-induced vascular oxidative stress is mediated by several mechanisms.1 Increased flux of glucose through the polyol pathway reduces antioxidant enzyme activity by depleting NADPH.2 Synthesis of endoperoxides in hyperglycemia leads to free radical formation.3,4 Acute increases in glucose alter Ca2+ homeostasis in vascular smooth muscle cells (VSMCs) through the production of excess O2·−.5 Finally, nonenzymatic glycation products of chronic hyperglycemia may provide oxidant stress, and glucose itself can auto-oxidize to form reactive oxygen species (ROS).6,7
Tesfamariam and Cohen8 have shown that incubating rabbit aortas in high glucose (44 mmol/L) impairs NO-mediated relaxation. Similar impairment in guinea pig aortas exposed to high glucose was mediated by excess O2·−.9 Thus, increased oxidative stress may be a common pathway of vasodilator dysfunction in diabetes.10,11
Although the role of ROS in NO-mediated endothelium-dependent dilation has been studied, little is known about the effect of hyperglycemia on hyperpolarization-mediated dilation. This form of dilation typically involves the opening of K+ channels in VSMC membranes and may play a critical compensatory vasodilator role in disease states in which NO-mediated dilation is impaired.12,13
VSMC membranes express several K+ channel gene families; however, voltage-gated K+ (Kv) channels and high-conductance Ca2+-activated K+ (BKCa) channels normally contribute the majority of K+ current.14 In preliminary studies, we demonstrated that Kv channels contribute to whole-cell K+ current in VSMCs from rat small coronary arteries (CAs).15 The purpose of the present study was to determine the effect of glucose-induced ROS generation on the function of Kv channels in rat small CAs by using a combination of histofluorescence, patch-clamp, and functional vasomotor methods. We examined the hypothesis that elevated glucose enhances the local production of O2·− and reduces Kv channel activity in rat coronary VSMCs.
Materials and Methods
Preparation of CAs
Seven-week-old male Sprague-Dawley rats (Harlan, Madison, Wis) were anesthetized with sodium pentobarbital (60 mg/kg IP). CAs (inner diameter 150 to 200 μm) were dissected from the ventricle and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum, 100 U/mL penicillin G, and 100 μg/mL streptomycin for 24 hours at 37°C. DMEM was supplemented with either 5.5 mmol/L d-glucose (normal glucose, NG), 23 mmol/L d-glucose (high glucose, HG), or 5.5 mmol/L d-glucose plus 17.5 mmol/L l-glucose (LG). l-Glucose, which is not metabolized, was used as an osmotic control. All media were filtered (0.2-μm filter) before use. After incubation, vessels were prepared for histofluorescence, patch-clamp, or videomicroscopic studies. Some CAs were used freshly after dissection.
Fluorescent Detection of Superoxide
The cell-permeable dye hydroethidine (HE, Molecular Probes) was used to evaluate the production of O2·−. When exposed to O2·−, HE is oxidized to ethidium bromide and trapped within the nucleus.16 Ethidium bromide fluoresces when excited by light at a 488-nm wavelength with an emission spectrum of 620 nm. CAs incubated in one of three solutions of glucose described above were exposed to HE (5 μmol/L) for 10 minutes, washed, and examined under confocal microscopy equipped with a krypton/argon laser. Fluorescence was detected with a 585-nm long-pass filter. A freshly isolated artery was used as a control to adjust for laser settings for use in all subsequent vessels. The fluorescence intensity/vascular area ratio of the central portion of the vessel was normalized to that obtained from the control vessel by using NIH image software. The ratio of the fluorescence (experimental vessel/control vessel) was compared among CAs incubated in each glucose solution.
Detection of Superoxide in Culture Media
Detection of O2·− production in vessel culture media was determined by monitoring increases in ferricytochrome c absorbance at 550 nm.17 Ferricytochrome c (final concentration 50 μmol/L) was added to the cuvette filled with either NG or HG culture media, and changes in absorbance were followed each minute for 20 minutes. Rates of O2·− anion production were calculated by the molar extinction coefficient of reduced ferricytochrome c [ε=21 000 cm−1×(mol/L)−1] and the portion that was inhibited by superoxide dismutase (SOD, 400 U/mL) and were expressed as micromoles of O2·− anion per liter of buffer per minute.
Patch-Clamp Recording of K+ Currents
Enzymatic isolation of single VSMCs was performed according to published methods.18 Patch-clamp recordings were obtained by using standard pulse protocols and instrumentation previously described for whole-cell and voltage-gated K+ current measurements.18 Briefly, families of K+ currents were generated by stepwise 10-mV depolarizing pulses (400-ms duration, 5-second intervals) from a holding potential of −60 mV in cells dialyzed with 10 nmol/L ionized Ca2+. Seal resistance was 2 to 10 GΩ. Peak current elicited at a single membrane potential was defined as the average of 500 sample points encompassing the maximal current point. In a single cell, Kv currents were defined as the difference between outward current recorded in drug-free bath solution and after superfusion with 3 mmol/L 4-aminopyridine (4-AP), a Kv channel blocker.19 Trials were performed in triplicate and averaged to estimate peak current amplitudes (picoamperes per picofarad) to normalize for cellular membrane area.20 For each cell, 10-mV hyperpolarizing steps were averaged to provide capacitance and leak compensation values. Whole-cell tail currents were elicited in symmetrical 145 mmol/L K+ by depolarizing cells from a constant holding potential of −60 in 10-mV increments to 60 mV, followed by an immediate repolarizing step back to −60 mV to generate families of tail currents.19
Segments of small CAs were placed into an organ chamber filled with physiological salt solution (PSS),13 cannulated with glass micropipettes, and secured with 10-0 nylon suture. Each pipette was attached to a pressure reservoir. The PSS in the organ chambers was warmed to 37°C, bubbled with 21% O2, 5% CO2, and 74% N2, and continually circulated with a rotary pump. Vessels were allowed to equilibrate for 60 minutes at an intraluminal pressure of 60 mm Hg. Internal diameters were measured with a calibrated manual videomicrometer with a resolution of 2 μm attached to an inverted microscope. Arteries that developed 15% to 25% spontaneous tone (percent reduction of initial internal diameter) in PSS were used. At the end of each experiment, vessels were maximally dilated with sodium nitroprusside (10−4 mol/L). In some vessels, endothelium was denuded with air.21 All chemicals were obtained from Sigma Chemical Co.
All data are expressed as mean±SEM. Percent constriction was defined as the percent reduction from control internal diameter in response to constrictor agents. Percent dilation was calculated as the percent change from control internal diameter (in the presence of established myogenic tone) to maximal diameter measured after sodium nitroprusside. Data from vessels incubated in NG, HG, or LG and studied by either patch clamp or videomicroscopy were compared by using a 1-way ANOVA with repeated measures for dose and condition (glucose exposure). Differences between individual means were determined by the Newman-Keuls test. The relative fluorescence (ethidium bromide technique) of arteries exposed to different levels of glucose was compared by using a 2-way ANOVA. All differences were judged to be significant at P<0.05.
Superoxide Production in CAs Incubated in HG
Figure 1A compares O2·− detected by HE between a freshly isolated (control) artery and arteries incubated in either NG, LG, or HG for 24 hours. Similar fluorescence intensities were observed in control, NG, and LG arteries. CAs incubated in HG showed greater fluorescence. Average fluorescence ratios, normalized to the control artery, are summarized in Figure 1B. Only incubation in HG stimulated the production of O2·− in rat small CAs. O2·− production in the incubation media alone was examined by using the ferricytochrome c method before and after a 24-hour incubation to mimic the conditions of the experiment. O2·− production before (0.88±0.02 and 0.5±0.25 μmol/L for 20 minutes in NG and HG media, respectively) and after (0±0.18 and 0±0.16 μmol/L for 20 minutes in NG and HG media, respectively) incubation was similar, indicating no O2·− generation in the incubation media. As a positive control, O2·− generation was 16±1.5 μmol/L for 20 minutes with the use of xanthine (XA)+xanthine oxidase (XO). Thus, the major source of radical production is likely from the interaction of glucose with coronary vessels.
Antioxidant Treatment Reduces Superoxide Production in Arteries Exposed to HG
The effect of antioxidants on O2·− generation in CAs exposed to HG was also examined by the histofluorescence method. Two CAs were incubated in parallel with HG for 24 hours. After incubation, one artery was treated with SOD and catalase (CAT) for 15 minutes, and the amount of O2·− generated was compared between arteries with and without antioxidant exposure. SOD and CAT greatly reduced the fluorescence intensity of CAs exposed to HG (Figure 2A) with average ratios of intensity of 5.2±1.6 for arteries exposed to HG and 0.5±0.1 for arteries exposed to HG with SOD+CAT (P<0.05, n=6) (Figure 2B). Fluorescence intensities of control and SOD+CAT–treated vessels were not different (P>0.05).
Comparison of Kv Current Densities
Figure 3A shows families of voltage-gated K+ currents generated by incremental 10-mV depolarizing steps from −60 to 60 mV in VSMCs isolated from rat CAs and incubated for 24 hours in DMEM containing NG, LG, or HG. Figure 3A shows that peak K+ current amplitude was similar between arteries exposed to NG or LG. The currents were reduced by 3 mmol/L 4-AP, identifying Kv channels as the primary conducting pathways. Whole-cell Kv current was suppressed in VSMCs from arteries exposed to HG, and 3 mmol/L 4-AP had less effect on total K+ current. Figure 3B compares K+ current densities between VSMCs from arteries exposed to NG, LG, and HG (n=10 each). Densities were significantly reduced in cells from arteries incubated in HG (28±6 pA/pF) compared with NG (41±7 pA/pF) and LG (42±5 pA/pF). In Figure 3C, it is seen that Kv current was reduced by 63% and 64% in VSMCs from arteries exposed to HG compared with those exposed to NG or LG, respectively. Glibenclamide (1 μmol/L), an ATP-sensitive K+ channel blocker, had little effect on total K+ currents (data not shown). Thus, 24 hours of exposure to HG suppresses Kv current in coronary VSMCs, and this impairment is not secondary to an osmotic effect.
Voltage dependence of K+ currents was determined by measuring the peak amplitude of tail currents generated at −60 mV as a function of activating prepulses (10-mV steps) to voltages as positive as 60 mV. Values obtained from four cells were fit by a Boltzmann function, Iact=Imax/[1+exp(Vm−V0.5)/k], where Imax is the maximal tail current amplitude at 60 prepulse, Iact is the tail current observed at a given prepulse voltage (Vm), V0.5 is the voltage for half of Imax, and k is the steepness factor (slope) indicative of voltage sensitivity. Figure 3D shows that HG reduced Imax but had little effect on the voltage dependence of K+ channels. V0.5 and k values were −3.3 mV and 18, respectively, in VSMCs exposed to NG and −8 mV and 16, respectively, in VSMCs exposed to HG.
Effect of Exogenous Generation of Superoxide on Kv Current
The direct effect of O2·− on Kv currents was examined with exogenous O2·− generated by the reaction of 0.1 mmol/L XA and 10 mU/mL XO. Figure 4A shows that the addition of XA had no effect on whole-cell current, whereas XA+XO suppressed K+ currents. The further addition of 4-AP had little effect, which would indicate that Kv current was already blocked by XA+XO. Notably, the more rapid activation of K+ current in Figure 4A compared with that in the earlier traces shown in Figure 3A was observed in some cells and may reflect the variable activation kinetics of different Kv channel subtypes.22 Current-voltage relationships in Figure 4B show that K+ current densities were 32±7 and 21±4 pA/pF in cells treated with XA versus XA+XO, respectively. The subsequent application of 3 mmol/L 4-AP in the presence of XA+XO produced no further reduction, consistent with the idea that O2·− decreases Kv current in coronary VSMCs. Similar to the results observed in cells from HG arteries, the results shown in Figure 4C demonstrate that O2·− decreased Imax but did not change the voltage dependence of K+ channel activation. V0.5 and k values were −10 mV and 17, respectively, in cells treated with XA and −9 mV and 18, respectively, in cells treated with XA+XO. XA+XO had no effect on the electrode offset evaluated by comparing electrode voltage (in cell-free conditions) between baths filled with drug-free solution, XA, or XA+XO.
Effect of Antioxidants on Kv Current
To determine whether the reduced Kv current after exposure to HG is associated with the generation of O2·−, K+ current density was compared among cells from arteries exposed to NG, LG, and HG before and after the application of SOD (150 U/mL) and CAT (500 U/mL). SOD and CAT partially restored peak K+ current density of cells from arteries incubated in HG (Figure 5C) but had little effect on K+ currents in cells from arteries exposed to NG or LG (Figures 5A and 5B, respectively). Thus, O2·− may contribute to the impaired Kv channel activity in small CAs exposed to HG.
Contractile Response to 4-AP
Resting diameters of small CAs incubated in NG, LG, and HG were not different (147±14, 159±19, and 140±16 μm, respectively; n=8 for each group). Figure 6A shows diameter changes in response to four increasing concentrations of 4-AP. A similar concentration-dependent contraction to 4-AP was seen in CAs incubated in NG or LG (maximum constriction 30±7% and 34±4%, respectively). In contrast, the maximal constriction to 4-AP in arteries exposed to HG was lower (13±5%). Passive diameters induced by sodium nitroprusside were 175±14, 182±16, and 164±13 μm (P>0.05) in CAs exposed to NG, LG, and HG, respectively. Maximal constriction to 4-AP was similar in vessels with and without endothelium (for NG, 28±7% before and 27±13% after denudation; for HG, 12±4% before and 9±3% after denudation; n=3 for denudation groups). Thus, elevations in glucose impair Kv channel function in coronary VSMCs.
Figure 6B shows similar KCl-induced constriction (20 to 40 mmol/L) between NG and HG arteries. Figure 6C indicates that arteries incubated in NG or HG showed similar dilator responses to the L-type Ca2+ channel antagonist nifedipine (10, 100, and 1000 nmol/L). These results indicate that 24-hour incubation in HG did not affect coronary vascular reactivity nonspecifically but appeared to selectively impair vasoactive responses dependent on functional Kv channels.
Effect of Antioxidants on Contractile Responses to 4-AP
Similar to electrophysiological observations, SOD and CAT restored constrictions of HG arteries to increasing concentrations of 4-AP but had little effect on arteries incubated in NG or LG (Figures 7A through 7C), further indicating that ROS contribute to the impaired function of Kv channels exposed to HG.
The present study demonstrates for the first time that short-term exposure of small CAs to HG suppresses Kv current in VSMCs. This impairment is manifested by a direct reduction of Kv current in coronary VSMCs and by reduced vasomotor responses to Kv channel activity. The novel findings of the present study are that exposure of small CAs to HG for 24 hours (1) increases O2·− production, (2) decreases 4-AP–sensitive K+ current in dissociated VSMCs, and (3) reduces 4-AP–induced contraction. Furthermore, the reduced Kv current and contractile response to 4-AP are linked to O2·− production, because treatment with radical scavengers partially restores these processes in arterial preparations exposed to HG. These findings may relate to coronary vasomotor regulation in diabetes, because NO-mediated dilation is impaired in DM and because compensatory dilator responses, including hyperpolarization factors, may rely on Kv channels.
Role of Kv Channels in the Circulation
Kv channels are highly expressed in VSMCs23,24 and contribute to membrane potential, particularly at lower levels of intracellular Ca2+, at which the BKCa channel is less active.14 In the present study, inhibition of Kv channels elicited a substantial vasoconstriction. Because Kv channels are activated by depolarization, they may serve to limit membrane depolarization during vasoconstriction. Kv channels also mediate pharmacological vasodilation. Forskolin, an activator of adenylate cyclase, increases 4-AP–sensitive K+ currents in rabbit CAs.25 The β-adrenoceptor agonist isoproterenol enhances 4-AP–sensitive currents in rabbit portal veins.26 In the coronary circulation, activation of histamine H1-receptors inhibits 4-AP–sensitive currents, consistent with a role for Kv channels in coronary vasospasm.27 The vasomotor effects of angiotensin II28 and pH29 are also in part mediated by Kv currents.
The predominant K+ current in cells from arteries either freshly isolated (no incubation) or incubated with NG or LG was sensitive to 4-AP. 4-AP (3 mmol/L) caused a 37% and 38% reduction of resting diameter of NG and LG arteries, respectively. Thus, Kv channels contribute to the resting membrane potential of rat small CAs and may be involved in coronary blood flow regulation.
Because the patch-clamp results show reduced Kv current in cells from HG arteries and because Kv is involved in resting tone, a reduction in resting diameter would be expected after incubation in HG media. However, resting diameters of arteries exposed to NG, LG, or HG were similar. This could be due to a compensatory increase in the contribution of other K+ channels, such as BKCa. Such compensation has been demonstrated for loss of NO during hypercholesterolemia.12
HG Increases Oxidative Stress
Elevated glucose induces ROS generation by several mechanisms. Glucose auto-oxidation forms O2·−, hydroxyl radicals, and hydrogen peroxide.7 Increased flux through glycolytic and polyol pathways results in an excess production of sorbitol. This is coupled with the NADPH-mediated generation of ROS. Acute elevations in glucose also depress natural antioxidant defenses. Incubation of purified bovine CuZn SOD with HG (10 to 100 mmol/L) reduces enzyme activity by 60%.30 In the present study, O2·− (fluorescence microscopy) was increased in small CAs incubated with high glucose. The increased fluorescence was reduced by SOD, confirming the involvement of O2·−.
The source of O2·− generation in the CA model is not known. In conduit arteries, the endothelium likely contributes,8 whereas other studies have implicated the adventitial layer.31 In hypercholesterolemia, elevated O2·− is seen throughout the vascular wall.32 Thus, multiple cell types may contribute to the generation of O2·− during exposure to HG. In the present study, auto-oxidation of glucose was not the source of O2·−, because virtually none was detected in the media alone. Future studies should examine the cellular source (endothelium, VSMCs, or adventitia) and the chemical reactions responsible, eg, XO and NAD(P)H oxidase, for O2·− generation in the coronary microcirculation in response to HG.
Effect of Superoxide Associated With HG on Kv Channel Activity
Little is known about the effect of O2·− on vascular mechanisms of hyperpolarization. Gating of large-conductance BKCa33 and Kv34 channels may be modified by the redox state of the cell. In the present study, the 4-AP–sensitive current was reduced in cells from arteries incubated with HG. In parallel, a reduced 4-AP contractile response was observed in the CA. Treatment with antioxidants partially restored Kv current and contraction to 4-AP, suggesting that O2·− generated by HG reduces Kv current and function. The importance of this finding is that Kv channels contribute largely to the total K+ current in rat CAs and may be responsible for physiological and pharmacological dilation.
The mechanism by which O2·− inhibits Kv channels is unknown. It may involve an increase in intracellular Ca2+ concentration by O2·−.35 Several reports indicate that high cytosolic Ca2+ levels inhibit Kv channel activity.36 Oxidation of specific amino acid residues within the Kv channel could also alter its gating.37
Kv channels have not been a major therapeutic focus because they represent a superfamily of K+ channels for which specific gene family blockers are unavailable for distinct gene family subtypes. Because 4-AP effectively blocks most Kv channels, the molecular composition of the specific subtype(s) of Kv channels in CAs affected by HG remains unclear. Future identification of Kv channel structure may provide new insight into potential sites of interaction between Kv channel subunits and ROS.
Although hyperosmolarity can alter vasoactive responses in CAs,38 the reduced Kv current caused by elevated glucose was not due to hyperosmotic effects in the present study, because substituting an equimolar concentration of the nonmetabolizable isomer, l-glucose, had no effect on either Kv currents or 4-AP constriction of isolated vessels.
It is possible that ROS other than O2·− participate in the vascular dysfunction associated with hyperglycemia. However, most data implicate O2·− as the primary culprit in DM and HG.39 The present study did not distinguish between endogenous production of O2·− or hydrogen peroxide because SOD and CAT were always given together. However, HE fluorescence and the effect of exogenous generation of O2·− suggest that this radical plays an important role in the oxidant stress associated with HG.
Because SOD does not readily penetrate cells, the restorative effect of SOD observed in the present study may underestimate the true role of O2·− in vasomotor abnormalities. This problem may be minimized in the present study by the small size of the vessels and, consequently, short diffusion distances. Furthermore, the effectiveness of combined SOD and CAT during vasomotor and fluorescence techniques suggests some degree of intracellular penetrance or removal of excess O2·− from within or near the cell membrane where the Kv channels are located. In support of this idea, others have used SOD effectively to improve vasomotor responses in conditions associated with the elevated production of O2·−.8,9 In the present study, the improvement with SOD was less than complete. The use of polyethylene glycol–SOD or a cell-permeable mimetic, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), might further improve Kv channel function after exposure to HG.
The reduction in K+ current density at membrane potentials between 0 and 30 mV is greater in cells from arteries exposed to HG than in cells treated with XA+XO. Possible explanations include the following: (1) O2·− generated by HG likely occurs inside the cell, whereas O2·− generated by XA+XO was primarily extracellular with subsequent entry into the cell. If inhibition of Kv channels depends on intracellular O2·−, this could explain the difference. Unfortunately, because O2·− detection by HE is not quantitative, we cannot determine the amount of O2·− generated in our experiments. (2) HG may act through multiple mechanisms to reduce Kv current, including the generation of other free radicals, such as peroxynitrite, to inhibit K+ currents.40
Finally, the reduction in 4-AP–sensitive K+ currents was observed at membrane potentials positive to 0 mV, whereas vessel constriction to 4-AP presumably occurred at more negative potentials, at which little Kv current was observed in patch-clamped VSMCs. This is likely due to the fact that although Kv channels show low open-state probabilities at negative voltage, small changes in their activation levels can significantly alter membrane potential (and, therefore, vasomotor tone) because of the high membrane resistance of VSMCs.
Our findings based on electrophysiological and functional data illustrate that the Kv channel importantly regulates the resting membrane potential of small CAs. In DM, in which NO-mediated dilation is reduced, the attendant hyperglycemia may also impair Kv channel–mediated dilation and thereby reduce myocardial perfusion. We speculate that β-adrenergic dilation, an important response to sympathetic activation, may also be impaired in DM, inasmuch as cAMP-mediated dilation involves the activation of Kv channels.25 Thus, a better understanding of the mechanisms by which hyperglycemia reduces K+ channel–mediated coronary vasodilation may enable the design of new therapies to improve myocardial perfusion in DM.
This study was supported by the National Institutes of Health (grants R01 HL-59238 to Dr Rusch and RO1 HL-52869 to Dr Gutterman), by a Veterans Administration Merit Award to Dr Gutterman, and by the Gutterman Foundation. Dr Liu is the recipient of a Scientist Development Grant from the American Heart Association.
Original received January 22, 2001; revision received May 18, 2001; accepted May 18, 2001.
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