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Electrophysiological effects of O₂^–· on the plasma membrane in vascular endothelial cells

Departments of Physiology and Anesthesiology and The Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin; and Department of Physiology, Louisiana State University, Health Sciences Center, New Orleans, Louisiana

Submitted 10 February 2005 ; accepted in final form 9 June 2005

	ABSTRACT

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Vascular dysfunction is a hallmark of many diseases, including coronary heart disease, stroke, and diabetes. The underlying mechanisms of these disorders are intimately associated with an increase in oxidative stress and excess generation of reactive oxygen species. Here, we report that the anionic free radical, superoxide (O₂^–·), directly affects the function of ion channels in vascular endothelial cells. Vascular endothelial cells were exposed to O₂^–· under physiological, symmetrical chloride and chloride-free conditions. Superoxide was generated from the reaction of xanthine (0.2 mM) and xanthine oxidase (0.1, 1, and 10 mU/ml) while its effects were determined with the whole cell mode of the patch-clamp technique. Inhibitors of K⁺ and Cl^– channels were used to determine the role of these ion channels in mediating the electrophysiological effects of superoxide. The addition of O₂^–· caused a dose-dependent depolarization of endothelial cells and activation of the whole cell current. Activation of superoxide-dependent current was observed in the presence of inhibitors of K⁺ channels, Ba²⁺ (100 µM) or iberiotoxin (100 nM), and was not affected by inhibitors of nonselective cation channels, La³⁺, or by inhibition of the Cl^–/HCO₃^– transporter by bumetanide. The inhibitors of the Cl^– channel, NPPB (0.1 mM) or DIDS (100 µM), partially prevented activation of superoxide-dependent current but were unable to reverse it. The effects of superoxide on the amplitude of whole cell current were prevented and reversed by superoxide dismutase. Taken together, these results suggest that superoxide directly affects the function of ion channels in vascular endothelium but the mechanisms of its modulatory effects remain unresolved.

vascular endothelium; chloride channels; 4,4'-diisothiocyanotostibene-2,2'-disulfic acid; superoxide dismutase

In vascular endothelium, ion channels play an important regulatory function in physical and metabolic processes. Production of nitric oxide (NO) depends on several cofactors and, importantly, on Ca²⁺ ions (13). Under physiological conditions, Ca²⁺ influx is regulated by the resting membrane potential of vascular endothelium, which depends on the activities of the inward rectifying K⁺ and Cl^– channels (28). Increased production of reactive oxygen species has been shown to affect endothelial cell polarization, which is directly related to the activity of ion channels (8). Moreover, organic peroxides and hydrogen peroxide induce depolarization that is consistent with a reduction of Ca²⁺ influx (5). Oxidants have been shown to exert effects such as activation of nonselective cationic channels, which may be a consequence of direct changes in the redox state of the cell (7, 8, 21, 22). The redox sensitivity of ion channels has been shown to be the result of an increase in the intracellular concentration of oxidized glutathione (GSSG). The modification of functional thiol groups within the channel protein might be responsible for an activation of the nonselective ion channels and other oxidative effects exerted on ion channel function (7). Therefore, the oxidative processes that occur inside and outside of the cells may affect vascular function by altering activity of the ion channels.

The role of formed byproducts of oxidative metabolism, e.g., superoxide, is unclear but may include vasoregulation (19, 20) in addition to its final, detrimental, effects on cell function. Our first goal was to determine whether superoxide generated from the enzymatic reaction between xanthine (X) and xanthine oxidase (XO) directly affects the level of depolarization of vascular endothelial cells. Second, we determined whether superoxide has modulatory effects on the activity of ion channels in human coronary artery endothelial cells. With the use of the patch-clamp technique, endothelial cells were voltage-clamped under a physiological gradient and under symmetrical chloride and chloride-free conditions. Superoxide, generated extracellularly, caused depolarization of endothelial cells and activated a superoxide-dependent current. The changes in the reversal potential were observed under physiological and under symmetrical Cl^– conditions. Superoxide-dependent current was activated independently of inhibitors of K⁺ channels or nonselective cation channels (NSCCs), and its activation was partially inhibited by chloride channel inhibitors. Our results support the idea that superoxide has direct actions on endothelial cell ion channels and chloride channels in particular.

	METHODS

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Materials. Chloride channel inhibitors, 4,4'-diisothiocyanoto-stibene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB); potassium channel inhibitor, BaCl₂; and the inhibitor of nonselective ion channels, La³⁺; and all salts were obtained from Sigma (St. Louis, MO). Iberiotoxin was from Calbiochem.

Endothelial cell culture. Human coronary artery endothelial cells (HCAECs) were purchased from Cambrex (San Diego, CA). Cells were subcultured in EGM-2MV media (Cambrex) with 5% FBS and maintained in the cell culture incubator in a humidified 5% CO₂ atmosphere. Cells were passaged every 3 days. When grown to 90% confluence, the cell monolayer exhibited typical cobblestone characteristics. Whole cell current was measured from single cells isolated by scraping with a cell scraper.

Treatment with superoxide. Extracellular generation of O₂^–· was accomplished by the enzymatic reaction of X (0.2 mM) and XO (0.1, 1, and 10 mU/ml). Catalase (500 U/ml) was added to prevent effects of H₂O₂, which is formed during reaction between X and XO. HCAECs were dispersed and loaded to a chamber of the patch-clamp baths. Cells were allowed to attach and the PSS was replaced with a solution containing 0.2 mM X (pH 7.4, see Solutions). XO was added to the bath as a bolus after the control recordings were taken. SOD was added as a bolus at 100 and 500 U/ml.

Solutions. For the physiological gradient bath, PSS contained (in mM) 145 NaCl, 5.4 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 HEPES, and 10 glucose (pH 7.3, adjusted with NaOH). In some experiments, calcium was omitted to form the Ca²⁺-free solution. The standard pipette solution contained (in mM) 145.0 K⁺ aspartate, 5.0 NaCl, 0.3 CaCl₂, 2.2 EGTA, 10 HEPES, and 7.5 glucose (pH 7.2, adjusted with KOH). The calculated E_rev for the K⁺ ions was –87.36 mV. Chloride-free solution (Na⁺ gluconate) contained (in mM) 145 Na⁺ gluconate, 1 K⁺ aspartate, 0.3 CaCl₂, 2.2 EGTA, 7.5 HEPES, and 10.0 glucose, pH 7.2. Chloride solution Na⁺ free contained (in mM) 100 N-methyl-D-glucamine-Cl (NMDG-Cl), 0.3 Ca²⁺ gluconate, 2.2 EGTA, 10 HEPES, and 7.5 glucose, pH 7.2. Chloride solution Na⁺ and Ca²⁺ free contained (in mM) 125 choline chloride, 4 TEA-Cl, 1 MgCl₂, and 5 HEPES, pH 7.2. Osmolarity of these solutions was checked with a freezing-point osmometer (Precision Systems) and adjusted with mannitol, as necessary, to maintain osmolarity at 300 ± 20 mosM. All experiments were performed at 37°C.

Electrophysiology. Measurements of ionic currents were performed using the patch-clamp technique in the whole cell configuration (15). Whole cell currents were recorded in response to successive voltage pulses of 500-ms duration, between –100 to +100 mV, in 10-mV increments, from a holding potential of –70 mV. Whole cell measurements were obtained using high-K⁺ solution in the pipette and PSS solution in the bath. Recording pipettes were pulled from borosilicate glass (no. 7052; Garner Glass Claremont) using a vertical puller (Narashige, Tokyo, Japan) and had resistance of 1–5 M when filled with high-K⁺ solution. Voltage protocols and data acquisition were carried out using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and a Digidata 1200A analog-to-digital converter (Axon Instruments). Currents were filtered by a 4-pole Bessel filter at 1 kHz and digitized at 5–10 kHz. The grounding electrode was an Ag-AgCl plug electrically connected to the bath via a 140 mM KCl agar bridge. During data acquisition, capacitative transients were not compensated. The temperature of the patch-clamp experiments was maintained at 37°C (Cell MicroControls, Virginia Beach, VA).

Data analysis. Analysis of whole cell data was carried out using pClamp 8.0 software (Axon Instruments). Unitary reversal potentials and conductance values were estimated by fitting the linear portion of each current-voltage (I-V) relationship. Data represent direct comparison between control and treated cells (superoxide, K inhibitors, Cl^– channel inhibitors) through the whole range of the potential. Differences within and between groups were determined using ANOVA and followed by analysis with the Tukey test. A value of P < 0.05 was considered statistically significant.

	RESULTS

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Superoxide-dependent whole cell current. Whole cell current was recorded in response to voltage steps from –100 to +100 mV, in 5-mV increments, using PSS in the bath and high-K⁺ solution in the pipette. Under these conditions, control cells displayed a zero-current potential (E_rev) of –30 mV (n = 22), a value consistent with that previously reported for human coronary artery endothelial cells (33), but almost +50 mV greater than the calculated equilibrium potential for K⁺ (E_K = –80 mV). Barium reduced the control whole cell current, but it did not completely inhibit it.

The electrophysiological effects of superoxide on vascular endothelial cells were determined using different concentrations of XO while maintaining a constant concentration of X (0.2 mM). In three sets of experiments, the level of X was maintained at 0.2 mM, whereas XO was used at 0.1, 1, and 10 mU/ml, respectively. Catalase (500 U/ml) was routinely added in all experiments that involved the reaction between X and XO. Whole cell current was recorded from endothelial cells treated with 0.1 mU/ml XO. Currents recorded before and after the addition of XO were not statistically different (n = 10; Fig. 1A), with E_rev shifted from –30 to 0 mV, a change statistically not significant.

View larger version (22K): [in this window] [in a new window]   Fig. 1. In vascular endothelial cells, superoxide dose dependently activates a whole cell current. Control currents were recorded in response to successive voltage pulses from –100 to +100 mV, under physiological gradient conditions. The amount of xanthine (X) was maintained at 0.2 mM while different concentrations of xanthine oxidase (XO) were added to the bath. A: current-voltage relationships of endothelial cells treated with 0.1 mU/ml of XO (n = 10; statistically not significant). B: whole cell currents in endothelial cells treated with 1 mU/ml of XO (n = 15, P < 0.05 vs. control). C: whole cell currents in endothelial cells treated with 10 mU/ml of XO (n = 10, P < 0.05 vs. control). Recordings were taken within 60 s after addition of X and XO in the presence of catalase (500 U/ml). D, E, and F: current traces recorded from control cells (top) and after addition of X and XO 0.1, 1, and 10 mU/ml, respectively. Data points represent mean values at each potential; error bars represent ± SE and are shown only when larger than the symbol size.

The addition of XO at 10 mU/ml caused an increase in the whole cell current. At –60 mV, the amplitude increased from –0.05 ± 0.02 to –0.56 ± 0.01 nA (n = 10, P < 0.01 vs. control). At +60 mV, amplitude increased from 0.15 ± 0.05 to 0.42 ± 0.04 nA (n = 10, P < 0.01 vs. control), corresponding to the I-V relationship of the current, which was linear between –40 to +40 mV, with E_rev shifted to +10 mV (Fig. 1C).

To determine the extent of electrophysiological effects of superoxide anion in the increase of whole cell current amplitude and cell polarization, the superoxide scavenger SOD was added to the bath. Whole cell current was recorded under control conditions, after addition of superoxide (XO: 10 mU/ml; X: 0.2 mM) and followed by SOD (500 U/ml). In the presence of superoxide, whole cell current increased 10-fold (at –60 mV, from –0.04 ± 0.002 to –0.48 ± 0.002 nA, P < 0.01 vs. control, n = 9). Subsequent addition of SOD (500 U/ml) reduced the whole cell current amplitude to the control levels. No statistical difference between whole cell current recorded under control conditions and current recorded after treatment with SOD was found (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Superoxide dismutase (SOD) reversed effects of superoxide on a whole cell current. A: current traces recorded in response to successive voltage pulses from –100 to +100 mV, under physiological gradient conditions, control cells, after addition of X and XO, followed by SOD. B: current-voltage relationships are plotted for currents sequentially recorded immediately after break-in, 60 s after addition of superoxide generating system (XO: 10 mU/ml), and after addition of SOD (500 U/ml). Data points represent mean values at each potential; error bars represent ± SE and are shown only when larger than the symbol size.

View larger version (19K): [in this window] [in a new window]   Fig. 3. SOD prevents the effects of superoxide on the amplitude of the whole cell current. A: control currents were recorded in response to successive voltage pulses from –100 to +100 mV, under physiological gradient conditions. Current traces represent whole cell currents recorded under control conditions, after addition of SOD (100 U/ml), and after addition of XO (10 mU/ml). B: current-voltage relationships are plotted for currents shown on the left. Data points represent mean values at each potential; error bars represent ± SE and are shown only when larger than the symbol size.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Inhibitors of cationic channels have no effect on the activation of the superoxide-dependent current. Under physiological gradient conditions, control currents were recorded in response to successive voltage pulses from –100 to +100 mV. Cells were treated with K+ channel inhibitors, Ba2+ (100 µM), iberiotoxin (IBX; 100 nM), or La3+ (100 µM).

To determine the role of anions in the superoxide-activated current, whole cell current was recorded using Na⁺-gluconate solution in the bath and in the pipette (see Solutions). This allowed for almost complete removal of Cl^– anion from the bath and the pipette. Under such experimental conditions, the whole cell current recorded from control cells displayed an E_rev at 0 mV and showed linear characteristics through the whole range of the potential studied (–100 to +100 mV). The addition of superoxide (XO: 1 mU/ml) activated a whole cell current with the magnitude of the amplitude similar to that observed under a physiological gradient (Fig. 1B). At the potential of –60 mV, current amplitude increased from –0.14 ± 0.01 for the control to –0.70 ± 0.07 nA (n = 8, P < 0.05 vs. control) after treatment with superoxide, and at +60 mV, it increased from 0.17 ± 0.01 to 0.92 ± 0.09 nA (n = 8, P < 0.05 vs. control). On addition of superoxide, no additional depolarization was observed (n = 8; Fig. 5). The whole cell current activated by superoxide was inhibited by SOD (100 U/ml, current amplitude returned to –0.17 ± 0.02) as well as by the addition of the inhibitor of chloride channels, a stilben derivative, DIDS (100 µM, current amplitude –0.47 ± 0.01 vs. control; Fig. 5).

View larger version (23K): [in this window] [in a new window]   Fig. 5. Superoxide activates a whole cell current under chloride-free conditions. A: current traces recorded immediately after break-in with PSS in the bath and after the change of the bath solution to sodium-gluconate solution (pipette contained sodium-gluconate solution, see Solutions). Under such experimental conditions, addition of superoxide increased the amplitude of the whole cell current. B: summarized data at the potentials of –60 and +60 mV, whole cell current was recorded, under symmetrical sodium-gluconate conditions, control conditions, in response to 1 mU/ml XO (0.2 mM X, catalase 500 U/ml). The addition of DIDS (100 µM, n = 5) or SOD (100 U/ml, n = 5) reduced superoxide-dependent whole cell current. C: increase in whole cell current in response to 1 mU/ml XO under physiological gradient.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Under symmetrical chloride gradient conditions, superoxide increased whole cell current amplitude. A: under symmetrical chloride gradient conditions [choline-chloride (Chol-Cl) and N-methyl-D-glucamine-Cl (NMDG-Cl), full composition see Solutions], currents were recorded in response to successive voltage pulses from –100 to +100 mV. Under such experimental conditions, addition of superoxide increased the amplitude of the whole cell current. The addition of the nonselective inhibitor of K+ channels (4 mM TEA) to the intracellular solution had no additional effect on activation of the superoxide-dependent current. B: butenamide (But), an inhibitor of Cl–/HCO3– transporter, had no effect on the superoxide-activated current.

Vascular endothelial cells have cationic (potassium, NSCCs) and anionic (chloride) channels. By virtue of its negative charge, superoxide is an anionic and reactive oxygen species. To delineate a role for chloride channels in the superoxide-dependent conductance, the whole cell current was recorded in the absence and presence of the chloride channel inhibitor, NPPB. Currents recorded from cells treated with NPPB (100 µM) had amplitudes similar to the control currents with an E_rev of –10 mV. Characteristically, addition of superoxide (XO: 1 mU/ml), to the NPPB-treated endothelial cells, had no effect on the magnitude of the whole cell current and did not affect the level of cell depolarization (n = 6, nonsignificant; Fig. 7). Second, inhibition of chloride channels with DIDS (100 µM) generated similar results. However, DIDS was less effective in preventing the activation of superoxide-dependent current (Fig. 7). Importantly, in vascular endothelial cells neither NPPB nor DIDS was able to reverse superoxide-activated whole cell current (not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Chloride channel inhibitors prevent activation of superoxide-dependent whole cell current. Control currents were recorded in response to successive voltage pulses from –100 to +100 mV, under physiological gradient conditions. Whole cell currents were recorded under control conditions, followed by the chloride channel inhibitor NPPB (n = 5, 100 µM) or DIDS (n = 7, 100 µM). No increase in the whole cell current amplitude in response to superoxide was observed after treatment with NPPB, and only a moderate but statistically significant increase was detected after treatment with DIDS (n = 5, P < 0.05 vs. control).

	DISCUSSION

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Superoxide is a free radical that is continuously generated in biological systems and results from a one electron reduction of molecular oxygen. A significant part of its biological significance can be traced to the formation of other more reactive species (3, 10, 11). However, superoxide may initiate a cascade of oxidative stress by a direct signaling mechanism. Therefore, as a molecule, superoxide may indirectly or directly affect the function of cells in the vascular wall.

Many functions of endothelial cells are regulated by levels of intracellular calcium, which are controlled by the electrochemical driving force for Ca²⁺ ions mediated through changes in membrane potential. The influx of Ca²⁺ may not only depend on the resting membrane potential but on the regulation of the ion channel function by the cell metabolism. In this respect, mechanical stress or osmotic swelling may lead to the release of ATP from endothelial cells; increased [ATP]_o has been shown to regulate calcium-dependent chloride channels, and [Ca²⁺]_i (17, 32). Intracellular ATP levels regulate the activity of chloride channels and may exert the regulatory function on calcium influx (27). Therefore, the calcium-dependent production of NO is likely regulated by both ATP and the electrochemical driving force for calcium.

Three types of channels that regulate the endothelial cell E_m are potassium, chloride, and NSCCs. In vascular endothelial cells, the resting membrane potential depends primarily on the activity of K_ir channels. The voltage dependence of the K_ir channels renders them inactive at potentials positive to –60 mV. The addition of superoxide caused a dramatic depolarization and increase in the magnitude of whole cell current. The treatment of endothelial cells with either Ba²⁺ ions or iberiotoxin did not prevent activation of the superoxide-dependent conductance. This implies that the alteration of the whole cell current caused by superoxide was independent of K_ir and K_Ca channels. Depolarization of the plasma membrane in endothelial cells was observed on the addition of oxidizing agents such as: hydrogen peroxide, tert-butyl-hydroperoxide, or peroxynitrite. These events were concomitant with activation of a NSCC, which is characterized by a linear I-V relationship and poor discrimination between Na:K:Ca ions (1:1:0.5) (4, 6, 22). The presence of the NSCC in the plasma membrane of endothelial cells, and their redox regulation during oxidative stress, implies the possibility of their activation in the presence of superoxide. However, La³⁺, an inhibitor of NSCC, did not affect the electrophysiological changes initiated by superoxide.

The role of chloride channels in vascular endothelial cells is not understood. Although their dysfunction is involved in several disease processes, their importance for cellular ionic and metabolic homeostasis is unknown. Because superoxide is a free radical that is also an anion, it is possible that its effects on the cell membrane and intracellular signaling may be exerted via chloride channels. A stilben derivative, DIDS, and another inhibitor of chloride channels, NPPB, prevented the activation of superoxide-dependent current, suggesting that chloride channels may play a role in superoxide conductance and mediate its effects on the plasma membrane. However, the same inhibitors were unable to reverse an already active superoxide-dependent current. This partial effect may suggest that the effects of superoxide on the plasma membrane are more complex than only the effects on the ion channels and/or that several processes may be contributing to the activation of the whole cell current. It is also possible that a longer exposure of the plasma membrane (>5 min) will lead to oxidative alterations in the ion channel protein rendering the binding site unavailable for DIDS or NPPB. Alternatively, the whole cell conductance activated by superoxide may be a multichannel event driven by the redox state and, hence, the metabolic state of cells.

Modification of bath solutions by excluding Cl^– anion or by imposing a symmetrical Cl^– gradient resulted in cell depolarization and activation of the chloride channels located on the plasma membrane of vascular endothelial cells. The addition of superoxide increased the amplitude of the whole cell current, which returned to normal after the addition of SOD. This also implies that the superoxide anion is a possible carrier of anionic whole cell current. In endothelial cells exposed to an oxidant, tert-butyl-hydroperoxide (0.4 mM), the inward ionic movement via the bumetanide-sensitive pathway is decreased, indicating that oxidant stress inhibits the Na⁺-K⁺-Cl^– cotransporter (9). Bumetanide, an inhibitor of the Cl^–/HCO₃^– transporter and Na⁺-K⁺-Cl^– cotransporter, had no effect on the control currents or on superoxide-activated whole cell current in vascular endothelial cells. Therefore, we find that chloride channels play an important role in the electrophysiological effects of superoxide in vascular endothelial cells.

In vascular endothelial cells, the electrophysiological effects we observe can be directly attributed to the presence of the superoxide molecule. The addition of SOD, a scavenger of superoxide, either abolished the increase or prevented activation of the whole cell current. However, membrane depolarization caused by superoxide could not be reversed by SOD, supporting the notion that extracellular superoxide can affect intracellular metabolic pathways. Because the cell is impermeable to SOD, it is possible that superoxide in the extracellular environment enters the cell via an anion channel and generates an intracellular metabolic signal either directly or through intermediate metabolites. Under pathophysiological conditions, the intracellular content of superoxide is drastically increased, which leads to an increase in oxidative stress. Importantly, superoxide has no ability to simply diffuse via a plasma membrane (1, 12, 14, 25); however, this free radical has been determined to be present in the extracellular milieu in both in vitro and in vivo studies. In vitro studies show that endothelial cells exposed to anoxia-reperfusion injury generate a large amount of superoxide that can be detected intracellularly and, more importantly, extracellularly (18). Second, in in vivo ischemic stimulation studies, superoxide was measured in the intraluminal space of the brain arteries (23, 24). Unfortunately, the mechanism of superoxide transport to the extracellular compartment remains unclear. However, chloride channels, which are widely distributed through the cells of the cardiovascular system, could contribute to the release of superoxide by endothelial cells.

The oxidative events mentioned above are always inhibited by SOD and, additionally, are dependent on the inhibitor of the chloride channels and transporters, DIDS. This implies that the protein that is involved in the transport of superoxide across the plasma membrane could belong to a group of chloride channels or transporters. Our results show that chloride channels may conduct superoxide across the plasma membrane in vascular endothelial cells.

	GRANTS

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American Heart Association beginning grant-in-aid (to A. K. Brzezinska) and National Institutes of Health Grants NS-38133, HL-65203, and HL-32788 (to W. M. Chilian) supported this work.

	ACKNOWLEDGMENTS

 
We thank J. Walczak for excellent technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. K. Brzezinska, Dept. of Physiology, LSU Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112 (e-mail: abrzez{at}lsuhsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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