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(Circulation. 1999;99:1719-1725.)
© 1999 American Heart Association, Inc.

Clinical Investigation and Reports

Hydrogen Peroxide, Potassium Currents, and Membrane Potential in Human Endothelial Cells

Rostislav Bychkov, PhD; Knud Pieper, MD; Christian Ried, PhD; Marianna Milosheva, PhD; Eugen Bychkov, PhD; Friedrich C. Luft, MD; Hermann Haller, MD

From the Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany, and MREID Université du Littoral, Dunkerque, France (E.B.).

Correspondence to Hermann Haller, MD, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany. E-mail haller{at}fvk-berlin.de

	Abstract

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Background—Hydrogen peroxide (H₂O₂) and reactive oxygen species are implicated in inflammation, ischemia-reperfusion injury, and atherosclerosis. The role of ion channels has not been previously explored.

Methods and Results—K⁺ currents and membrane potential were recorded in endothelial cells by voltage- and current-clamp techniques. H₂O₂ elicited both hyperpolarization and depolarization of the membrane potential in a concentration-dependent manner. Low H₂O₂ concentrations (0.01 to 0.25 µmol/L) inhibited the inward-rectifying K⁺ current (K_IR). Whole-cell K⁺ current analysis revealed that H₂O₂ (1 mmol/L) applied to the bath solution increased the Ca²⁺-dependent K⁺ current (K_Ca) amplitude. H₂O₂ increased K_Ca current in outside-out patches in a Ca²⁺-free solution. When catalase (5000 µ/mL) was added to the bath solution, the outward-rectifying K⁺ current amplitude was restored. In contrast, superoxide dismutase (1000 u/mL) had only a small effect on the H₂O₂-induced K⁺ current changes. Next, we measured whole-cell K⁺ currents and redox potentials simultaneously with a novel redox potential-sensitive electrode. The H₂O₂-mediated K_Ca current increase was accompanied by a whole-cell redox potential decrease.

Conclusions—H₂O₂ elicited both hyperpolarization and depolarization of the membrane potential through 2 different mechanisms. Low H₂O₂ concentrations inhibited inward-rectifying K⁺ currents, whereas higher H₂O₂ concentrations increased the amplitude of the outward K⁺ current. We suggest that reactive oxygen species generated locally increases the K_Ca current amplitude, whereas low H₂O₂ concentrations inhibit K_IR via intracellular messengers.

Key Words: nitric oxide • potassium • free radicals • endothelium

	Introduction

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Hydrogen peroxide (H₂O₂) is important in inflammation, ischemia-reperfusion injury, and atherosclerosis.^{1 2 3} The endothelium is exposed to local reactive oxygen species generated by neutrophils and monocytes. In vitro, oxidative stress can be produced either by direct application⁴ of H₂O₂ or by continuous H₂O₂ generation in enzymatic systems.⁵ An interaction between H₂O₂ and the endothelium may modulate the cellular redox balance. H₂O₂ may also trigger multiple functions, such as the release of lactate dehydrogenase⁶ and tissue factor.¹ Exposure of endothelial cells to H₂O₂ decreases cellular ATP,^{6 7} disrupts DNA strands,⁶ and inhibits protein synthesis.⁸ H₂O₂ stimulates adherence of neutrophils to isolated canine carotid arteries, veins, and cultured endothelial cells.⁹ Reactive oxygen species (H₂O₂, O₂^-, and OH^-) disturb Ca²⁺ homeostasis in renal tubular epithelial cells¹⁰ and mammalian ovarian cells.¹¹ In addition, H₂O₂ was recently found to regulate intracellular Ca²⁺ signaling by stimulating Ca²⁺ release from the inositol triphosphate–sensitive stores in venous endothelial cells,¹² bovine pulmonary endothelial cells,^{7 13} and human endothelial cells.^{2 4} Although H₂O₂ has been shown to regulate intracellular and intercellular signal transduction, few data are available on the regulation of endothelial cell membrane potential by H₂O₂. Membrane potential contributes to the regulation of the release of vasoactive compounds^{14 15} and influences the contractile state of underlying vascular smooth muscle.^{16 17 18} Furthermore, control of the membrane potential is an essential requirement in cell growth.^{19 20 21} We investigated the effects of H₂O₂ on endothelial cell membrane potential, ion currents, and membrane polarization. Because H₂O₂ can both oxidize and reduce the cellular membrane components, we also examined links between ionic currents and the redox potential of whole endothelial cells.

	Methods

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Materials
Iberiotoxin was obtained from RBI (Natick). DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene), niflumic acid, EGTA, 4-aminopyridine, HEPES, and A23187 were purchased from Sigma-Aldrich. All salts and H₂O₂ were obtained from Merck. Human umbilical vein endothelial cells were isolated from umbilical cords by chymotrypsin treatment.²² Cells were subcultured in EGM (Clonetics) with 2% FCS and were used between passages 1 and 2.

K⁺ Current Recordings
Whole-cell K⁺ currents were measured by conventional patch clamp²³ or by perforated patch with nystatin.²⁴ The external solution E1 contained (in mmol/L) 140 NaCl, 1.8 CaCl₂, 1 MgCl₂, 5.4 KCl, and 10 Na-HEPES, pH 7.4. The E2 solution contained (in mmol/L) 90.4 NaCl, 1.8 CaCl₂, 1 MgCl₂, 50 KCl, and 10 Na-HEPES, pH 7.4. The patch pipette (resistance, 3x10⁶ to 8x10⁶ ) was filled with a solution I1 containing (in mmol/L) 80 K-aspartate, 30 KCl, 20 NaCl, 1 MgCl₂, 3 Mg-ATP, 10 EGTA, and 5 K-HEPES, pH 7.4. The Cs⁺-dialyzing pipette solution I2 contained (in mmol/L) 80 Cs-aspartate, 40 CsCl, 10 tetraethylammonium chloride, 1 MgCl₂, 3 Mg-ATP, 10 EGTA, and 5 Cs-HEPES, pH 7.4. In the experiments recording single channel activity in the outside-out configuration, 1.5 mmol/L EGTA was used in the intracellular solution. Ca²⁺ was added to the solution to obtain 3 µmol/L free Ca²⁺ concentration. Na⁺ ions replaced Ca²⁺ ions when Ca²⁺-free solution was used. Solutions were perfused through the chamber by gravity at a rate of 1 mL per 10 seconds. Experiments were done at room temperature. Nystatin (Sigma) was dissolved in dimethyl sulfoxide and diluted in the pipette solution to give a final concentration ranging from 50 to 100 µg/mL. Whole-cell access was achieved by nystatin within 10 to 20 minutes of seal formation. Whole-cell K⁺ currents were recorded at 5 to 10 kHz with an Axopatch 200A or a List EPC-7 amplifier, filtered at 1 kHz with an 8-pole low-pass Bessel filter instrument (Frequency Devices), digitized with a CED1401 interface (Cambridge Electronic Design Ltd), and analyzed with CED Patch and Voltage Clamp Software version 6.08. We performed 2 electrode recordings from 1 endothelial cell with 2 amplifiers. One was used in the voltage-clamp mode to record K⁺ currents. The second was connected to the redox sensor in the current-clamp mode. Series resistance and total cell capacitance were calculated from uncompensated capacitative transients, from 10-ms hyperpolarizing linear ramp pulses (10 mV), or by adjustment of the series resistance and whole-cell capacitance controls of the Axopatch 200A amplifier to eliminate the resulting current transitions. The membrane input resistance of the cells was measured with small hyperpolarizing voltage pulses (10 mV for 10 ms) from a holding potential of -40 mV. A UV lamp (Braun HUV1) was used to expose endothelial cells treated with H₂O₂ to UV flashes for 2 to 6 seconds. Voltage ramps were used routinely to measure instantaneous current-voltage relations (I/V curves). Ramps from –150 to 150 mV for 500 ms were applied from a holding potential of 0 mV.

Redox Sensor Preparation
Redox-sensitive glass electrodes were synthesized by the melting of appropriate quantities of transition metal and glass-forming oxides in a furnace at 800°C to 900°C for 12 to 30 hours with a subsequent quenching of the melt.^{25 26} The synthesized glasses were annealed for 24 hours at 20°C to 30°C below the glass transition temperature to remove the internal stress of vitreous membranes. Bulk sensor membranes were cut from the melt and thoroughly polished with an alumina powder and then a diamond-polishing paste. Platinum or gold was sputtered on the back side of the membrane, and a metallic wire was attached to it by a silver microadhesive. The membranes were then sealed into PVC tubes. Redox microsensors for single cell measurements were prepared by use of a metallic wire coated with a redox-sensitive thin-layer glass electrode. Two techniques were used for coating: sputtering of the bulk glass and dipping of a metallic wire into the glass-forming melt with subsequent quenching and annealing. Redox calibration measurements of the prepared bulk glass sensors and redox microsensors were performed with potassium hexacyanoferrate (II)/(III) solutions K3,4[Fe(CN)6] with a ratio of oxidized to reduced species of 0.01, 0.1, 1, 10, and 100. The total redox concentration varied between 0.05 and 5x10^-7 mol/L.

Data Analysis
All values are given as mean±SEM. Wilcoxon rank sum or Mann-Whitney-Wilcoxon tests were used to determine significant differences. A value of P<0.05 was considered statistically significant. The Nernst equation, E=2.303xRT/F[log10(A)0/(A)I], with R, T, and F having their usual meanings and (A)0 and (A)I being the extracellular and intracellular concentrations of an ion, respectively, was used to calculate the equilibrium potentials.

	Results

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K⁺ Currents in Cultured Endothelial Cells
The current-voltage relationship for the unstimulated endothelial cells was characterized by a pronounced inward rectification at potentials negative to the K⁺ reversal potential (E_rev) and relatively small outward currents at potentials positive to the E_rev (Figure 1A). The inward K⁺ current (K_IR) showed strong rectification at potentials negative to –80 mV with a voltage dependency reported elsewhere.^{20 24 27} K_IR current was recorded in 64 of the 75 cells examined. Second K⁺ current amplitude increased at voltages positive to 27±12 mV under physiological K⁺ gradient and had strong outward rectification. Reversal potential of both the K_IR current and outward K⁺ current was dependent on the extracellular K⁺ concentration. Varying the [K⁺]_o from 5.4 to 50 to 140 mmol/L shifted the K⁺ current reversal potential from –75±8 to 28±5 and 0±0.2 mV, respectively (n=20). The whole-cell inward rectifier K⁺ current was reversibly inhibited by Ba²⁺ (100 µmol/L), a typical blocker of the K_IR current, to 8±6% at –120 mV. Tetramethylammonium (TEA) was added to the bath solution after the inward rectifier inhibition by 100 µmol/L Ba²⁺. TEA (1 mmol/L) blocked the outward K⁺ current up to 17±6% (n=6) in the resting endothelial cells. Ca²⁺ ionophore A23187 (1 µmol/L) added to the bath solution ([Ca²⁺]_o=1.8 mmol/L) increased the K⁺ current amplitude to 187±10% at 100 mV (n=10) (Figure 1B). Increased intracellular Ca²⁺ concentration elicited an increase in K⁺ current amplitude and shifted the apparent threshold of the current-voltage relation from 27±4 to -14±8 mV. Measurements were performed in perforated patch configuration. Iberiotoxin (100 nmol/L) inhibited the K_Ca current stimulated by Ca²⁺ influx (n=8) to 15±3% of the control values.

View larger version (18K): [in this window] [in a new window]   Figure 1. A, K+ currents recorded under physiological K+ gradient ([K+]o 5.4 mmol/L/[K+]i 140 mmol/L) (Con) after replacement of bath solution with 140 mmol/L K+-containing solution (140 mmol/L ([K+]o) and after application of 100 µmol/L Ba2+. B, Current-voltage relation recorded under physiological K+ gradient in control after application of 100 µmol/L Ba2+. Ca2+ ionophore A23187 (1 µmol/L) elicited activation of KCa current, and TEA (1 mmol/L) inhibited KCa current elicited by Ca2+ influx.

H₂O₂-Induced Inhibition of K_IR
Cells were pretreated with 100 nmol/L iberiotoxin to prevent activation of the large-conductance BK_Ca channels. Experiments were performed in 140 mmol/L of K⁺-containing and Ca²⁺-free solution. Cells were dialyzed with 10 mmol/L EGTA to prevent the interfering influence of the intracellular Ca²⁺ transients to K⁺ currents. K_IR was obtained by 10-mV voltage steps over the range of 0 to –150 mV at a holding potential at 0 mV or by ramp changing from –150 to 150 mV. First, K_IR was recorded under physiological K⁺ gradients (5.4 mmol/L_o · 140 mmol/L_i^-1). Then, the extracellular solution was changed to symmetrical K⁺ solution (140 mmol/L_i · 140 mmol/L_o^-1). H₂O₂ applied to the bath solution decreased K_IR in a dose-dependent manner (Figure 2B). H₂O₂ (250 µmol/L) decreased the K_IR peak and steady-state current (Figure 2C). The amplitude of the current was measured at -150 mV and plotted against the corresponding H₂O₂ concentration (Figure 2A). IC₅₀ and curve slope were estimated from the best fit of the logistic function and were 110±3 µmol/L and 0.83±0.07, respectively. Interestingly, low H₂O₂ concentrations (0.25 mmol/L) decreased the slope of the I/V curve. In contrast, the high H₂O₂ concentrations (up to 0.5 mmol/L) elicited, in addition to the decreasing slope, a parallel shift of the current-voltage relation toward negative potentials. However, this shift of about 13±4 mV was independent of the extracellular ionic composition. The shift was observed when the bath solution contained 5.4 mmol/L K⁺. When cells were pretreated with iberiotoxin, H₂O₂ elicited an increase in the K⁺ current amplitude from 52±4 to 81±7 pA at 150 mV. Stimulation of the iberiotoxin-insensitive K⁺ current was observed in 4 of the 19 cells examined.

View larger version (23K): [in this window] [in a new window]   Figure 2. A, Endothelial cells were pretreated with iberiotoxin (100 nmol/L) for 10 minutes. Dose-response curve of KIR current is plotted on semilogarithmic scale. Points represent mean current amplitude measured from 5 to 8 cells at -150 mV. Data were fitted with logistic function Y=A1+(A1-A2)/[1+(x/x0)p], where A1 corresponds to initial value, A2 to final value, x to H2O2 concentration, x0 to half-maximal activation (IC50), and p to slope of curve. Values obtained from best fit were as follows: A1=100%; A2=34±5%; x0=0.11±0.003 mmol/L; P=0.83±0.07. B, Superimposed current-voltage (I/V) relations of KIR current were recorded in 140 mmol/L K+-containing solution in control (Con) and after application of different H2O2 concentrations. Concentrations are in mmol/L range. C, Control I/V relationships were recorded in 5 and 140 mmol/L K+-containing solutions. H2O2 (250 µmol/L) elicited decrease in peak and steady-state KIR current (lower traces). Currents were elicited by voltage step pulses applied from holding potential of 0 to –150 mV in 10-mV steps.

H₂O₂- and Ca²⁺-Dependent K+ Channels
Next, we examined the effect of H₂O₂ on iberiotoxin-sensitive K⁺ current. The amplitude of the whole-cell K⁺ current induced by H₂O₂ was concentration dependent. The dose-response relationship to the amplitude of the K_Ca current was examined in the Ca²⁺-free solution containing 140 mmol/L K⁺. Cells were dialyzed with 10 mmol/L EGTA. K_IR current was blocked by 100 µmol/L Ba²⁺. Niflumic acid (100 µmol/L) and DIDS were used to block Cl^- currents that were described previously in human endothelial cells.²⁰ DIDS (100 µmol/L) elicited an inhibition in the same range as niflumic acid. Thus, we used niflumic acid in subsequent experiments to prevent possible activation of the Cl^- current. Niflumic acid (100 µmol/L) in the bath solution inhibited the outward current to 41±5 from 58±4 pA (Figure 3A). These subsequent experiments were performed in the presence of 100 µmol/L Ba²⁺ and 100 µmol/L niflumic acid in the bath solution. H₂O₂ activated the outward-rectifying K⁺ current under these conditions in a dose-dependent manner. The current amplitude was measured at 150 mV and plotted against the corresponding H₂O₂ concentration with a semilogarithmic scale (Figure 3C). The slope and half-maximal activation, calculated from the best fit, were 1.3±0.01 and 0.71±0.05 mmol/L, respectively. Three repetitive H₂O₂ applications in the Ca²⁺-free bath solution elicited repetitive increases in the outward K⁺ current amplitude with subsequent washout.

View larger version (15K): [in this window] [in a new window]   Figure 3. A, I/V relationship was recorded in 140 mmol/L K+-containing solution in control experiments (Con), after application of 100 µmol/L Ba2+ to bath solution, and after application of 100 µmol/L Ba2+ and niflumic acid (NA). B, Recordings were done in presence of 100 µmol/L Ba2+ and 100 µmol/L niflumic acid. Superimposed traces of I/V relations recorded in 140 mmol/L K+-containing solution: in control (c) and after application of different H2O2 concentrations. Concentrations are shown in mmol/L range. C, Dose-response curve of KCa current and H2O2 are plotted on semilogarithmic scale. Points represent mean current amplitude measured from 5 to 8 cells at 150 mV. Data were fitted with logistic function Y=A1+(A1-A2)/[1+(x/x0)p] (see Figure 2). Values obtained from best fit were as follows: A1=100%; A2=554±22%; x0=0.71±0.05 mmol/L; P=1.3±0.01.

Effects of Superoxide Dismutase and Catalase
We then investigated the effects of superoxide dismutase (SOD) and catalase on these 2 different K⁺ currents using a current-voltage relationship elicited by ramp from –150 mV to 150 mV applied from 0-mV holding potential. We used 1 mmol/L H₂O₂ because at that concentration we observed all effects described previously. SOD (1000 µ/mL) did not prevent inhibition of the K_IR current elicited by 1 mmol/L H₂O₂ (Figure 4A). The amplitude of the outward component of the K⁺ current decreased slightly to 88±6% of the maximal activation level. In contrast to SOD, catalase (5000 µ/mL) decreased the amplitude of the outward-rectifying K⁺ current to control values (Figure 4B). Catalase also inhibited the parallel shift of the K_IR current-voltage relationship toward negative potentials. However, catalase did not reverse the inhibition of the K_IR current elicited by H₂O₂.

View larger version (25K): [in this window] [in a new window]   Figure 4. Effect of catalase and SOD on K+ currents altered by H2O2. Current-voltage relationships were obtained by linear ramp voltage applied from 0-mV holding potential. Currents were recorded in symmetrical K+ solution (140 mmol/Li · 140 mmol/Lo-1). A, H2O2 (1 mmol/L)-elicited inhibition of inward-rectifying K+ current occurred with parallel shift of current voltage relationship of I/V curve to left. SOD added to bath solution with H2O2 elicited small decrease in H2O2-activated current (H2O2+SOD). Control current (Con) corresponds to K+ current altered by 1 mmol/L H2O2. B, Catalase added with H2O2 decreased amplitude of activated outward K+ current (catalase+H2O2). Control traces in both panels are shown by thick lines; traces that correspond to application of H2O2 are shown with thin lines; traces that correspond to H2O2 together with SOD or catalase are shown by dotted lines.

In the next series of experiments, we examined the stimulatory effect of H₂O₂ on BK_Ca channels in the membrane patches using the outside-out configuration. The BK_Ca currents were recorded under a physiological K⁺ gradient in Ca²⁺-free solution. A single channel activity was recorded in 6 patches (Figure 5A). Amplitudes of the single K⁺ channels were plotted against the membrane potential. The data were fitted with linear regression analysis (Figure 5B). The slope (181±4 pS) indicated that we recorded large-conductance Ca²⁺-dependent K⁺ channels. The single channel activity was recorded continuously at 0-mV holding potential. H₂O₂ (0.5 mmol/L) elicited a sustained increase in BK_Ca channel activity (Figure 5C) without changing the amplitude (Figure 5D). Catalase (5000 u/mL) decreased sustained H₂O₂-mediated activation of the BK channels.

View larger version (26K): [in this window] [in a new window]   Figure 5. A, Typical activity of BKCa channel recorded from patch in outside-out configuration at different holding potentials. B, Amplitudes of channels are measured and plotted against corresponding voltage. Data were fitted by linear regression. Slope estimated from best fit was 181±4 pA. C, Activity of channel recorded in control (Con), after application of H2O2, and after addition of catalase to bath solution. Recordings were made at 0-mV holding potential. D, Amplitude distribution obtained in control (left) and in after application of H2O2 (right). Amplitude distributions were fitted by double gaussian function. Peaks were at 0.11±0.01 and 4.95±0.03 pA for control and 0.13±0.02 and 5.01±0.03 after H2O2 application to bath solution.

Depolarization and Hyperpolarization of Whole Endothelial Cells
The membrane potential was recorded in the current-clamp mode. The values at rest ranged from -70 to -20 mV. The average membrane potential was –52±17 mV (n=60). H₂O₂ (0.5 to 2 mmol/L) added to the bath solution elicited fully reversible hyperpolarization (Figure 6A). The rate of hyperpolarization depended on the H₂O₂ concentration. We estimated the H₂O₂ effect on the membrane potential by plotting the difference between membrane potential under control conditions and after H₂O₂ application to the bath solution. The semilogarithmic plot (Figure 6B) showed a near-Nernstian response of the membrane potential hyperpolarization elicited by H₂O₂, with a slope of 64±7 mV per decade. The repetitive H₂O₂ application to the bath solution elicited repetitive hyperpolarizations of the membrane potential. H₂O₂ applied to the bath solution at concentrations <0.25 mmol/L elicited depolarization of the membrane potential from –59±11 to 32±7 mV (n=11) (Figure 6C). Barium (100 µmol/L) elicited depolarization from –57±14 to 28±9 mV (n=6).

View larger version (10K): [in this window] [in a new window]   Figure 6. A, H2O2-elicited sustained membrane potential hyperpolarization. Lines show time of H2O2 application to bath solution. Endothelial cells were dialyzed with 10 mmol/L EGTA. Membrane potential was recorded in Ca2+-free solution. B, Difference between control membrane potential [V(Con)] and membrane potential [V(H2O2)] recorded after H2O2 application is plotted on semilogarithmic scale. Points represent mean values from 8 experiments. Data were fitted with linear function Y=A+BX, where Y is membrane potential difference, X is concentration of H2O2, A=-30±1.6, and B=-64.19±6.62. Values were obtained from best fit. C, Depolarization of strongly hyperpolarized endothelial cell elicited by H2O2 (250 µmol/L). Time of application is shown by line.

Whole-Cell Redox Potential and Steady-State K⁺ Currents With H₂O₂
We hypothesized that H₂O₂ might act through the generation of free radicals, in part by producing the superoxide ion. H₂O₂ or products of its degradation could oxidize some components of the endothelial cell plasma membrane and thereby modulate the K⁺ currents. To examine this possibility, we used UV flash. UV light triggered H₂O₂ destruction and thereby increased the amount of superoxide ion in the bath solution.²⁸ Outside-out configuration was used to perform the following experiment. H₂O₂ (0.5 mmol/L), at a concentration lower than the previously estimated IC₅₀, was added first to the bath solution. H₂O₂ elicited an increase in the K_Ca current. Next, we exposed the experimental chamber to a UV flash for 2 to 6 seconds, when the K_Ca current activation reached a steady-state level (Figure 7A). We observed an increase in the K_Ca current amplitude of 291±42% at 150 mV (n=6). UV light alone, without H₂O₂, did not augment the K⁺ current amplitude.

View larger version (18K): [in this window] [in a new window]   Figure 7. A, Current-voltage relationships recorded under control (Con) conditions, after application of H2O2 (0.5 mmol/L), and after exposure of endothelial cells to UV light. B, Calibration of redox sensor made in standard solution contained different ratios of redox pairs. Redox sensor response (y axes) was plotted on semilogarithmic scale vs Fe(III)/Fe(II) ratio (x axes). Data were fitted with linear function. Slope was calculated from best fit and was equal to 56.9 mV per decade. B, Simultaneous recording of whole-cell redox response and steady-state K+ current at 0-mV holding potential. Line shows H2O2 application time (1 mmol/L) in bath solution.

We also recorded the K⁺ current and redox potential of whole cells simultaneously with a novel electrode, selectively sensitive to the redox pairs ratio, in aqueous solutions.^{25 26} The redox glass electrode was calibrated in standard solutions containing different ratios of oxidizing to reducing species.^{2 24} Redox potential formed by the Fe(II)/Fe(III) redox pair was measured with the measurement mode of the zero current. The sensor potential increased with increasing ratio of the oxidizing species. The sensor potential was close to the equilibrium redox potential²⁸ with a near-Nernstian slope of 56.9±0.3 mV per decade (Figure 7C). The calibrated redox electrode was connected directly with the patch-clamp preamplifier. A glass pipette filled with the I1 solution with nystatin was placed on the redox electrode. First, we recorded the K⁺ current in a whole-cell configuration. Second, the glass pipette with the redox sensor was placed on the cytoplasmic membrane of the endothelial cell after the whole-cell recording was established. We waited 5 to 10 minutes, until the membrane potential transients elicited by the second pipette attachment to the cell surface vanished. The action of H₂O₂ on whole-cell redox potential and K⁺ currents was examined only after the membrane potential was stabilized. We used the measurement mode of the zero current to record redox potential from endothelial cells. The steady-state K⁺ current was recorded at 0-mV holding potential in the physiological K⁺ gradient. Application of H₂O₂ (1 mmol/L) to the bath solution elicited an increase in K⁺ current and simultaneously decreased the redox potential (Figure 6C) (n=5). However, the kinetics of the responses were different. The K⁺ current increased exponentially, whereas the redox potential decreased in a stepwise fashion. The redox potential decreased by about -28±5 mV (n=5). The K⁺ current increase and the decrease in redox potential elicited by H₂O₂ were fully reversible.

	Discussion

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H₂O₂ secretion by activated neutrophils suggests that H₂O₂ serves as an intercellular messenger.^{2 9 12} How much H₂O₂ is produced by neutrophils in vivo is unknown; however, within the microenvironment, the concentration might be quite high^{12 13} and could reach 1 to 2 mmol/L if such an area represents a limited diffusion space. The H₂O₂ concentrations used in our study are similar to those used in previous studies on cell function.^{2 7 12 13} We focused only on very early events in response to H₂O₂, which occurred within the first 5 minutes after application of H₂O₂ to the bath solution. Two main K⁺ currents were present in our cells: the inward-rectifying K_IR and Ca²⁺-dependent K⁺ currents. Both currents have been characterized in endothelial cells previously.^{10 19 20 21 24 27 29 30} We found that H₂O₂ inhibited K_IR and elicited an increase in the K_Ca current. Thus, H₂O₂ could regulate the membrane potential by 2 different mechanisms. Low H₂O₂ concentrations inhibited K_IR and depolarized the membrane. Inward-rectifying K⁺ currents were previously reported to regulate the resting membrane potential of endothelial cells.^{21 24} H₂O₂ at concentrations >0.5 mmol/L increased the amplitude of the K_Ca current and thus elicited membrane potential hyperpolarization.

H₂O₂ induced membrane depolarization in cells that had hyperpolarized membrane potentials from –55 to –75 mV. The hyperpolarization elicited by high H₂O₂ concentrations was resolute in cells with intermediately polarized membrane potentials from –25 to –50 mV. An H₂O₂-elicited increase in intracellular Ca²⁺ could lead to K_Ca current activation.^{2 12} However, we also observed K_Ca current stimulation by H₂O₂ in cells dialyzed with 10 mmol/L EGTA in a Ca²⁺-free solution. High intracellular EGTA concentration buffered all Ca²⁺ release from the intracellular stores triggered by 50 µmol/L histamine. We examined 10 cells, and histamine elicited no changes in K_Ca current amplitude when cells were dialyzed with 10 mmol/L EGTA. We also observed stimulation of BK_Ca channels by H₂O₂ in the outside-out membrane patches in a Ca²⁺-free solution. BK_Ca channels with similar conductances have been found in other endothelial cell preparations.²⁹ However, whether BK_Ca is indeed the unique Ca²⁺-dependent K⁺ channel stimulated by H₂O₂ is still unknown. Three different types of Ca²⁺-dependent K⁺ channels (large, intermediate, and small conductance) were found previously in different preparations of endothelial cells.^{8 29 30}

K_IR was inhibited by H₂O₂, in contrast to the Ca²⁺-dependent K⁺ current. However, the IC₅₀ concentration for the inward rectifier was 10 times smaller than for the K_Ca current. We hypothesized that superoxide ion, the product of H₂O₂ degradation in aqueous solutions, might increase the K_Ca current amplitude. Superoxide ion may oxidize some plasma membrane components and thereby could modulate membrane permeability. Recently, oxidation of voltage-gated K⁺ channels was demonstrated.³¹ Concentration of the superoxide ion in bath solution is significantly lower than the H₂O₂ concentration, which might explain why high H₂O₂ concentrations stimulated K_Ca current, compared with inward-rectifying K⁺ current. The experiments performed with UV light support this hypothesis. UV flash dramatically increased the amplitude of the K_Ca current, whereas H₂O₂ applied to the bath solution at low concentrations did not increase it. The next line of evidence for a chemical redox reaction came from the simultaneous measurement of the whole-cell K_Ca current and redox potential. Simultaneous measurements of the K⁺ current and redox potential revealed a strong correlation between K⁺ current increase and redox response. The kinetics of the K⁺ current activation and the increase in redox potential were different. The current increased in an exponential fashion, whereas the redox potential changed in a stepwise manner. The effect was fully reversible and could be repeated. Future studies of endothelial cell responses to different reactive oxygen species, including H₂O₂, will clarify under which conditions H₂O₂-mediated effects are physiological or injurious to endothelial cells.

	Acknowledgments

 
This work was supported by a grant in aid from the Deutsche Forschungsgemeinschaft to Dr Haller.

	Footnotes

 
Dr R. Bychkov is now at the College de France INSERM U114, 11 PL Marcelin, Berthelot, Paris, France.

Received May 28, 1998; revision received December 1, 1998; accepted December 29, 1998.

	References

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