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(Circulation. 1998;97:268-275.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Hydrogen Peroxide Induces Intracellular Calcium Oscillations in Human Aortic Endothelial Cells

Qinghua Hu, PhD; Stefano Corda, MD; Jay L. Zweier, MD; Maurizio C. Capogrossi, MD; ; Roy C. Ziegelstein, MD

From the Department of Medicine (Q.H., J.L.Z., M.C.C., R.C.Z.), Division of Cardiology, Johns Hopkins Bayview Medical Center, Johns Hopkins University School of Medicine, Baltimore, Md; Laboratory of Cardiovascular Science (S.C., M.C.C.), Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Md; and Laboratorio di Patologia Vascolare (M.C.C.), Istituto Dermopatico dell' Immacolata, Rome, Italy.

Correspondence to Roy C. Ziegelstein, MD, Department of Medicine, Division of Cardiology, Johns Hopkins Bayview Medical Center, 4940 Eastern Ave, Baltimore, MD 21224-2780. E-mail rziegels{at}welchlink.welch.jhu.edu

	Abstract

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Background—Because the vascular endothelium is exposed to oxidant stress resulting from ischemia/reperfusion and from the products of polymorphonuclear leukocytes or monocytes, studies were performed to examine the effect of hydrogen peroxide (1 µmol/L to 10 mmol/L) on endothelial Ca²⁺ signaling.

Methods and Results—At low concentrations (1 to 10 µmol/L), hydrogen peroxide did not affect intracellular Ca²⁺ concentration in subconfluent, indo 1–loaded human aortic endothelial monolayers. At a concentration of 100 µmol/L hydrogen peroxide, intracellular free Ca²⁺ gradually increased from 125.3±6.8 to 286.3±19.9 nmol/L over 4.2±0.9 minutes before repetitive Ca²⁺ oscillations were observed, consisting of an initial large, transient spike of 1 µmol/L followed by several spikes of decreasing amplitudes at a frequency of 0.7±0.1 min^-1 over 12.0±1.1 minutes. After these oscillations, intracellular Ca²⁺ reached a plateau of 543.4±64.0 nmol/L, which was maintained above baseline levels for >5 minutes and then partially reversible on washout of hydrogen peroxide in most monolayers. Intracellular Ca²⁺ oscillations were typically observed when monolayers were exposed to 100 to 500 µmol/L hydrogen peroxide. Higher concentrations of hydrogen peroxide (1 and 10 mmol/L) increased intracellular Ca²⁺ but only rarely (2 of 6 monolayers at 1 mmol/L) or never (at 10 mmol/L) stimulated intracellular Ca²⁺ oscillations. Removal of Ca²⁺ from the buffer either before hydrogen peroxide stimulation or during an established response did not block intracellular Ca²⁺ oscillations in response to 100 µmol/L hydrogen peroxide, but prior depletion of an intracellular Ca²⁺ store with either caffeine, histamine, or thapsigargin abolished Ca²⁺ oscillations.

Conclusions—Hydrogen peroxide induces concentration-dependent intracellular Ca²⁺ oscillations in human endothelial cells, which results from release of an endoplasmic reticulum Ca²⁺ store. Because oxidant production appears to occur in the micromolar range in the postischemic/anoxic endothelium and is associated with impaired endothelium-dependent relaxation, the effects of micromolar concentrations of hydrogen peroxide on endothelial Ca²⁺ signaling described in the present study may be important in the pathogenesis of postischemic endothelial dysfunction.

Key Words: calcium • endothelium • free radicals

	Introduction

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The endothelium in vivo may be exposed to oxidant stress resulting from ischemia and reperfusion or from the products of PMNs or monocytes. The endogenous oxidant hydrogen peroxide (H₂O₂), which is derived from PMNs and monocytes, has been shown to affect endothelium-dependent relaxation,¹ decrease endothelial energy stores,^{2 3} induce DNA strand breaks,^{3 4} enhance PMN/endothelial cell adhesion,^{5 6} increase endothelial permeability,⁷ and stimulate the release of prostaglandin I₂ from the endothelium.⁸ H₂O₂ may also stimulate release of Ca²⁺ from the InsP₃-sensitive Ca²⁺ store in endothelial cells,⁹ suggesting that H₂O₂ acts as an intracellular messenger in this cell type.

Release of Ca²⁺ from the InsP₃-sensitive store in HAECs has also been demonstrated during adhesion of PMNs or monocytes,¹⁰ suggesting that the effect of PMN adhesion on endothelial [Ca²⁺]_i may be mediated through released oxidants. The alteration in Ca²⁺ signaling during PMN adhesion has been linked to an increase in monolayer permeability that may regulate transendothelial migration of PMNs.¹¹ A similar link between an H₂O₂-induced increase in endothelial [Ca²⁺]_i and enhanced permeability also has been established recently.⁷ The agonist histamine enhances monolayer permeability¹² and stimulates dose-dependent endothelial [Ca²⁺]_i oscillations (at low concentrations of agonist) that merge into sustained [Ca²⁺]_i increases (at high concentrations of agonist) to provide a sensitive frequency-modulated control of the functional response to the agonist.¹³ We hypothesized that H₂O₂ similarly may produce [Ca²⁺]_i oscillations at lower (submillimolar) concentrations. The present study reports the first observation of oxidant stress-induced [Ca²⁺]_i oscillations in vascular endothelial cells.

	Methods

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Culture of HAECs
HAECs were obtained as proliferating quaternary cultures (Clonetics) and grown to confluence to passages 5 to 9 in endothelial cell growth medium supplemented with 2% fetal bovine serum, 10 µg/L human recombinant epidermal growth factor, 1 mg/L hydrocortisone, 50 µg/mL gentamicin, 50 ng/mL amphotericin-B, and 12 µg/mL bovine brain extract (Clonetics) in a 37°C humidified atmosphere of 95% air/5% CO₂. To measure [Ca²⁺]_i, HAEC were plated at a concentration of 1x10⁵/mL and grown to 70% confluence on 25-mm-diameter circular glass coverslips (VWR Scientific) precoated with 2% gelatin solution (Sigma Chemical).

Measurement of Intracellular Free Ca²⁺ Concentration in Response to H₂O₂
HAEC monolayers on glass coverslips were incubated with culture medium containing 10 µmol/L indo 1 (acetoxymethyl ester form; Molecular Probes) in a room temperature, 95% air/5% CO₂ atmosphere for 30 minutes.¹⁴ The coverslips were then gently washed for 30 minutes with indicator-free HBS composed of (in mmol/L) NaCl 137, KCl 4.9, CaCl₂ 1.5, MgSO₄ 1.2, NaH₂PO₄ 1.2, D-glucose 15, and HEPES 20, pH 7.40, at room temperature to allow deesterification of the indicator. Monolayers were exposed to H₂O₂ in HBS (made from a 3% stock solution; Sigma) at a flow rate of 1.8 mL/min until a plateau [Ca²⁺]_i was achieved (15 to 30 minutes). In some experiments, monolayers were pretreated with TG (Calbiochem), histamine, or caffeine (Sigma) before H₂O₂ stimulation. Indo 1 fluorescence was recorded a field of two or three connected cells of a subconfluent HAEC monolayer on a coverslip in a perfusion chamber mounted on the stage of a modified Nikon Diaphot inverted epifluorescence microscope. Indo 1 fluorescence was excited at 350±50 nm using a xenon short arc lamp (UXL-75 XE; Ushio Inc) and bandpass interference filters (Omega Optical) with selected wavelength bands of emitted fluorescence at 405±10 and 485±10 nm, corresponding to the Ca²⁺-bound and -free forms of the indicator, respectively. Emitted indo 1 fluorescence was collected and measured using a spectrofluorimeter (PTI; Deltascan). Autofluorescence from unloaded HAECs was subtracted automatically from indo 1 fluorescence recordings.

The intracellular minimum and maximum ratios (R_min and R_max, respectively) were determined as described previously¹⁴ and used to calculate [Ca²⁺]_i according to the formula: [Ca²⁺]_i=K_d(R-R_min)/(R_max-R)(S_f2/S_b2),¹⁵ where K_d is the dissociation constant of indo-1, and S_f2 and S_b2 are the fluorescence intensities at 490 nm of the Ca²⁺-free and Ca²⁺-saturated indicator, respectively. K_d was determined to be 207 nmol/L under the present experimental conditions through an in vitro calibration method.

Data Analysis and Statistics
Data are reported as mean±SEM. Statistical comparisons were made using the Student's t test for paired and unpaired groups. Comparisons among treatment groups for experiments with different concentrations of H₂O₂ were analyzed with ANOVA. A difference was considered significant at P<.05.

	Results

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Effects of Hydrogen Peroxide on [Ca²⁺]_i in HAECs
In Ca²⁺-containing solution, the basal [Ca²⁺]_i level was 117.2±5.9 nmol/L (n=41) and was not affected by exposure to 1 or 10 µmol/L H₂O₂ over a 20-minute period. In all monolayers studied (n=8), 100 µmol/L H₂O₂ increased [Ca²⁺]_i from 125.3±6.8 to 286.3±19.9 nmol/L and then caused [Ca²⁺]_i oscillations (Fig 1A). The typical pattern of the changes in [Ca²⁺]_i was triphasic and consisted of (1) the latency period, the interval between exposure to H₂O₂ and the first large peak of the [Ca²⁺]_i transient,¹⁶ (2) oscillations, the repetitive, transient [Ca²⁺]_i spikes, and (3) plateau, the baseline level of [Ca²⁺]_i after [Ca²⁺]_i oscillations. The average latency period was 4.2±0.9 minutes at this concentration. [Ca²⁺]_i oscillations consisted of rapid, transient, and large increases in [Ca²⁺]_i, each followed by a rapid decrease to a [Ca²⁺]_i level that was slightly above the preceding baseline. The initial transient [Ca²⁺]_i increase averaged 1113.6±265.4 nmol/L, with 6.6±0.6 oscillatory [Ca²⁺]_i increases at a frequency of 0.7±0.1 min^-1, with a mean [Ca²⁺]_i increase of 779.1±121.1 nmol/L over the next 12.0±1.1 minutes. After these [Ca²⁺]_i oscillations, a plateau [Ca²⁺]_i level was reached (543.4±64.0 nmol/L), which was maintained above baseline levels for >5 minutes and then partially reversible on washout of H₂O₂ in 6 of 8 monolayers examined.

View larger version (16K): [in this window] [in a new window]   Figure 1. Effect of H2O2 on HAEC [Ca2+]i. A, Representative indo 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 (arrow) in HBS with 1.5 mmol/L Ca2+. After an initial gradual increase in [Ca2+]i, this concentration of H2O2 induced [Ca2+]i oscillations in all monolayers studied (n=8). The increase in [Ca2+]i was partially reversible in 6 of 8 monolayers. B, Representative indo 1 fluorescence from an HAEC monolayer exposed to 250 µmol/L H2O2 (arrow) in HBS with 1.5 mmol/L Ca2+. After the initial increase in [Ca2+]i, oscillations were observed in 5 of 6 monolayers studied. The increase in [Ca2+]i was partially reversible in 4 of 6 monolayers studied. C, Representative indo 1 fluorescence from an HAEC monolayer exposed to 500 µmol/L H2O2 (arrow) in HBS with 1.5 mmol/L Ca2+. After an increase in [Ca2+]i, [Ca2+]i oscillations were observed in 5 of 6 monolayers studied. The increase in [Ca2+]i was partially reversible in 4 of 6 monolayers studied. D, Representative indo 1 fluorescence from an HAEC monolayer exposed to 1 mmol/L H2O2 (arrow) in HBS with 1.5 mmol/L Ca2+. After an increase in [Ca2+]i (n=6), oscillations were observed in only 2 monolayers. In the remaining 4 monolayers, [Ca2+ ]i reached a plateau without oscillations. The increase in [Ca2+]i was partially reversible in 3 of 6 monolayers.

As shown in Fig 1B and 1C, higher concentrations of H₂O₂ (250 and 500 µmol/L) also stimulated [Ca²⁺]_i oscillations (in 5 of 6 monolayers studied at each concentration) with some differences from those observed at 100 µmol/L H₂O₂. There was a nonsignificant trend for the latency period to be shorter at the higher concentrations of H₂O₂ (3.9±0.5 minutes for 250 µmol/L and 3.5±0.6 minutes for 500 µmol/L H₂O₂). Although there was no difference in the magnitude of the average [Ca²⁺]_i oscillations at these concentrations (773.1±109.2 nmol/L for 250 µmol/L H₂O₂ and 791.4±89.6 nmol/L for 500 µmol/L, P=NS compared with each other and with 100 µmol/L H₂O₂), the oscillation frequency was greater at the higher concentrations of H₂O₂ (0.9±0.1 min^-1 for each versus 0.7±0.1 min^-1 at 100 µmol/L H₂O₂, P<.05). There was a nonsignificant trend toward a shorter duration of oscillations at 250 µmol/L H₂O₂ (10.7±0.7 minutes) compared with 100 µmol/L H₂O₂; this was significant at 500 µmol/L H₂O₂ (5.6±0.6 minutes; P<.05 versus 100 and 250 µmol/L H₂O₂). The [Ca²⁺]_i level reached before oscillations occurred was not different for each of the three concentrations of H₂O₂ (286.3±19.9 nmol/L for 100 µmol/L H₂O₂, 299.2±38.7 nmol/L for 250 µmol/L H₂O₂, 293.6±12.4 nmol/L for 500 µmol/L H₂O₂; P=NS). There was a nonsignificant trend for the plateau [Ca²⁺]_i level after oscillations to increase with increasing concentrations of H₂O₂ (543.4±64.0 nmol/L for 100 µmol/L H₂O₂, 603.8±99.2 nmol/L for 250 µmol/L H₂O₂, 698.9±122.4 nmol/L for 500 µmol/L H₂O₂; P=NS, Fig 2). After washout, [Ca²⁺]_i was partially reversible in 4 of 6 monolayers studied at 250 and 500 µmol/L H₂O₂.

View larger version (10K): [in this window] [in a new window]   Figure 2. Effect of H2O2 on HAEC [Ca2+ ]i plateau level. Averaged data show the effect of H2O2 concentration (1 µmol/L to 10 mmol/L) on the [Ca2+ ]i plateau. The change in [Ca2+]i from baseline ([Ca2+]i) is shown on the y axis. There was no significant effect of H2O2 on [Ca2+]i at 1 to 10 µmol/L, but at higher H2O2 concentrations, plateau [Ca2+]i progressively increased (*P<.05 vs 100 µmol/L H2O2; P<.05 vs 100 to 500 µmol/L, n=4 to 8 monolayers).

Although [Ca²⁺]_i oscillations were noted in most monolayers at 100, 250, and 500 µmol/L H₂O₂, oscillations were noted in only 2 of 6 monolayers exposed to 1 mmol/L H₂O₂, and the more typical response was for [Ca²⁺]_i to reach plateau directly (Fig 1D). At this concentration of H₂O₂, the plateau [Ca²⁺]_i was 988.7±105.7 nmol/L (P<.05 versus the plateau [Ca²⁺]_i level at 100 µmol/L H₂O₂; Fig 2), and this was partially reversible in 5 of 6 monolayers examined. In the 2 monolayers in which [Ca²⁺]_i oscillations were observed, the level of [Ca²⁺]_i before oscillations (303.7±13.4 nmol/L) and the average magnitude of the transient [Ca²⁺]_i increase (775.0±127.5 nmol/L) were similar to those at lower H₂O₂ concentrations; the oscillation frequency was 1.0±0.0 min^-1. When monolayers were exposed to 10 mmol/L H₂O₂, [Ca²⁺]_i increased to 1252.5±128.2 nmol/L (P<.05 versus the plateau [Ca²⁺]_i level for 100, 250, and 500 µmol/L H₂O₂; Fig 2); this was then partially reversible in 3 of 6 monolayers studied. [Ca²⁺]_i oscillations were not observed in any of the 6 monolayers examined.

Role of Extracellular Ca²⁺ in H₂O₂-Induced Ca²⁺ Signaling in HAECs
In Ca²⁺-free/EGTA buffer, the basal [Ca²⁺]_i was 88.3±7.5 nmol/L (n=25). Under these conditions, in which influx of Ca²⁺ from the extracellular space does not contribute to an increase in [Ca²⁺]_i, stimulation with 100 µmol/L H₂O₂ increased [Ca²⁺]_i and caused [Ca²⁺]_i oscillations (Fig 3). As shown in Fig 4, there was no difference in the [Ca²⁺]_i level before oscillations (286.3±19.9 versus 248.3±21.4 nmol/L, P=NS) or in the latency period (4.2±0.9 versus 3.8±0.5 minutes, P=NS) in the presence and absence of buffer Ca²⁺, respectively. After this latency period, [Ca²⁺]_i oscillations were observed for an average of 9.0±0.9 minutes, which is 3 minutes shorter than that in the presence of 1.5 mmol/L Ca²⁺ (12.0±1.1 minutes, P<.05). The oscillation frequency was less in the absence than in the presence of extracellular Ca²⁺ (0.4±0.1 versus 0.7±0.1 min^-1, P<.05). The average increase in [Ca²⁺]_i during the oscillations was slightly, although not significantly, lower in the absence of buffer Ca²⁺ (619.2±74.6 nmol/L) than in the presence of 1.5 mmol/L Ca²⁺ (779.1±121.1 nmol/L; P=NS), and the plateau [Ca²⁺]_i level was lower in the absence than in the presence of buffer Ca²⁺ (378.9±40.1 versus 543.4±64.0 nmol/L; P<.05).

View larger version (13K): [in this window] [in a new window]   Figure 3. Effect of H2O2 on HAEC [Ca2+]i in Ca2+-free buffer. Representative indo 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 (arrow) in HBS without added Ca2+ and with 1 mmol/L EGTA (present throughout). After an initial gradual increase in [Ca2+]i, [Ca2+]i oscillations were observed in all monolayers studied (n=11).

View larger version (16K): [in this window] [in a new window]   Figure 4. Effect of H2O2 on HAEC [Ca2+]i in Ca2+-free buffer. Averaged data compare the effects of 100 µmol/L H2O2 on latency (minutes; see text for definition); amplitude (nmol/L), frequency (min-1), duration of [Ca2+]i oscillations (minutes), and plateau [Ca2+]i level (nmol/L) in buffer with 1.5 mmol/L Ca2+ (filled bars, n=8) and Ca2+-free buffer with 1 mmol/L EGTA (open bars, n=11). A significant decrease in [Ca2+]i oscillation frequency and duration and on the plateau [Ca2+]i increase was found in Ca2+-free buffer (*P<.05 vs buffer with 1.5 mmol/L Ca2+).

Roles of Intracellular Ca²⁺ Pool in H₂O₂-Induced Ca²⁺ Signaling
To further characterize the contribution of an intracellular Ca²⁺ store to the effect of H₂O₂ on HAEC [Ca²⁺]_i, HAEC monolayers were first exposed for 30 minutes to Ca²⁺-free/EGTA HBS to inhibit extracellular Ca²⁺ influx. The monolayers were then stimulated by one of three agonists to release the intracellular Ca²⁺ pool before exposure to 100 µmol/L H₂O₂. In some studies, supramaximal concentrations of histamine were used to release the InsP₃-sensitive ER Ca²⁺ store.^{16 17} In other experiments, HAEC monolayers were exposed to caffeine, which releases Ca²⁺ from an intracellular store in endothelial cells¹⁸ that is pharmacologically distinct from the InsP₃-releasable Ca²⁺ store and may affect the ryanodine-sensitive Ca²⁺ pool in endothelial cells.¹⁹ Finally, in a third set of experiments, intracellular Ca²⁺ stores were released by the ER Ca²⁺-ATPase inhibitor TG,²⁰ which largely depletes the InsP₃- and agonist-sensitive Ca²⁺ pools without activating the InsP₃ pathway.²¹

As shown in Fig 5, in Ca²⁺-free solution, 100 µmol/L histamine stimulated a large, transient increase in [Ca²⁺]_i of 1608.4±48.1 nmol/L (n=3), which has been shown to result from release of the ER Ca²⁺ pool mediated by stimulation of InsP₃ production.²² This concentration of histamine appears to deplete the histamine-releasable pool in HAECs because a subsequent application of histamine after washout did not affect [Ca²⁺]_i. After release of the histamine-sensitive Ca²⁺ pool in Ca²⁺-free solution, 100 µmol/L H₂O₂ increased [Ca²⁺]_i by only 66.8±2.2 nmol/L (versus 261.2±33.9 nmol/L in Ca²⁺-free buffer without histamine pretreatment; P<.05), and [Ca²⁺]_i oscillations were abolished.

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of H2O2 on HAEC [Ca2+]i after depletion of the histamine-sensitive intracellular Ca2+ pool in Ca2+-free buffer. Representative indo 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 after exposure to 100 µmol/L histamine (His) in HBS without added Ca2+ and with 1 mmol/L EGTA (present throughout). This concentration of histamine appeared to deplete the histamine-releasable intracellular Ca2+ pool because a subsequent application of histamine after washout (Wash) did not affect [Ca2+]i. H2O2 increased [Ca2+]i, but no oscillations were observed (n=3).

When HAEC monolayers were exposed to 10 mmol/L caffeine in Ca²⁺-free buffer for 20 minutes (Fig 6A), there was a gradual, slow, and persistent increase in [Ca²⁺]_i of 48.8±7.9 nmol/L (n=5; P<.001) that was fully reversible on washout of caffeine and was similar to the effect previously reported in this cell type.¹⁸ After caffeine exposure, 100 µmol/L H₂O₂ stimulated an additional increase in [Ca²⁺]_i (Fig 6B) to 252.5±33.5 nmol/L. The magnitude of this [Ca²⁺]_i increase (132.2±22.3 nmol/L, n=5) was reduced compared with Ca²⁺-free solution without caffeine pretreatment (261.2±33.9 nmol/L; P<.05), and [Ca²⁺]_i oscillations were again abolished.

View larger version (17K): [in this window] [in a new window]   Figure 6. Effect of H2O2 on HAEC [Ca2+]i after release of a caffeine-sensitive intracellular Ca2+ pool in Ca2+-free buffer. A, Representative indo 1 fluorescence obtained from an HAEC monolayer during stimulation with 10 mmol/L caffeine in HBS without added Ca2+ and with 1 mmol/L EGTA. A gradual increase in [Ca2+]i was observed (n=6) that was reversible on washout (Wash). B, Representative indo 1 fluorescence from an HAEC monolayer during exposure to 100 µmol/L H2O2 after caffeine (10 mmol/L) stimulation in HBS without added Ca2+ and with 1 mmol/L EGTA. After caffeine stimulation, 100 µmol/L H2O2 initiated a further increase in [Ca2+]i, but no [Ca2+]i oscillations were observed (n=5).

The ER Ca²⁺-ATPase inhibitor TG (1 µmol/L, Fig 7) stimulated a large, transient increase in [Ca²⁺]_i in Ca²⁺-free buffer (1434.7±224.7 nmol/L, n=3) and appeared to empty the TG-sensitive intracellular Ca²⁺ pool because a subsequent application of TG after washout did not affect [Ca²⁺]_i. The subsequent application of 100 µmol/L H₂O₂ increased [Ca²⁺]_i by an additional 108.8±30.1 nmol/L (versus 261.2±33.9 nmol/L in Ca²⁺-free solution without TG pretreatment; P<.05) but did not stimulate [Ca²⁺]_i oscillations.

View larger version (12K): [in this window] [in a new window]   Figure 7. Effect of H2O2 on HAEC [Ca2+]i after depletion of the TG-sensitive intracellular Ca2+ store. Representative indo 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 after stimulation with 1 µmol/L TG in HBS without added Ca2+ and with 1 mmol/L EGTA (present throughout). This concentration of TG appeared to empty the TG-sensitive intracellular Ca2+ pool because a subsequent application of TG after washout (Wash) did not affect [Ca2+]i. H2O2 increased [Ca2+]i, but no [Ca2+]i oscillations were observed (n=3).

Similar results were observed when two different agonists were used to deplete the intracellular Ca²⁺ store (Fig 8) to avoid the possibility of receptor desensitization, which may occur during sequential stimulation by the same agonist in vascular endothelial cells.²³ TG (1 µmol/L) increased [Ca²⁺]_i from 89.9±13.2 to 1402.9±170.8 nmol/L (n=4) and appeared to deplete the histamine-sensitive intracellular Ca²⁺ store because the subsequent application of 100 µmol/L histamine failed to increase [Ca²⁺]_i. Stimulation with 100 µmol/L H₂O₂ after histamine exposure still increased [Ca²⁺]_i, but the magnitude of the [Ca²⁺]_i increase was reduced compared with that observed in experiments in Ca²⁺-free buffer in which no pretreatment was used (90.1±15.1 versus 261.2±33.9 nmol/L; P<.01) and [Ca²⁺]_i oscillations were abolished.

View larger version (12K): [in this window] [in a new window]   Figure 8. Effect of H2O2 on HAEC [Ca2+]i after depletion of the TG- and histamine-sensitive intracellular Ca2+ stores. Representative indo 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 after stimulation first with 1 µmol TG (TG) and then with 100 µmol/L histamine (His) in HBS without added Ca2+ and with 1 mmol/L EGTA (present throughout). After TG stimulation and washout (Wash), application of histamine did not increase [Ca2+]i. H2O2 increased [Ca2+]i, but no [Ca2+]i oscillations were observed (n=4).

Comparison of Histamine- and H₂O₂-Stimulated [Ca²⁺]_i Oscillations in Ca²⁺-Free Buffer
In HUVECs,¹³ histamine stimulates [Ca²⁺]_i oscillations that rapidly decline in frequency and amplitude in the absence of buffer Ca²⁺. When buffer Ca²⁺ is withdrawn during an established response, the frequency and amplitude of histamine-induced [Ca²⁺]_i oscillations are immediately reduced in HUVECs.¹³ Fig 9A shows that removal of buffer Ca²⁺ has the same effect on histamine-induced [Ca²⁺]_i oscillations in HAECs. In contrast, removal of buffer Ca²⁺ has no obvious effect on established [Ca²⁺]_i oscillations induced by H₂O₂ (Fig 9B). Given the transient nature of the [Ca²⁺]_i oscillations in response to H₂O₂ even in the continuous presence of buffer Ca²⁺, it may be more difficult to accurately determine the effect of withdrawal of buffer Ca²⁺ during an established response.

View larger version (23K): [in this window] [in a new window]   Figure 9. Effect of buffer Ca2+ removal on established [Ca2+]i oscillations. A. Representative indo 1 fluorescence from an HAEC monolayer exposed to 1 µmol/L histamine (His) first in HBS with 1.5 mmol/L Ca2+ (noted as Ca2+ at the top of the tracing). After [Ca2+]i oscillations occurred, the buffer was changed to Ca2+-free solution with 1 mmol/L EGTA (Ca2+-free). The gap between Ca2+-containing and Ca2+-free conditions (70 seconds) is the time required for Ca2+-free buffer to fully replace Ca2+-containing buffer in the chamber. [Ca2+]i oscillations were not observed after removal of buffer Ca2+ but were rapidly restored when cells were again exposed to Ca2+- containing solution (n=3). B, Representative indo 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 first in HBS with 1.5 mmol/L Ca2+ (noted as Ca2+ at the top of the tracing). After the initial [Ca2+]i response, the buffer was changed to Ca2+-free solution with 1 mmol/L EGTA (Ca2+-free) (n=6).

	Discussion

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The present study is the first to document H₂O₂-induced [Ca²⁺]_i oscillations HAECs. Previous authors have reported that oxidant stress increases endothelial [Ca²⁺]_i^24–27 and inhibits agonist-stimulated Ca²⁺ influx.²⁸ In addition to differences in species or vascular bed of origin between these studies and the present study, prior work examined confluent or postconfluent monolayers as opposed to the subconfluent monolayers examined in the present study. Endothelial cell culture conditions and degree of confluence may influence the appearance of [Ca²⁺]_i oscillations^{29 30} and may explain the failure to observe [Ca²⁺]_i oscillations in prior work.

After the initial report of [Ca²⁺]_i oscillations in cultured HUVECs in response to histamine,¹³ endothelial [Ca²⁺]_i oscillations were also reported after stimulation with thrombin,³¹ bradykinin,³² and ATP.³² When HUVECs are exposed to low concentrations of histamine (0.1 to 3 µmol/L), oscillatory changes in [Ca²⁺]_i occur¹³ that are similar in many respects to the [Ca²⁺]_i oscillations reported here in HAECs after stimulation with 100 µmol/L H₂O₂. In both situations, oscillations typically occur at a frequency of 0.7 min^-1 and consist of an increase in [Ca²⁺]_i from 100 nmol/L to 1 µmol/L before [Ca²⁺]_i rapidly declines again. The frequency of histamine-induced [Ca²⁺]_i oscillations in HUVECs¹³ and of H₂O₂-stimulated [Ca²⁺]_i oscillations in HAECs both increase with increasing agonist concentration until (at higher concentrations) oscillations appear to "fuse"^{13 17} (Fig 1C) before a sustained elevation of [Ca²⁺]_i is observed. The [Ca²⁺]_i oscillations in response to low concentrations of histamine in HUVECs reported by Jacob et al¹³ were also observed when HAEC monolayers were exposed to 0.3 µmol/L (n=4) and 1 µmol/L (n=5) histamine (data not shown). At high concentrations of both agonists (100 µmol/L histamine or 10 mmol/L H₂O₂), oscillations were never observed in HAECs (data not shown).

There are differences between the spiking behavior of the histamine-induced [Ca²⁺]_i oscillations observed in HUVECs¹³ and the spiking behavior of the H₂O₂-induced [Ca²⁺]_i oscillations reported in the present study. In Ca²⁺-containing buffer, histamine-stimulated [Ca²⁺]_i oscillations in HUVECs persist for 25 to 30 minutes without an appreciable decrease in oscillation amplitude.¹³ [Ca²⁺]_i oscillations similarly persisted when HAECs were exposed to 1 µmol/L histamine (n=5) but steadily declined in amplitude when experiments were performed in Ca²⁺-free solution (n=5, data not shown). In contrast, H₂O₂-induced [Ca²⁺]_i oscillations decreased in amplitude even in the presence of buffer Ca²⁺ (Fig 1A) before oscillations were no longer observed. This suggests that in contrast to what occurs with histamine, the source of Ca²⁺ for H₂O₂-induced [Ca²⁺]_i oscillations becomes depleted even in the presence of external Ca²⁺. Alternatively, H₂O₂ may stimulate two distinct and opposite intracellular signaling pathways, one of which initiates [Ca²⁺]_i oscillations, and another (which may be preferentially activated at higher H₂O₂ concentrations) that inhibits this spiking behavior.

The different spiking behavior observed for H₂O₂- and histamine-induced [Ca²⁺]_i oscillations implies different mechanisms for these oscillations. Agonist-stimulated repetitive [Ca²⁺]_i spikes observed in HUVECs are believed to be initiated by increases in the level of InsP₃,^{13 17} and extracellular Ca²⁺ appears to be required to replete the InsP₃-sensitive intracellular Ca²⁺ store responsible for the oscillations.¹³ Stimulation of InsP₃ has been well documented in response to several agonists that induce oscillations, such as histamine and thrombin (in HUVECs),²² ATP (in bovine aortic endothelial cells),³³ and bradykinin (in porcine aortic endothelial cells).³⁴ The model suggested for agonist-induced [Ca²⁺]_i oscillations¹⁶ explains the observations that [Ca²⁺]_i oscillations in response to histamine persist without a decrement in oscillation amplitude in the presence of external Ca²⁺ and that a rapid decrease in both frequency and amplitude of histamine-induced oscillations occurs when Ca²⁺ is removed from the buffer.¹³

The situation appears to be different for the [Ca²⁺]_i oscillations stimulated by H₂O₂. There is no evidence that H₂O₂ stimulates InsP₃ production at the micromolar concentrations that stimulated [Ca²⁺]_i oscillations in the present study.²⁵ In contrast to the situation with histamine,¹³ H₂O₂-induced [Ca²⁺]_i oscillations decrease in amplitude and persist for 12.0 minutes in Ca²⁺-containing buffer, indicating that the intracellular Ca²⁺ store responsible for the oscillations is not sufficiently repleted by external Ca²⁺ to maintain continued oscillatory [Ca²⁺]_i changes.

The initial, gradual increase in [Ca²⁺]_i after H₂O₂ stimulation occurred at least in part via release of an intracellular Ca²⁺ store sensitive to histamine, caffeine, or TG, because the H₂O₂-induced [Ca²⁺]_i increase during the latency period was lower after pretreatment with these agonists than it was when they were not used. TG depletes the ER Ca²⁺ store in endothelial cells,^{20 26} and both the caffeine- and InsP₃-sensitive Ca²⁺ stores appear to be part of the endothelial ER Ca²⁺ store.¹⁸ The complete inhibition of H₂O₂-induced [Ca²⁺]_i oscillations by prior emptying of these stores suggests that there is, at least in part, some overlap between these stores and the intracellular Ca²⁺ pool in HAECs that is responsible for [Ca²⁺]_i oscillations in response to H₂O₂. In support of this, stimulation of HAEC monolayers in Ca²⁺-free buffer with either histamine (100 µmol/L, n=4) or TG (1 µmol/L, n=3) after H₂O₂ stimulation and washout failed to increase [Ca²⁺]_i (data not shown).

H₂O₂ increased [Ca²⁺]_i to a level of 250 to 300 nmol/L before oscillations occurred. This [Ca²⁺]_i level of 250 to 300 nmol/L appears to be a "threshold" level reached before [Ca²⁺]_i oscillations because it was fairly constant over an H₂O₂ concentration range of 100 to 500 µmol/L and invariably was observed before [Ca²⁺]_i oscillations in both the presence and absence of external Ca²⁺. The threshold [Ca²⁺]_i rise may be necessary to trigger [Ca²⁺]_i oscillations, but it is not sufficient because millimolar concentrations of H₂O₂ increase [Ca²⁺]_i above the threshold without triggering [Ca²⁺]_i oscillations. It does not appear that depletion of the ER Ca²⁺ store indirectly blocks H₂O₂-induced [Ca²⁺]_i oscillations by preventing the threshold [Ca²⁺]_i increase during the initial latency period because caffeine pretreatment abolished H₂O₂-induced [Ca²⁺]_i oscillations but did not prevent H₂O₂ from increasing [Ca²⁺]_i to threshold (252.5±33.5 versus 248.3±21.4 nmol/L in Ca²⁺-free buffer without caffeine pretreatment; P=NS).

Thus, H₂O₂ induces [Ca²⁺]_i oscillations in HAECs that result from release of an ER Ca²⁺ store. Although further studies will be required to elucidate the molecular basis by which H₂O₂ and other oxidants affect endothelial ER Ca²⁺ storage and release mechanisms, previous work indicates that oxygen radicals may inhibit normal ER Ca²⁺-ATPase function. The H₂O₂-generating enzyme xanthine oxidase depletes the InsP₃-sensitive Ca²⁺ store in vascular endothelial cells,³⁵ even though H₂O₂ itself does not appear to stimulate InsP₃ production.²⁵ This suggests that H₂O₂ may release an intracellular (ER) Ca²⁺ store in an InsP₃-independent manner, much like the ER Ca²⁺-ATPase inhibitor TG.²⁰ Indeed, previous work indicates that H₂O₂ reduces the TG-mobilizable Ca²⁺ pool in WEH17.2 and W.Hb13 cells³⁶ and inhibits the (sarco)plasmic reticulum Ca²⁺-ATPase in a variety of cell types.^{37 38} This effect of oxidants on the Ca²⁺-ATPase may be mediated by direct attack on the ATP binding site of the enzyme.³⁹ Alternative mechanisms by which H₂O₂ and other oxidants affect endothelial ER Ca²⁺ storage and release mechanisms must be considered given the effects of H₂O₂ on critical cellular components. Because elevated oxidant production occurs in postischemic/anoxic human aortic,⁴⁰ human umbilical vein,⁴¹ and bovine pulmonary artery⁴² endothelial cells in the high-to-low micromolar range and is associated with impaired endothelium-dependent relaxation,¹ the effects of micromolar concentrations of H₂O₂ on endothelial Ca²⁺ signaling described in the present study may be important in the pathogenesis of postischemic endothelial dysfunction.

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular Ca2+ concentration ER = endoplasmic reticulum HAEC = human aortic endothelial cell HBS = HEPES-buffered saline HUVEC = human umbilical vein endothelial cell InsP3 = inositol trisphosphate PMN = polymorphonuclear leukocyte TG = thapsigargin

	Acknowledgments

 
This work was supported in part by National Heart, Lung, and Blood Institute grants HL-03102 and HL-52315.

Received July 15, 1997; revision received September 22, 1997; accepted September 25, 1997.

	References

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