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(Circulation. 2000;102:2541.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Hypoxia/Reoxygenation Stimulates Intracellular Calcium Oscillations in Human Aortic Endothelial Cells

Qinghua Hu, PhD; Roy C. Ziegelstein, MD

From the Department of Medicine, Division of Cardiology, Johns Hopkins Bayview Medical Center, Johns Hopkins University School of Medicine, Baltimore, Md.

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 rziegel{at}jhmi.edu

	Abstract

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Background—We have previously shown that hydrogen peroxide stimulates endothelial [Ca²⁺]_i oscillations. This study was performed to determine whether posthypoxic reoxygenation stimulates [Ca²⁺]_i oscillations in vascular endothelial cells.

Methods and Results—Hypoxia (glucose-free 95% N₂/5% CO₂ bicarbonate buffer for 60 minutes) stimulated an increase in [Ca²⁺]_i from 111.9±7.9 to 161.7±17.7 nmol/L (n=12, P<0.01) in indo 1–loaded human aortic endothelial cells. On reoxygenation (glucose-containing 95% air/5% CO₂ bicarbonate buffer), 13 of 16 cells responded with repetitive [Ca²⁺]_i oscillations with an average amplitude of 570.6±59.3 nmol/L, occurring at a mean interval of 0.28±0.04/min and persisting for 60 minutes. [Ca²⁺]_i oscillations were still observed in 4 of 7 cells studied in Ca²⁺-free buffer but did not occur when the intracellular Ca²⁺ store was first depleted during hypoxia by either 1 µmol/L thapsigargin or by 10 mmol/L caffeine (n=6 for each). Reoxygenation-induced [Ca²⁺]_i oscillations were abolished by 10 µmol/L diphenyleneiodonium, an inhibitor of NAD(P)H oxidase (n=7), and by polyethylene glycol (PEG)–catalase (5000 U/mL, n=4) but were not prevented by inhibitors of xanthine oxidase (n=5), cyclooxygenase (n=4), nitric oxide synthase (n=5), the mitochondrial electron transport chain (n=4), or by PEG–superoxide dismutase (n=5).

Conclusions—Posthypoxic reoxygenation stimulates repetitive [Ca²⁺]_i oscillations that are dependent on Ca²⁺ release from an intracellular pool and require extracellular Ca²⁺ to be maintained. These oscillations may be initiated by NAD(P)H oxidase–derived hydrogen peroxide and may play a role in signal transduction during ischemia/reperfusion in vivo.

Key Words: calcium • endothelium • hypoxia

	Introduction

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The restoration of blood flow to ischemic tissue can impair endothelium-dependent vasodilation,^{1 2} increase the tendency for endothelial cells to adhere to circulating leukocytes by upregulating the expression of cell adhesion molecules,^{3 4} and increase vascular permeability.⁵ Reactive oxygen species (ROS) play an important role in the initiation and amplification of postischemic endothelial dysfunction.⁶ Several sources of ROS generation by endothelial cells exposed to hypoxia/reoxygenation (H/R) have been reported,^{7 8 9 10 11} including an H₂O₂-generating NAD(P)H oxidase.^{9 11}

Hypoxia was previously shown to increase endothelial cytosolic calcium ([Ca²⁺]_i), followed by a gradual return to basal [Ca²⁺]_i levels after reoxygenation.¹² We have shown¹³ that H₂O₂ stimulates repetitive [Ca²⁺]_i oscillations in human aortic endothelial cells (HAECs). Because H/R stimulates ROS generation by endothelial cells and H₂O₂ initiates endothelial [Ca²⁺]_i oscillations, we hypothesized that H/R stimulates [Ca²⁺]_i oscillations in HAECs. The present study reports the first observation of H/R-stimulated [Ca²⁺]_i oscillations in vascular endothelial cells and the possible role of NAD(P)H oxidase–derived H₂O₂ in this type of Ca²⁺ signaling.

	Methods

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Culture of HAECs
HAECs were obtained as proliferating cultures at the fourth passage (Clonetics) and were grown as previously described¹³ in endothelial cell growth medium supplemented with 2% FBS, with addition of 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. The cells are obtained from a single donor, and each group of cells has been shown by Clonetics to test positive for acetylated LDL and von Willebrand factor and negative for smooth muscle -actin. After reaching confluence, cells were passaged an additional 1 to 5 times; HAECs were not used after the ninth passage. To study the effects of H/R on HAEC [Ca²⁺]_i, HAECs were grown on 25-mm-diameter circular glass coverslips (VWR Scientific) precoated with 2% gelatin solution (Sigma Chemical Co) for at least 2 hours at 37°C. The glass coverslips were washed 3 times with PBS (Quality Biological, Inc) before cell seeding. After exposure to a solution of 0.025% trypsin and 0.01% hydroxyethyl-EDTA (Sigma) for 1 to 2 minutes, cells detached, and the suspension was then centrifuged at 1000 rpm for 10 minutes at room temperature. The supernatant was discarded, the cells were then resuspended in growth medium, and 0.5 mL of the cell suspension (1x10⁵ cells/mL) was plated on each glass coverslip. Cells were used for experiments after reaching 70% confluence after incubation for 1 to 2 days at 37°C in a humidified atmosphere of 95% air/5% CO₂.

Measurement of Intracellular Free Ca²⁺ Concentration During H/R
HAEC [Ca²⁺]_i was measured as previously described¹³ with the fluorescent Ca²⁺ probe indo 1. An indo 1 stock solution was made by dissolving 50 µg of the acetoxymethyl ester (AM) form of indo 1 (Molecular Probes) in 49.5 µL DMSO (Sigma). HAEC monolayers on glass coverslips were incubated with culture medium containing 10 µmol/L indo 1-AM (final DMSO concentration 1 vol%) in a room-temperature 95% air/5% CO₂ atmosphere for 30 minutes. The coverslips were then maintained for at least 30 minutes in indicator-free bicarbonate-buffered saline (BBS) containing (in mmol/L) NaCl 116.4, KCl 5.4, MgSO₄ 1.6, CaCl₂ 1.5, NaH₂PO₄ 1.0, D-glucose 5.6, and NaHCO₃ 26.2 continuously gassed with 95% air and 5% CO₂ to allow for deesterification of the indicator. Indo 1 fluorescence was recorded from a single cell within a group of 2 to 3 connected cells of a subconfluent HAEC monolayer on a coverslip. The coverslip was placed in a perfusion chamber (volume of solution 2.5 mL) mounted on the stage of a modified Nikon Diaphot inverted epifluorescence microscope. The perfusion chamber was covered with a plastic top to decrease diffusion of gas, and the chamber itself was gassed through a side port with the same gas mixture that was used to continuously gas the perfusate. HAECs were perfused at a flow rate of 6.2 mL/min, allowing the solution in the chamber to be completely replaced in 30 seconds. To simulate ischemia-reperfusion, HAECs were first exposed to hypoxia by switching the perfusion solution from BBS to glucose-free BBS continuously gassed with 95% N₂/5% CO₂ for 60 minutes. After this period, cells were reoxygenated in glucose-containing BBS balanced with 95% air and 5% CO₂. Indo 1 fluorescence was excited at 350±50 nm with a xenon short-arc lamp (UXL-75 XE, Ushio Inc), and bandpass interference filters (Omega Optical) selected wavelength bands of emitted fluorescence at 405±10 nm and 485±10 nm, corresponding to the Ca²⁺-bound and Ca²⁺-free forms of the indicator, respectively. Emitted indo 1 fluorescence was collected and measured with a spectrofluorimeter (PTI, Deltascan). Autofluorescence from unloaded HAECs at each wavelength was generally <10% of indo 1–loaded HAECs. Because autofluorescence changed little during the course of the experiment, a single value measured at the start of the experiment was subtracted automatically from indo 1 fluorescence recordings.

To determine [Ca²⁺]_i from indo 1 fluorescence ratios, the intracellular minimum and maximum ratios (R_min and R_max, respectively) were determined as previously described.¹⁴ To determine R_min, indo 1–loaded HAEC monolayers on the glass coverslips were perfused with a solution containing (in mmol/L) NaCl 137, KCl 5.0, MgSO₄ 1.2, NaH₂PO₄ 1.2, D-glucose 16, HEPES 10, and EGTA 2, pH 7.40. HAEC monolayers were then exposed to a solution of similar composition except with 10 mmol/L EGTA and 0.05% Triton X-100. An intracellular R_max was determined by first perfusing HAEC monolayers with a solution containing 132 mmol/L KCl, 10 mmol/L K-HEPES, 1 mmol/L MgSO₄, 2 µmol/L rotenone (Sigma), 2 µmol/L FCCP (Sigma), and 10 ng/mL valinomycin (Calbiochem). HAECs were then exposed to a similar solution containing 2 µmol/L ionomycin (Sigma), 69.2 mmol/L CaCl₂, and 100 mmol/L HEDTA (free [Ca²⁺] of 5900 nmol/L). The values of intracellular R_min and R_max were used to calculate [Ca²⁺]_i according to the following equation: [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 indicators, respectively. K_d was determined to be 207 nmol/L under the present experimental conditions by use of an in vitro calibration method.

Data Analysis and Statistics
Data are reported as mean±SEM. Reported n values indicate the number of single cells from which separate fluorescence recordings were obtained in each experimental protocol. Statistical comparisons were made by use of Student’s t test for the paired and the unpaired groups. ANOVA was used when multiple group comparisons were performed. A difference was considered significant at a value of P<0.05.

	Results

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Effects of H/R on [Ca²⁺]_i in HAECs
In Ca²⁺-containing BBS, basal [Ca²⁺]_i (obtained immediately before the onset of hypoxia) was 111.9±7.9 nmol/L, and it increased to 161.7±17.7 nmol/L during a 60-minute period of hypoxia (n=12, P<0.01, Figure 1). The increase in [Ca²⁺]_i occurred over the first 4.5±0.8 minutes of hypoxia, after which [Ca²⁺]_i reached a plateau that was maintained for the duration of hypoxic exposure. The reported value for [Ca²⁺]_i during hypoxia was obtained after [Ca²⁺]_i reached this plateau. In BBS without added Ca²⁺ and with 1 mmol/L EGTA, basal [Ca²⁺]_i was 86.8±13.6 nmol/L under normoxic conditions and did not increase during the 60-minute period of hypoxia (peak [Ca²⁺]_i=94.4±13.3 nmol/L, n=4, P=NS versus baseline).

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of hypoxia on HAEC [Ca2+]i. Representative indo 1 fluorescence from an HAEC monolayer exposed to glucose-free hypoxia (95% N2/5% CO2, indicated by H) after a period of normoxia (N, only 3 minutes shown). Hypoxia produced an increase in [Ca2+]i (n=12, P<0.01), which occurred shortly after transition from normoxia and then reached a plateau that was maintained for duration of hypoxic exposure.

After 60 minutes of hypoxia in Ca²⁺-containing BBS, repetitive [Ca²⁺]_i oscillations were observed during reoxygenation in 13 of 16 HAECs examined (Figure 2A). Oscillations typically occurred after a latency period (the interval between reoxygenation and the first large peak of the [Ca²⁺]_i transient¹⁶ ) of 3.2±0.5 minutes. [Ca²⁺]_i oscillations consisted of transient, large increases in [Ca²⁺]_i, each followed by a rapid decrease to a [Ca²⁺]_i level that was approximately the same as the preceding baseline. [Ca²⁺]_i oscillations demonstrated some heterogeneity in amplitude and interspike interval (Figure 2A) but had an average amplitude of 570.6±59.3 nmol/L, occurred at a mean interval of 0.28±0.04/min, and persisted for the 60 minutes of posthypoxic observation. [Ca²⁺]_i oscillations were still observed in 4 of 7 HAECs in which reoxygenation occurred in Ca²⁺-free buffer with 1 mmol/L EGTA. In the 4 HAECs in which oscillations were observed, the [Ca²⁺]_i spikes were maintained for only 20 minutes, after which no further [Ca²⁺]_i oscillations were observed (Figure 2B).

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of hypoxia/reoxygenation on HAEC [Ca2+]i. A, Representative indo 1 fluorescence from an HAEC monolayer exposed to hypoxia (95% N2/5% CO2, indicated by H) and then reoxygenated (REOX, 95% air/5% CO2), both after a period of normoxia (N, only 2 minutes shown) in buffer with 1.5 mmol/L Ca2+. (Conditions also apply to B and C.) Repetitive [Ca2+]i oscillations were observed during reoxygenation in 13 of 16 HAEC monolayers studied. Note difference in time scale during hypoxia and remainder of tracing. B, Indo 1 fluorescence from an HAEC monolayer exposed to hypoxia and then reoxygenated, both after a period of normoxia in buffer without added Ca2+ and with 1 mmol/L EGTA. Trace is representative of 2 of 3 monolayers reoxygenated in Ca2+-free buffer; third did not demonstrate oscillations. Note difference in time scale during hypoxia and remainder of tracing. C, Representative indo 1 fluorescence from an HAEC monolayer exposed to endoplasmic reticulum Ca2+-ATPase inhibitor TG (1 µmol/L) during hypoxia and reoxygenation, both after a period of normoxia. [Ca2+]i oscillations were not observed during reoxygenation when intracellular Ca2+ store was depleted during preceding period of hypoxia (n=3). Note difference in time scale during hypoxia and remainder of tracing.

To further examine the mechanism of [Ca²⁺]_i oscillations during reoxygenation, HAECs were pretreated with either thapsigargin (TG, 1 µmol/L) or 10 mmol/L caffeine during hypoxia to deplete the intracellular Ca²⁺ store before reoxygenation. The endoplasmic reticulum Ca²⁺-ATPase inhibitor TG largely depletes the inositol 1,4,5-trisphosphate (InsP₃)– and agonist-sensitive Ca²⁺ pools without activating the InsP₃ pathway.¹⁷ Caffeine releases Ca²⁺ from an intracellular store in endothelial cells that is pharmacologically distinct from the InsP₃-releasable Ca²⁺ pool.¹⁸ When HAECs were exposed to TG during hypoxia, an increase in [Ca²⁺]_i of 439.5±65.7 nmol/L was observed, which then returned to a plateau [Ca²⁺]_i of 257.5±30.5 nmol/L during continued exposure to TG (n=6, Figure 2C). The prior depletion of an intracellular Ca²⁺ store with TG prevented [Ca²⁺]_i oscillations during reoxygenation, even with 1.5 mmol/L Ca²⁺ present in the buffer solution. Similar results were observed when the intracellular store was first depleted with caffeine during hypoxia (n=6, data not shown). Thus, the initiation of [Ca²⁺]_i oscillations during reoxygenation appears to be dependent on Ca²⁺ release from an intracellular pool and independent of extracellular Ca²⁺. Maintenance of oscillations during reoxygenation appears to require extracellular Ca²⁺, possibly because of a requirement of the intracellular pool to be refilled.

Effect of Inhibitors of ROS-Generating Enzyme Systems on Reoxygenation-Stimulated [Ca²⁺]_i Oscillations in HAECs
To examine the effect of inhibitors of ROS-generating enzymes on reoxygenation-stimulated [Ca²⁺]_i oscillations, HAECs were pretreated with one of the following enzyme inhibitors for 30 minutes before hypoxia, and the inhibitors were present both during hypoxia and subsequent reoxygenation: (1) the flavoprotein inhibitor diphenyleneiodonium (DPI, 10 µmol/L), which is a known inhibitor of the NAD(P)H oxidase; (2) the xanthine oxidase inhibitor oxypurinol (100 µmol/L); (3) the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L); (4) the cyclooxygenase inhibitor indomethacin (10 µmol/L); or (5) the mitochondrial electron transport chain inhibitor rotenone (100 µmol/L). In each case, the concentration of the inhibitor used was based on previous use of enzyme inhibitors in studies of the effects of H/R^{4 9 19 20} or of ROS production by endothelial cells.²¹ The effectiveness of these inhibitor concentrations was also verified by measuring the ability for DPI, oxypurinol, indomethacin, and rotenone at these concentrations to block the intracellular production of ROS during H/R (measured by the fluorescent indicator 2',7'-dichlorofluorescin diacetate, diacetoxymethyl ester, DCFH-DA) and by measuring the ability for this concentration of L-NAME to block histamine-stimulated NO production using the fluorescent probe 4,5-diaminofluorescein diacetate (data not shown).

None of the 5 enzyme inhibitors affected basal [Ca²⁺]_i. As shown in Figure 3A, DPI prevented the generation of [Ca²⁺]_i oscillations on reoxygenation (n=7) but did not affect the generation of [Ca²⁺]_i oscillations stimulated by exogenous H₂O₂ (100 µmol/L). In contrast, [Ca²⁺]_i oscillations persisted in HAECs treated with oxypurinol, L-NAME, indomethacin, and rotenone (n=4 to 5 for each, Figure 3B through 3E). Of note, the decline in the amplitude of H₂O₂-induced [Ca²⁺]_i oscillations shown in Figure 3A is a consistent observation even under control conditions.¹³

View larger version (26K): [in this window] [in a new window]   Figure 3. Effect of inhibitors of ROS-generating enzyme systems on reoxygenation-stimulated [Ca2+]i oscillations in HAECs. A, Representative indo 1 fluorescence from an HAEC monolayer pretreated for 30 minutes with NAD(P)H oxidase inhibitor DPI (10 µmol/L, present throughout) and then exposed to hypoxia (95% N2/5% CO2, indicated by H) and reoxygenation (REOX, 95% air/5% CO2), both after a period of normoxia (N, only 2 minutes shown). (Conditions also apply to B through E.) DPI prevented reoxygenation-stimulated [Ca2+]i oscillations without affecting [Ca2+]i oscillations triggered by 100 µmol/L H2O2 (n=7). Note difference in time scale during hypoxia and remainder of tracing. B, Representative indo 1 fluorescence from an HAEC monolayer pretreated for 30 minutes with xanthine oxidase inhibitor oxypurinol (100 µmol/L, present throughout) and then exposed to hypoxia and reoxygenation, both after a period of normoxia. Oxypurinol did not inhibit reoxygenation-stimulated [Ca2+]i oscillations (n=5). Note difference in time scale during hypoxia and remainder of tracing. C, Representative indo 1 fluorescence from an HAEC monolayer pretreated for 30 minutes with NO synthase inhibitor L-NAME (1 mmol/L, present throughout) and then exposed to hypoxia and reoxygenation, both after a period of normoxia. L-NAME did not inhibit reoxygenation-stimulated [Ca2+]i oscillations (n=5). Note difference in time scale during hypoxia and remainder of tracing. D, Representative indo 1 fluorescence from an HAEC monolayer pretreated for 30 minutes with cyclooxygenase inhibitor indomethacin (10 µmol/L, present throughout) and then exposed to hypoxia and reoxygenation, both after a period of normoxia. Indomethacin did not inhibit reoxygenation-stimulated [Ca2+]i oscillations (n=4). Note difference in time scale during hypoxia and remainder of tracing. E, Representative indo 1 fluorescence from an HAEC monolayer pretreated for 30 minutes with mitochondrial inhibitor rotenone (100 µmol/L, present throughout) and then exposed to hypoxia and reoxygenation, both after a period of normoxia. Rotenone did not inhibit reoxygenation-stimulated [Ca2+]i oscillations (n=4). Note difference in time scale during hypoxia and remainder of tracing.

Effect of Free Radical Scavengers on Reoxygenation-Stimulated [Ca²⁺]_i Oscillations in HAECs
Because we previously demonstrated that H₂O₂ stimulates [Ca²⁺]_i oscillations in HAECs, we examined the effects of polyethylene glycol (PEG)–catalase and PEG–superoxide dismutase (SOD) on reoxygenation-stimulated [Ca²⁺]_i oscillations in HAECs. For these experiments, HAECs were incubated for 24 hours with either PEG-SOD (1000 U/mL, Sigma) or PEG-catalase (5000 U/mL, Sigma) before exposure to H/R. The concentrations of PEG-SOD and PEG-catalase were chosen on the basis of previous experiments in endothelial cells,²² and the effectiveness of this concentration of PEG-catalase was verified by measuring the DCFH oxidation rate during posthypoxic reoxygenation in HAECs. As shown in Figure 4A, PEG-SOD did not affect H/R-stimulated Ca²⁺ signaling in HAECs. During hypoxia, [Ca²⁺]_i increased from 107.1±15.6 to 182.6±12.4 nmol/L ([Ca²⁺]_i= 75.5±16.2 nmol/L, n=5, P=NS versus control). On reoxygenation, PEG-SOD did not affect the latency period before oscillations were observed (3.4±0.9 minutes) or the average amplitude (725.0±92.1 nmol/L) or mean interval of [Ca²⁺]_i oscillations (0.18±0.02/min, P=NS for each). In contrast, PEG-catalase pretreatment prevented the generation of [Ca²⁺]_i oscillations on reoxygenation of HAECs (Figure 4B) without affecting the increase in [Ca²⁺]_i during hypoxia (122.4±20.6 to 195.4±5.2 nmol/L, [Ca²⁺]_i=73.0±25.1 nmol/L, n=4, P=NS versus control) or the response to the Ca²⁺ ionophore ionomycin. PEG-catalase also did not affect the peak amplitude of the increase in [Ca²⁺]_i stimulated by 100 µmol/L histamine in Ca²⁺-free buffer (892.6±63.5 versus 901.1±53.1 nmol/L, n=3, P=NS), suggesting that its effect was not related to a more general effect on intracellular Ca²⁺ release and agonist-stimulated [Ca²⁺]_i oscillations.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effect of free radical scavengers on reoxygenation-stimulated [Ca2+]i oscillations in HAECs. A, Representative indo 1 fluorescence from an HAEC monolayer pretreated for 24 hours with free radical scavenger PEG-SOD (1000 U/mL) and then exposed to hypoxia (95% N2/5% CO2, indicated by H) and reoxygenation (REOX, 95% air/5% CO2), both after a period of normoxia (N, only 2 minutes shown). (Conditions also apply to B.) PEG-SOD did not inhibit reoxygenation-stimulated [Ca2+]i oscillations (n=5). Note difference in time scale during hypoxia and remainder of tracing. B, Representative indo 1 fluorescence from an HAEC monolayer pretreated for 24 hours with free radical scavenger PEG-catalase (5000 U/mL) and then exposed to hypoxia and reoxygenation, both after a period of normoxia. PEG-catalase abolished reoxygenation-stimulated [Ca2+]i oscillations without affecting response to ionomycin (n=4). Note difference in time scale during hypoxia and remainder of tracing.

As summarized in Figure 5, [Ca²⁺]_i oscillations during reoxygenation were abolished by DPI and PEG-catalase. Rotenone decreased the mean interval of [Ca²⁺]_i oscillations during reoxygenation (0.11±0.01/min versus 0.28±0.04/min, P<0.05, n=4) without affecting latency or oscillation amplitude. Neither PEG-SOD nor any of the enzyme inhibitors except rotenone affected latency, amplitude, duration, rate of rise or rate of fall of the [Ca²⁺]_i spike, or mean interval of [Ca²⁺]_i oscillations (n=4 to 5, P=NS for each). The peak amplitude of the increase in [Ca²⁺]_i stimulated by 100 µmol/L histamine (892.6±63.5 nmol/L in Ca²⁺-free buffer) was not different in HAECs pretreated with DPI (911.9±253.7 nmol/L) or rotenone (810.3±90.0 nmol/L, n=3, P=NS for each), suggesting that the effect of DPI and rotenone on [Ca²⁺]_i oscillations during reoxygenation was not due to a more general effect of these inhibitors on intracellular Ca²⁺ release and agonist-stimulated [Ca²⁺]_i oscillations.

View larger version (16K): [in this window] [in a new window]   Figure 5. Effect of inhibitors of ROS-generating enzyme systems and free radical scavengers on reoxygenation-stimulated [Ca2+]i oscillations in HAECs. A, Averaged data comparing effect of inhibitors of ROS-generating enzyme systems and free radical scavengers on latency (interval between reoxygenation and first large peak of [Ca2+]i transient) of reoxygenation-induced [Ca2+]i oscillations in HAECs. Except for DPI and PEG-catalase (PEG-CAT), which abolished oscillations, neither PEG-SOD nor any inhibitors (n=4 to 5 for each) affected latency period compared with control (n=13, P=NS for each). B, Averaged data comparing effect of inhibitors of ROS-generating enzyme systems and free radical scavengers on amplitude of reoxygenation-induced [Ca2+]i oscillations in HAECs. Except for DPI and PEG-catalase (PEG-CAT), which abolished oscillations, neither PEG-SOD nor any inhibitors (n=4 to 5 for each) affected [Ca2+]i oscillation amplitude compared with control (n=13, P=NS for each). C, Averaged data comparing effect of inhibitors of ROS-generating enzyme systems and free radical scavengers on mean interval of reoxygenation-induced [Ca2+]i oscillations in HAECs. Other than DPI and PEG-catalase (PEG-CAT), which abolished oscillations, only rotenone (n=4) decreased [Ca2+]i oscillation frequency compared with control (n=13, *P<0.05).

	Discussion

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The present study is the first to document that reoxygenation stimulates [Ca²⁺]_i oscillations in HAECs after a period of hypoxia. Although the baseline interspike [Ca²⁺]_i remained in the 150 to 200 nmol/L range, [Ca²⁺]_i periodically exceeded 500 nmol/L during repetitive [Ca²⁺]_i oscillations that lasted for up to 60 minutes in the presence of buffer Ca²⁺. On the basis of experiments in which Ca²⁺ influx was prevented in Ca²⁺-free buffer and others in which the intracellular Ca²⁺ store was pharmacologically depleted before reoxygenation, it appears that the intracellular Ca²⁺ store is responsible for the initiation of [Ca²⁺]_i oscillations and that extracellular Ca²⁺ is necessary for the maintenance of [Ca²⁺]_i oscillations, probably by refilling the endoplasmic reticulum Ca²⁺ store. This mechanism is similar to that described for the [Ca²⁺]_i oscillations stimulated by histamine in human endothelial cells.²³

Multiple ROS-generating systems are activated during reoxygenation, including xanthine oxidase,^{7 8 10 22} NO synthase,¹⁰ cyclooxygenase,²⁴ the mitochondrial electron transport chain,²⁵ and NAD(P)H oxidase.^{9 11} Our data suggest that [Ca²⁺]_i oscillations during reoxygenation may be stimulated by NAD(P)H oxidase–derived H₂O₂. This is consistent with previous work showing that H/R stimulates an H₂O₂-generating NAD(P)H oxidase⁹ and that intracellular ROS generation during H/R in endothelial cells requires the small GTP-binding protein rac1, a component of NADPH oxidase.¹¹ Because DPI is not a specific inhibitor of NAD(P)H oxidase, however, it is possible that alternative sources of ROS may be responsible for, or at least may modulate, [Ca²⁺]_i oscillations observed on reoxygenation after a period of hypoxia.

It is now known that gene expression driven by Ca²⁺-sensitive proinflammatory transcription factors like nuclear factor-B is regulated by the frequency of [Ca²⁺]_i oscillations in HAECs.²⁶ [Ca²⁺]_i oscillations may be an adaptive form of cell signaling during relatively brief exposures to H/R, avoiding the lethal effects of sustained [Ca²⁺]_i elevations, which have been implicated in oxidant-mediated cytotoxicity in vascular endothelial cells.²⁷ These more brief and sublethal exposures to H/R may result in the generation of [Ca²⁺]_i oscillations characterized by peak [Ca²⁺]_is, which only periodically exceed the threshold for activation and thereby avoid cell injury. Thus, our finding that [Ca²⁺]_i oscillations occur during posthypoxic reoxygenation of HAECs may be important in many of the physiological and pathophysiological responses that occur during ischemia/reperfusion in vivo.

	Acknowledgments

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

Received May 2, 2000; revision received June 15, 2000; accepted June 15, 2000.

	References

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