Critical Role of NADPH Oxidase-derived Reactive Oxygen Species in Generating Ca2+ Oscillations in Human Aortic Endothelial Cells Stimulated by Histamine*

There is increasing evidence that intracellular reactive oxygen species (ROS) play a role in cell signaling and that the NADPH oxidase is a major source of ROS in endothelial cells. At low concentrations, agonist stimulation of membrane receptors generates intracellular ROS and repetitive oscillations of intracellular Ca2+ concentration ([Ca2+] i ) in human endothelial cells. The present study was performed to examine whether ROS are important in the generation or maintenance of [Ca2+] i oscillations in human aortic endothelial cells (HAEC) stimulated by histamine. Histamine (1 μm) increased the fluorescence of 2′,7′-dihydrodichlorofluorescin diacetate in HAEC, an indicator of ROS production. This was partially inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 μm), by the farnesyltransferase inhibitor H-Ampamb-Phe-Met-OH (2 μm), and in HAEC transiently expressing Rac1N17, a dominant negative allele of the protein Rac1, which is essential for NADPH oxidase activity. In indo 1-loaded HAEC, 1 μm histamine triggered [Ca2+] i oscillations that were blocked by DPI or H-Ampamb-Phe-Met-OH. Histamine-stimulated [Ca2+] i oscillations were not observed in HAEC lacking functional Rac1 protein but were observed when transfected cells were simultaneously exposed to a low concentration of hydrogen peroxide (10 μm), which by itself did not alter either [Ca2+] i or levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3). Thus, histamine generates ROS in HAEC at least partially via NADPH oxidase activation. NADPH oxidase-derived ROS are critical to the generation of [Ca2+] i oscillations in HAEC during histamine stimulation, perhaps by increasing the sensitivity of the endoplasmic reticulum to Ins-1,4,5-P3.

There is increasing evidence that intracellular reactive oxygen species (ROS) 1 play an important role in cell signaling (1)(2)(3)(4). Both vascular smooth muscle cells and endothelial cells are capable of generating ROS (5). A number of enzyme systems likely contribute to ROS generation in endothelial cells, including arachidonic acid-metabolizing systems (6), the mitochondrial electron transport chain (7), xanthine oxidase (8), nitric-oxide synthase (9), the cytochrome P450 enzyme system (10), and endothelial NADPH oxidase (11). The endothelial NADPH oxidase shares some structural features of the multicomponent NADPH oxidase of phagocytes, most of which have been identified in endothelial cells at the RNA or protein level (12)(13)(14). Among these include the small GTP-binding protein Rac1, which is necessary for enzyme function. Whereas the oxidase of phagocytes generates large quantities of ROS that are necessary to eliminate engulfed microorganisms, the NADPH oxidase of non-phagocytic cells generates low levels of ROS that appear to have a cell signaling function.
At low concentrations, agonists like histamine (30), bradykinin (31), and thrombin (32) stimulate repetitive oscillations of intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) in endothelial cells. The initiation of each Ca 2ϩ spike appears to be due chiefly to the generation of Ins-1,4,5-P 3 (33), and the maintenance of oscillations may be related to repetitive cycles of fast activation and slow inactivation of the Ins-1,4,5-P 3 receptor by Ca 2ϩ (34,35). Thus, the observation, that NADPH oxidase-generated ROS increases the sensitivity of intracellular Ca 2ϩ stores to Ins-1,4,5-P 3 , suggests a potential link between oxidase stimu-* This work was supported by a Beginning-Grant-in-Aid 0060165U from the American Heart Association Mid-Atlantic Affiliate (to Q. H.) and by National Institutes of Health Grant R01 HL63720 (to R. C. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18

MATERIALS AND METHODS
Endothelial Cell Culture-Human aortic endothelial cells (HAEC) were obtained as proliferating quaternary cultures (Clonetics, San Diego, CA) and were grown to confluence to passages 5-9 in endothelial cell growth medium supplemented with 2% fetal bovine serum, 10 g/liter human recombinant epidermal growth factor, 1 mg/liter hydrocortisone, 50 g/ml gentamicin, 50 ng/ml amphotericin-B, 12 g/ml bovine brain extract (Clonetics) in a 37°C humidified atmosphere of 95% air-5% CO 2 . To measure [Ca 2ϩ ] i , HAECs were grown on 25-mmdiameter circular glass coverslips (VWR Scientific, Media, PA) precoated with 2% gelatin solution (Sigma Chemical Co.) for at least 2 h at 37°C. The glass coverslips were washed three times with phosphatebuffered saline (Quality Biological, Inc., Gaithersburg, MD) before cell seeding. After exposure to a solution of 0.025% trypsin and 0.01% EDTA (Sigma), HAECs were plated at an approximate concentration of 1 ϫ 10 5 /ml on the glass coverslips. Cells were used for experiments after reaching ϳ70% confluence after incubation for 1-2 days at 37°C in a humidified atmosphere of 95% air-5% CO 2 .
Measurement of Intracellular ROS Generation in HAEC-Detection of intracellular ROS was performed by a previously established method using the ROS-sensitive fluorescent probe 2Ј,7Ј-dihydrodichlorofluorescin diacetate (DCF-DA) and confocal microscopy (1,36). For measurement of intracellular ROS, HAECs were plated in a chamber slide system (Fisher Sciences, Newark, DE) at a density of 1 ϫ 10 5 cells/ml and then cultured for 3 days. The cells were washed with HEPESbuffered saline (HBS) and were loaded with 5 g/ml of DCF-DA (Molecular Probes, Eugene, OR) for 5 min at 37°C. The fluorescent dichlorofluorescin was quantified by using a laser-scanning confocal microscope (Leica TCS-4D, Heidelberg, Germany) with the excitation and emission wavelengths of 488 and 520 nm, respectively.

Measurement of [Ca 2ϩ ] i -HAEC [Ca 2ϩ
] i was measured as previously described (37) using the fluorescent Ca 2ϩ probe indo 1. Briefly, HAEC in tissue culture dishes were exposed to a solution of 0.025% trypsin and 0.01% EDTA (Sigma Chemical Co., St. Louis, MO) and were then plated at a concentration of ϳ1 ϫ 10 5 /ml on 25-mm-diameter circular glass coverslips (VWR Scientific, Media, PA) precoated with 2% gelatin solution (Sigma) for at least 2 h at 37°C. Cells were used for experiments after reaching ϳ70% confluence after incubation for 1-2 days at 37°C in a humidified atmosphere of 95% air-5% CO 2 . To measure [Ca 2ϩ ] i , HAEC monolayers on glass coverslips were incubated with culture medium containing 10 M of the ester derivative (acetoxymethyl ester form) of indo 1 (Molecular Probes, Eugene, OR) in a room temperature 95% air-5% CO 2 atmosphere for 30 min. The coverslips were then gently washed three times with indicator-free HBS of the following composition (in millimolar): NaCl 137, KCl 4.9, CaCl 2 1.5, MgSO 4 1.2, NaH 2 PO 4 1.2, D-glucose 15, HEPES 20 (pH adjusted to 7.40 at room temperature with NaOH). The cells were maintained in HBS for at least 30 min before the beginning of the experiment to allow for de-esterification of the indicator. The fluorescence of indo 1 was recorded from single HAEC on coverslips in a perfusion chamber mounted on the stage of a modified Nikon Diaphot inverted epifluorescence microscope. The fluorescence of indo 1 was excited at 350 Ϯ 50 nm using a xenon short arc lamp (UXL-75 XE, Ushio Inc., Japan), and bandpass interference filters (Omega Optical, Brattleboro, VT) with selected wavelength bands of emitted fluorescence at 405 Ϯ 10 nm and 485 Ϯ 10 nm, corresponding to the Ca 2ϩ -bound and Ca 2ϩ -free forms of the indicator, respectively. Emitted indo 1 fluorescence was collected and measured using a spectrofluorometer (PTI, Deltascan). The photometer had a series of fixedpinhole diaphragms to regulate the recording field area. Autofluorescence from unloaded HAEC was generally Ͻ5% of indo 1-loaded HAEC and was subtracted automatically from indo 1 fluorescence recordings.
To determine [Ca 2ϩ ] i from indo 1 fluorescence ratios, the intracellular minimum and maximum ratios (R min and R max , respectively) were determined as previously described (37). To determine R min , indo 1-loaded HAECs on the glass coverslips were perfused with a solution containing (in millimolar): NaCl 137, KCl 5.0, MgSO 4 1.2, NaH 2 PO 4 1.2, D-glucose 16, HEPES 10, and EGTA 2, pH 7.40. HAECs were then exposed to a solution of similar composition except with 10 mM EGTA and 0.05% Triton X-100. An intracellular R max value was determined by first perfusing HAEC with a solution containing 132 mM KCl, 10 mM K-HEPES, 1 mM MgSO 4 , 2 M rotenone (Sigma), 2 M carbonyl cyanide p-trifluoromethoxyphenylhydrozone (Sigma), and 10 ng/ml valinomycin (Calbiochem, La Jolla, CA). HAECs were then exposed to a similar solution containing 2 M ionomycin (Sigma), 69.2 mM CaCl 2 , and 100 mM HEPES (free [Ca 2ϩ ] of 5900 nM). The values of intracellular R min and R max were used to calculate [Ca 2ϩ ] i according to the following formula (38) where K d is the dissociation constant of indo 1, and S f2 and S b2 are the fluorescence intensities at ϳ490 nm of the Ca 2ϩ -free and Ca 2ϩ -saturated indicator, respectively. K d was determined to be 207 nM under the present experimental conditions using an in vitro calibration method.
HAEC Transiently Expressing the Dominant Negative Allele of Rac1-An adenovirus encoding the Myc epitope-tagged, dominant negative Rac1 cDNA containing a substitution at position 17 (Rac1 N17 ) was used as described previously (4). Expression of the Rac1 N17 mutant was confirmed by protein immunoblotting with an antibody to the Myc epitope (9E10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Ins-1,4,5-P 3 Measurement-To measure Ins-1,4,5-P 3 levels, HAECs were stimulated with histamine or other agonists, the reaction was stopped by adding 1 M ice-cold trichloroacetic acid, and the cells were maintained for 15 min on ice. The cells were then scraped and centrifuged at 1000 ϫ g for 10 min at 4°C. The supernatant was removed and incubated for 15 min at room temperature. Levels of 1,4,5-InsP 3 in each supernatant were determined using a 3 H-label Radioreceptor Assay kit (PerkinElmer Life Sciences, Inc., Boston, MA) according to the manufacturer's instructions. Duplicate measurements were performed for each separate experiment. Cellular Ins-1,4,5-P 3 levels were normalized as -fold increase of paired-control experiments in unstimulated HAEC.
Statistical Analysis-Data are reported as mean Ϯ S.E. Statistical comparisons were made using Student's t test for the paired and the unpaired groups. An analysis of variance was used when multiple comparisons were performed. A difference was considered significant at p Ͻ 0.05.

Histamine Stimulates Intracellular ROS Generation in HAEC-
The effect of 1 M histamine on intracellular ROS generation was examined in DCF-loaded HAEC by confocal microscopy. This concentration of histamine triggers [Ca 2ϩ ] i oscillations in human endothelial cells (30,39). As shown in Fig. 1, 1 M histamine stimulated intracellular ROS generation in a time-dependent manner (Fig. 1A). Histamine increased DCF fluorescence intensity in HAEC by 21.4 Ϯ 8.9%, 54.3 Ϯ 13.8%, and 213.8 Ϯ 33.3% at 1, 5, and 10 min, respectively (p Ͻ 0.01 versus control at 5 and 10 min, n ϭ 9 for each). As shown in Fig. 1B, the increase in DCF fluorescence stimulated by histamine was markedly attenuated by the NADPH oxidase inhibitor DPI and by the farnesyltransferase (FTase) inhibitor H-Ampamb-Phe-Met-OH (LC Laboratories, Woburn, MA). FTase inhibitors inhibit the post-translational modification (the covalent addition of a 15-carbon farnesyl group) of Ras family GTP-binding proteins, including Rac1, which is essential for NADPH oxidase activity. Farnesylation is necessary for membrane association and biologic activity of these proteins (40,41). In the presence of 10 M DPI or 2 M FTase inhibitor, concentrations previously shown to inhibit ROS generation in HAEC (42) and fibroblasts (4), respectively, histamine-stimulated DCF fluorescence was inhibited by ϳ60 -70% (60.0 Ϯ 12.0% for DPI and 85.6 Ϯ 18.8% for FTase inhibitor versus 213.8 Ϯ 33.3% at 10 min, p Ͻ 0.05 for each). Histaminestimulated DCF fluorescence was also markedly attenuated in HAEC transiently expressing Rac1 N17 , a dominant negative allele of Rac1 (4). As shown in Fig. 1C was employed for these studies rather than a superoxide-generating system, because our previous work showed that the increased sensitivity of intracellular Ca 2ϩ stores to Ins-1,4,5-P 3 stimulated by NADPH oxidase activity was also blocked by catalase but was unaffected by superoxide dismutase (29). As shown in Fig. 2C ( (Fig. 2C, bottom), and in another a single [Ca 2ϩ ] i spike was observed. To determine whether the effect of H 2 O 2 was related to an increase in the sensitivity of intracellular Ca 2ϩ stores to Ins-1,4,5-P 3 (29) or to an effect on Ins-1,4,5-P 3 levels, Ins-1,4,5-P 3 levels were measured in HAEC treated with 1 M histamine or 10 M H 2 O 2 alone or with the two together. As shown in Fig. 2D, 1 M histamine alone produced a rapid increase in Ins-1,4,5-P 3 levels, with a 1.56 Ϯ 0.25-fold increase evident 1 min after stimulation. H 2 O 2 did not affect Ins-1,4,5-P 3 levels by itself and did not alter the effect of histamine on Ins-1,4,5-P 3 levels (1.63 Ϯ 0.16-fold increase at 1 min, p ϭ NS compared with histamine alone, n ϭ 3 for each).
Additional experiments were performed to assess whether blocking the generation of ROS by the NADPH oxidase affects histamine-stimulated [Ca 2ϩ ] i oscillations in HAEC. In these experiments (Fig. 3), [Ca 2ϩ ] i oscillations were generated and then cells were exposed to histamine-free buffer either alone (Fig. 3A), with DPI ( Fig. 3B), or with the FTase inhibitor (Fig.  3C) before a second exposure to histamine. After [Ca 2ϩ ] i oscillations were generated in the presence of histamine, the washout of histamine resulted in the cessation of oscillations. When the cell was stimulated again with histamine after approximately a 10-min washout period, [Ca 2ϩ ] i oscillations recurred without any significant difference in oscillation amplitude (⌬indo 1 ratio ϭ 1.20 Ϯ 0.21 versus 1.14 Ϯ 0.24, p ϭ NS) or frequency (0.28 Ϯ 0.02 versus 0.23 Ϯ 0.04 min Ϫ1 , p ϭ NS) when compared with that observed before the 10-min washout (Fig.  3A). By contrast, repetitive [Ca 2ϩ ] i oscillations were not observed when either 10 M DPI (Fig. 3B) or 2 M FTase inhibitor (Fig. 3C) was present during and after histamine washout. Of note, DPI (up to 20 min) did not affect the content of cellular ATP, which is used for the biosynthesis of Ins-1,4,5-P 3 , as measured by bioluminescent method (data not shown). DISCUSSION This study shows that histamine, like many other agonists (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), stimulates the production of ROS in endothelial cells. Histamine-stimulated ROS production results, at least in part, from activation of the NADPH oxidase and Rac1 signaling, because it is inhibited by the NADPH oxidase inhibitor DPI, by an FTase inhibitor, and in cells lacking functional Rac1 protein.
The generation of ROS by the NADPH oxidase was previously reported when endothelial cells were stimulated by thrombin (28), vascular endothelial growth factor (21), or tumor necrosis factor-␣ (7,(22)(23)(24)(25). In the case of tumor necrosis factor, NADPH oxidase-derived ROS were found to be important in activating nuclear factor-B (NF-B) in endothelial cells (24). NF-B activation was previously shown to be redox-sensitive in HeLa cells (36). In HeLa cells, transient expression of a constitutively active Rac1 mutant increased NF-B transcriptional activity, whereas basal and cytokine-stimulated NF-B activity was inhibited in dominant negative Rac1 mutants. This is interesting, because we previously showed that [Ca 2ϩ ] i oscillations regulate NF-B activity during histamine stimulation in HAEC (39), and now show that NADPH oxidase-derived ROS are critical to the generation of [Ca 2ϩ ] i oscillations during histamine stimulation.
The effect of NADPH oxidase activation on [Ca 2ϩ ] i oscillations may derive from the sensitization of the ER Ins-1,4,5-P 3 receptor by oxidase-derived ROS. Ins-1,4,5-P 3 is critical to the generation of [Ca 2ϩ ] i oscillations in non-excitable cells (33). Like many other agonists, histamine binds to membrane receptors and activates phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate and to generate diacylglycerol and Ins-1,4,5-P 3 (44). The generation of Ins-1,4,5-P 3 after agonist stimulation leads to the release of ER Ca 2ϩ . [Ca 2ϩ ] i oscillations are believed to depend on Ins-1,4,5-P 3 receptors releasing Ca 2ϩ in "hotspots" in the ER (45) and the subsequent diffusion of this Ca 2ϩ to adjacent sites in the ER, increasing the local sensitivity of the Ins-1,4,5-P 3 receptor and inducing further Ca 2ϩ release. Changes in the sensitivity of the ER to Ins-1,4,5-P 3 are likely to be important in the generation of repetitive [Ca 2ϩ ] i spikes. Redox sensitivity of the Ins-1,4,5-P 3 receptor has previously been reported in hepatocytes (46 -48), and we previously showed that NADPH oxidase activation increases the sensitivity of intracellular Ca 2ϩ stores to Ins-1,4,5-P 3 in HAEC. The finding, that 10 M H 2 O 2 "restores" [Ca 2ϩ ] i oscillations in histamine-stimulated HAEC expressing the dominant negative form of Rac1 but does not affect Ins-1,4,5-P 3 levels in histamine-stimulated HAEC, is consistent with the notion that histamine-stimulated ROS increase Ins-1,4,5-P 3 receptor sensitivity and thereby affect the generation of [Ca 2ϩ ] i oscillations.
Histamine-stimulated ROS may also affect upstream signaling pathways in HAEC. For example, we recently showed that activation of phospholipase D (PLD), which exhibits redox sensitivity in endothelial cells (49), regulates [Ca 2ϩ ] i oscillation frequency in HAEC during histamine stimulation (50). It is not likely that the effect of histamine-stimulated ROS on the generation of [Ca 2ϩ ] i oscillations is related to PLD signaling, however, because time-dependent activation of PLD by histamine in HAEC is not rapid enough to affect generation of oscillations. Stimulation of HAEC by histamine activates PLD by 5 min, but no significant effect is observed 1 min after stimulation. Moreover, inhibition of PLD decreases oscillation frequency, but does not inhibit the generation of [Ca 2ϩ ] i oscillations during histamine stimulation (50). Alternatively, histamine-stimulated ROS may modulate [Ca 2ϩ ] i oscillations by an effect on PLC-␥, because generation of Ins-1,4,5-P 3 by agonists like bradykinin appears to be secondary to tyrosine phosphorylation of PLC-␥1 (51) and H 2 O 2 is known to activate PLC-␥ (52). We do not believe this mechanism is likely to play a role during histamine stimulation, because 1 M histamine stimulated only weak tyrosine phosphorylation of PLC-␥1 and histamine-stimulated tyrosine phosphorylation of PLC-␥2 was not inhibited by DPI, by an FTase inhibitor, or in cells lacking functional Rac1 protein (data not shown).
ROS may also be important in the generation of [Ca 2ϩ ] i oscillations, because of an effect on other redox-sensitive Ca 2ϩ release mechanisms that are activated by histamine. For example, the ryanodine receptor (RyR) may be important in histamine-stimulated Ca 2ϩ signaling. It was previously shown that blocking ryanodine-sensitive Ca 2ϩ release inhibits [Ca 2ϩ ] i oscillations in endothelial cells stimulated by histamine (53). Redox regulation of the RyR is well-established in cardiac and skeletal muscle, with sulfhydryl oxidation, S-nitrosylation, or modification of sulfhydryl groups of the RyR by other oxidants increasing Ca 2ϩ release channel activity (54). Hyper-reactive cysteine moieties may represent biochemical components of a transmembrane redox sensor within the RyR channel complex that conveys information about localized changes in redox potential produced by different stimuli (55,56).
The finding that ROS play a role in agonist-stimulated Ca 2ϩ signaling is novel and important in cell biology, reinforcing the relatively new concept that ROS are not only important pathophysiologically but also play major roles in cell signaling. It was recently shown, for example, that epidermal growth factor increases [Ca 2ϩ ] i in fibroblasts without affecting inositol phosphates but rather via the production of H 2 O 2 regulated by Rac and RhoA (57). This finding is interesting in light of our previous work showing that H 2 O 2 increases the sensitivity of the ER Ca 2ϩ store to Ins-1,4,5-P 3 (29) and that H 2 O 2 generates [Ca 2ϩ ] i oscillations in endothelial cells (43), even though H 2 O 2 itself does not stimulate Ins-1,4,5-P 3 production, as shown in this work and by others (58).
The present study shows that elevated levels of Ins-1,4,5-P 3 alone may not be sufficient to initiate repetitive [Ca 2ϩ ] i spiking behavior in HAEC during histamine stimulation. Although histamine increases levels of Ins-1,4,5-P 3 (44), the generation of ROS during histamine stimulation is necessary for oscillations to be triggered. More specifically, it appears that NADPH oxidase-derived ROS are important in histamine-triggered Ca 2ϩ signaling, because oscillations were not observed in the presence of DPI, an FTase inhibitor or in dominant negative Rac1 transfected cells. The critical role of ROS in the generation of [Ca 2ϩ ] i oscillations is further supported by the finding that 10 M H 2 O 2 "restored" [Ca 2ϩ ] i oscillations in histaminestimulated HAEC expressing the dominant negative form of Rac1, even though this concentration of H 2 O 2 alone did not affect Ca 2ϩ signaling or Ins-1,4,5-P 3 levels. Taken together, these data suggest that histamine activates an NADPH oxidase in HAEC, resulting in the production of ROS that increase the sensitivity of the Ins-1,4,5-P 3 receptor to Ins-1,4,5-P 3 and thereby play a role in the generation of [Ca 2ϩ ] i oscillations.