Effects of Superoxide Anions on Endothelial Ca²⁺ Signaling Pathways

From the Department of Medical Biochemistry, University of Graz, Graz, Austria.

Correspondence to Dr Wolfgang F. Graier, Department of Medical Biochemistry, University of Graz, Harrachgasse 21/III, A-8010 Graz, Austria. E-mail wolfgang.graier{at}kfunigraz.ac.at

	Abstract

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Abstract—Although the involvement of free radicals in the development of endothelial dysfunction under pathological conditions, like diabetes and hypercholesterolemia, has been proposed frequently, there is limited knowledge as to how superoxide anions (O₂^-) might affect endothelial signal transduction. In this study, we investigated the effects of preincubation with the O₂^--generating system xanthine oxidase/hypoxanthine (XO/HX) on mechanisms for Ca²⁺ signaling in cultured porcine aortic endothelial cells. Incubation of cells with XO/HX yielded increased intracellular Ca²⁺ release and capacitative Ca²⁺ entry in response to bradykinin and ATP in a time- and concentration-dependent manner. This effect was prevented by superoxide dismutase but not by the tyrosine kinase inhibitor tyrphostin A48. In addition, capacitative Ca²⁺ entry induced by the receptor-independent stimulus 2,5-di-(tert-butyl)-1,4-benzohydroquinone or thapsigargin was enhanced in O₂^--exposed cells (+38% and +32%, respectively). Increased Ca²⁺ release in response to bradykinin in XO/HX-pretreated cells might be due to enhanced formation of inositol-1,4,5-trisphosphate (+140%). Exposure to XO/HX also affected other signal transduction mechanisms involved in endothelial Ca²⁺ signaling, such as microsomal cytochrome P450 epoxygenase and membrane hyperpolarization to Ca²⁺ store depletion with thapsigargin (+103% and +48%, respectively) and tyrosine kinase activity (+97%). A comparison of bradykinin-initiated intracellular Ca²⁺ release and thapsigargin-induced hyperpolarization with membrane viscosity modulated by XO/HX (decrease in viscosity) or cholesterol (increase in viscosity) reflected a negative correlation between bradykinin-initiated Ca²⁺ release and membrane viscosity. Because intracellular Ca²⁺ is a main regulator of endothelial vascular function, our data suggest that O₂^- anions are involved in regulation of the vascular endothelium.

Key Words: cytochrome P450 epoxygenase • inositol-1,4,5-trisphosphate • membrane fluidity • membrane potential • tyrosine kinase

	Introduction

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The generation of free radicals has been proposed to play a crucial role in the development of endothelial dysfunction under certain pathological conditions, including diabetes mellitus,¹ hypercholesterolemia,² reoxygenation/reperfusion injury,³ and septic shock.⁴ Among the oxygen free radicals, superoxide anions have been demonstrated to be involved in hyperglycemia-induced impairment of endothelium-dependent relaxation,⁵ endothelial cell–mediated LDL oxidation,⁶ decreased endothelial cell proliferation⁷ initiated by hyperglycemia, and upregulation of adhesion molecules.⁸ In addition, superoxide anions neutralize endothelium-derived NO, resulting in diminished endothelium-dependent relaxation.⁹

Few studies have examined the effect of superoxide anions on endothelial signal transduction mechanisms. Franceschi et al¹⁰ showed that superoxide anions produced by the xanthine oxidase/hypoxanthine (XO/HX) reaction resulted in short-term membrane hyperpolarization in endothelial cells due to stimulation of Ca²⁺ entry through a non–voltage-dependent mechanism.^{10 11} In agreement with the findings of increased [Ca²⁺]_i, enhanced intracellular prostaglandin I₂ formation after incubation with XO/HX was described in human umbilical vein endothelial cells.¹² In most of these studies, very high concentrations of superoxide anions were used. For example, in coronary artery smooth muscle cells, attenuation of angiotensin II–initiated contraction by such high superoxide anion concentrations (>1 mU/mL XO) did not differ from that induced by H₂O₂ (>30 µmol/L).¹³ We have shown that increased formation of superoxide anion during exposure to a high D-glucose concentration elicits changes in endothelial Ca²⁺ signaling.¹⁴ However, the superoxide anion levels mediated by high D-glucose treatment,¹⁴ for example, were much lower than those used in the other studies mentioned above while exposure time was prolonged for several hours. Under such conditions, superoxide anion had no short-term effects on resting endothelial [Ca²⁺]_i, whereas the mobilization of Ca²⁺ evoked by bradykinin was enhanced.¹⁴ So far, no detailed studies have been performed to understand how physiological concentrations of superoxide anions affect endothelial Ca²⁺ signaling. As shown in Figure 1, endothelial cell stimulation with an agonist, such as bradykinin or ATP, results in G protein–mediated activation of phospholipase C and the formation of inositol-1,4,5-trisphosphate (IP₃), which in turn releases Ca²⁺ from intracellular Ca²⁺ pools. In addition to the intracellular Ca²⁺ release, an extracellular Ca²⁺ influx is activated due to the depletion of intracellular Ca²⁺ pools,¹⁵ thus representing a so-called "capacitative Ca²⁺ entry" pathway.^{16 17}

View larger version (22K): [in this window] [in a new window]   Figure 1. Proposed mechanisms involved in endothelial Ca2+ signaling. Interaction of agonist (eg, bradykinin or ATP) with its receptor (R) results in formation of IP3, which in turn releases Ca2+ from intracellular Ca2+ pool(s). Owing to Ca2+ store depletion, endothelial capacitative Ca2+ entry becomes activated. Involvement of TK (Tk)55 or cytochrome P450 epoxygenase (P450) as the link between Ca2+ store depletion and activation of membrane Ca2+-entry pathways is discussed in text. Inhibition of endoplasmic Ca2+ ATPase (gray circles) by TG or BHQ also results in activation of Ca2+ entry, again based on depletion of intracellular Ca2+ stores.56 Thus, these compounds activate the same Ca2+ entry pathway as bradykinin, without formation of IP3. Gp indicates G protein linked to the membrane receptor and the PLC, phospholipase C.

In the current study, the mechanism whereby prolonged exposure to small superoxide anion concentrations affects endothelial Ca²⁺ signaling was investigated further. The effect of pretreatment with superoxide anions on endothelial Ca²⁺ signaling (ie, intracellular Ca²⁺ mobilization¹⁸ and capacitative Ca²⁺ entry¹⁹ ) and mechanisms previously shown to be involved in endothelial Ca²⁺ signaling (Figure 1), such as formation of IP₃,¹⁸ cytochrome P450 epoxygenase,^{19 20} membrane hyperpolarization,²¹ tyrosine kinase (TK),²² and membrane fluidity, were assessed.

	Methods

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Materials
1-Ethoxypyrene-3,6,8-tris (dimethylsulfonamide), 1-hydroxypyrene-3,6,8-tris (dimethylsulfonamide), fura 2–acetoxymethyl ester (fura 2-AM), DPH, and 1-(4-trimethylammoniumphenyl)-6-phenyl-DPH (TMA-DPH) were obtained from Lambda Fluorescence Technology. Cell culture materials were from Life Technologies, and FCS was from PAA Laboratories. Bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC₄(5)) and SNARF acetoxymethyl ester (SNARF-1/AM) were from Molecular Probes Inc. Petri dishes were from Corning, and 6- and 24-well plates were from Falcon. The protein TK assay kit was purchased from Calbiochem-Novabiochem International. All other chemicals were obtained from Sigma.

Cell Culture
Endothelial cells were isolated from porcine aortae by enzymatic digestion with 200 U/mL collagenase (type II) in Dulbecco's minimal essential medium (DMEM) plus dilutions of (vol/vol) 0.02 amino acids and 0.01 vitamins plus trypsin inhibitor (soybean type I, 1 mg/mL) as described previously.¹⁹ Cells were cultured in Opti-DMEM containing 3% FCS. Only cells from passage 1 or 2 were used for experiments (10 to 14 days in culture).

Ca²⁺ Measurement
Free [Ca²⁺]_i was determined in porcine aortic endothelial cells in suspension or monolayer as indicated by the fura 2 technique as previously described.¹⁹ In brief, cells were incubated with DMEM containing 2 µmol/L fura 2-AM in the dark for 45 minutes (suspended cell experiments) or 30 minutes (single-cell experiments) at 37°C. Afterward, the cells were centrifuged and resuspended in DMEM. Just before the experiment, cells were centrifuged and resuspended in nominal Ca²⁺-free (ie, 10 µmol/L free extracellular Ca²⁺) HEPES-buffered solution containing (in mmol/L) 145 NaCl, 5 KCl, 1 MgCl₂, and 10 HEPES, pH 7.4. [Ca²⁺]_i was monitored every 0.25 to 2.0 seconds (depending on the instrument used) as the ratio of 340 and 380 nm excitation at 510 nm emission.

To study specifically the effects of superoxide anions on the stimulation of capacitative Ca²⁺ entry, Mn²⁺ quench experiments were performed as previously described.^{19 23 24} In brief, this approach is based on use of the surrogate divalent cation Mn²⁺, which enters the cells through the channels involved in capacitative Ca²⁺ entry. However, the entry of Mn²⁺ into fura 2–loaded cells induces a decrease in fluorescence at 360 nm excitation and 510 nm emission (ie, the isosbestic, Ca²⁺-insensitive wavelength of fura 2). The amount of Mn²⁺ entering the cells is proportional to the fractional decrease in fluorescence relative to the initial intensity.

Data Acquisition
In view of the reported problems concerning [Ca²⁺]_i calibration in our system¹⁹ and the general uncertainties of the calibration techniques,²⁵ [Ca²⁺]_i in each experiment was expressed as the 340- to 380-nm emission ratio. Because of minor differences between the instruments used (Hitachi F2000, Hitachi F4500, and Perkin-Elmer LS-50B/FFA), caution is necessary when comparing given ratio units between different figures. Thus, for each experimental series, results were compared with those obtained in control cells (ie, preincubated in the absence of XO) performed daily and shown in each figure.

Superoxide Anion Treatment
Superoxide anions were generated by the reaction of XO with HX in DMEM (containing 1.8 mmol/L Ca²⁺) for incubation with the cells; however, phenol red–free DMEM was used for superoxide anion measurements. The generation of superoxide anions was determined as the difference in the reduction of ferricytochrome c (10 µmol/L, horse heart type III) in the absence or presence of SOD (476 U/mL). The reduction of ferricytochrome c was monitored at 550 nm. The difference in absorption between samples in the absence and presence of SOD directly shows extinction due to superoxide anion–related reduction of ferricytochrome c. Concentrations of superoxide anions were calculated by using the molar extinction coefficient of the reduced form of ferricytochrome c (=21 000).¹⁴ Production of superoxide anions was controlled for each cell pretreatment procedure in phenol red–free DMEM without added cells.

Cells were incubated in DMEM containing 1 mmol/L HX with or without XO at the concentrations indicated for 1 to 3 hours. Experiments were performed in the absence of XO/HX after a 45-minute equilibration (for IP₃ measurements, 15 minutes) of the cells in normal DMEM. The percentage of XO-treated cells responding to agonist stimulation was comparable to that of cells treated with HX alone. Likewise, XO up to 1000 µU/mL did not affect cell viability or size, as determined by trypan blue incorporation (viability only) and the Schärfe cell counter (viability and cell size, Casy-1).

IP₃ Formation
IP₃ was determined by using a customized radioactive binding assay (Biotrak, Amersham International) as described previously.²⁶ In brief, endothelial cells were cultured to confluence in 6-well plates. Before experimentation, the cells were washed twice with HEPES-buffered solution (plus 2.5 mmol/L CaCl₂) and equilibrated at 37°C in 1 mL of the salt solution. After 15 minutes, the compound to be tested was added at a dilution of 1:100. After a 30-second incubation, the experiment was stopped by the addition of 200 µL of 20% chilled HClO₄. After 20 minutes on ice, the pH of the supernatant was adjusted to 7.5 by adding KOH. After 15 minutes at 4°C, the samples were centrifuged for 15 minutes at 2000g, and the resulting supernatant was used for determination of IP₃ content with the radioactive binding assay.

Microsomal Cytochrome P450 Monooxygenase (CYP450 MO)
Microsomal CYP450 MO was measured as previously described.²⁰ In brief, cells were suspended in intracellular-like buffer containing (in mmol/L) 150 KCl, 10 MgCl₂ , and 50 Tris, with pH adjusted to 7.5. 1-Ethoxypyrene-3,6,8-tris-(dimethylsulfonamide) (25 µmol/L) was added under constant stirring, and cells were permeabilized with 1 mg/mL saponin in the presence of an NADPH-regenerating system (25 IU isocitric dehydrogenase [NADP⁺], 8 mmol/L DL-isocitric acid, and 1 mmol/L NADP⁺). As shown recently, enzyme activity was further enhanced by depletion of intracellular Ca²⁺ stores with thapsigargin (TG; 2 µmol/L). Activity of microsomal CYP450 MO was recorded at 495 nm excitation and 550 nm emission and was calculated by using a standard calibration curve.²⁰

Membrane Potential
Variations in membrane potential were measured by using DiBAC₄(5) as previously described.²⁰ In brief, cultured endothelial cells were suspended in a buffer (9.3x10⁶ cells/mL) containing (in mmol/L) choline chloride 145, KCl 5, HEPES-free acid 10, and MgCl₂ 1, adjusted with KOH to pH 7.4. Under constant stirring, DiBAC₄(5) (1 µmol/L) was added. After an equilibration period, the compound to be tested was added. Membrane potential was monitored at 590 nm excitation and 616 nm emission. Calibration of the DiBAC₄(5) signal was performed by cumulatively adding KCl (final concentration, 5 to 70 mmol/L) to the solution in the presence of gramicidin D (800 nmol/L) as described recently.²⁰ The membrane potential for each KCl concentration was calculated according Vieira et al²⁷ and was correlated with the fluorescence readings. Resting potential against 5 mmol/L KCl was expected to be -34 mV,²⁸ owing to the known problems for estimation of absolute membrane potentials with the potentially sensitive fluorescent probes.²⁹ All data were expressed as changes in millivolts from resting potentials.

TK Activity
Endothelial TK activity was measured by using a customized photometric protein TK assay kit from Calbiochem-Novabiochem International as previously described.²⁰ In brief, cell lysates were obtained by sonication of the cell suspension on ice in a buffer containing (in mmol/L) Tris 20, NaCl 50, EDTA 1, EGTA 1, PMSF 0.2, Na₃VO₄ 0.2 , and mercaptoethanol 5; 1 µg/mL pepstatin; and 0.5 µg/mL leupeptin, with the pH adjusted to 7.4. Mg²⁺ and ATP were added to cell extract aliquots, and the mixture was incubated in the absence or presence of XO/HX as indicated. Phosphorylation of an immobilized substrate was determined by a horseradish peroxidase–labeled phosphotyrosine-specific antibody, and the conversion of tetramethylbenzidine as a substrate of horseradish peroxidase was monitored in a plate-reader photometer at 450 nm.

Membrane Viscosity
Membrane viscosity was measured by using a technique based on the depolarization of fluorescence light emitted from TMA-DPH.³⁰ In brief, cultured endothelial cells were washed twice and harvested by enzymatic digestion (trypsin), centrifuged, resuspended in HEPES buffer (see above), and placed in a thermostatically controlled cuvette in a Hitachi F-2000 spectrofluorometer equipped with polarizers at the excitation site and an analyzer at the emission site. Before the addition of the dye (TMA-DPH, 20 µmol/L), autofluorescence was monitored at each setting of the polarizer and analyzer. After addition of TMA-DPH, cells were equilibrated at 37°C in the dark. Fluorescence intensity was monitored at 355 nm excitation and 450 nm emission at settings I_vv (both polarizer and analyzer in the vertical position) and I_vh (polarizer in the vertical position and analyzer in the horizontal position). After subtraction of autofluorescence at each setting, membrane viscosity was calculated according to the following equation³⁰ : h=[(I_vv/I_vh)-1]/[0.73-0.27(I_vv/I_vh)], where h is the viscosity expressed in poises.

Additional studies were performed in cell monolayers. Endothelial cells were grown on glass coverslips (1-cm diameter) for 16 hours. For the experiments, the coverslip was mounted in the cuvette at an angle of 60°. The experimental procedure was identical to that mentioned above.

Intracellular pH
Intracellular pH was measured as previously described.²⁶ In brief, endothelial cells were loaded in DMEM for 45 minutes with 10 µmol/L SNARF-1/AM in the dark. An in situ calibration after each experiment was performed in the presence of 10 µmol/L nigericin for the calculation of pH.

Statistics
All data represent the mean±SEM. Experiments were performed in triplicate with at least 3 different cell preparations. Data evaluation was performed by ANOVA. Differences, estimated by Scheffé's F test, were considered statistically significant at P<0.05.

	Results

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Superoxide Anion Generation
The time-dependent generation of superoxide anions induced by the XO/HX mixture (150 µU/mL and 1 mmol/L, respectively) is illustrated in Figure 2. Over 60 minutes, the rate of production of superoxide anions by this system was 35.2±0.7 nmol/(Lxmin) (n=15). By comparison, this rate was 10-fold higher than that for the spontaneous secretion of superoxide anions by cultured endothelial cells, as determined previously.¹⁴ Furthermore, the increased secretion of superoxide anions induced by prolonged exposure of endothelial cells to high concentrations of D-glucose was 3-fold less than that generated by the mixture of 150 µU/mL XO and 1 mmol/L HX.¹⁴ However, to adopt the latter conditions for this study, we reasoned that levels of superoxide anions in the supernatant were necessarily much lower than those achieved within the endothelial cells themselves, particularly after exposure to high D-glucose.

View larger version (17K): [in this window] [in a new window]   Figure 2. Production of superoxide anions by XO in the presence of HX under experimental conditions. In phenol red–free DMEM-like solution containing 1 mmol/L HX, superoxide anions were produced by 150 µU/mL XO and monitored by reduction of ferricytochrome c (10 µmol/L) at 550 nm in the absence or presence of 476 U/mL SOD. Amount of superoxide anion produced was calculated by differences in absorption in the sample with and without SOD as measured by molar extinction coefficient. Linear equation for correlation of time versus superoxide anion production is y=0.03523x+0.08028 (R=0.91, n=15).

Ca²⁺ Signaling: Receptor-Dependent Agonists
The effects of XO/HX pretreatment on the mobilization of Ca²⁺ induced by the endothelial agonists bradykinin (Figure 3A) and ATP (Figure 3B) were examined. For this series of experiments, the cells were first incubated with or without 150 µU/mL XO for 1 hour in DMEM containing 1 mmol/L HX. The effects of the agonists were initiated in nominally Ca²⁺-free solution to assess the release of Ca²⁺ from intracellular stores. Afterward, in the continuous presence of the agonist (bradykinin or ATP), 2.5 mmol/L Ca²⁺ was added to the bath to monitor the elevation in cytosolic Ca²⁺ due to an influx from the extracellular medium. As shown in Figure 3A and 3B, both the release of intracellular Ca²⁺ and the influx stimulated by bradykinin and ATP were significantly enhanced in cells treated with XO/HX. By contrast, the magnitude of Ca²⁺ entry obtained after addition of 2.5 mmol/L Ca²⁺ in the absence of an agonist was not changed significantly by the XO/HX treatment (Figure 3B). Coincubation with SOD (250 U/mL) during exposure to the anion-generating system normalized endothelial Ca²⁺ signaling to ATP (Figure 3B), whereas SOD did not affect endothelial Ca²⁺ signaling in control cells (no XO present during preincubation procedure; data not shown). In contrast to SOD, coincubation with cycloheximide during the XO/HX preincubation period had no effect on the enhancement of endothelial Ca²⁺ signaling by superoxide anion (data not shown). There was no detectable effect on either ATP-induced Ca²⁺ release or capacitative Ca²⁺ entry when the cells were preincubated with 150 µU/mL XO in the absence of HX (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 3. Effect of exposure of porcine aortic endothelial cells to superoxide anion on bradykinin (Bk)- (A) and ATP- (B) stimulated intracellular Ca2+ release and capacitative Ca2+ entry. Cultured cells were preincubated in DMEM with 1 mmol/L HX for 1 hour without (open symbols) or with (closed symbols) 150 µU/mL XO and with 150 µU/mL XO and 250 U/mL SOD (+SOD; B only). Experiments were performed in cell suspension without HX and XO after an equilibration time of 45 minutes. As indicated by horizontal lines, 100 nmol/L bradykinin, 100 µmol/L ATP, or solvent (no ATP) was added, followed by addition of 2.5 mmol/L CaCl2. Intracellular Ca2+ was expressed as the ratio of fura 2 fluorescence at 340 and 380 nm excitation at 510 nm emission, mean±SEM (A, n=17; B, n=28 for ATP/XO; n=16 for ATP; and n=8 for ATP/XO+SOD, no ATP, and no ATP/XO).

When endothelial cells were stimulated with bradykinin (100 nmol/L) in the presence of 2.5 mmol/L extracellular Ca²⁺, both the initial spike and the plateau phase, which remained constant for at least 10 minutes, were enhanced in cells pretreated for 1 hour with XO/HX (150 µU/mL and 1 mmol/L, respectively) by 63% and 84%, respectively (data not shown). In agreement with our findings that pretreatment with XO/HX did not affect basal endothelial Ca²⁺ levels, preincubation with XO/HX (150 µU/mL and 1 mmol/L) for 1 hour did not affect basal intracellular pH (control, 7.53±0.13; with XO/HX, 7.41±0.09; n=7; NS versus control).

Figure 4A and 4B illustrates the time dependence of the enhancement of Ca²⁺ signaling induced by preincubating the cells with XO/HX. After 30 minutes, a slight increase in Ca²⁺ signaling was detectable, and a 1-hour preincubation showed the maximal effect, which was not enhanced further by incubation durations up to 3 hours (data not shown). Conversely, endothelial Ca²⁺ signaling was restored 12 hours after removal of XO/HX after a 1-hour treatment.

View larger version (30K): [in this window] [in a new window]   Figure 4. Effect of exposure to superoxide anion on bradykinin- and ATP-initiated Ca2+ signaling in cultured endothelial cells. Cells were preincubated with XO/HX in DMEM as indicated. After a 45-minute equilibration in DMEM without HX and XO, experiments were performed with suspended cells as indicated. A, Time course of exposure to 150 µU/mL XO in the presence of 1 mmol/L HX on bradykinin- (100 nmol/L) initiated signaling (n=33). B, Time course of exposure to 150 µU/mL XO in the presence of 1 mmol/L HX on endothelial Ca2+ signaling stimulated by 100 µmol/L ATP (n=12). C, Concentration-response relationship of various XO activities during 1-hour preincubation in DMEM containing 1 mmol/L HX on bradykinin- (100 nmol/L) stimulated intracellular Ca2+ release (open symbols) and Ca2+ entry (filled symbols) (n=8). D, Effect of 1-hour preincubation with 150 µU/mL XO in the presence of 1 mmol/L HX on bradykinin-stimulated Mn2+ entry, expressed as Mn2+-mediated quenching of fura 2 fluorescence in response to the addition of 100 nmol/L bradykinin in the presence of 200 µmol/L MnCl2. Each point represents mean±SEM (n=10).

The concentration dependence of XO in the presence of 1 mmol/L HX was assessed on bradykinin-induced Ca²⁺ signaling, according to the protocols shown in Figure 4A and 4B (Figure 4C). Threshold potentiation could be observed with a concentration of XO as low as 100 µU/mL. To demonstrate the effect of superoxide anions on bradykinin-stimulated capacitative Ca²⁺ entry more precisely, Mn²⁺ quench studies were performed (Figure 4D). In agreement with the results on capacitative Ca²⁺ entry, Mn²⁺ quench in response to stimulation with 100 nmol/L bradykinin was significantly enhanced in cells preincubated with 150 µU/mL XO in the presence of 1 mmol/L HX for 1 hour (Figure 4D). Coincubation with SOD completely prevented the effect of the XO/HX treatment on the bradykinin-induced Mn²⁺ quench (data not shown).

In contrast to SOD, coincubation with the TK inhibitor tyrphostin A48 (714 nmol/L) failed to affect XO/HX-mediated alterations in endothelial Ca²⁺ signaling in response to 100 nmol/L bradykinin (Figure 5). In agreement with these findings, coincubation with 5 µmol/L erbstatin during preincubation with 150 µU/mL XO in the presence of 1 mmol/L HX also failed to increase endothelial Ca²⁺ signaling in response to 100 nmol/L bradykinin (data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 5. Coincubation with TK inhibitor tyrphostin A48 during preincubation with superoxide anion had no effect on changes in bradykinin-induced Ca2+ signaling. Cells were incubated in DMEM containing 1 mmol/L HX for 1 hour (control), plus 714 nmol/L tyrphostin A48 (control+Tyr.A48), plus 150 µU/mL XO (XO/HX), and plus 714 nmol/L tyrphostin A48 and 150 µU/mL XO (XO/HX+Tyr.A48). After equilibration for 45 minutes in DMEM without both XO/HX and Tyr.A48, cells were resuspended in HEPES buffer in the absence of XO and Tyr.A48. As indicated by horizontal lines, cells were stimulated with 100 nmol/L bradykinin (Bk) followed by 2.5 mmol/L CaCl2. Points represent mean±SEM (n=6).

Because agonist-induced Ca²⁺ release was shown to be due to the formation of IP₃,¹⁸ the effect of preincubation with the superoxide anion–generating system on endothelial IP₃ formation was studied (Figure 6). A 1-hour preincubation with 150 µU/mL XO in the presence of HX had no effect on basal IP₃ levels, whereas IP₃ formation due to a 30-second stimulation with 100 nmol/L bradykinin was increased significantly (Figure 6).

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of exposure of endothelial cells to superoxide anion on bradykinin-stimulated formation of IP3. Cultured endothelial cells were exposed for 1 hour in DMEM containing 1 mmol/L HX in the absence (control, open columns) or presence (XO/HX, filled columns) of 150 µU/mL XO. After equilibration for 15 minutes, basal IP3 level and IP3 levels after 30-second stimulation with 100 nmol/L bradykinin were measured by radioactive binding assay. Levels of IP3 are shown as pmol IP3/106 cells, mean±SEM (n=4 to 6). *P<0.05 versus cells not preincubated with XO.

IP₃-Independent Capacitative Ca²⁺ Entry
Endothelial capacitative Ca²⁺ entry depends on the depletion of IP₃-sensitive Ca²⁺ stores.¹⁵ To investigate whether the effect of superoxide anions on endothelial Ca²⁺ entry was due only to increased Ca²⁺ store depletion by IP₃, the effect of XO/HX treatment on endothelial Ca²⁺ signaling induced by the IP₃-independent Ca²⁺ mobilizers 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ; Figure 7A)¹⁹ and thapsigargin (TG; Figure 7B)¹⁹ was studied. Whereas pretreatment with XO/HX had no effect on intracellular Ca²⁺ release induced by either BHQ (Figure 7A) or TG (Figure 7B), Ca²⁺ entry after stimulation with either BHQ (Figure 7A) or TG (Figure 7B) was significantly enhanced in cells pretreated with XO/HX.

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of preincubation of endothelial cells with XO/HX on endothelial Ca2+ signaling initiated by BHQ (A) or TG (B). Cells were exposed for 1 hour in DMEM containing 1 mmol/L HX in the absence (control, open symbols) or presence (XO/HX, filled symbols) of 150 µU/mL XO. After equilibration for 45 minutes in DMEM, suspended cells were stimulated in the nominal absence of extracellular Ca2+ with 15 µmol/L BHQ (A) or 2 µmol/L TG (B), followed by addition of 2.5 mmol/L CaCl2 as indicated by horizontal lines. Endothelial Ca2+ signaling was measured with fura 2 as described in Methods. Each point represent mean±SEM (A, n=16, controls; n=24, HX/XO; B, n=8). Identical results were obtained on single cells (n=12).

Microsomal CYP450 MO Activity
The importance of microsomal CYP450 MO activity for endothelial Ca²⁺ entry and membrane potential has been demonstrated in endothelial cells.^{19 20} Thus, the effect of treatment with superoxide anions on CYP450 MO activity in endothelial cells was tested (Figure 8A). Endothelial cells were preincubated for 1 hour with 150 µU/mL XO in the presence of 1 mmol/L HX, and CYP450 MO was activated by depletion of IP₃-sensitive stores with 2 µmol/L TG.³¹ In cells preincubated with XO/HX, CYP450 MO activity was augmented by 64% (Figure 8A). Coincubation with SOD (450 U/mL) prevented the effect of XO/HX on endothelial CYP450 MO (data not shown). Inhibition of endothelial NO synthase with N^G-nitro-L-arginine (300 µmol/L) had no effect on the influence of XO/HX on endothelial CYP450 MO activity (Figure 8A).

View larger version (62K): [in this window] [in a new window]   Figure 8. Effect of exposure of endothelial cells to XO in the presence of HX on TG-induced microsomal CYP450 MO activity (A) and membrane hyperpolarization (B). Endothelial cells were preincubated with or without NG-nitro-L-arginine (L-NNA, 300 µmol/L) for 1 hour in DMEM containing 1 mmol/L HX in the absence (control) or presence (XO/HX) of 150 µU/mL XO for 1 hour. After equilibration for 45 minutes in DMEM, effect of 2 µmol/L TG on CYP450 MO activity (A) and membrane potential (B) was measured in cell suspensions. A, CYP450 MO activity 4 minutes after addition of TG (2 µmol/L) is given as pmol of 1-hydroxypyrene-3,6,8-tris-(dimethylsulfonamide produced per minute by 106 cells, mean±SEM (without L-NNA, n=8; with L-NNA, n=6). *P<0.05 versus cells not preincubated with XO. B, Membrane potential was monitored after 2 minutes of TG stimulation by 1 µmol/L DiBAC4(5) as described in Methods. Changes in membrane potential are expressed in mV, mean±SEM (n=8). *P<0.05 versus cells not preincubated with XO.

Membrane Hyperpolarization
In addition to CYP450 MO, membrane hyperpolarization is known to play a crucial role in endothelial capacitative Ca²⁺ entry.^{20 21} Thus, the effect of preincubation with the superoxide anion–generating system XO/HX on TG-induced membrane hyperpolarization was studied (Figure 8B). Although TG-induced intracellular Ca²⁺ release was not altered by XO/HX (see above), the latter augmented membrane hyperpolarization by 2 µmol/L TG in nominal Ca²⁺-free solution by 48% (Figure 8B). Membrane hyperpolarization to TG in untreated as well as XO/HX-pretreated endothelial cells was abolished in the presence of 10 mmol/L tetrabutylammonium chloride (data not shown).

TK
Preincubation with XO in the presence of 1 mmol/L HX yielded increased Ca²⁺-activated TK activity in cultured endothelial cells. Use of 150 and 300 µU/mL XO during the preincubation period of 45 minutes increased endothelial TK activity by 97% and 156%, respectively (Figure 9A). The TK inhibitors erbstatin (100 µmol/L) and tyrphostin A48 (714 nmol/L) strongly inhibited all TK activity in lysates of untreated and HX/XO-pretreated cells (control, 4.59±1.06 and 3.33±1.31 U/mg protein; XO/HX, 3.66±0.76 and 4.73±1.27 U/mg protein; n=3).

View larger version (56K): [in this window] [in a new window]   Figure 9. Effect of exposure of endothelial cells to XO (150 and 300 µU/mL) in the presence of 1 mmol/L HX on TK activity (A) and membrane viscosity measured in cell suspension (B). A, TK was measured as described in Methods and is expressed as U/mg protein. Each column represents mean±SEM (n=4). *P<0.05 versus control. Differences between 150 and 300 µU/mL were not statistically different. B, Cultured endothelial cells were preincubated for 1 hour in DMEM containing 1 mmol/L HX in the absence of XO (Control), in the presence of 150 µU/mL XO (XO/HX), or in the presence of 150 µU/mL XO and 300 U/mL SOD (XO/HX+SOD). After a 45-minute equilibration, membrane viscosity was monitored in suspended cells by using 20 µmol/L TMA-DPH as described in Methods. Membrane viscosity is expressed in poises, mean±SEM (n=9). *P<0.05 versus cells not preincubated with XO. Similar results were obtained in cell monolayer experiments.

Membrane Viscosity
Exposure of endothelial cells to 150 µU/mL XO in the presence of 1 mmol/L HX significantly reduced membrane viscosity (Figure 9B). This effect was completely prevented when 300 U/mL SOD was present during XO/HX treatment (Figure 9B) or when no HX was present (data not shown).

To investigate whether increased membrane stiffness might result in an effect opposite to increased membrane fluidity, endothelial cells were loaded with cholesterol. As shown in Figure 10A, incubating the cells for 30 or 60 minutes with 250 µmol/L cholesterol (from 25 mmol/L stock in ethanol) yielded increases in membrane viscosity of 113% and 135%, respectively. Moreover, cholesterol loading for 30 or 60 minutes attenuated bradykinin-induced intracellular Ca²⁺ release and capacitative Ca²⁺ entry by 26% and 47% or 63% and 68%, respectively (Figure 10B).

View larger version (47K): [in this window] [in a new window]   Figure 10. Effect of exposure to cholesterol on endothelial membrane viscosity (A) and bradykinin-initiated Ca2+ signaling (B). Cultured endothelial cells were exposed in DMEM with 1% ethanol (solvent, control) or with 250 µmol/L cholesterol (from 25 mmol/L stock solution in ethanol, cholesterol) for 30 and 60 minutes. No difference was found between control cells after 30- and 60-minute exposures. As indicated by horizontal lines, endothelial Ca2+ signaling was evoked in suspended cells by 100 nmol/L bradykinin in the nominal absence of extracellular Ca2+ followed by addition of 2.5 mmol/L CaCl2. Columns and points represent mean±SEM (A, n=6; B, n=8).*P<0.05 versus cells not preincubated with cholesterol. Similar results were obtained in cell monolayer experiments (n=7).

The correlation between membrane viscosity and agonist-induced intracellular Ca²⁺ release (100 nmol/L bradykinin) was negative (Figure 11A). Endothelial cells were incubated for 20 or 60 minutes in DMEM containing 150 µU/mL XO and 1 mmol/L HX to decrease cell membrane viscosity. To increase cell membrane viscosity, cells were exposed to 25 or 250 µmol/L cholesterol for 30 or 60 minutes. Similar results were obtained when endothelial capacitative Ca²⁺ entry activity was correlated with membrane viscosity (data not shown). In agreement with these findings on bradykinin-induced Ca²⁺ signaling, alterations of TG-induced hyperpolarization were negatively correlated with changes in membrane viscosity (Figure 11B).

View larger version (14K): [in this window] [in a new window]   Figure 11. Correlation of bradykinin-initiated Ca2+ release (A) and TG-induced hyperpolarization (B) in cultured endothelial cells with membrane viscosity measured after exposure to cholesterol or the superoxide anion–generating system. In untreated cells (open squares) and in cells preincubated with either cholesterol (open symbol) or XO in the presence of HX (filled symbols), membrane viscosity (y axis) and peak intracellular Ca2+ release to 100 nmol/L bradykinin (A, n=6) and peak membrane hyperpolarization to TG (2 µmol/L; B, n=3) in the nominal absence of extracellular Ca2+ (in percent of response in control cells) were measured.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Among the oxygen free radicals produced in the vascular wall, only peroxides (H₂O₂, 2,5-butylhydroperoxide) have been intensively studied for their effect on endothelial Ca²⁺ signaling. Thus, peroxides in concentrations >30 µmol/L attenuate agonist-induced intracellular Ca²⁺ release and capacitative Ca²⁺ entry in a time-dependent manner.³² However, prolonged incubation with peroxides initiates Ca²⁺ entry per se.³³ In contrast, we demonstrated that hyperglycemia enhances endothelial Ca²⁺ signaling owing to the formation of superoxide anions.¹⁴ In agreement with this previous report, the current study shows that preincubation of endothelial cells with a superoxide anion–generating system mimics the effects of hyperglycemia. Because XO had no effect on endothelial Ca²⁺ signaling in the absence of HX, the changes reported herein are due to the formation of superoxide anions. This conclusion is further supported by abolition of the effect of XO/HX in the presence of SOD.

Tan et al³⁴ showed that oxygen-derived free radicals stimulate adenylyl cyclase via TK activation in A10 cells. In endothelial cells, the TK inhibitor tyrphostin A48 had no effect on superoxide anion–mediated augmentation of agonist-initiated Ca²⁺ signaling. These findings do not support the involvement of tyrphostin A48-sensitive TKs in XO/HX-evoked changes in Ca²⁺ signaling. However, because it is possible that 1 single TK inhibitor is not able to prevent all TK activity, the involvement of TK in XO/HX-mediated changes in Ca²⁺ signaling cannot be excluded. In spite of this, superoxide anions actually enhanced endothelial TK activity. Fleming and coworkers²² have suggested that TK activity is involved in endothelial capacitative Ca²⁺ entry. On the contrary, Vostal and Shafer³⁵ found that capacitative Ca²⁺ entry was independent of any TK activity. Although our results show that an increase in tyrphostin A48-sensitive TK activity may not be involved in the enhancement of capacitative Ca²⁺ entry after XO/HX treatment, these findings do not give any indication whether TKs are involved in endothelial Ca²⁺ signaling. Moreover, it needs to be investigated whether the observed increase in TK activity by XO/HX might influence other TK-mediated cellular functions, like shear stress–activated NO formation.³⁶

The concentration-response relationship of superoxide anions with respect to amplification of intracellular Ca²⁺ release was identical to that for capacitative Ca²⁺/Mn²⁺ entry. The augmented intracellular Ca²⁺ release in response to the IP₃-generating compounds bradykinin and ATP after superoxide anion treatment might be due to enhanced IP₃ formation on addition of bradykinin to XO/HX-treated cells. Similar findings were described in airway epithelium.³⁷ These data indicate that the superoxide anion might affect either phospholipase C activity or receptor–G protein–phospholipase C coupling. Radical-mediated changes in G protein activity/coupling have been reported for NO on G_o proteins in neurons³⁸ and for oxyradicals for isoproterenol receptor–coupled G_i and G_S proteins in ischemic/reperfused hearts.³⁹ Additional studies are needed to clarify whether the reported changes in IP₃ production by superoxide anion preincubation are due to changes in G protein activity/coupling or phospholipase C activity.

Because intracellular Ca²⁺ store depletion regulates the activity of capacitative Ca²⁺ entry pathways in endothelial cells,¹⁵ one might speculate that the effect of superoxide anions on capacitative Ca²⁺/Mn²⁺ entry is due to the pronounced depletion of Ca²⁺ pools. Our finding that BHQ- and TG-induced capacitative Ca²⁺ entry was also enhanced in XO/HX-treated cells while intracellular Ca²⁺ release remained unchanged after superoxide anion treatment indicates that in addition to its effect on IP₃ formation, XO/HX directly affects the mechanism(s) involved in capacitative Ca²⁺/Mn²⁺ entry. The mechanisms of capacitative Ca²⁺ entry regulation in endothelial cells are still poorly understood (Figure 1).^{40 41} We have provided evidence for the involvement of CYP450 MO–derived arachidonic acid metabolites, the epoxyeicosatrienoic acids (EETs), in autacoid-stimulated capacitative Ca²⁺ entry and membrane hyperpolarization.^{19 20} Although membrane hyperpolarization does not open the capacitative Ca²⁺ entry pathway in endothelial cells, it provides the driving force for Ca²⁺ to enter the cell.^{21 42} Because CYP450 MO activity was enhanced by preincubation with XO/HX, one might expect that the enhanced formation of EETs results in augmentation of endothelial capacitative Ca²⁺/Mn²⁺ entry due to enhanced EET-mediated membrane hyperpolarization. Such enhancement of capacitative Ca²⁺/Mn²⁺ entry by enhanced membrane hyperpolarization in endothelial cells was described for the K_ATP channel opener Hoe-234⁴³ and for K_Ca channel activation by cAMP.⁴⁴ In addition, direct effects of EETs on the capacitative Ca²⁺ entry pathway itself are also possible.

In agreement with our findings, in atherosclerotic vessels agonist-induced (ie, Ca²⁺-mediated) production of NO is augmented,⁴⁵ although reduced NO-mediated relaxation is observed owing to degradation of NO by oxygen free radicals.⁴⁶ It needs to be investigated whether superoxide anions may serve as mediators of a compensatory adaptation of the endothelium in states with increased oxygen radical production (eg, hypercholesterolemia or hyperglycemia) to maintain the Ca²⁺-mediated release of vasodilator mediators, although the bioactivity of NO is diminished owing to its degradation by free radicals.

Because cycloheximide had no effect on superoxide anion–initiated changes in endothelial Ca²⁺ signaling, the involvement of nuclear responses via superoxide anion–initiated transcription factors (eg, nuclear factor-B) followed by changes in gene expression resulting in an altered Ca²⁺ signaling cascade might be excluded in the short-term model used in this study.

It is possible that each mechanism described might have specific superoxide anion–sensitive elements or that superoxide anions affect 1 parameter common to all mechanisms studied. Such a parameter common to all phenomena studied could be membrane fluidity. In agreement with reports from the literature,⁴⁷ incubation with XO/HX decreased membrane viscosity in endothelial cells. This mechanism was prevented by SOD. In contrast, cholesterol loading of endothelial cell membranes yielded an increase in membrane viscosity,⁴⁸ which was associated with a decrease in bradykinin-initiated Ca²⁺ signaling (ie, intracellular Ca²⁺ release and capacitative Ca²⁺ entry) and TG-induced membrane hyperpolarization. In the current work, a negative correlation between membrane viscosity and agonist-stimulated intracellular Ca²⁺ release and TG-induced hyperpolarization could be demonstrated, suggesting that the superoxide anion–mediated decrease in membrane viscosity might have significant impact on endothelial Ca²⁺ signaling mechanisms. In agreement with our findings, increased phospholipase C activity in response to agonist by decreased membrane viscosity was shown in the mouse brain, heart, and liver⁴⁹ and in rat ventricular myocytes.⁵⁰ Similar to changes in plasmalemmal enzyme/channel activity by modulations in cell membrane viscosity, microsomal enzyme activities are altered by changes in fluidity of the microsomal membrane.⁵¹ Such increased microsomal membrane fluidity might explain the enhanced microsomal CYP450 MO activity after exposure of endothelial cells to XO/HX presented here. Other than membrane-bound enzymes, changes in membrane viscosity have also been demonstrated to modulate Na⁺, K⁺, and Ca²⁺ channel activity.^{52 53} Additional studies are necessary to clarify whether the observed increase in membrane hyperpolarization in XO/HX-pretreated cells is due to direct modulation of membrane channel activity (eg, K_Ca channels) or an increase in activity of microsomal CYP450 MOs.

In this study, an additional role for superoxide anions beside their involvement in the development of cellular/vascular dysfunction is proposed. We have demonstrated that superoxide anions modulate the mechanisms involved in endothelial Ca²⁺ signaling shown in Figure 1 (phospholipase C, membrane hyperpolarization, TK, and CYP450 MO). These effects of superoxide anions might result in enhanced NO biosynthesis during enhanced superoxide formation,^{14 45} whereas on the other hand, superoxide anions attenuate NO bioactivity.^{45 54} Additional studies are necessary to understand under which circumstances the deleterious properties of superoxide anions overcome the beneficial effects reported here.

	Acknowledgments

 
This work was supported by the Austrian Science Fund (P12341-MED), the Austrian National Bank (6568), the Austrian Heart Foundation (ÖHF), and the Franz Lanyar Foundation (all to W.F.G.). The authors wish to thank Dr Jean-Vivien Mombouli for critically reviewing this manuscript. The excellent technical assistance of Beatrix Petschar and Andreas Maier are appreciated by all authors.

Received October 16, 1997; accepted March 27, 1998.

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