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Circulation Research. 1999;84:1203-1211

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(Circulation Research. 1999;84:1203-1211.)
© 1999 American Heart Association, Inc.

Original Contribution

Chemiluminescent Detection of Oxidants in Vascular Tissue

Lucigenin But Not Coelenterazine Enhances Superoxide Formation

Margaret M. Tarpey, C. Roger White, Edward Suarez, Gloria Richardson, Rafael Radi, Bruce A. Freeman

From the Departments of Anesthesiology (M.M.T., G.R., B.A.F.), Medicine (C.R.W.), and Biochemistry and Medical Genetics (B.A.F.), and the Center for Free Radical Biology (M.M.T., C.R.W., B.A.F.), University of Alabama at Birmingham, Birmingham, Ala, and Department of Biochemistry (E.S., R.R.), Universidad de la Republica, Montevideo, Uruguay.

Correspondence to Margaret M. Tarpey, Department of Anesthesiology, University of Alabama at Birmingham, 619 19th St S, Birmingham, AL 35233. E-mail margaret.tarpey{at}ccc.uab.edu


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Lucigenin-amplified chemiluminescence has frequently been used to assess the formation of superoxide in vascular tissues. However, the ability of lucigenin to undergo redox cycling in purified enzyme-substrate mixtures has raised questions concerning the use of lucigenin as an appropriate probe for the measurement of superoxide production. Addition of lucigenin to reaction mixtures of xanthine oxidase plus NADH resulted in increased oxygen consumption, as well as superoxide dismutase–inhibitable reduction of cytochrome c, indicative of enhanced rates of superoxide formation. Additionally, it was revealed that lucigenin stimulated oxidant formation by both cultured bovine aortic endothelial cells and isolated rings from rat aorta. Lucigenin treatment resulted in enhanced hydrogen peroxide release from endothelial cells, whereas exposure to lucigenin resulted in inhibition of endothelium-dependent relaxation in isolated aortic rings that was superoxide dismutase inhibitable. In contrast, the chemiluminescent probe coelenterazine had no significant effect on xanthine oxidase–dependent oxygen consumption, endothelial cell hydrogen peroxide release, or endothelium-dependent relaxation. Study of enzyme and vascular systems indicated that coelenterazine chemiluminescence is a sensitive marker for detecting both superoxide and peroxynitrite.


Key Words: superoxide • free radical • lucigenin • coelenterazine • peroxynitrite


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Superoxide (O2) and other oxidants play a central role in the maintenance of vascular homeostasis and injury. Nitric oxide (·NO)-dependent cell signaling, including endothelium-dependent relaxation, is modulated by both O2 and superoxide dismutases (SODs), the family of enzymes that catalyze the formation of hydrogen peroxide (H2O2) and molecular oxygen from O2.1 2 3 4 Alterations in both the rates of formation and extents of scavenging of O2 have been implicated in the vascular dysfunction seen in atherosclerosis, hypertension, diabetes, and chronic nitrate tolerance.5 6 7 8 9 10 Finally, increased rates of vascular production of O2 contribute to the initiation of proinflammatory events, with transcriptional regulation of the gene expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 sensitive to changes in cellular oxidant production.11 12

Measurement of the vascular production of O2 is problematic for several reasons. Low intracellular steady-state concentrations occur as a result of the balance between the endogenous rates of partial reduction of oxygen to O2 and the scavenging of O2 by highly efficient cytoplasmic and mitochondrial SODs, resulting in intracellular O2 concentrations estimated to rarely exceed 10 nmol/L.13 Extracellular release of small proportions of intracellularly formed O2 may occur via diffusion through anion channels.14 15 Also, O2 formed from plasma membrane-bound oxidases remains at relatively low levels in vascular tissues because of serum and extracellular fluid components, including low molecular weight oxidant scavengers and the heparin-binding extracellular SOD.3 16 17 18 19

Lucigenin-enhanced chemiluminescence has been used extensively as an indicator of O2 production by O2-generating enzyme systems and activated leukocytes20 21 22 23 and has been adopted for use in the estimation of O2 production by cultured cell systems, tissue homogenates, and intact vascular tissue.2 24 25 26 Initial studies of mechanisms of O2-dependent lucigenin chemiluminescence showed that lucigenin must first undergo univalent reduction, before a secondary reaction with O2, to ultimately result in light emission.27 More recently, lucigenin has been demonstrated to undergo cycles of univalent reduction followed by auto-oxidation to yield O2, thus accelerating rates of O2 generation by purified enzyme-substrate complexes.28 29 30 31 Although no lucigenin-dependent stimulation of cell O2 production was apparent from activated macrophages undergoing respiratory burst,32 this is in contrast to results obtained when low levels of O2 were generated,32 a condition more reflective of oxidant production in both normal and diseased vascular tissues. The concentration-dependent enhancement by lucigenin of O2 formation is particularly troublesome in studies using vascular tissue, in which concentrations of lucigenin from 50 to 500 µmol/L are typically used in an attempt to amplify the chemiluminescent signal obtained from low rates of O2 production. These concerns led us to investigate the potential for lucigenin to enhance endogenous rates of vascular oxidant production in 2 model systems, bovine aortic endothelial cells (BAECs) in vitro and isolated rings from rat aorta, as well as its ability to undergo redox cycling during enzymatic generation of low levels of O2. From these studies, we reveal the limitations extant in the use of lucigenin to quantify vascular O2 production. In addition, the luminophore coelenterazine (2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo[1,2-{alpha}]pyrazin-3-one) has been reported to be useful in the detection of inflammatory cell-derived O2.33 Thus, purified enzymatic sources of O2 and H2O2 were used to describe the oxidant-dependent chemiluminescence properties of coelenterazine and its response to ·NO-derived metabolites as previously for lucigenin and luminol.27 28 31 34 Both cell culture and isolated aortic ring systems, in contrast to lucigenin, demonstrate the absence of accelerated O2 generation on exposure to up to 50 µmol/L coelenterazine, a concentration almost 20-fold in excess of that required for maximal chemiluminescence yield in purified enzymatic systems, affirming the utility of this cell-permeant probe for the estimation of O2 and peroxynitrite (ONOO-) in biological systems.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Chemicals and Reagents
FCS and iron-supplemented calf serum were acquired from Hyclone. Cell culture reagents were from GIBCO. SOD and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) were obtained from OXIS, Inc. Coelenterazine was from Molecular Probes. Glucose oxidase (GO) and xanthine oxidase (XO) were obtained from Calbiochem. Manganese tetrakis (N-ethylpyridinium-2-yl)porphyrin (T2E), an SOD mimetic, was a gift from Dr Irwin Fridovich (Duke University). All other chemicals were purchased from Sigma.

Animals
Ten-week-old male Sprague-Dawley rats (300 to 325 g) were obtained from Harlan, Inc (Wilmington, MA), for vascular function analysis. All rats were maintained at constant humidity (60±5%), temperature (24±1°C), and light cycle (6 AM to 6 PM) and were fed a standard rat pellet diet (Ralston Purina Diet) ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham and were consistent with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1985).

ONOO– Synthesis
ONOO– was synthesized as previously described and treated with MnO2 to eliminate residual H2O2.35 The concentration of ONOO- was determined spectrophotometrically ({epsilon}302=1.67 mmol/L-1 · cm-1),36 with working solutions of ONOO- prepared by dilution in 0.1N NaOH and used within 15 minutes.

Biochemical Analyses
XO activity was monitored spectrophotometrically by measuring rates of uric acid production at 295 nm ({epsilon}=11 mmol/L-1 · cm-1) in 50 µmol/L xanthine and 50 mmol/L potassium phosphate buffer, pH 7.4. GO activity was determined by polarographic measurement of oxygen consumption of 5 to 10 U · mL-1 enzyme in 1 mmol/L glucose and 50 mmol/L potassium phosphate buffer, pH 7.4. Extracellular release of H2O2 from BAEC was measured in cells incubated in HBSS by reaction of medium removed from cell monolayers with ammonium ferrous sulfate and potassium thiocyanate.37 38 Superoxide generation was measured by the SOD-inhibitable reduction of 10 µmol/L oxidized cytochrome c monitored spectrophotometrically at 550 nm, using {epsilon}M=21 mmol/L-1 · cm-1.39

Chemiluminescence Measurements
Lucigenin- and coelenterazine-enhanced chemiluminescence was monitored using a Thorn EMI photon counter equipped with an EMI Fact 50 Mk III photomultiplier cooled to –20°C. The voltage in the photomultiplier was kept at 1500 V. Reactions were carried out in continuously stirred glass tubes containing 3-mL reaction volumes maintained at 37°C. Data acquisition and analysis were performed with DQ software (DATAQ Instruments).

Cell Culture
BAECs were cultured in medium 199 with 5% newborn calf serum, 5% iron-supplemented calf serum, 100 U · mL-1 penicillin, and 100 µg · mL-1 streptomycin. Experiments were carried out on subcultured passage 5 to passage 10 cells within 36 hours of reaching confluence. The purity of cultures was determined on the basis of their characteristic cobblestone morphology, positive immunostaining for factor VIII antigen, and selective incorporation of fluorescently labeled acetylated LDL. Cellular protein content was measured by reaction with bicinchoninic acid.40

Vessel Reactivity Studies
The effects of lucigenin and coelenterazine on endothelium-dependent relaxation was assessed in isolated rat aortic ring segments. Briefly, the aorta was excised and placed in prewarmed and oxygenated Krebs-Henseleit buffer and dissected free of fat and adhering tissue. Individual ring segments (2 to 3 mm in width) were cut from vessels, and each vascular ring was mounted on 2 stainless steel hooks and suspended from a force-displacement transducer in a water-jacketed tissue bath. The ring was anchored to a hook at the base of the chamber, and a passive load of 2.0 g was applied to vessels. Ring segments were bathed in a Krebs-Henseleit solution of the following composition (in mmol/L): NaCl 118, KCl 4.6, NaHCO3 27.2, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.75, Na2-EDTA 0.03, and glucose 11.1 (pH 7.4). The solution was continuously aerated with a 95% O2, 5% CO2 gas mixture unless specified and maintained at 37°C. Changes in isometric tension were measured using capacitative force transducers (Radnoti Glass Technology, Inc). Real-time data were acquired and digitized using an IBM-compatible computer and stored for later analysis using commercially available software (Experimenter's Workbench, Datawave, Inc).

After an equilibration period of 45 minutes, ring segments were depolarized with KCl (70 mmol/L) to determine the contractile capacity of the vessel. Rings were then thoroughly washed and allowed to equilibrate for an additional 45 minutes. All experiments were performed in the presence of indomethacin (5 µmol/L) to eliminate the effects of cyclooxygenase-derived vasoactive molecules. In initial studies, ring segments were submaximally contracted with the {alpha}-adrenergic agonist phenylephrine (PE). When tension development reached a plateau, ·NO-dependent relaxation was tested in PE-contracted ring segments by cumulative exposure to acetylcholine (ACh; 1x10-9 to 3x10-6 mol/L), an endothelium-dependent vasodilator. Vessels were then thoroughly washed and allowed to recover for 1 hour. In the next series of experiments, lucigenin (100 and 250 µmol/L) was added to the tissue bath before the initiation of the PE/ACh protocol. No effect of lucigenin on basal arterial tone was noted under these conditions. In some experiments, the effect of lucigenin on ·NO-dependent relaxation was assessed in tissues that were treated with 95% compressed air, 5% CO2. Recombinant chimeric heparin binding human CuZn SOD (100 U · mL-1)10 or native bovine CuZn SOD (200 U · mL-1) was added in some experiments to the tissue bath 5 minutes before the addition of lucigenin. In parallel experiments, the effects of coelenterazine (10 and 50 µmol/L) on ·NO-dependent relaxation were also tested. As for experiments studying lucigenin effects on vascular function, coelenterazine was added to the tissue bath before exposure to PE. All experiments were performed in the dark to minimize potential photochemical reactions of buffers, lucigenin, and coelenterazine mediated by fluorescent light.

Vessel Chemiluminescence Studies
Similarly isolated vascular ring segments were incubated with DMNQ (100 µmol/L) in the presence or absence of the SOD mimetic MnT2E (100 µg · mL-1) in 50 mmol/L potassium phosphate and 100 µmol/L EDTA, pH 7.4, for 3 hours at 37°C. Control rings were incubated with 0.95% ethanol, the vehicle used to dissolve DMNQ. Ring segments were rinsed thoroughly, and 10 µmol/L coelenterazine was added to assess vascular chemiluminescence.

Statistical Analysis
Comparisons between groups were made by use of 1-way ANOVA. When significance was indicated, Student-Newman-Keuls post hoc analysis was performed. P<0.05 was considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Lucigenin-Dependent Oxidant Production
To study the effect of lucigenin on low rates of O2 formation, as might be expected in vascular tissues in contrast to the high-output O2 production associated with activated leukocytes, the reduction of XO by NADH was used as an exemplary reduced flavoprotein.32 Figure 1Down demonstrates the effect of lucigenin on XO plus NADH–dependent oxidation to yield O2, as measured by cytochrome c reduction. In the presence of 150 µmol/L NADH, 50 mU · mL-1 XO reduced cytochrome c at a rate of 0.32 µmol/L · min-1, with the rate of cytochrome c reduction diminished 93% by 30 U · mL-1 SOD. The basal rate of cytochrome c reduction was increased 5-fold, to 1.64 µmol/L · min-1 by 20 µmol/L lucigenin. Lucigenin-enhanced cytochrome c reduction was inhibited 43% by SOD, to 0.94 µmol/L · min-1. Addition of coelenterazine (10 and 50 µmol/L) had no effect on the rate of XO plus NADH–mediated cytochrome c reduction (data not shown). Table 1Down depicts the rates of oxygen consumption by reaction systems under these conditions. Both 20 and 250 µmol/L lucigenin enhanced the rate of XO plus NADH–dependent oxygen consumption, whereas 10 µmol/L coelenterazine had no effect. Lucigenin also mediated direct (ie, O2-independent) cytochrome c reduction by XO, as indicated by the significant fraction (57%) of SOD-resistant cytochrome c reduction (Figure 1Down). This direct reduction of cytochrome c by lucigenin was also investigated using GO plus glucose, as before.28 At pH 7.4, GO plus glucose divalently reduces oxygen to H2O2, with no O2 formation detected by assessment of rates of cytochrome c reduction. However, addition of lucigenin resulted in dose-dependent increases in reduction of cytochrome c (Table 2Down). Addition of up to 300 U · mL-1 SOD had no effect on rates of cytochrome c reduction, again revealing direct O2-independent electron transfer from lucigenin to cytochrome c. In contrast, up to 50 µmol/L coelenterazine did not significantly enhance rates of cytochrome c reduction.



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  		Figure 1. SOD-inhibitable reduction of cytochrome c by XO. Reaction mixtures contained 150 µmol/L NADH, 50 mU · mL-1 XO in 50 mmol/L potassium phosphate and 100 µmol/L EDTA buffer, pH 7.4. Addition of 20 µmol/L lucigenin (LUC) increased the rate of cytochrome c reduction 5-fold. In the presence of 30 U · mL-1 SOD, lucigenin-enhanced cytochrome c reduction was inhibited 43%.


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  		Table 1. Influence of Lucigenin on Xanthine Oxidase–Dependent O2 Consumption


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  		Table 2. Influence of Lucigenin or Coelenterazine on GO–Dependent Cytochrome c Reduction

Lucigenin-Enhanced Vascular Production of Oxidants
Endothelial cell monolayer O2 production measured via SOD-inhibitable cytochrome c reduction, in the presence or absence of lucigenin, could not be determined reliably because of complications induced by very low rates of cell O2 release, SOD-insensitive direct reduction of cytochrome c by cellular components, cytochrome c adsorption to cell monolayers, and the reoxidation of reduced cytochrome c by accumulating H2O2. Therefore, the extracellular release of H2O2, the product of both enzymatic and spontaneous dismutation of O2, was used to reflect changes in cellular O2 formation.41 Preliminary studies revealed that lucigenin interfered with assay systems for H2O2 that relied on peroxidase-mediated oxidation of phenolic compounds, eg, p-hydroxyphenylacetic acid. Thus, extracellular release of H2O2 from BAEC was measured by reaction of medium removed from cell monolayers with ammonium ferrous sulfate and potassium thiocyanate.37 38

Exposure of BAEC to 50 and 250 µmol/L lucigenin resulted in significant increases in the extracellular release of H2O2 over time (Figure 2Down), demonstrating a lucigenin-dependent acceleration in the rates of either extracellular or intracellular partial reduction of oxygen to O2 and its dismutation product, H2O2. In the absence of cells, there was no increase in H2O2 concentrations over time (not shown), establishing lucigenin-dependent enhancement of H2O2 formation as a cell-associated event. The addition of catalase (200 U · mL-1) to reactions completely inhibited H2O2 accumulation (not shown).



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  		Figure 2. Lucigenin-dependent H2O2 release from BAECs. BAECs were thoroughly rinsed and then incubated with lucigenin for 3 hours at 37°C. Medium was removed and acidified for measurement of H2O2. Cells were assayed for protein content. Data are mean±SEM of 3 separate experiments repeated in triplicate. *P<0.05 compared with control.

The addition of lucigenin (100 and 250 µmol/L) to isolated aortic rings inhibited endothelium-dependent relaxation (Figure 3ADown). The presence of SOD in the organ bath restored responsiveness to ACh, indicating lucigenin-enhanced vascular production of O2, which in turn reacted with the added SOD.5 Because of the concern that the hyperoxic environment (95% O2, 5% CO2) in which vascular relaxation studies are typically performed may enhance O2 formation42 43 44 and artifactually increase vascular reduction of lucigenin and thus rates of formation of partially reduced oxygen species, studies were also conducted in 21% O2, 5% CO2 mixtures, with similar results (not shown). In contrast, addition of coelenterazine to cultured endothelial cells had no effect on extracellular H2O2 release (Table 3Down) or endothelium-dependent relaxation (Figure 3BDown).



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  		Figure 3. Influence of lucigenin or coelenterazine on endothelium-dependent relaxation of thoracic aortic rings. A, Lucigenin (100 and 250 µmol/L) was added to the tissue bath, and vascular relaxation responses to ACh were determined. CuZn SOD (200 U · mL-1)–restored lucigenin (250 µmol/L) inhibited endothelium-dependent relaxation. {diamondsuit}P<0.05 compared with control; *P<0.05 compared with control and 100 µmol/L lucigenin. B, Addition of coelenterazine (10 and 50 µmol/L) had no effect on the response to ACh. Numbers in parentheses refer to number of animals from which vessels were used to construct curves. Data are mean±SEM.


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  		Table 3. Effect of Coelenterazine on H2O2 Release From BAECs

Coelenterazine-Dependent Chemiluminescence
To characterize the utility of coelenterazine as a probe for O2 and other biologically relevant oxidants, chemiluminescence experiments were performed. The O2 and H2O2 generating system of acetaldehyde and XO was used to limit the accumulation of purine oxidation products (eg, urate) that scavenge ·NO- and XO-derived reactive species.45 Coelenterazine-enhanced chemiluminescence yields were linearly related to coelenterazine concentrations ranging from 0.5 to 3 µmol/L (Figure 4Down). Chemiluminescence was directly proportional to increasing rates of O2 and H2O2 formation by XO up to 14 µmol/L · min-1 O2 (Figure 5Down). Addition of catalase had no effect on coelenterazine chemiluminescence from acetaldehyde and XO (Figure 6Down), nor did bolus addition of up to 1 mmol/L H2O2 (not shown). In contrast, 20 U · mL-1 SOD fully inhibited chemiluminescence (Figure 6Down). Together, these data confirm that under these conditions coelenterazine chemiluminescence was dependent on O2 generation alone and not H2O2, as previously reported for an XO plus hypoxanthine reaction mixture.33



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  		Figure 4. Superoxide-dependent chemiluminescence as a function of coelenterazine concentration. Reactions consisted of 120 µmol/L acetaldehyde, 8 mU · mL-1 XO (generating 7 µmol/L · min-1 O2) and varying concentrations of coelenterazine in 50 mmol/L potassium phosphate buffer, pH 7.4, at 37°C.



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  		Figure 5. Coelenterazine-enhanced chemiluminescence as a function of rate of superoxide generation. Reaction mixtures contained 4 µmol/L coelenterazine, 120 µmol/L acetaldehyde, and varying concentrations of XO in 50 mmol/L potassium phosphate buffer, pH 7.4, at 37°C.



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  		Figure 6. Influence of hydrogen peroxide and superoxide on coelenterazine-enhanced chemiluminescence. Reaction mixtures contained 8 µmol/L coelenterazine, 120 µmol/L acetaldehyde, and 8 mU · mL-1 XO (generating 7 µmol/L · min-1 O2). Where indicated, 200 U · mL-1 catalase and 20 U · mL-1 SOD were added.

Repeated bolus addition of ·NO to this O2-generating system resulted in an initial decrease in the chemiluminescent signal, followed by a transient increase (Figure 7ADown). This suggests that ·NO inhibits the chemiluminescence yield of O2 and coelenterazine, whereas the product of ·NO and O2 reaction, ONOO-, also stimulated coelenterazine chemiluminescence. This was confirmed by bolus addition of 10 µmol/L ONOO-, which resulted in a rapid increase and subsequent decay of coelenterazine chemiluminescence (Figure 7BDown), whereas the decomposition products of ONOO-, primarily nitrate and nitrite, did not stimulate chemiluminescence.



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  		Figure 7. The effect of ·NO and ONOO- on superoxide-dependent coelenterazine chemiluminescence. A, Reaction mixture contained 1 µmol/L coelenterazine, 120 µmol/L acetaldehyde, and 2 mU · mL-1 XO (generating 1.6 µmol/L · min-1 O2). Where indicated, 1.6 µmol/L ·NO was added. B, Reaction mixture contained 1 µmol/L coelenterazine and bolus addition of 10 µmol/L ONOO- (solid line) or decomposed ONOO- (dashed line), as indicated by the arrow.

To test the utility of coelenterazine as a detector of vascular oxidant production, isolated rat aortic segments were incubated with DMNQ, a cell-permeant redox-cycling quinone that stimulates O2 and H2O2 production.46 After a 3-hour incubation, coelenterazine (10 µmol/L) was added and chemiluminescence was measured. Table 4Down demonstrates an increase in coelenterazine-enhanced chemiluminescence in rings treated with DMNQ, compared with control rings. The membrane-permeable low molecular weight SOD mimetic MnT2E47 decreased chemiluminescence below control levels, suggesting ongoing oxidant production under basal conditions, as noted previously.2 4


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  		Table 4. Coelenterazine-Enhanced Chemiluminescence of Vascular Rings


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Lucigenin, added in concentrations that are commonly used to assess O2 production in vascular systems (50 to 250 µmol/L), increased both endothelial cell H2O2 production and O2-dependent inhibition of endothelium-dependent relaxation. This demonstrates that lucigenin undergoes sequential cycles of reduction and reoxidation by cellular oxidoreductases with an ultimate acceleration of electron transfer to oxygen, leading to enhanced rates of vascular cell production of O2 and accumulation of its dismutation product, H2O2. Reaction of lucigenin-derived O2 with ·NO was manifest by the acute impairment of agonist-induced vascular relaxation by lucigenin. In aggregate, these properties of lucigenin will lead to inaccurate and artifactual overestimation of rates of tissue O2 production.

Although there are several approaches that sensitively detect the reduction of oxygen to O2, the low intracellular steady-state levels of O2 typically present in vascular tissues have presented methodological challenges. First, the efficient and redundant systems that have evolved to scavenge O2 require that any detection technique must be sensitive enough to effectively compete with these intracellular and extracellular antioxidant components for reaction with O2. Second, there is often inadequate access of probes to accurately assess changes in rates of O2 production at intracellular sites. For example, cytochrome c and native CuZn SOD are excluded from the intracellular milieu and differentially interact with cell surfaces on the basis of charge repulsion and size exclusion.48 Third, many O2 detection reactions are not sufficiently specific, eg, ferricytochrome c reduction can occur by O2-independent direct electron transfer (Table 2Up),49 and reduced cytochrome c can be reoxidized by cell-derived reactive species such as H2O2 and ONOO-.50 51 Finally, the potential toxicity of some O2 probes limits extended application in cell or tissue-based systems, as with some spin traps used for electron paramagnetic resonance spectroscopy.52

Lucigenin-enhanced chemiluminescence has been used because of its potential to overcome the issues of intracellular access, purported specificity for reaction with O2, and minimal cellular toxicity. The mechanism of O2-dependent lucigenin chemiluminescence requires an initial 1-electron reduction of the lucigenin di-cation to the mono-cation radical (Equation 1Down), with the mono-cation radical then reacting with O2 to yield a dioxetane (Equation 2Down). Spontaneous decomposition of the unstable dioxetane results in the formation of an electronically excited acridone whose return to ground state is accompanied by light emission. However, the mono-cation radical can be formed via O2-independent electron transfer reactions to then directly reduce oxygen, resulting in the accelerated production of O2 and reformation of the lucigenin di-cation (Equation 3Down). Equation 2Down may predominate under conditions that produce large amounts of O2, such as in XO plus xanthine reaction mixtures or during the oxidative activation of neutrophils or macrophages.32 The contribution of Equation 3Down is significant, however, especially for systems that have lower rates of O2 production.28 29 32 It was therefore important to ascertain the potential for lucigenin-dependent modulation of O2 generation in vascular tissues.

(1)

(2)

(3)
Numerous enzymes, including XO, GO, aldehyde oxidase, lipoamide dehydrogenase, and endothelial ·NO synthase, have been noted to directly reduce the lucigenin di-cation to its mono-cation radical28 29 32 (Equation 1Up). Several of these systems result in lucigenin-dependent stimulation of the partial reduction of oxygen, as indicated by accelerated rates of oxygen consumption, O2 production, and H2O2 accumulation after O2 dismutation (Figures 1Up and 3Up, Tables 1Up and 3Up).28 31 32 In vascular diseases, there is an enhanced infiltration of inflammatory cells containing high levels of NADPH oxidase and expression by vascular cells of not only neutrophil-like oxidases, but other oxidases as well (eg, 15-lipoxygenase, XO), all capable of mediating direct electron transfer to lucigenin.9 53 54 55 56 57 58 59 60 Within an intact cellular or tissue environment, it has not been determined with precision the enzyme(s) that are responsible for the observed "lucigenin reductase" activity. Thus, in conditions that have been associated with elevated lucigenin chemiluminescence, the apparent increase in rates of O2 production may in part be due to enhanced rates of direct reduction of lucigenin by electron transfer systems, rather than a true increase in tissue O2 generation. For example, the hypercholesterolemic rabbit, a model for atherosclerosis, displays an increase in vascular XO activity59 and enhanced lucigenin-dependent chemiluminescence,25 59 61 with increased rates of vascular O2 production proposed on the basis of these increases in lucigenin chemiluminescence. Importantly, other lines of evidence also infer an actual increase in steady-state O2 concentrations in this model, such as the partial normalization of impaired endothelium-dependent relaxation by cell-permeant SODs and inhibitors of XO.5 62 However, the present observations reveal that it is not feasible to estimate actual rates of vascular O2 production using lucigenin. It remains possible that lucigenin chemiluminescence may serve as a nonspecific redox-sensitive marker for the enhanced electron transfer activities that are present in various pathologies, with the caveat that (from the present and previous observations) the estimation of O2 formation from such electron transfer processes may be artifactually enhanced. This is especially the case for mitochondria, organelles rich in flavoproteins that can directly reduce lucigenin to the mono-cation radical. This will enhance nonrespiratory mitochondrial oxygen consumption32 because of lucigenin-mediated redirection of reducing equivalents to O2 and H2O2 formation.30 Thus, it would be expected that a significant fraction of mitochondrial lucigenin-dependent chemiluminescence is due to direct respiratory chain reduction of lucigenin, extramitochondrial diffusion of the mono-cation radical, and subsequent auto-oxidation to O2. This lucigenin-dependent chemiluminescence would be inhibitable by various electron transport and flavoprotein inhibitors, as well as by exogenously added SOD.63 Added SOD would not be expected to inhibit lucigenin chemiluminescence induced by "true" respiratory chain-derived O2, since it is a mitochondria-impermeable macromolecule and would not be anticipated to outcompete the already significant levels of respiratory chain-associated endogenous mitochondrial MnSOD.

Coelenterazine is a lipophilic luminophore originally isolated from the coelenterate Aequorea and is the light-producing chromophore in the aequorin complex.64 Coelenterazine and its analogs, including CLA (2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-{alpha}] pyrazin-3-one) and MCLA (2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-{alpha}] pyrazin-3-one), have been used to reflect O2 production in both purified enzyme reactions and activated leukocytes.33 65 66 67 Studies of the chemical nature of the excited species responsible for the chemiluminescence of pyrazin-3-one compounds are incomplete but suggest that O2-stimulated chemiluminescence occurs after direct oxidation to the acetamidopyrazine anion, which can then also be protonated to form additional neutral light-emitting excited species.68

There has not been a systematic evaluation of the potential for coelenterazine or its analogs to undergo cycles of reduction and reoxidation analogous to lucigenin, nor has there been a description of the impact of other reactive species such as ·NO or ONOO- on coelenterazine-dependent chemiluminescence. Unlike lucigenin, there was no apparent enhancement of O2 generation by coelenterazine in vessel preparations, using the exquisitely sensitive modulation of ·NO-dependent signaling by O2 as an indicator (Figure 3BUp). Similar benefits have been observed for the coelenterazine analog, CLA.69 Additional confirmation that coelenterazine does not undergo cyclic auto-oxidation reactions comes from lack of an effect by coelenterazine on rates of partial reduction of oxygen by XO and cultured vascular endothelial cells (Tables 1Up and 3Up). In aggregate, these observations support the concept that coelenterazine-dependent chemiluminescence is an oxidative process that does not require an initial reduction of the luminescent probe.66 68

The linear correlations between peak chemiluminescence response, coelenterazine concentrations between 0.5 and 3 µmol/L, and rates of SOD-inhibitable O2 generation demonstrate that coelenterazine is indeed a sensitive indicator of O2 (Figures 4 through 6UpUpUp). The coelenterazine chemiluminescence responses to ·NO and ONOO- illustrate the care that must be exercised when interpreting the measurement of reactive species in biological tissues (Figure 7AUp and 7BUp). In systems in which there is the likelihood of cogeneration of both O2 and ·NO, additional control reactions using SODs, ·NO synthase inhibitors, and ONOO- scavengers, such as urate, ebselen, or metalloporphyrins,34 70 must be performed to differentiate the reactive species responsible for coelenterazine chemiluminescence.

In summary, the use of lucigenin-enhanced chemiluminescence to estimate rates of O2 formation in vascular tissue must be viewed with caution, as its ability to undergo cycles of reduction and oxidation contributes to O2 production, confirming previous studies using purified enzyme-substrate reaction mixtures.28 29 31 Additionally, the properties of oxidant-dependent coelenterazine chemiluminescence suggest its utility as a sensitive and more reliable alternative probe in place of lucigenin for the detection of O2 and related oxidants in biological systems.


*    Acknowledgments
 
This study was supported by grants from the National Institutes of Health (K08 HL-03457; R01 HL-58115; R01 HL-51245, R01 HL-54815, and P60 HL-58418) and the American Heart Association (96015430).

Received January 15, 1999; accepted February 24, 1999.


*    References
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