© 1999 American Heart Association, Inc.
Original Contribution |
Chemiluminescent Detection of Oxidants in Vascular Tissue
Lucigenin But Not Coelenterazine Enhances Superoxide Formation
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
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Abstract |
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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
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Introduction |
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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-
]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.
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Materials and Methods |
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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 (
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 (=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
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
-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.
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Results |
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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 1
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 1 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 1). 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 2).
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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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 2), 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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The addition of lucigenin (100 and 250 µmol/L) to isolated
aortic rings inhibited endothelium-dependent relaxation
(Figure 3A). 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 3) or
endothelium-dependent relaxation (Figure 3B).
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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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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 4). Chemiluminescence was directly
proportional to increasing rates of
O2 and
H2O2 formation by XO up to
14 µmol/L · min-1
O2 (Figure 5). Addition of catalase had no effect on
coelenterazine chemiluminescence from acetaldehyde and XO (Figure 6), nor did bolus addition of up to
1 mmol/L H2O2 (not
shown). In contrast, 20 U · mL-1 SOD
fully inhibited chemiluminescence (Figure 6). 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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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 7A). 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 7B), whereas the decomposition products of
ONOO-, primarily nitrate and nitrite, did not
stimulate chemiluminescence.
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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 4 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 2
),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 1),
with the mono-cation radical then reacting with
O2 to yield a dioxetane (Equation 2). 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 3). Equation 2 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 3 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) |
).
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 1 and 3, Tables 1 and 3).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-]
pyrazin-3-one) and MCLA
(2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-]
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 3B). 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 1 and 3). 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 6). 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 7A and 7B). 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.
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Acknowledgments |
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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.
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