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2 Division of Pulmonary and Critical Care Medicine, Departments of 1 Anesthesia and Critical Care and Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21224
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Apocynin
(4-hydroxy-3-methoxy-acetophenone) inhibits NADPH oxidase in activated
polymorphonuclear (PMN) leukocytes, preventing the generation of
reactive oxygen species. To determine if apocynin attenuates
ischemia-reperfusion lung injury, we examined the effects of apocynin
(0.03, 0.3, and 3 mM) in isolated in situ sheep lungs. In
diluent-treated lungs, reperfusion with blood (180 min) after 30 min of
ischemia (ventilation 28% O2, 5% CO2) caused
leukocyte sequestration in the lung and increased vascular permeability [reflection coefficient for albumin (
alb) 0.47 ± 0.10, filtration coefficient (Kf) 0.14 ± 0.03 g · min
1 · mmHg1
· 100 g1] compared with nonreperfused lungs
(alb 0.77 ± 0.03, Kf
0.03 ± 0.01 g · min1 · mmHg1 · 100 g1; P < 0.05). Apocynin attenuated the increased protein permeability at 0.3 and 3 mM (alb 0.69 ± 0.05 and 0.91 ± 0.03, respectively, P < 0.05); Kf was
decreased by 3 mM apocynin (0.05 ± 0.01 g · min1 · mmHg1 · 100 g1, P < 0.05). Diphenyleneiodonium (DPI,
5 µM), a structurally unrelated inhibitor of NADPH oxidase, worsened
injury (Kf 0.32 ± 0.07 g · min1 · mmHg1 · 100 g1, P < 0.05). Neither apocynin nor DPI
affected leukocyte sequestration. Apocynin and DPI inhibited whole
blood chemiluminescence and isolated PMN leukocyte-induced resazurin
reduction, confirming NADPH oxidase inhibition. Apocynin inhibited
pulmonary artery hypertension and perfusate concentrations of
cyclooxygenase metabolites, including thromboxane B2. The
cyclooxygenase inhibitor indomethacin had no effect on the increased
vascular permeability, suggesting that cyclooxygenase inhibition was
not the explanation for the apocynin results. Apocynin prevented
ischemia-reperfusion lung injury, but the mechanism of protection
remains unclear.
sheep; diphenyleneiodonium; pulmonary edema; vascular permeability
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ISCHEMIA-REPERFUSION LUNG injury is a well-known clinical phenomenon characterized by increased pulmonary vascular permeability, edema, and resistance to blood flow (31). The pathogenesis of this injury appears to involve the generation of reduced oxygen species (ROS), which can be detected during both ischemia (3, 25) and reperfusion (17). The ability of superoxide dismutase to attenuate ischemia-reperfusion lung injury suggests that superoxide anion production represents a critical step in the process (31). One of the major sources of superoxide anion is the NADPH-dependent oxidase present in the plasma membrane of phagocytic cells such as polymorphonuclear (PMN) leukocytes and macrophages. The enzyme consists of a heterodimeric transmembrane protein, flavocytochrome b245, formed by the combination of gp91-phox and p22-phox and multiple cytoplasmic components, including the proteins p47-, p67-, and p40-phox (19). With appropriate stimuli, the cytosolic and membrane proteins assemble, and the enzyme generates superoxide anion by a one-electron transfer from NADPH to O2.
An injurious role for PMN leukocyte-derived NADPH oxidase has been inferred from the protective effect of PMN leukocyte depletion (30) and adhesion molecule inhibition (25, 26) on the increased pulmonary vascular permeability after ischemia (25) and ischemia-reperfusion (26, 30). For example, in ischemic rat lungs, ROS production and increased capillary protein permeability were observed in the vicinity of adherent PMN leukocytes (25). Other investigators have suggested that ROS production during pulmonary ischemia may originate from a NADPH oxidase in pulmonary endothelial cells (3). Pulmonary endothelial or macrophage-derived ROS production from this enzyme complex could explain the presence of a PMN leukocyte-independent component of lung ischemia-reperfusion injury (2, 22, 36). Alternatively, ROS from sources other than NADPH oxidase, such as xanthine oxidase (1) or cytochrome P-450 (6), may play a role.
Apocynin is a methoxy-substituted catechol derived from the root extract of the medicinal herb Picrorhiza kurroa (39). Apocynin has been shown to confer protection in animal models of arthritis (43) and in ozone (37)- and endotoxin-induced (40, 48) lung injury. These protective effects were attributed to the ability of apocynin to inhibit NADPH oxidase in PMN leukocytes by reacting with thiol groups required for enzyme assembly (39). Apocynin is thought to require activation by reacting with hydrogen peroxide and extracellular peroxidase, possibly limiting the effect of apocynin to stimulated PMN leukocytes (39, 44).
We hypothesized that apocynin would attenuate ischemia-reperfusion lung injury. To test this hypothesis, we determined the effect of 0.03, 0.3, and 3 mM apocynin on the pulmonary vascular injury caused by 30 min of ischemia and 180 min of reperfusion in isolated sheep lungs (32). Additional experiments were performed with diphenyleneiodonium (DPI), a compound that is structurally unrelated to apocynin (16) and that impairs NADPH oxidase by flavoprotein inhibition (9), and indomethacin, a blocker of cyclooxygenase, to elucidate the mechanisms of the observed apocynin effects.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Blood Studies
Effects of apocynin and DPI on whole blood luminol-enhanced
chemiluminescence.
To compare the effects of apocynin (Aldrich Chemical, Milwaukee, WI)
and DPI (Colour Your Enzyme, Ontario, Canada) on the capacity of whole
sheep blood to generate ROS, heparinized blood was diluted 1:10 in
Hanks' balanced salt solution (HBSS), and 0.78 ml was added to the
stirred, warmed (37°C) cuvette of a Chronolog 560-CA luminometer
(Chronolog Instruments, Haverton, PA). Apocynin (3, 0.3, and 0.03 mM),
DPI (5 µM), or diluent (0.1 ml) was added, and the mixture was
allowed to incubate for 30 min. Luminol (Sigma Chemical, St. Louis, MO)
stock solution (62.5 mM in DMSO) was added to achieve a final
concentration of 0.5 mM, and baseline chemiluminescence was recorded
for 2 min. Serum-treated zymosan (STZ) was added to achieve a final
solution of 1.2 mg/ml, and peak chemiluminescence was recorded. STZ was
prepared by boiling zymosan A (Sigma Chemical) in HBSS for 20 min. It
was then incubated two times (30 min each) in serum (normal human
donors) and reconstituted in HBSS (10 mg/ml). STZ was frozen and stored
at 
80°C until use.
Effects of apocynin and DPI on resazurin reduction by activated PMN leukocytes. Luminol-enhanced chemiluminescence detects both intra- and extracellular ROS but is dependent on the generation of hypochlorous acid from superoxide-derived hydrogen peroxide (46). Therefore luminol-enhanced chemiluminescence could be attenuated by mechanisms other than NADPH oxidase inhibition, such as scavenging hydrogen peroxide or hypochlorous acid. We therefore examined the effects of apocynin and DPI on resazurin reduction. Resazurin (Sigma Chemical) is directly reduced by activated NADPH oxidase to form the fluorescent product resorufin (13). Isolated PMN leukocytes (5 × 106) were incubated at 37°C for 75 min with resazurin (0.03 mg/ml), STZ (1 mg/ml), and apocynin or diluent to achieve concentrations of 3, 0.3, 0.03, or 0 mM apocynin. The effect of apocynin was compared with increasing concentrations of DPI (0.003-0.3 mM). To determine if the assay was affected by superoxide anion scavenging, the effect of superoxide dismutase (bovine erythrocyte; Sigma Chemical) was also examined. Resorufin production was monitored (excitation 530 nm, emission 590 nm) with a Cytofluor 2350 fluorometer (Millipore, Bedford, MA).
PMN isolation technique.
PMN leukocytes were isolated from peripheral blood of healthy human
donors. Briefly, whole blood was collected in a syringe containing 10 U
heparin/ml and diluted 1:2 with HBSS. Diluted whole blood (6 ml) was
centrifuged (1,850 rpm) over a Ficoll-Histopaque gradient (3 ml
Histopaque 1077 over 3 ml Histopaque 1119) for 30 min. The PMN
leukocyte layer was washed two times in HBSS and reconstituted in
NaHCO3- and MgCl2-containing HBSS.
Lung Studies
Isolated lung preparation. ISCHEMIC-REPERFUSED LUNGS.
Studies were performed in isolated sheep lungs perfused and ventilated in situ. Young sheep (20-37 kg) were anesthetized with intramuscular ketamine (30 mg/kg) and atropine (0.5 mg). Anesthesia was maintained by intravenous infusion of pentobarbital sodium (20 mg · kg1 · h1). A tracheostomy was
performed, and mechanical ventilation was begun with
oxygen-supplemented room air at a tidal volume of 12 ml/kg and
respiratory rate of 15 breaths/min. A sternotomy was performed, heparin
(10,000 units) was injected intravenously, and the animal was
exsanguinated from a left atrial cannula into the perfusion circuit.
After exsanguination, ventilation was maintained with warmed,
humidified gas at a tidal volume of 12 ml/kg and a frequency of 10 breaths/min. Inspired O2 and CO2 concentrations were 28 and 5%, respectively. End-expiratory tracheal pressure was
maintained at 5 mmHg.
The pulmonary artery was cannulated, and the pulmonary vasculature was
flushed with 300 ml of warmed 3% dextran 70 (Sigma Chemical) in normal
saline. The solution was allowed to dwell for 3 min before being
drained by gravity from the left atrium. After 30 min of ischemia, the
lungs were perfused in situ with a mixture of autologous blood
(~1,000 ml) and 3% dextran in Ringer lactate solution (~300 ml)
through an extracorporeal circuit described previously
(32). The perfusate was warmed (38-39°C) and
continuously filtered of clots and bubbles. The desired flow was
achieved over 3-5 min by gradually increasing roller pump speed.
The lungs were perfused for 180 min, with time 0 defined as
the time a flow of 90 ml · kg1 · min1 was achieved. Tracheal, pulmonary arterial
(Ppa), and left atrial pressures were measured with Statham
P50 transducers referenced to the level of the left atrium.
Ppa was determined at end expiration while the lung was
mechanically ventilated. Left atrial pressure was kept subatmospheric.
Pressures were continuously recorded (Grass model 7D polygraph;
Astro-Med, West Warwick, RI). Perfusate O2 and
CO2 tensions and pH were measured at regular intervals using standard electrode techniques. Perfusate pH was adjusted to
7.35-7.45 at 30-min intervals with 1 N NaHCO3.
Perfusate glucose concentrations were monitored with Dextro-stix and
kept within 90-130 mg/dl by periodic addition of 50% glucose in water.
Perfusate samples were obtained from the reservoir immediately before
reperfusion and from the left atrium at fixed intervals during
reperfusion and were analyzed for total leukocyte, PMN leukocyte, and
platelet counts as previously described (30). Plasma
samples were frozen for later analysis of thromboxane B2 (TxB2), the stable metabolite of thromboxane
A2, 6-keto-PGF1
, the stable metabolite of
prostacyclin, PGF2, PGE2, and
PGD2 by gas chromatography-mass spectrometry as previously
described (33).
Pulmonary vascular permeability was assessed after reperfusion by
estimation of the filtration coefficient (Kf)
and reflection coefficient for albumin (alb) as
previously described (32, 34). Briefly,
perfusion and ventilation were stopped, and the lungs were rapidly
removed from the chest and suspended from a force transducer.
Ventilation was resumed, the pulmonary arterial and left atrial
cannulas were connected to a pressurized stirred reservoir filled with
perfusate, and intravascular pressure (referenced to the level of the
middle of the lung) was increased from 20 mmHg to 30, 35, and 40 mmHg
at 10-min intervals. The rate of lung weight gain over the last 5 min
at each vascular pressure was plotted against vascular pressure, and
the slope of this relationship was used to estimate
Kf. The alb was determined by the
filtered volumes method, as modified for a nonflowing system, by Becker and co-workers (5). The right lung was then resected for
measurement of extravascular lung water (EVLW) and blood-free dry
weight (BFDW) as previously described (30). In
some experiments, tissue sections from the left lung were obtained for
light microscopy after fixing the lung via the vasculature with 4%
paraformaldelyde while maintaining a constant alveolar pressure of 10 mmHg. The lung tissue was imbedded in paraffin and sectioned and
stained with hematoxylin and eosin. Note that all EVLW measurements and
lung histology were performed on lung tissue subjected to the period of
increased static vascular pressure required to measure
alb and Kf.
NONREPERFUSED LUNGS.
Pulmonary vascular permeability and EVLW were measured in 10 lungs that
were excised without reperfusion. Ventilation was maintained throughout
an ischemic time of ~30 min of ischemia, and
Kf and alb were measured as
previously described (32, 34).
Pharmacological intervention. Six groups of ischemic-reperfused lungs were studied. In three groups, apocynin was added to the vascular flush solution and the reservoir blood before reperfusion to achieve pulmonary vascular concentrations of 3 mM (n = 6), 0.3 mM (n = 5), or 0.03 mM (n = 5). A fourth group of lungs (n = 4) was treated with 5 µM DPI in the vascular flush and perfusate. Because apocynin is known to inhibit thromboxane formation, an additional group of five lungs was treated with 56 µM indomethacin (Sigma Chemical). Drug-treated lungs were compared with five diluent-treated control lungs.
Apocynin and indomethacin were dissolved in the 300-ml vascular flush solution by heating. To achieve the appropriate perfusate concentrations, additional drug was dissolved in 100 ml normal saline containing 10 mM NaHCO3, which was added to the ~1,500 ml perfusion circuit just before initiating reperfusion. A stock solution of DPI (0.5 mg/ml) was prepared in distilled water by sonication for 30-60 s. Two milliliters of this stock solution were added to the vascular flush solution; 10 ml were added to the reservoir just before reperfusion.Statistics
All time course data were compared with a two-factor (group, time), split-plot ANOVA (38). The peak pulmonary artery pressure and perfusate prostanoid concentrations and lung water and pulmonary vascular permeability were analyzed by one-way ANOVA. The mean values of the prostanoid concentrations were found to be directly proportional to SD, indicating nonhomogeneity of variance; therefore, these data were transformed to logarithms before statistical analysis to comply with the assumptions of the ANOVA. When significant variance ratios were obtained, least-significant differences were calculated to allow comparison of individual means. Values presented in the text are means ± SE. Differences were considered significant at P
0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Sheep body weight averaged 26.5 ± 0.7 kg and was not different among groups. There were no differences in perfusate cell concentrations between groups of reperfused lungs. Perfusate hematocrit and total leukocyte, PMN leukocyte, and platelet concentrations averaged 21.5 ± 0.7% and 3,710 ± 268, 1,165 ± 111, and 463,000 ± 57,000 cells/mm3, respectively, in reservoir blood at time 0. As in previous studies (32), perfusate PMN leukocytes fell to near 0 cells/mm3 in all lungs by 30 min of reperfusion. Platelet concentrations were unchanged.
Blood Studies
As shown in Fig. 1, all three doses of apocynin significantly inhibited luminol-enhanced chemiluminescence of zymosan-stimulated sheep blood in a dose-dependent fashion. For example, 3 mM apocynin inhibited 97 ± 1% of the peak chemiluminescence generated by zymosan in diluent-treated blood, whereas 0.3 mM attenuated the signal by 75 ± 4%. DPI (5 µM) produced an effect similar to 3 mM apocynin, blocking 97 ± 1% of the luminol-enhanced signal.
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Figure 2 shows the effects of apocynin,
DPI, and superoxide dismutase on the reduction of resazurin by
STZ-stimulated PMN leukocytes. Both apocynin and DPI inhibited
resazurin reduction in a dose-dependent manner. Similar to the results
obtained with whole blood chemiluminescence, 0.03 and 0.3 mM apocynin
attenuated resazurin reduction by 31 ± 7 and 83 ± 3%,
respectively. Unlike whole blood chemiluminescence, 3 mM apocynin
(82 ± 2%) provided no additional inhibition compared with 0.3 mM. DPI was less effective at inhibiting resazurin reduction than whole
blood chemiluminescence; 3 µM DPI inhibited resazurin reduction by
69 ± 4% compared with 97 ± 1% inhibition of
chemiluminescence by 5 µM DPI. Superoxide dismutase had no
significant effect on PMN leukocyte-induced resazurin reduction at
either concentration studied.
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Lung Studies
Effect of ischemia and reperfusion.
Figure 3 shows the effect of ischemia and
reperfusion on pulmonary vascular permeability. Compared with
nonreperfused lungs (mean and 95% confidence limits indicated in Fig.
3), the diluent-treated ischemic-reperfused lungs had decreased
alb (0.47 ± 0.10 vs. 0.77 ± 0.03) and
increased Kf (0.14 ± 0.03 vs. 0.03 ± 0.01 g · min1 · mmHg1
· 100 g1), indicating increased vascular permeability
to protein and water. The increase in water permeability was
corroborated by an increase (P < 0.05) in EVLW
normalized to BFDW in the ischemic-reperfused compared with the
nonreperfused lungs (7.97 ± 0.28 vs. 4.77 ± 0.20 g/g BFDW,
data not shown).
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, and PGF2 occurred at 30 min
and are shown in Fig. 5. Measurable
amounts of neither PGD2 nor PGE2 were
detected. Ppa peaked at 30 min of reperfusion (33 ± 7 mmHg; Fig. 5). Ppa decreased to 16 ± 3 mmHg
at 60 min and remained at this level for the remainder of the
reperfusion period (data not shown).
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Effect of apocynin.
Apocynin inhibited the increased pulmonary vascular permeability caused
by ischemia and reperfusion in a dose-dependent manner (Fig. 3).
Treatment with 0.3 mM apocynin was associated with a alb
of 0.69 ± 0.05 and a Kf of 0.07 ± 0.01 g · min1 · mmHg1
· 100 g1, values near the 95% confidence limits for
the nonreperfused lungs. Moreover, 3 mM apocynin completely prevented
the increase in permeability, as evidenced by a alb of
0.91 ± 0.03, which exceeded the 95% confidence limits for
nonreperfused lungs. The Kf in the 3 mM apocynin
group (0.05 ± 0.01 g · min1 · mmHg1 · 100 g1) was significantly
less than diluent-treated lungs and was not different from the
Kf for nonreperfused lungs. Apocynin treatment also decreased EVLW, with 3 mM apocynin resulting in 5.34 ± 0.26 g/g BFDW, which was less than the diluent-treated lungs
(P < 0.05) and not different from the nonreperfused
group (P = 0.06, data not shown). As shown in Fig. 4,
lungs treated with 3 or 0.3 mM apocynin had no histological evidence of
peribronchial hemorrhage compared with multiple areas of extravascular
erythrocytes observed on tissue sections from diluent-treated lungs.
Although 0.03 mM apocynin significantly attenuated luminol-enhanced
chemiluminescence of sheep blood (29 ± 9%, Fig. 1) and resazurin
reduction by activated PMN leukocytes (31 ± 7%, Fig. 2), this
dose had no effect on the decrease in alb (0.48 ± 0.05) and increase in Kf (0.17 ± 0.04 g · min1 · mmHg1
· 100 g1) caused by ischemia and reperfusion. Similar
to the diluent-treated lungs, two of the 0.03 mM apocynin groups were
reperfused for <60 min because of massive edema formation, whereas all
of the lungs treated with 0.3 or 3 mM apocynin were reperfused for 180 min.
,
whereas only 3 mM apocynin decreased perfusate 6- keto-PGF1 concentration. Although TxB2
production was significantly attenuated by 0.03 mM apocynin, the
30-min Ppa was decreased only by 0.3 and 3 mM apocynin
to 20 ± 4 and 11 ± 2 mmHg, respectively.
To determine if the ameliorating effect of apocynin on the vascular
permeability change was secondary to inhibition of NADPH oxidase, we
studied six lungs treated with the structurally unrelated inhibitor
DPI. Unlike apocynin, DPI did not attenuate the pulmonary hypertension
and increased vascular protein permeability; Ppa at 30 min
(45 ± 4 mmHg) and alb (0.42 ± 0.13) were not
different from the diluent-treated lungs, whereas
Kf (0.32 ± 0.07 g · min1 · mmHg1 · 100 g1) and EVLW (9.97 ± 1.25 g/g BFDW) were
significantly greater than diluent-treated lungs (Figs. 3 and 5).
To determine if any of the protective effects of apocynin were
secondary to inhibition of cyclooxygenase, we studied five lungs
treated with indomethacin. The indomethacin dose was previously shown
in this preparation to decrease perfusate levels of
TxB2 and 6-keto- PGF1 to near
0 ng/ml and to eliminate the early transient pulmonary hypertension
(29). As shown in Fig. 3, indomethacin treatment did not
prevent vascular injury inasmuch as alb
(0.51 ± 0.06) and Kf (0.10 ± 0.03 g ·
min1 · mmHg1 · 100
g1) were not different from diluent-treated lungs.
As expected, indomethacin blocked the early pulmonary hypertension
(Fig. 5) previously shown to be dependent on the generation of
thromboxane (29).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The major finding of the present study was that apocynin, a novel
inhibitor of NADPH oxidase, prevented the increased vascular permeability caused by ischemia and reperfusion in isolated sheep lungs. As shown in Fig. 3, 30 min of ischemia followed by 180 min of
reperfusion decreased alb and increased
Kf compared with a separate group of
nonreperfused lungs. Apocynin treatment attenuated both the decrease in
alb and the increase in Kf caused
by ischemia-reperfusion injury. alb is a dimensionless
index that describes the ability of the vascular endothelium to
maintain an oncotic pressure gradient during convective fluid movement;
a alb of zero indicates free movement of albumin across
the vessel wall, whereas a alb of one indicates
impermeability to albumin. We have found alb to be more
sensitive to small changes in vascular permeability than Kf (47). Kf
is an index of water permeability which, unlike alb, is
affected by changes in vascular surface area, confounding the
interpretation of Kf as an index of injury
(16). We previously showed that the decrease in
alb in this preparation was dependent on the combination
of ischemia and reperfusion; neither ischemia (30 min with ventilation)
without reperfusion nor extracorporeal perfusion without ischemia
decreased alb (32) compared with estimates
of alb in intact sheep lungs (28).
The effect of apocynin on the changes in vascular permeability after
ischemia-reperfusion was dose dependent. Of note, the 3 mM
apocynin-treated lungs had a alb that was significantly greater than the nonreperfused lungs and comparable to a measurement of
alb in intact sheep lungs (0.84) (28). This
suggests that 3 mM apocynin was able to maintain normal endothelial
albumin permeability during ischemia and reperfusion. In addition, 3 mM apocynin decreased the Kf to within the 95%
confidence limits of the nonreperfused lungs, suggesting that the
component of increased water permeability from the interaction of
ischemia and reperfusion was also completely eliminated.
Although we did not quantify lung hemorrhage, the histology suggested that apocynin decreased erythrocyte extravasation into the interstitium (Fig. 4). The peribronchial hemorrhage observed in the diluent-treated lungs most likely corresponds to the nearly 100% incidence of blood-stained lung lymph we previously reported in this preparation (30). Moreover, reperfusion with PMN leukocyte-depleted blood significantly decreased the incidence of hemorrhagic lung lymph (30), supporting the possibility that the attenuating effect of apocynin on lung hemorrhage was mediated by an inhibitory effect on PMN leukocyte function. Of note, the protective effect of apocynin was not due to inhibition of PMN leukocyte sequestration in the lung because this occurred to the same degree in all lungs (data not shown).
The protective effect conferred by apocynin on ischemia-reperfusion lung injury correlated with the capacity of apocynin to inhibit inducible chemiluminescence in our whole blood studies (Fig. 1). Luminol-enhanced chemiluminescence reflects, predominantly, the free radical production by PMN leukocytes (46). Apocynin, at concentrations similar to those used in the present study, has indeed been shown to inhibit the ability of isolated PMN leukocytes to utilize oxygen after activation as well as the capacity of isolated PMN leukocytes to induce lucigenin-enhanced luminescence after stimulation by STZ (39). The resazurin assay data (Fig. 2) provided further support that apocynin directly impairs the activity of the NADPH oxidase enzyme. This assay, which measures the reduction of resazurin to resorufin by the NADPH oxidase enzyme, is a direct measure of NADPH oxidase activity (13). The lack of effect of superoxide dismutase on resazurin reduction confirmed that this assay is not affected by superoxide anion scavenging.
The ROS scavenging effect of apocynin remains undefined. Stolk et al. (39) showed that apocynin did not scavenge superoxide anion generated by xanthine oxidase. They concluded, however, that apocynin scavenged hydrogen peroxide because of a dose-response inhibition of luminol-enhanced chemiluminescence generated by hydrogen peroxide. But these studies, in which hydrogen peroxide was diluted with PBS buffer, did not rule out the possibility that the effect of apocynin was on metal ions or peroxidases contaminating the PBS buffer. Hydrogen peroxide does not cause luminol-enhanced chemiluminescence in the absence of contaminating metal ions or peroxidase (46).
Interestingly, the incremental protection from ischemia-reperfusion lung injury conferred by the 3 mM apocynin vs. 0.3 mM apocynin was not paralleled by an incremental inhibition of resazurin reduction by 3 mM apocynin. This disparity suggests that the protective qualities of apocynin were not limited to its effects on PMN leukocyte NADPH oxidase. A ROS scavenging effect of 3 mM apocynin, for example, would explain both its incremental effect on ischemia-reperfusion lung injury and its incremental capacity to attenuate luminol-enhanced luminescence of whole blood. Alternatively, the additional inhibitory effect of 3 mM apocynin on vascular injury and inducible whole blood luminescence may reflect the capacity of higher concentrations of apocynin to block NADPH oxidase activity in non-PMN leukocyte cells (14, 39). Monocytes (45), lymphocytes (7), macrophages (15), pulmonary vascular smooth muscle cells (24), and, possibly, endothelial cells (3) were shown to contain an NADPH oxidase-enzyme complex. For example, experiments in ischemic rat and mouse lungs have implicated an endothelial NADPH oxidase as a major source of ROS production during ischemia (3). Al-Mehdi et al. (3) demonstrated ROS production from the parenchyma of ischemic rat and mouse lungs that appeared to be of endothelial origin. The ROS signal was attenuated by PR-39 and diphenyliodonium, structurally unrelated inhibitors of NADPH oxidase, and was absent in lungs from gp91-phox knockout mice, suggesting a NADPH oxidase source. Pulmonary vascular permeability was not measured during ischemia, however, and the lungs were not subjected to reperfusion, so the relationship between endothelial ROS production and ischemia-reperfusion lung injury was unclear.
In contrast to apocynin, DPI not only failed to prevent the increase in
vascular permeability, but it may have enhanced the vascular injury
associated with ischemia-reperfusion, as evidenced by an increase
in Kf (Fig. 3) and EVLW compared with
diluent-treated lungs. This concentration of DPI was as potent an
inhibitor of whole blood chemiluminescence as 3 mM apocynin (Fig. 1),
although larger concentrations of DPI were necessary to produce a
comparable inhibition of resazurin reduction (Fig. 2). Therefore, DPI
both blocked NADPH oxidase function and either nonspecifically
interfered with the chemiluminescence reaction or scavenged ROS.
Nevertheless, based on its NADPH oxidase inhibitory activity as
determined by the resazurin assay, we expected this dose of DPI to
attenuate vascular injury to a similar degree as 0.3 mM apocynin. The
inability of DPI to do so suggests that either the protection of
apocynin was independent of its effect on NADPH oxidase or that the
toxicity of DPI overshadowed the benefits of NADPH oxidase inhibition. In addition to NADPH oxidase inhibition, DPI inhibits nitric oxide synthase (NOS) activity (41). The NOS enzyme complex is,
in fact, more sensitive to DPI-induced inhibition than is the NADPH oxidase enzyme (41). Grimminger et al. (14)
found that NOS inhibition by DPI enhanced pulmonary vasoconstriction
from the thromboxane analog U-46619, and we previously showed that
inhibition of constitutive NOS increased pulmonary vascular resistance
in our in situ lung preparation (34). In the present
study, however, the tendency toward exacerbating pulmonary hypertension
was transient (Fig. 5), peaking at 30 min, and was followed by a
tendency toward increased vasodilation at 180 min of reperfusion in the
apocynin-treated compared with diluent-treated lungs (6 ± 1 vs.
16 ± 5 mmHg, respectively, P = 0.10). A similar
biphasic effect of DPI on vascular tone was observed in preconstricted
systemic vessels (8). Pulmonary vasodilation would
increase the vascular surface available for fluid movement and may
partially explain the increase in Kf caused by
DPI treatment. This could not have been the entire explanation, however, because the maximal fractional change in hematocrit from the
fluid-exchanging region of the DPI-treated lungs, measured for the
calculation of alb, was significantly greater than that in diluent-treated lungs (0.93 ± 0.19 vs. 0.42 ± 0.03, respectively; P < 0.05). The magnitude and duration of
the increased static vascular pressure during the alb
and Kf measurements were the same in all lungs.
Thus an increase in the maximal fractional change in hematocrit
indicates an increase in vascular water conductance (34).
Loss of constitutive NOS activity may explain the injurious effect of
DPI on vascular permeability, independent of its effect on vascular
resistance (23, 27). NOS inhibition did not
increase vascular permeability during ventilated ischemia in the sheep lung (34), but we have not determined the role of nitric
oxide in the reperfusion phase of the injury. Other cellular functions inhibited by DPI include mitochondrial electron transport
(20), cytochrome P-450 enzyme activity
(35), and ion channel open probability (49).
Inhibition of mitochondrial respiration was previously shown to
exacerbate pulmonary edema in isolated rat lungs, although neither
alb nor Kf was measured
(4). We did not exceed 5 µM DPI because this
concentration was shown to inhibit 80% of macrophage NADPH oxidase
activity without affecting mitochondrial respiration (15).
Nevertheless, the DPI-treated lungs required more NaHCO3 to
maintain a normal perfusate pH (data not shown), suggesting that 5 µM
DPI may have caused partial inhibition of mitochondrial respiration.
The lactic acidosis resulting from ventilation with nitrogen during
ischemia and reperfusion in a previous study (32) was more
severe than that produced by DPI in the present study. Nitrogen
ventilation did not worsen vascular permeability (32),
arguing against mitochondrial dysfunction as the cause for increased
Kf with DPI treatment in this model. Finally,
DPI inhibits potassium and calcium channels in vascular smooth muscle
(49). Administration of cromakalim, a potassium channel
opening drug, attenuates ischemia-reperfusion injury in isolated rat
lungs (18), suggesting that closed potassium channels may
worsen the injury. Although glibenclamide, a potassium channel closing
agent, did not exacerbate ischemia-reperfusion lung injury in the rat
lung model (18), species differences could exist.
Regardless of the mechanism behind the enhanced injury caused by DPI, the lack of protection raises the possibility that NADPH oxidase did not play a major role in the ischemia-reperfusion lung injury. The injurious role of PMN leukocyte NADPH oxidase in ischemia-reperfusion lung injury remains controversial, with recent studies showing no effect of PMN leukocyte depletion on this injury (22, 36). Other investigators have identified a PMN leukocyte-independent injury early in reperfusion (<30 min), followed by a later injury (4 h) mediated by PMN leukocytes (11). The early phase of injury appeared to be mediated by macrophage-derived cytokines. The later phase of injury is consistent with our previous observation that reperfusion of untreated ischemic sheep lungs with leukocyte-depleted blood decreased lung hemorrhage after 3 h of reperfusion (30). Unfortunately, vascular permeability was not measured in that study, so a direct comparison of leukopenic reperfusion with apocynin treatment cannot be made.
The protective effect of apocynin may not be limited to NADPH oxidase inhibition. Apocynin has been shown to inhibit cytochrome P-450 (35) and thromboxane synthase (10). Interestingly, both cytochrome P-450 (6) and thromboxane (21, 22) have been implicated as mediators of ischemia-reperfusion lung injury. Cytochrome P-450 inhibition is shared by DPI (42), making this pathway a less likely explanation for the effect of apocynin. As shown in Fig. 5, apocynin inhibited prostanoid production and pulmonary hypertension during reperfusion. Thromboxane production appeared to be more sensitive to the inhibitory effects of apocynin compared with the other measured cyclooxygenase products. This pattern of prostanoid inhibition is similar to that shown by others (10). Indomethacin treatment, with a dose previously shown to inhibit cyclooxygenase in this preparation (29), had no effect on the increased vascular permeability in the present study. These data suggest that the protection conferred by apocynin was not due to thromboxane synthesis inhibition and are consistent with our previous findings of an inverse correlation between the thromboxane-induced pulmonary hypertension and vascular permeability in this model (32). In contrast, the increased vascular protein permeability associated with ischemia-reperfusion was attenuated by indomethacin in rat lungs (21) and thromboxane receptor blockade in rabbit lungs (22), suggesting that the role of thromboxane in this form of injury may depend on the species being studied.
Apocynin was recently shown to attenuate the pulmonary artery vasoconstriction from either hypoxia or the thromboxane analog U-46619 (14), suggesting that apocynin may have an inhibitory effect on vascular smooth muscle contraction. ATP-regulated potassium channel opening agents attenuate ischemia-reperfusion lung injury (18) and inhibit pulmonary vasoconstriction from either hypoxia (50) or U-46619 (12). To our knowledge, the effect of apocynin on endothelial or smooth muscle cell ion channels has not been studied, but an effect on potassium channels could represent an alternative explanation for its remarkable ability to prevent ischemia-reperfusion lung injury.
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ACKNOWLEDGEMENTS |
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We thank Teresa Privett and Laura Welch for expert technical assistance and Wanda Moran for excellent secretarial support.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-50504 (D. B. Pearse) and by Grants-In-Aid from the American Heart Association (J. M. Dodd-o and D. B. Pearse) with funds contributed in part by the American Heart Association, Maryland Affiliate. D. B. Pearse is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: D. B. Pearse, Div. of Pulmonary and Critical Care Medicine, Johns Hopkins Bayview Medical Center, 5501 Hopkins Bayview Cir., Baltimore, MD 21224 (E-mail: dpearse{at}welch.jhu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 30 November 1999; accepted in final form 19 January 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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