Ca2+-dependent
p47phox translocation in
hydroperoxide modulation of the alveolar macrophage respiratory
burst
Huanfang
Zhou,
Roger F.
Duncan,
Timothy W.
Robison,
Lin
Gao, and
Henry Jay
Forman
Department of Molecular Pharmacology and Toxicology, University
of Southern California, Los Angeles, California 90033
 |
ABSTRACT |
Oxidative stress produces dual effects on the
respiratory burst of rat alveolar macrophages. Preincubation with
hydroperoxide concentrations
[H2O2
or tert-butyl hydroperoxide
(t-BOOH); <50 µM] enhances
stimulation of the respiratory burst, whereas higher concentrations
inhibit stimulation. Both the enhancement and inhibition are markedly
attenuated by buffering t-BOOH-induced
changes in intracellular Ca2+
concentration
([Ca2+]i).
Phosphorylation of the NADPH oxidase component
p47phox and its translocation from
cytoplasm to plasma membrane are essential in respiratory burst
activation. Phorbol 12-myristate 13-acetate (PMA)-stimulated
p47phox phosphorylation was
negligibly affected by 25 or 100 µM
t-BOOH. Nonetheless, 25 µM
t-BOOH increased PMA-stimulated
p47phox translocation, whereas 100 µM t-BOOH decreased PMA-stimulated translocation. In unstimulated cells, however, neither phosphorylation nor translocation of p47phox was
affected by t-BOOH. Buffering of the
t-BOOH-mediated changes of
[Ca2+]i
abolished the effects of t-BOOH on
PMA-stimulated translocation in parallel to effects upon the
respiratory burst. The results suggest that the dual effects of
hydroperoxides are mediated, in part, by
Ca2+-dependent processes affecting
the assembly of the respiratory burst oxidase at steps that are
separate from p47phox
phosphorylation.
p47phox phosphorylation; oxidative stress; reduced nicotinamide adenine dinucleotide phosphate
oxidase assembly; calcium ion; rat
 |
INTRODUCTION |
ALVEOLAR MACROPHAGES function as the first line of
defense against bacteria and foreign particles in the lungs (11). Part of the mechanism by which phagocytic cells kill microbes depends on the
generation of superoxide in a metabolic process known as the
respiratory burst (22). The respiratory burst oxidase includes several
cytosolic components, p47phox,
p67phox,
p40phox, rac1 or rac2, and the
transmembrane electron carrier flavocytochrome b558, a heterodimer of
p22phox and
gp91phox. During the activation of
the respiratory burst oxidase of neutrophils, p47phox is phosphorylated at
multiple sites by protein kinases at the COOH-terminal domain (7, 8,
14, 26). The phosphorylated protein is then translocated from the
cytoplasm to the plasma membrane for the assembly of the oxidase
complex (4, 9). Failure to phosphorylate or translocate
p47phox results in failure to
produce superoxide in stimulated cells (8).
Exposure of alveolar macrophages to air pollutants (e.g., nitrogen
dioxide and ozone) or prolonged exposure to hyperoxia in vivo leads to
significant inhibition of the respiratory burst (1, 12). Nevertheless,
alveolar macrophages, after a brief exposure to hyperoxia in vivo, have
an enhanced response to subsequent stimulation of the respiratory burst
(28). We used tert-butyl hydroperoxide
(t-BOOH), a relatively stable and
water-soluble oxidant, to mimic both the enhancing and inhibitory
effects of oxidant exposure on the rat alveolar macrophage respiratory
burst. Because of their rapid metabolism, hydroperoxides cannot be
precisely measured in vivo. Nonetheless, evidence for the generation of significant concentrations of hydroperoxides and their breakdown products in exposure to various oxidative stresses is well documented (27, 29). Previous studies from our laboratory have shown that low
concentrations of t-BOOH
(<50 µM) enhance the respiratory burst stimulated by phorbol
12-myristate 13-acetate (PMA), whereas higher concentrations (but still
sublethal; <100 µM) inhibit the burst (20). In this study, we
examined the possibility that the alteration of the respiratory burst
by t-BOOH could involve effects upon
p47phox phosphorylation
and/or p47phox
translocation.
Although PMA does not stimulate changes in intracellular
Ca2+ concentration
([Ca2+]i),
PMA stimulation of the respiratory burst can be modulated by changes in
[Ca2+]i
(5, 16). Studies from our laboratory have shown that 25 µM
t-BOOH caused a transient elevation in
[Ca2+]i
in alveolar macrophages, whereas 100 µM
t-BOOH caused a sustained elevation in
[Ca2+]i
(19). This dual effect of t-BOOH on
[Ca2+]i
correlated with the effects on the alveolar macrophage respiratory burst (15). Furthermore, buffering of hydroperoxide-mediated changes in
[Ca2+]i,
using the intracellular Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'tetraacetic
acid (BAPTA), suppressed the enhancement of the respiratory burst by 25 µM t-BOOH and attenuated the
inhibition by 100 µM t-BOOH (16). These data suggest that changes in
[Ca2+]i
induced by t-BOOH are involved in the
process that modulates the respiratory burst. Thus the effect of
buffering
[Ca2+]i
upon t-BOOH-mediated changes on
p47phox translocation was also
investigated.
 |
EXPERIMENTAL PROCEDURES |
Materials. PMA, leupeptin, pepstatin
A, aprotinin, phenylmethylsulfonyl fluoride (PMSF), sodium
deoxycholate, and Nonidet P-40 (NP-40) were purchased from Sigma.
Bio-lyte 3/10 ampholyte, piperazine diacrylamide,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and two-dimensional (2-D) sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) standards were from
Bio-Rad. Precast 10% tris(hydroxymethyl)aminomethane
(Tris)-glycine gels (15 well and 2-D) and SeeBlue prestained standard
were from Novex. A rabbit polyclonal antibody against the COOH-terminal
decapeptide of p47phox was
generously provided by Dr. Bernard M. Babior of the Scripps Research
Institute.
Preparation of alveolar macrophages.
Alveolar macrophages were obtained by bronchoalveolar lavage of
Sprague-Dawley rats (specific pathogen free, male, 250-350 g) with
sodium phosphate-buffered saline (pH 7.4, 0.01 M, 100 ml). Macrophages
were resuspended and stored at 4°C in Krebs-Ringer-phosphate buffer
(KRPH, pH 7.4; 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, 125 mM NaCl, 5 mM KCl, 10 mM sodium phosphate, 5 mM glucose, 1.3 mM CaCl2, and 1.0 mM
MgSO4). Macrophage yield was ~1 × 107 cells/rat (viability
>98%).
Measurement of alveolar macrophage respiratory
burst. Alveolar macrophages (1 × 106 cells) were incubated
(37°C, 15 min) in 1 ml of KRPH buffer. The respiratory burst was
stimulated by the addition of PMA (100 ng/ml) in the presence of 80 µM ferricytochrome c. Superoxide production was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome c, which
was monitored continuously at 550-540 nm in a dual-wavelength
spectrophotometer.
Analysis of p47phox phosphorylation by
2-D gel electrophoresis.
Alveolar macrophages (2 × 106 cells/ml) were incubated
(37°C, 15 min) in 1 ml of KRPH buffer with or without
t-BOOH (25 or 100 µM
t-BOOH) and, subsequently, were
stimulated with or without PMA (100 ng/ml) for 5 min, at which time the
rate of superoxide anion production is maximal. Cells were isolated
using a microcentrifuge for 10 s and were dissolved in 0.1 ml of
first-dimension sample buffer (0.1 g dithiothreitol, 0.4 g CHAPS, 5.4 g
urea, 0.5 ml Bio-lyte 3/10 ampholyte, and 6 ml
H2O). Aliquots (40 µl of each sample) were subjected to nonequilibrium pH gradient isoelectric focusing in a minicapillary gel (monomer solution: 9.2 M urea, 4%
acrylamide, 4% Bio-lyte 3/10 ampholyte, 1.5% CHAPS, and 0.5% NP-40).
The top and bottom chambers of the mini-Protean II electrophoresis apparatus contained 10 mM
H3PO4
and 100 mM NaOH, respectively. The samples were electrophoresed for 4 h
(100 V, 10 min; 200 V, 10 min; 400 V, 3.5 h; and 600 V, 10 min),
and the tube gels were extruded and transferred to the top of the
minislab 2-D gel for the second run.
p47phox (nonphosphorylated and
phosphorylated isoforms) was detected by immunoblotting (see
Immunoblotting).
Translocation of p47phox from cytosol to
plasma membrane.
Alveolar macrophages (1 × 107 cells/ml) were treated (15 min, 37°C) with or without t-BOOH
and then were activated by PMA (1 µg/ml) for 5 min. Cells were
collected using a microcentrifuge for 10 s, resuspended in 0.5 ml of
ice-cold lysis buffer [20 mM Tris · HCl, pH
7.4, 150 mM NaCl, 5 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 5 mM EDTA, 50 µM leupeptin, 25 µM pepstatin A, 25 µM
aprotinin, and 2 mM PMSF], sonicated 3 times for 10 s, and
centrifuged (2,000 g, 10 min) to
remove nuclei and unbroken cells. The postnuclear lysates were
ultracentrifuged further (100,000 g,
60 min). The resulting supernatant was designated the cytosolic
fraction. The membrane pellet was resuspended in 200 µl of the lysis
buffer supplemented with sodium deoxycholate (1%) and NP-40 (1%). The
protein concentration of each sample was measured using the
bicinchoninic acid protein assay (Pierce). Cytosolic (5 µg
protein/sample) and membrane (50 µg protein/sample) proteins were
suspended in 20 µl of Laemmli sample buffer, heated in boiling water
for 5 min, separated by SDS-PAGE on a precast 10% polyacrylamide gel,
and analyzed by immunoblotting.
Immunoblotting. Separated proteins
were transferred to a nitrocellulose membrane and were incubated with
the anti-p47phox antibody (diluted
1:5,000) overnight at 4°C. The
p47phox-antibody complex was
visualized using an enhanced chemiluminescence detection system
(Amersham, Arlington Heights, IL) after a 1-h incubation with goat
anti-rabbit immunoglobulin G (Kirkegaard and Perry, Gaithersburg,
MD). The developed film was scanned using a Hewlett-Packard ScanJet
4c/T scanner and accompanying software. The images were then analyzed
for relative density using SigmaScan software (Jandel Scientific
Software, San Rafael, CA).
Statistical analysis. Data are
expressed as means ± SE. The amounts of
p47phox translocation from all
groups were compared with the PMA stimulation group using one-way
analysis of variance following Dunnett's method. Differences in two
groups were considered statistically significant at
P < 0.05.
 |
RESULTS |
Effect of t-BOOH on
p47phox phosphorylation.
The relationship of p47phox
phosphorylation to the activation of the respiratory burst oxidase in
rat alveolar macrophages was examined. As a frame of reference, Fig.
1 shows the time course of superoxide
production of alveolar macrophages stimulated by PMA. Superoxide
production had a lag phase of ~1 min, reached a maximal rate at ~5
min, and then became progressively slower until ~30 min. Figure
2 shows
p47phox phosphorylation in
alveolar macrophages stimulated by PMA for 0, 1, 5, 7, or 10 min, at
which time point lysates were prepared for analysis by 2-D gel
electrophoresis and immunoblotting. Resting alveolar macrophages
contained one isoform of p47phox
predominantly. A single more acidic
p47phox isoform was distinctly
apparent after 1 min of stimulation. Several additional
p47phox isoforms were apparent at
5 min. The more acidic p47phox
phosphoprotein forms reached a maximum at ~7 min.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1.
Superoxide production by alveolar macrophages stimulated with phorbol
12-myristate 13-acetate (PMA). Typical time course of superoxide
production is shown. Respiratory burst was measured as described in
EXPERIMENTAL PROCEDURES.
|
|

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
Time course of p47phox
phosphorylation stimulated with PMA. Alveolar macrophages were
stimulated with PMA for 1, 5, 7, or 10 min.
p47phox isoforms were analyzed by
2-dimensional gel electrophoresis as described in EXPERIMENTAL
PROCEDURES. Data are representative of 2 experiments with
different cell preparations.
|
|
The effect of t-BOOH treatment on the
level of p47phox phosphorylation
was examined next. Two concentrations of
t-BOOH were chosen [25 µM,
which causes maximum enhancement (41 ± 5%) of the PMA-stimulated respiratory burst, and 100 µM, which causes a 96 ± 2% inhibition without cytotoxicity; see Ref. 16]. These effects were verified. Nonetheless, treatment of alveolar macrophages with 25 or 100 µM
t-BOOH alone did not lead to the
phosphorylation of p47phox (Fig.
3). Furthermore, pretreatment with either
25 or 100 µM t-BOOH did not markedly
change the pattern of p47phox
phosphorylation induced by PMA (Fig. 3). In other words, PMA induced
the phosphorylation of p47phox,
but t-BOOH did not alter this
phosphorylation to any clear extent and certainly not in a manner
consistent with its effect on the respiratory burst. Thus modulation of
the phosphorylation of p47phox
does not appear to be the hydroperoxide-mediated event for either the
enhancement or the inhibition of the burst.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of tert-butyl hydroperoxide
(t-BOOH) on
p47phox phosphorylation. Alveolar
macrophages were treated with t-BOOH
(0, 25, or 100 µM) for 15 min. Shown is phosphorylation of
p47phox from unstimulated alveolar
macrophages and alveolar macrophages stimulated with PMA for 5 min.
p47phox phosphorylation was
analyzed as in Fig. 2. Data are representative of 2 experiments with
different cell preparations.
|
|
Modulation of p47phox translocation by
t-BOOH.
During the activation of the respiratory burst oxidase, phosphorylated
p47phox translocates from the
cytosol to the plasma membrane. Such translocation of the cytosolic
oxidase components is an essential step in the activation of the
oxidase. Figure 4 shows that stimulation
with PMA decreased the amount of
p47phox in the cytosolic fraction
and increased the amount of
p47phox in the membrane fraction
compared with the control as a function of time.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 4.
Time course of p47phox
translocation stimulated with PMA. Alveolar macrophages were stimulated
with PMA for 1, 5, or 10 min before sonication and preparation of
cytosolic and membrane fractions. Cytosolic (5 µg protein/sample) and
membrane (50 µg protein/sample) proteins were analyzed by
immunoblotting as described in EXPERIMENTAL PROCEDURES. 0, Control; 1, 1 min; 5, 5 min; and 10, 10 min. Results
are representative of 3 experiments using different cell
preparations.
|
|
To determine whether t-BOOH modulates
the translocation of p47phox,
alveolar macrophages were treated with or without
t-BOOH, and the amount of
p47phox in the membrane fraction
was detected by immunoblotting (Fig. 5).
Neither 25 nor 100 µM t-BOOH alone
affected p47phox distribution.
However, 25 µM t-BOOH increased the
translocation of p47phox from the
cytosol to the membrane upon stimulation with PMA, whereas 100 µM
t-BOOH decreased the translocation.
These results suggest that hydroperoxide modulation of the respiratory
burst involves, in part, the alteration of the translocation of
p47phox at a step(s) separate from
the phosphorylation of p47phox.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of t-BOOH on
p47phox translocation. Alveolar
macrophages were treated with or without
t-BOOH (0, 25, or 100 µM) for 15 min. Then macrophages were treated with or without PMA for 5 min. Shown
is p47phox in the membrane
fractions (50 µg protein/sample) analyzed as in Fig. 4.
Lane 1, control (no
t-BOOH or PMA); lane
2, 25 µM t-BOOH
only; lane 3, 100 µM
t-BOOH only; lane
4, PMA only; lane 5,
25 µM t-BOOH then PMA; and
lane 6, 100 µM
t-BOOH then PMA. Figure is
representative of 4 experiments from different cell preparations. Mean
and SE compared with density of PMA-stimulated alveolar macrophages are
shown in bottom.
* P < 0.05 vs. PMA stimulation
by analysis of variance (ANOVA).
|
|
Ca2+-dependent
p47phox translocation during exposure to
t-BOOH.
Incubation of alveolar macrophages with
t-BOOH causes reversible elevation of
[Ca2+]i
(17, 19). Buffering of this
t-BOOH-induced elevation of [Ca2+]i
with the Ca2+ chelator BAPTA
attenuates the dual effects of t-BOOH
on the burst (16). Although incubation with BAPTA-acetoxymethyl ester
(AM) alone does not affect the PMA-stimulated respiratory burst,
pretreatment with BAPTA-AM depresses the enhancement of the
PMA-stimulated respiratory burst by 25 µM
t-BOOH from 41 ± 5 to 8 ± 7%
and reduces the inhibition of the burst by 100 µM
t-BOOH from 96 ± 2 to 42 ± 10% (16). Thus there are
Ca2+-dependent and
Ca2+-independent components to the
inhibition of the respiratory burst by 100 µM
t-BOOH.
To examine whether the modulation of
p47phox translocation by
t-BOOH resulted from
t-BOOH-induced elevation of
[Ca2+]i,
alveolar macrophages were incubated for 15 min with 5 µM BAPTA-AM before exposure to t-BOOH. Figure
6 shows that, after treatment of alveolar
macrophages with BAPTA-AM, 25 µM
t-BOOH did not enhance translocation
after PMA stimulation. Furthermore, after treatment with BAPTA-AM,
p47phox translocation was no
longer decreased by 100 µM t-BOOH.
Thus comparison of the results in the presence (Fig. 6) or absence (Fig. 5) of BAPTA suggests that hydroperoxide-induced changes in
[Ca2+]i
modulate the translocation of
p47phox. The other component of
inhibition of the respiratory burst, which is unaffected by BAPTA,
apparently does not involve
p47phox translocation.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of buffering changes of intracellular
Ca2+ concentration
([Ca2+]i)
mediated by t-BOOH on
p47phox translocation. Alveolar
macrophages were treated with 5 µM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA) for 15 min before treatment with
t-BOOH (25 or 100 µM) for 15 min.
Amounts of p47phox in membrane
fractions (50 µg protein/sample) were analyzed as in Fig. 4.
Lane 1, control (no BAPTA, no
t-BOOH, and no PMA);
lane 2, BAPTA only;
lane 3, PMA only;
lane 4, BAPTA plus PMA;
lane 5, BAPTA, 25 µM
t-BOOH, and PMA; and
lane 6, BAPTA, 100 µM
t-BOOH, and PMA. Figure is
representative result of 3 experiments from different cell
preparations. Mean and SE compared with PMA-stimulated alveolar
macrophages from all 3 experiments are shown in
bottom.
* P < 0.05 vs. PMA stimulation
by ANOVA.
|
|
 |
DISCUSSION |
The goal of this investigation was to determine whether the modulation
of the respiratory burst by exposure to hydroperoxide is due to changes
in the phosphorylation of p47phox
and/or its translocation to the plasma membrane. Various
protein kinases, including protein kinase C, guanosine
3',5'-cyclic monophosphate-dependent protein kinase, and
p72syk tyrosine kinase, can be
activated by hydroperoxides (18, 23, 30). In addition, both
serine-threonine and tyrosine protein phosphatases may be inactivated
under similar conditions (30). Therefore, it seemed reasonable to
investigate whether the phosphorylation of
p47phox, an apparently obligatory
step in the assembly of the respiratory burst oxidase (8), was affected
under conditions in which hydroperoxides modulate the respiratory
burst.
Although the phosphorylation of
p47phox appears to be a step
required for the assembly of the NADPH oxidase in alveolar macrophages (Fig. 2), it is only marginally affected by
t-BOOH (Fig. 3). Thus alteration of
the pattern of p47phox
phosphorylation does not appear to play a role in the hydroperoxide modulation of the respiratory burst. This does not imply that protein
phosphorylation is unaffected by oxidative stress in alveolar macrophages but only that one particularly important step in the activation of the respiratory burst was not the target of hydroperoxide modulation. Rather, many of the dual effects of sublethal
concentrations of t-BOOH on the
alveolar macrophage respiratory burst result from an increase in
p47phox translocation by 25 µM
t-BOOH and the decrease in
p47phox translocation by 100 µM
t-BOOH (Fig. 5). Buffering of the
t-BOOH-mediated changes of
[Ca2+]i
using BAPTA, which markedly attenuates the effects of
t-BOOH on the respiratory burst (16),
abolishes the dual effects of t-BOOH
on p47phox translocation (Fig. 6).
The effect of BAPTA in eliminating the effect of 100 µM
t-BOOH on translocation was more
dramatic than its effect upon inhibition of the respiratory burst. This
suggests that 100 µM t-BOOH had an
additional Ca2+-independent
inhibitory effect on the respiratory burst. Taken together, these
results suggest that oxidative stress induces 1)
Ca2+-dependent processes that
modulate the assembly of the respiratory burst oxidase at a step that
is separate from p47phox
phosphorylation and 2) a
Ca2+-independent process that
inhibits the respiratory burst separately from both
p47phox phosphorylation or its
translocation.
Recently, our laboratory demonstrated that sublethal concentrations of
t-BOOH causes disassociation of
annexin VI, a major Ca2+-binding
protein in macrophages, from the plasma membrane to the cytosol (17).
This dissociation apparently results in the initial increase in
[Ca2+]i.
t-BOOH at 25 µM causes a transient
elevation in
[Ca2+]i,
whereas 100 µM t-BOOH induces a
sustained elevation in
[Ca2+]i
because of reversible oxidation of the thiol group in
Ca2+-adenosinetriphosphatase of
the plasma membrane (17). Although the
t-BOOH-induced elevation in
[Ca2+]i
alone is not sufficient to activate the respiratory burst oxidase, this
elevation in
[Ca2+]i
can potentially modulate various components of signal pathways (e.g.,
phospholipases, protein kinases, and protein phosphatases).
Two possible mechanisms through which the elevation in
[Ca2+]i
may then modulate the assembly of the respiratory burst oxidase may be
hypothesized. First, an increase in
[Ca2+]i
can result in the production of diacylglycerol, which augments both
p47phox translocation and
superoxide anion production in a cell-free system (24). Diacylglycerol
can be produced by the action of phospholipase C or by the sequential
action of phospholipase D and phosphatidic acid phosphohydrolase (2).
Phospholipase D can be activated by an increase in
[Ca2+]i
or can be attenuated by the chelation of intracellular
Ca2+ using BAPTA (21). Preliminary
results have indicated the activation of phospholipase D by
t-BOOH. Second, phospholipase
A2 can also be activated by both
an increase in
[Ca2+]i
and dissociation of annexin VI from the membrane to the cytosol (3, 6).
We have previously shown that t-BOOH
releases arachidonic acid through the activation of phospholipase
A2 in alveolar macrophages (25).
Arachidonic acid can directly activate NADPH oxidase by causing
p47phox translocation without
phosphorylation in a cell-free system (13); however, the relevance of
this to physiological stimulation of the respiratory burst has been
questioned (10).
In summary, our results demonstrated that the dual effects of
t-BOOH on PMA-stimulated
p47phox translocation account for
much of the dual effects on the alveolar macrophage respiratory burst.
The regulation of p47phox
translocation results from the elevation of
[Ca2+]i
caused by t-BOOH. This increase in
[Ca2+]i
apparently results in an increase in
p47phox translocation with low
oxidative stress but decreased translocation with greater oxidative
stress. The remaining component of
t-BOOH-induced inhibition is
independent of changes in
[Ca2+]i
and does not involve an effect upon translocation. Although the precise
mechanism for the effect of the hydroperoxide-induced elevation in
[Ca2+]i
on the translocation of p47phox is
unknown, it represents an example of the mimicry of physiological signaling that is becoming more apparent in low-level oxidative stress.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Jamel El Benna and Bernard M. Babior for rabbit
anti-p47phox polyclonal antibody
and Dr. Carolyn Hoyal, Dr. Nalini Kaul, and Jinah Choi for helpful
comments.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-37556.
Address for reprint requests: H. J. Forman, Dept. of Molecular
Pharmacology and Toxicology, School of Pharmacy, PSC 516, University of
Southern California, 1985 Zonal Ave., Los Angeles, CA 90033.
Received 3 April 1997; accepted in final form 20 August 1997.
 |
REFERENCES |
1.
Amoruso, M. A.,
G. Witz,
and
B. D. Goldstein.
Decreased superoxide anion radical production by rat alveolar macrophages following inhalation of ozone or nitrogen dioxide.
Life Sci.
28:
2215-2221,
1981[Medline].
2.
Billah, M. M.,
S. Eckel,
T. J. Mullmann,
R. W. Egan,
and
M. I. Siegel.
Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils.
J. Biol. Chem.
264:
17069-17077,
1989[Abstract/Free Full Text].
3.
Coméra, C.,
B. Rothhut,
and
F. Russo-Marie.
Identification and characterization of phospholipase A2 inhibitory proteins in human mononuclear cells.
Eur. J. Biochem.
188:
139-146,
1990[Abstract].
4.
De Mendez, I.,
A. G. Adams,
R. A. Sokolic,
H. L. Malech,
and
T. L. Leto.
Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells.
EMBO J.
15:
1211-1220,
1996[Abstract].
5.
Dorio, R. J.,
J. Nelson,
and
H. J. Forman.
A dual role for calcium in regulation of superoxide generation by stimulated rat alveolar macrophages.
Biochim. Biophys. Acta
928:
137-143,
1987[Medline].
6.
Edwards, A. M.,
and
C. M. Lucas.
Induction of gamma-glutamyl transpeptidase in primary cultures of normal rat hepatocytes by liver tumor promotors and structurally related compounds.
Carcinogenesis
6:
733-739,
1987[Abstract].
7.
El Benna, J.,
L. P. Faust,
and
B. M. Babior.
The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation.
J. Biol. Chem.
269:
23431-23436,
1994[Abstract/Free Full Text].
8.
El Benna, J.,
L. P. Faust,
J. L. Johnson,
and
R. M. Babior.
Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping.
J. Biol. Chem.
271:
6374-6378,
1996[Abstract/Free Full Text].
9.
El Benna, J.,
J. M. Ruedi,
and
B. M. Babior.
Cytosolic guanine nucleotide-binding protein rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase.
J. Biol. Chem.
269:
6729-6734,
1994[Abstract/Free Full Text].
10.
Ely, E. W.,
M. C. Seeds,
F. H. Chilton,
and
D. A. Bass.
Neutrophil release of arachidonic acid, oxidants, and proteinases: causally related or independent.
Biochim. Biophys. Acta
1258:
135-144,
1995[Medline].
11.
Fels, A. O.,
N. A. Pawlowski,
E. B. Cramer,
T. K. King,
Z. A. Cohn,
and
W. A. Scott.
Human alveolar macrophages produce leukotriene B4.
Proc. Natl. Acad. Sci. USA
79:
7866-7870,
1982[Abstract].
12.
Forman, H. J.,
J. J. Williams,
J. Nelson,
R. P. Daniele,
and
A. B. Fisher.
Hyperoxia inhibits stimulated superoxide release by rat alveolar macrophages.
J. Appl. Physiol.
53:
685-689,
1982[Abstract/Free Full Text].
13.
Henderson, L. M.,
S. K. Moule,
and
J. B. Chappell.
The immediate activator of the NADPH oxidase is arachidonate not phosphorylation.
Eur. J. Biochem.
211:
157-162,
1993[Abstract].
14.
Heyworth, P. G.,
and
J. A. Badwey.
Continuous phosphorylation of both the 47 and the 49 kDa proteins occurs during superoxide production by neutrophils.
Biochim. Biophys. Acta
1052:
299-305,
1990[Medline].
15.
Hoyal, C. R.,
E. Gozal,
and
H. J. Forman.
Hydroperoxide mediated modulation of the alveolar macrophage respiratory burst is independent of thapsigargin effects (Abstract).
FASEB J.
8:
A666,
1994.
16.
Hoyal, C. R.,
E. Gozal,
H. Zhou,
K. Foldenauer,
and
H. J. Forman.
Modulation of the rat alveolar macrophage respiratory burst by hydroperoxide is calcium dependent.
Arch. Biochem. Biophys.
326:
166-171,
1996[Medline].
17.
Hoyal, C. R.,
A. P. Thomas,
and
H. J. Forman.
Hydroperoxide-induced increases in intracellular calcium due to annexin VI translocation and inactivation of plasma membrane Ca2+-ATPase.
J. Biol. Chem.
271:
29205-29210,
1996[Abstract/Free Full Text].
18.
Landgraf, W.,
S. Regulla,
E. Meyer,
and
F. Hofmann.
Oxidation of cysteines activates cGMP-dependent protein kinase.
J. Biol. Chem.
266:
16305-16311,
1991[Abstract/Free Full Text].
19.
Livingston, F. R.,
E. M. K. Lui,
G. A. Loeb,
and
H. J. Forman.
Sublethal oxidant stress induces a reversible increase in intracellular calcium dependent on NAD(P)H oxidation in rat alveolar macrophages.
Arch. Biochem. Biophys.
299:
83-91,
1992[Medline].
20.
Murphy, J. K.,
C. R. Hoyal,
F. R. Livingston,
and
H. J. Forman.
Modulation of the alveolar macrophage respiratory burst by hydroperoxides.
Free Radic. Biol. Med.
18:
37-45,
1995[Medline].
21.
Natarajan, V.,
and
J. G. Garcia.
Agonist-induced activation of phospholipase D in bovine pulmonary artery endothelial cells: regulation by protein kinase C and calcium.
J. Lab. Clin. Med.
121:
337-347,
1993[Medline].
22.
Nathan, C. F.,
and
S. Tsunawaki.
Enzymatic basis of macrophage activation.
J. Biol. Chem.
259:
4305-4312,
1984[Abstract/Free Full Text].
23.
O'Brian, C. A.,
N. E. Ward,
I. B. Weinstein,
A. W. Bull,
and
L. J. Marnett.
Activation of rat brain protein kinase C by lipid oxidation products.
Biochem. Biophys. Res. Commun.
155:
1374-1380,
1988[Medline].
24.
Park, J. W.,
and
B. M. Babior.
The translocation of respiratory burst oxidase components from cytosol to plasma membrane is regulated by guanine nucleotides and diacylglycerol.
J. Biol. Chem.
267:
19901-19906,
1992[Abstract/Free Full Text].
25.
Robison, T. W.,
A. Sevanian,
and
H. J. Forman.
Inhibition of arachidonic acid release by nordihydroguaiaretic acid and its antioxidant action in rat alveolar macrophages and Chinese hamster lung fibroblasts.
Toxicol. Appl. Pharmacol.
105:
113-122,
1990[Medline].
26.
Rotrosen, D.,
and
T. L. Leto.
Phosphorylation of neutrophil 47-kDa cytosolic oxidase factor.
J. Biol. Chem.
265:
19910-19915,
1990[Abstract/Free Full Text].
27.
Sagai, M.,
and
T. Ichinose.
Lipid peroxidation and antioxidative protection mechanism in rat lungs upon acute and chronic exposure to nitrogen dioxide.
Environ. Health Perspect.
73:
179-189,
1987[Medline].
28.
Smith, R. M.,
and
P. Mohideen.
One hour in 1 ATA oxygen enhances rat alveolar macrophage chemiluminescence and fungal cytotoxicity.
Am. J. Physiol.
260 (Lung Cell. Mol. Physiol. 4):
L457-L463,
1991[Abstract/Free Full Text].
29.
Thomas, H. V.,
P. K. Mueller,
and
R. L. Lyman.
Lipoperoxidation of lung lipids in rats exposed to nitrogen dioxide.
Science
159:
532-534,
1968[Medline].
30.
Whisler, R. L.,
M. A. Goyette,
I. S. Grants,
and
Y. G. Newhouse.
Sublethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppresses protein phosphatases in Jurkat T cells.
Arch. Biochem. Biophys.
319:
23-35,
1995[Medline].
AJP Lung Cell Mol Physiol 273(5):L1042-L1047
1040-0605/97 $5.00
Copyright © 1997 the American Physiological Society