(Received for publication, July 22, 1994; and in revised form, November 14, 1994)
From the
The effects of the trivalent arsenical phenylarsine oxide (PAO)
on the activity of NADPH oxidase in human neutrophils were studied. PAO
caused a rapid dosedependent inhibition of superoxide generation which
was maximal at a concentration of 1 µM, irrespective of
the stimulating agent. This inhibitory effect was not due to impaired
transduction of activation signals since neither degranulation nor
phagocytosis were modified. When cytosolic and membrane fractions from
resting neutrophils were combined to reconstitute the NADPH oxidase,
Ogeneration was inhibited by PAO while
translocation of the NADPH oxidase components to the plasma membrane
fraction was not affected. The inhibition was completely and
specifically reversed by 2,3-dimercaptopropanol, not by dithiothreitol
or
-mercaptoethanol, indicating that PAO binds covalently to
spatially vicinal thiol groups. PAO inhibited the plasma
membrane's capacity to initiate O
generation while it apparently did not affect the
cytosol. When PAO was added subsequently to NADPH oxidase activation,
no inhibition was observed, indicating that PAO cannot reach its target
once the oxidase is functionally assembled. In conclusion, PAO is the
first complete and reversible inhibitor of NADPH oxidase which could
provide the basis for new therapeutical approaches in inflammatory
diseases.
Reactive oxygen species such as superoxide, hydrogen peroxide, hypohalides, and hydroxyl radicals, formed by neutrophils play a primary role in host defense against disease. They are also implicated in the pathogenesis of many diseases, e.g. respiratory distress syndromes, rheumatoid arthritis, and ischemia-reperfusion injury(1) . Therefore, there is great interest in the development of methods for control of their production.
The enzyme
responsible for neutrophil-derived oxygen radical production is NADPH
oxidase. This multicomponent electron-transfer complex, found
exclusively in phagocytes and B-lymphocytes, catalyses the reduction of
oxygen to superoxide anions (O) using NADPH as
the electron donor (see (2) for review). The oxidase is
dormant in resting neutrophils but acquires catalytic activity and
releases O
outside the cells or inside
phagosomes when the cells are exposed to appropriate stimuli (see (3) and (4) for review). The known components
necessary for the respiratory burst include a unique low potential
b-type cytochrome, cytochrome b
, present in the
membrane of both specific granules (5) and secretory
vesicles(6) , and at least three cytosolic proteins
p47
, p67
, and
p21
(7, 8, 9) .
Recently, an additional cytosolic protein, p40
, has been
proposed to modulate the activity of NADPH oxidase(10) . Except
for p21
, for which the recruitment to the plasma
membrane is still
debated(11, 12, 13, 14, 15) ,
the other oxidase components undoubtedly associate together at the
plasma membrane upon cell activation.
The molecular details of the
pathways regulating the activation, the assembly, and the deactivation
to NADPH oxidase are still unknown. Distinct pathways for transduction
of activation signals to the NADPH oxidase have been identified (see (16) for review). Inhibitors of several transducing proteins (17, 18, 19) only partially inhibit O generation. Protease inhibitors such
as antichymotrypsin(20, 21, 22) , arylating
agents such as iodonium halides (23, 24) , or the
chrysotherapeutic agent auranofin (25) have been proposed to
inhibit O
generation. However, these
molecules often provided incomplete and irreversible inhibition and
they did not directly and specifically affect the NADPH oxidase complex (26) .
Recent data suggest that phosphorylation of proteins
on tyrosine residues may play an important role in the activation of
neutrophils (27, 28) . We sought to manipulate the
tyrosine phosphorylation pathway by inhibiting tyrosine phosphatase
activity to increase the level of tyrosine-phosphorylated proteins in
human neutrophils and study whether this can modify the NADPH oxidase
activity. A recent study of Oetken and co-workers (29) on the
effects of phenylarsine oxide (PAO) ()on phosphorylations
showed that PAO augments tyrosine phosphorylations in hematopoietic
cells. PAO, a membrane-permeable trivalent arsenical that specifically
complexes vicinal sulfhydryl groups of proteins to form stable ring
structures, has been widely used as an inhibitor of phosphotyrosine
phosphatases (30, 31) . Although PAO cannot be
considered as a specific inhibitor of tyrosine phosphatases since it
also affects glucose transport(32, 33) , inhibits
receptor endocytosis(34, 35) , and blocks
insulin-mediated activation of p21
(36) ,
we studied its effect on the O
generation in human neutrophils. Surprisingly, we observed that,
when PAO is used at concentrations well below those which produce the
effects described above, it inhibits fully and reversibly the NADPH
oxidase without any other detectable effect.
Markers (Class I HLA molecules for the
plasma membrane, lactoferrin for the specific granules, and
-glucuronidase for the azurophil granules) were assayed as
described previously (11) to verify that the membrane fraction
was essentially free (<10%) of contaminating granules. Protein
concentration was measured by the Bio-Rad protein assay as described by
the supplier.
In some experiments, cytosol and membrane fractions were
treated separately for 10 min at 4 °C with or without 1 µM PAO. Unbound PAO was then eliminated by two consecutive
centrifugations of the membrane fraction and by running the cytosol on
a Sephadex G-25 column as described by the manufacturer. Briefly, 2.5
ml of PBS containing cytosolic proteins (140 µg/ml) were loaded
onto Pharmacia PD-10 columns. Proteins were eluted with 3.5 ml of PBS.
The final concentration of cytosolic proteins was 100 µg/ml
(dilution factor 1.4) indicating that proteins were not retained on the
column. We verified that the amount of p47 and
p67
recovered in the eluted cytosol did not change
compared to control. The reconstitution assay was made by combining 0.5
ml of the eluted cytosolic proteins with 25 µg of membrane proteins
in the presence of ATP, MgCl
, CaCl
, NADPH, and
arachidonic acid as described above. Addition of 0.5 mM GTP
and/or 10 µM GTP
S did not improve the assay.
Figure 1:
PAO
inhibits dose dependently the Ogeneration
initiated by different stimuli. A, neutrophils were incubated
with different concentrations of PAO for 10 min at 37 °C and
subsequently stimulated. NADPH oxidase activity was measured in resting
cells and in cells stimulated with PMA (100 ng/ml). The results of four
experiments are expressed as mean ± S.D. Inset, resting
and PMA-stimulated neutrophils were pretreated or not (none) with 1
µM PAO and O
production was
measured. In the last line (PAO/wash), cells were treated with
1 µM PAO, washed free of PAO by dilution of the incubation
medium (1/5, v/v), centrifugation, and resuspension of the cells in
PAO-free medium. B, neutrophils were incubated with different
concentrations of PAO for 10 min at 37 °C, and subsequently
stimulated with PMA (100 ng/ml), A23187 (2.5 µM),
opsonized zymosan (OZ) (3 mg/ml), fMLP (2.5 µM),
or a combination of PMA and A23187 (50 ng/ml and 2.5 µM,
respectively). One experiment representative of three is
shown.
To further characterize
the inhibitory effect of PAO on Ogeneration,
cells were stimulated by a variety of stimuli described to activate the
NADPH oxidase through distinct pathways of signal
transduction(37, 43, 44) . We observed that
O
generation initiated by either surface
receptor-mediated agonists (fMLP and opsonized zymosan) or stimuli
which act at sites distal to plasma membrane receptors (PMA and A23187)
did not show different sensitivity to PAO (Fig. 1B).
These data suggested that either a step common to these different
transduction pathways or the NADPH oxidase itself was sensitive to PAO.
Figure 2:
PAO
does not modify the phosphatase activity, the profile of
tyrosine-phosphorylated proteins, or the intracellular concentration of
ATP. A, neutrophils were treated with or without (control) 1
µM PAO for 10 min at 37 °C before PMA stimulation and
submitted to subcellular fractionation as described under
``Experimental Procedures.'' The cytosolic (1),
membrane (2), and granular fractions (3) (5
10
cell equivalent) were then assayed for their phosphatase
activity. B, neutrophils pretreated or not with 1 µM PAO and stimulated with PMA were incubated with 100 µM vanadate and solubilized in SDS sample buffer containing 1 mM vanadate. Proteins were separated by gel electrophoresis and
transferred to nitrocellulose membrane. The tyrosine-phosphorylated
proteins were revealed with antiphosphotyrosine antibodies. Molecular
weight standards are shown on the left. C, cytosols were
isolated from resting and PMA-activated neutrophils pretreated or not
with PAO (1 µM) and intracellular ATP levels were measured
by the luciferin/luciferase assay. The results of three experiments are
expressed as mean ± S.D.
PAO was reported
to decrease the intracellular ATP concentration in several cell
systems. Although this was generally observed when PAO was used at
concentrations well above 1 µM(33, 48) , e.g. at 18 µM it had a 10-15% inhibitory
effect on adipocyte ATP content(49) , we examined whether
inhibition of O production could result from
the decrease in cellular ATP. As shown on Fig. 2C, 1
µM PAO did not cause any modification in neutrophil ATP
levels in resting or PMA-activated cells.
In intact cells, the oxidase
activity is maintained by the continuous recruitment of new functional
enzyme units(52, 53, 54) . We examined the
effect of PAO when added after the activating agent on intact cells (Fig. 3). We observed that when PAO was added to cell
suspensions 30 s after PMA (during the lag phase, i.e. before
the generation of O ), O
production was strongly inhibited.
When added 2 or 5 min after PMA, when the oxidase was active, the
cytochrome c reduction flattened but did not plateau
immediately.
Figure 3:
Addition of PAO on PMA-activated cells
inhibits the formation of new functional oxidase units. The effect of
PAO on the O production was measured
in PMA-stimulated neutrophils. PAO was added at different times after
the stimulus (30 s, 2 min, and 5 min).
Figure 4:
PAO does not inhibit translocation of
p22, p91
, p47
, and
p67
to the plasma membrane in intact cells or in the
cell-free system. A, neutrophils pretreated or not with 1
µM PAO and stimulated or not with PMA were fractionated by
differential centrifugation. Proteins from the cytosol and the plasma
membrane-enriched fraction were immunoblotted with affinity-purified
anti-p47
antibodies and anti-p67
antiserum (left panel) or with anti-p22
and anti-p91
(right panel) antibodies. An
additional unidentified band at approximately 42 kDa (right
panel) was observed. In parallel, the inhibitory effect of PAO on
the generation of O
by intact cells was measured
(93 ± 9%, n = 3). B, cytosol and
membrane fractions from resting neutrophils were obtained by
centrifugation on discontinuous Percoll gradient. These fractions were
coincubated in the presence or absence of 1 µM PAO
followed or not by the addition of arachidonic acid for 2 min.
Cytochrome c and NADPH were added and the generation of
O
was measured (see Table 4, lines 1 and
2). After activation, membrane and cytosolic fractions were separated
by centrifugation and proteins were immunoblotted as in A.
The small G protein p21 is
required for NADPH oxidase activation(7, 8) , however,
we did not test it in the above assays since we recently demonstrated
that it is not recruited to the plasma membrane when intact neutrophils
are activated(11) . However, we explored the possibility that
PAO could inhibit the O
production by
interacting with the GTP binding site of p21
since
vicinal thiols, critical for the enzyme activity, have been identified
in the GTP-binding site of phosphoenolpyruvate
carboxykinase(57) . Reconstitution experiments of the oxidase
activity were performed using cytosolic fractions preincubated with 10
µM GTP
S to hypothetically protect the GTP-binding
site from the subsequent addition of PAO. Under these conditions the
inhibitory effect of 1 µM PAO was unchanged suggesting
that the GTP-binding site of p21
is not the target of PAO
(data not shown).
In several cell systems, when PAO is used at concentrations
over 10 µM, it has been described to affect multiple
sites(30, 31, 32, 33, 34, 35, 36) ,
such as modification of the phosphatase activities, the pattern of
tyrosine-phosphorylated proteins, or the ATP concentration which are
potentially involved in the regulation of NADPH oxidase(58) .
Here in neutrophils, PAO was used at 1 µM, a concentration
which provided a complete inhibition of the O generation without detectable effect
on the above mentioned PAO targets. In addition, it did not affect
other neutrophil functions which are initiated upon cell activation, i.e. phagocytosis and degranulation. Therefore, using PAO at 1
µM in neutrophils appeared to have a limited effect on O
generation. Furthermore, PAO also
prevented the O
generation in human
monocytes (data not shown). We observed that PAO inhibited
indiscriminately the generation of O
initiated by several stimuli reported to activate the oxidase
through different signaling
pathways(37, 43, 44) . It also inhibited the
O
production by the reconstituted
oxidase in the cell-free system in which transduction signals such as
phosphorylation events are
bypassed(59, 60, 61, 62) . From
these results, it emerged that PAO could directly affect the NADPH
oxidase complexes.
Whether PAO was affecting the membrane or the
cytosolic components of the oxidase was determined. We report that,
although chromatography through Sephadex G-25 columns induced a
decrease in the O generating capacity
of the cytosol, no inhibitory effect of PAO was detectable in this
fraction. This eliminates the possibility that PAO could interact with
cytosolic proteins including the recently identified
p40
, a cytosolic protein not required for activity of
the oxidase in the cell-free system. In contrast, the capacity of the
membrane fraction to induce O
generation in the cell-free assay was affected by PAO. This
indicated that one or more of the membrane components of the oxidase
were targeted by PAO such as cytochrome b
, a
membrane-associated heterodimer which constitutes the electron
transport apparatus of the NADPH oxidase(63) . Other proteins
which may regulate the oxidase activity such as rap1 or
membrane-associated cytoskeletal
proteins(14, 64, 65) , have also to be
considered as potential PAO targets. The identity of the PAO-sensitive
protein(s) remains to be established.
In our attempt to characterize
the inhibitory mechanism of PAO, the following results were obtained:
1) PAO had no effect on the generation of O when added to isolated membranes containing the activated oxidase
complex; 2) when PAO was added to the cell suspension after the O
generation had initiated, the
production of O
rapidly decreased but
did not cease, indicating that new units of active oxidase are not
formed anymore, whereas functional enzymes remained active for few more
minutes; 3) the components of the NADPH oxidase appeared to associate
in a similar extent with the plasma membrane in the presence or absence
of PAO in both intact cells or the cell-free system while the
generation of O
was fully inhibited;
4) the inhibitory effect of PAO which reacts specifically with vicinal
sulfhydryl groups to form stable five-ring structures (50) was
reversed by the vicinal dithiol competitor DMP, while monothiols, or
dithiols which do not have the correct space (49, 50) between the sulfhydryl groups, did not react
efficiently with PAO. Taken together, these observations suggest that
PAO may act at a step of the activation process distal to the
translocation of the oxidase subunits and proximal to the starting
point of O
generation by complexing
spatially vicinal sulfhydryl groups. This step could correspond to a
conformational change needed to initiate of the electron transport
chain activity. This hypothesis is supported by the observation that
the activated oxidase is PAO-insensitive, suggesting that the
PAO-reactive sulfhydryl groups are not vicinal anymore. As an
alternative hypothesis, it can be proposed that assembly of the oxidase
components in the plasma membrane is sufficient to drive the electron
transport but, once assembled, a steric hindrance impairs PAO to reach
its target and inhibit the electron transfer.
N-Ethylmaleimide (NEM), a known monothiol alkylating
compound, has been described to inhibit irreversibly the generation of
O in neutrophils(66) . Its
mechanism of action can be distinguished from that of PAO according to
the following observations: (i) NEM inhibits fMLP-induced production of
O
but is a poor inhibitor when the
cells are stimulated with serum-opsonized zymosan(67) , (ii)
NEM irreversibly inhibits the capacity of the cytosol to drive the O
generation in the cell-free
assay(66) , and (iii) in the presence of NEM, translocation of
p47
and p67
to the plasma membrane during
neutrophil activation is inhibited(54) . Therefore, PAO and NEM
do not act on the same targets. In addition, NEM was used at 150
µM to induce a complete inhibition of the O
generation in intact neutrophils, a
concentration producing multiple effects in cells(50) .
In
conclusion, we describe the first complete and reversible inhibitor of
the human NADPH oxidase. Reversal of the inhibition by DMP indicates
that PAO affects the NADPH oxidase through its association with a
protein, or a protein complex, containing vicinal thiol groups. This
PAO-sensitive site is likely to be membrane-associated. The observation
that PAO did not inhibit the O generation once the oxidase is activated suggests that the
vicinal thiol groups are no longer accessible, due to a steric
hindrance, or adjacent, as the result of a potential conformational
change occurring to activate the enzyme.
The respiratory burst of phagocytes has a critical role in tissue damage in inflammatory diseases. The prevention of inappropriate generation of oxygen reactive species is therefore likely to be an important homeostatic mechanism. Arsenical compounds have been applied for human therapy of a variety of protozoan infections(50) . Whether PAO produces anti-inflammatory effects in vivo is currently in progress in the laboratory. In addition, the possibility to use a complete and reversible inhibitor of the oxidase will be an important tool to progress in the understanding of the role of oxygen radicals as mediators of signal transduction in phagocytes.