(Received for publication, October 18, 1994; and in revised form, December 16, 1994)
From the
The human neutrophil NADPH oxidase-associated H channel acts as a charge compensator for the electrogenic
generation of superoxide (O
). The
expression of the channel activity was found to increase in parallel
with that of the stimulatable generation of O
in differentiated HL60 cells. HL60 cells induced to differentiate
in the presence of succinyl acetone (a inhibitor of heme synthesis)
were unable to generate O
, failed to
express p22-phox but retained H
channel
activity. EBV transformed B lymphocyte cell lines from normal and CGD
patients lacking expression of either p47-phox or p67-phox all expressed unaltered channel activity; however, the activity
was completely absent in the lymphocyte cell line lacking
gp91-phox. CHO cells and undifferentiated HL60 cells
transfected with gp91-phox cDNA expressed H
channel activity correlating with the expression of
gp91-phox. We therefore conclude that the large subunit of the
NADPH oxidase cytochrome b (gp91-phox) is the
arachidonate activable H
channel of human neutrophils.
The activation of the NADPH oxidase is associated with a rapid
depolarization of the membrane potential (: -60 mV
-15 mV), a slight fall in pH
(0.1),
and the generation of superoxide
(O
)(1, 2, 3, 4, 5, 6, 7, 8, 9) .
The superoxide is generated as a result of a single electron transfer
from internal NADPH, through the oxidase complex, to external oxygen.
We have previously shown that this is an electrogenic process and that
an efflux of H
ions through a channel provides the
necessary charge compensation(1, 2) . The opening of
the channel is not synchronous with the initiation of
O
generation; either the H
channel or O
precedes; in either
case rapid depolarization would occur, as is observed. As described for
H
conductance in the snail neuron the channel is
inhibited by Cd
and Zn
ions. The
tight coupling of the activities of the oxidase and the H
channel can only be overcome by the provision of an alternative
charge compensation pathway, e.g. valinomycin and
K
(10) .
In resting neutrophils the channel
is closed and the opening is associated with the initiation of
O generation as a result of a stimulus.
We have shown that the channel is not opened by depolarization of
, by an imposed change in the pH gradient or by a
combination of the two. However in all cases the H
channel was opened following the addition of arachidonate (8
µM). The direction and extent of H
flux
observed is that dictated by the proton motive force (
p =
+ RTln[H
]
/F[H
]
) (11) .
The NADPH oxidase complex consists of a transmembrane, heterodimeric cytochrome b (gp91-phox, p22-phox) located in the plasma membrane and membrane of the specific granules. A number of cytosolic factors (p67-phox, p47-phox, a small monomeric GTP-binding protein, and possibly p40-phox, a recently described protein with similarity to p47-phox)(9, 12, 13, 14, 15) . Upon activation these cytosolic factors have been shown to translocate partially to the membrane where they may interact with the cytochrome b(16) .
Although the H channel has
been identified functionally and characterized, the protein(s) which
bestow this property on the cell has/have not been identified. By
definition a channel or carrier must be made up of a transmembrane
protein(s), which may be regulated by cytosolic protein(s). We have
investigated the involvement of the oxidase components
(gp91-phox, p67-phox, p47-phox,
p22-phox) in the H
channel activity and have
shown that the expression of the channel activity correlates with the
expression of gp91-phox, but not with the expression of
p67-phox, p47-phox, or p22-phox.
The human
myeloid cell line (HL60) and the Epstein-Barr virus (EBV)-transformed B
Lymphocyte cell lines were maintained in a CO-independent
medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum
(FCS) (Life Technologies, Inc.) and 2 mML-glutamine
(Life Technologies, Inc.). The HL60 cells were induced to differentiate
by the addition of 105 mM dimethyl formamide (DMF) to cultures
at 2
10
cell/ml. The cells were harvested (day
5-7) by centrifugation at 800
g for 10 min,
washed in 1 volume of Na
medium, and finally
resuspended in Na
medium at 2
10
cells/ml. In the investigation of the role of heme, the HL60
cells were maintained and induced to differentiate (105 mM DMF), in a CO
-independent medium, in the absence of
FCS, but supplemented with Nutridoma-SP (Boehringer Mannheim) and 2
mML-glutamine. Succinyl acetone (SA) (10 µg/ml)
was added with the DMF and was present through out differentiation.
Chinese hamster ovary cells (CHO) were maintained Ham's F-12
nutrient mixture (Life Technologies, Inc.) supplemented with 10% FCS,
50 units/ml penicillin and 50 µg/ml streptomycin (Life
Technologies, Inc.). The medium was replaced every 2-3 days, and
the cells were divided once a week as described below. For harvesting
or splitting of the cells the medium was discarded, cells washed twice
with filter sterilized phosphate-buffered saline prior to treatment
with trypsin:EDTA (Life Technologies, Inc.) for 3-4 min. 37
°C. The cells were pelleted by centrifugation (800 g for 10 min) following the addition of fresh medium (10% FCS). They
were either resuspended in the assay buffer (harvesting) or 20 ml fresh
medium and divided equally between two flasks.
Fluxes
of H were monitored as changes in pH
determined by ratioing the fluorescence intensity after
alternative excitation at 490 and 455 nm, at 37 °C as described
previously(2) . An inward
p was imposed on the
cells in a Na
medium by the addition of valinomycin
(2.7 µM) and Hepes (pH
= 6.7). Whereas
an outward acting
p was imposed on cells in a
K
medium in the presence of valinomycin (2.7
µM) and Tris (pH
= 8.2). Opening of the
channel was measured following the addition of 8-12 µM arachidonate (Fig. SI).
Scheme I:
The AA-activated flux of H through the channel can be monitored as the fall in pH
of BCECF-loaded cells suspended in either a Na
or K
medium. In a Na
medium the
addition of valinomycin (a) resulted in a hyperpolarization of
the membrane potential. The subsequent addition of Hepes (b)
rapidly dropped the external pH, providing a proton motive force for
the influx of H
ions and hence a fall in internal pH,
following the addition of arachidonate (c). The addition of
valinomycin (a) to cells suspended in a K
medium results in a rapid membrane potential depolarization,
which following the addition of Tris (b) provides a proton
motive force for the efflux of H
upon the addition of
arachidonate (c). The measurements were performed in the
presence of excess diphenylene iodonium and amiloride to exclude the
contribution of superoxide generation and the
Na
:H
exchanger,
respectively.
The pH changes were
calibrated in a K
medium in the presence of 10
µM nigericin, as described previously (17) .
Scheme II: The gp91-phox cDNA (i) was inserted as a KpnI/HindIII fragment into the multiple cloning site of the plasmid pMEP-4 (ii), downstream from the human metallothionein IIa promoter.
The HL60 cells in 1 ml of CO-independent
medium, in the absence of FCS, were transfected by electroporation (250
V, 960 µF) in the presence of 10 µg of pMEM-4-91kDa or 10
µg of pMEP-4. The cells were allowed to recover, diluted, and
maintained in CO
-independent medium with 10% FCS for 48 h
before initiating selection by supplementing the medium with 300
µg/ml hygromycin b. The cells were maintained for 4 weeks before
selection of transfectants was judged to have occurred.
Induction of
expression from the metallothionein promoter was stimulated by the
addition of 10 µM Cd to the culture
medium 24 h prior to harvesting.
The flux of H through the channel was
monitored as the fall of pH
of cells suspended in a
Na
medium in the presence of valinomycin. In the
presence of this ionophore, the K
gradient results in
a large
, negative inside. This together with a favorable pH
gradient (pH
< pH
) provides the driving
force for H
entry. H
egress was
studied by suspending cells in K
medium again in the
presence of valinomycin and the imposition of a pH gradient with
pH
> pH
. In both situations the counter
movement of K
provides charge compensation for
H
movements (Fig. SI). The measurements of
pH
changes were carried out in the presence of excess
diphenylene iodonium (an inhibitor of the NADPH oxidase) and amiloride
in order to exclude the contribution of O
generation and Na
/H
exchanger,
respectively(11) .
Figure 1:
Induction of NADPH oxidase and
H channel activity in HL60 cells. The
generation of superoxide (a, b) and the expression of the
AA-activable H
channel (c, d) were monitored
in a K
medium, as described under ``Experimental
Procedures,'' in HL60 cells induced and differentiated (a,
c) and uninduced (b, d). The generation of superoxide was
stimulated by the addition of 50 nM phorbol 12-myristate
13-acetate (a, b), and 66 µg/ml superoxide dismutase was
added where indicated. Additions of 10 mM Tris, 2.7 µM valinomycin, and 4 µM arachidonate were made where
indicated (c, d). A downward deflection corresponds to a rise
in pH
.
The level of
expression of many proteins is reported to alter during the induction
of the differentiation of HL60 cells(28) . However as both
subunits of cytochrome b (gp91-phox and
p22-phox) are reported to be transmembrane
proteins(19, 20, 29) , their possible role in
functioning of the H
channel was investigated.
Figure 2:
Superoxide generation and H channel activity in succinyl acetone-treated HL60 cells. The generation of superoxide (a-c) and the
H
channel activity (e-g) were monitored
in a K
medium, as described under ``Experimental
Procedures'' in HL60 cells which had been induced and
differentiated (a, e), induced and differentiated in the
presence of 10 µg/ml SA (b, f), and uninduced (c,
g). Additions of 50 nM phorbol 12-myristate 13-acetate,
66 µg/ml superoxide dismutase were made as indicated in a-c and 10 mM Tris, 2.7 µM valinomycin, and 8 µM arachidonate were made in e-g. A downward deflection corresponds to a rise in
pH
. d, the immunocytochemistry was
performed on HL60 cells which had been induced and differentiated
(first column), induced and differentiated in presence of 10 µg/ml
SA (second column), uninduced (third column), and uninduced in the
presence of 10 µg/ml SA (fourth column) as described under
``Experimental Procedures.'' They were immunostained with
polyclonal anti-oxidase component antibodies: anti-gp91-phox (row 1), anti-p67-phox (second row), anti-p47-phox (third row), and anti-p22-phox (fourth row). The images
were obtain using a Bio-Rad MRC 500 confocal
microscope.
It is possible that the absence of the heme moiety may inhibit the expression of either or both cytochrome b subunits or may render their expression unstable. Therefore we investigated the expression of the cytochrome b subunits as well as the cytosolic factors (p67-phox, p47-phox) in HL60 cells differentiated in the presence and absence of succinyl acetone. This was done by using anti-oxidase component polyclonal antibodies in combination with confocal optical microscopy. In agreement with previous reports, the differentiation of HL60 cells was associated with a clear cut increase in the expression of gp91-phox, p67-phox, p47-phox, and p22-phox (Fig. 2d). However in the presence of succinyl acetone, the increase in expression of p22-phox was not observed (Fig. 2d, fourth row), whereas the increased expression of gp91-phox and p67-phox was unaffected (Fig. 2d first and second rows), the expression of p47-phox was far greater in the cells differentiated in the presence of succinyl acetone (Fig. 2d, third row). Therefore in the absence of heme, stable expression of p22-phox, in induced HL60 cells, does not occur.
Since the cells
differentiated in the presence of succinyl acetone lack
p22-phox, but not gp91-phox, they were utilized to
investigate the role of p22-phox in the H channel. HL60 cells differentiated in the presence or absence of
10 µg/ml succinyl acetone showed a similar increase in expression
of the H
channel compared with the undifferentiated
HL60 cells (Fig. 2, e-g). Therefore we conclude
that p22-phox does not act as the H
channel,
nor is it involved in the regulation of its activity. The presence of a
fully functional H
channel in HL60 cells
differentiated in the presence of succinyl acetone which are unable to
generate O
also demonstrates that the
channel is functionally independent of the activity of the oxidase.
Chronic grandulomatous disease (CGD) is a genetically
inherited disorder in which the expression of at least one of the
oxidase components is absent. These patients are unable to generate
O and show an increased susceptibility
to infection(34) . Although the cytosolic factors cannot, by
definition, act as the H
channel, it has been
suggested that p47-phox may regulate its
activity(35) . CGD-derived EBV-transformed B lymphocyte cell
lines lacking specific components of the NADPH oxidase were assayed for
H
channel activity as described above.
Normal
EBV-transformed B lymphocytes express an arachidonate-activable
H channel (Fig. 3a) as do the cell
lines lacking expression of p67-phox (Fig. 3b)
and p47-phox (Fig. 3c). No major differences
in the AA-stimulated H
channel were observed between
normal and either of the cell lines lacking the cytosolic factors. This
suggests that neither is involved in the regulation of channel
activity. In contrast to this, no channel activity was observed in the
cell line lacking gp91-phox (Fig. 3d). The
expected pH
change was only observed following the addition
of an uncoupler. Thus the failure to observe an AA induced pH
fall is the consequence of an absence of a pathway for the flux
of H
ions. This strongly suggests that gp91-phox may have a role in the H
channel or that it may
be crucial to the regulation of its activity.
Figure 3:
H channel activity in
EBV-transformed B lymphocytes cell lines from normal and CGD patients. The fall in pH
under imposed pH gradient
and negative membrane potential were monitored in a Na
medium as described under ``Experimental Procedures.''
The responses were monitored in cell lines established from normal (a) and in CGD cell lines established from patients lacking
expression of p67-phox (b), p47-phox (c), and gp91-phox (d) following the
addition of 8 µM arachidonate. 10 mM Hepes, 2.7
µM valinomycin, and 50 µM CCCP were added as
indicated in the figure.
Figure 4:
Expression of gp91-phox and the
H channel in transfected and nontransfected CHO cells. a, CHO cells transfected with pMEM-4-91 (i-iv) or just the plasmid pMEP-4 (v, vi) were
immunostained with the polyclonal anti gp91-phox as described
under ``Experimental Procedures.'' Expression from the
metallothionein IIa promoter was activated by treating the cells with
10 µM Cd
for 24 h prior to
immunostaining (i-iii, v). The images were obtained
using a Bio-Rad MRC 500 confocal microscope. The fall in
pH
under imposed pH gradient and negative membrane
potential were monitored in a Na
medium as described
under ``Experimental Procedures.'' The responses were
monitored in CHO cell (b), CHO cells transfected with
pMEP-4-91 (c), CHO cells transfected with
pMEP-4-91 and treated with 10 µM Cd
for 24 h (d), and a CCCP valinomycin control, in CHO
cells transfected with pMEP-4-91 (e). Additions of 10
mM Hepes, 2.7 µM valinomycin, 8 µM arachidonate, and 50 µM CCCP were made as indicated
in the figure. A downward deflection corresponds to a rise in
pH
.
CHO cells are impermeable to H ions and do not
express an AA activable H
channel (Fig. 4b). We also failed to detect the H
channel in CHO cell lines transfected with pMEP-4 ±
inducer (data not shown) and in CHO-91 grown in the absence of inducer (Fig. 4c). In all cases the addition of CCCP gave rise
to the expected pH
change. However in CHO-91 cells in which
the expression of gp91-phox had been induced, a clear
AA-activable H
channel was observed (Fig. 4d). A similar set of results was observed when
using the HL60-91 cell line (data not shown).
We have previously
shown that at these concentrations AA is not a protonophore, it can
only induce an H conductance in the presence of
protein(11) . However a greater problem is that AA has
detergent like properties and at sufficient concentrations can cause
cell lysis. The failure of AA to induce a pH
change in
nonexpressing CHO-91 cells argues against AAinduced cell lysis as the
cause of the observed pH
changes. However it is possible
that the expression of large amounts of a foreign protein results in
the CHO-91 cells being much more susceptible to lysis by AA. That this
is not the case is demonstrated by the requirement for AA in
concentration > 45 µM to allow access of propidium
iodide (a cell-impermeant fluorescent dye(39) ) in both the
CHO-91 and induced CHO-91 cells. Therefore the pH
changes
observed above (Fig. 4d) following the addition of 8
µM AA are not the result of cell lysis.
The HL60-91 and
CHO-91 cells induced to express gp91-phox did not express
p22-phox, p47-phox, or p67-phox as
determined by immunostaining using the anti-oxidase-component
polyclonal antibodies (Table 1). As the polyclonal antibodies
readily cross-react with neutrophils from Chinese hamsters (Table 1), it may be concluded that the expression of the
H channel requires the expression of gp91-phox but that p22-phox, p47-phox, and p67-phox are not involved.
In this paper we have shown that the functional expression of
the H channel correlates with the expression of
gp91-phox (the large subunit of cytochrome b
) upon induction of HL60 cells and in
EBV-transformed B lymphocyte cell lines derived from CGD patients. As
CHO cells transfected with gp91-phox cDNA express an
AA-activable H
channel only associated with the
expression of gp91-phox, we conclude that the large subunit
cytochrome b
functions as an essential part of
the NADPH oxidase associated H
channel.
A number of
recent electrophysiological studies have described a H conductance in differentiated HL60 cells, murine peritoneal
macrophages, and human neutrophils (40, 41, 42) which have very similar
properties to those of the NADPH oxidase-associated H
channel originally described by us. The conductances were blocked
by Cd
and Zn
ions (reversibly) and
show a delay in opening (1-2 s) following the imposition of a
depolarizing pulse(40) , in keeping with our observation that
opening of the channel is not synchronous with the initiation of
superoxide generation(1, 2) . We have previously
reported that depolarization of
, by the addition of
valinomycin to cells suspended in a high K
medium,
failed to open the channel and that it was only opened by the addition
of arachidonate(11) . In agreement with this the patch clamp
studies found no conductance in cells clamped at 0 mV, even in the
presence of a 1
1.5 pH unit imposed transmembrane pH
gradient(41) . The described H
conductance was
measured in cells at +60 mV, in the presence of a gradient of 1 pH
unit or greater. In addition to this DeCoursey and Cherny (42) have shown that the presence of 50 µM arachidonate the H
conductance is greatly
enhanced and is observed in cells clamped at less positive potentials,
suggesting that AA facilitates the opening and/or functioning of the
channel.
Grinstein and colleagues (35, 43) have
reported that the H conductance is absent in cells
from CGD patients lacking either gp91-phox, p47-phox,
or p22-phox, is reduced in CGD carriers (50%) and that the
activity is slightly reduced (30%) in an X-linked CGD patient with a
point mutation, but expressing gp91-phox at a reduced level.
From this they conclude that the channel is not part of the oxidase,
although it requires ``the assembly of the components of the
respiratory burst oxidase''(43) . However in contradiction
to this conclusion they also report normal H
conductances in CGD cells lacking p67-phox(43) .
As the authors failed to adequately distinguish between
superoxide-generating (H
liberating) cells and
nongenerating cells, clear interpretation of their data is difficult.
The authors have recently published a paper in which they report to
demonstrate H
channel activity in a number of patients
with X-linked CGD(44) . This is in strong disagreement with
their earlier reported defective channel activity in these patients (35, 43) and with their stated requirement for the
assembly of the oxidase(43) . In this paper the channel is
absent in CDG patient cells by functional assay but present as defined
by electrophysiological techniques. Again clear interpretation of
contradictory data is difficult.
There has been some debate as to which of the cytochrome b subunits carries the heme moiety. The amino acid sequence of neither contains a region of high homology to the heme binding consenus sequence(45) . The heme spectrum suggests a low spin heme with bisimidazole co-ordination(46, 47) ; however p22-phox, the preferred candidate, only contains 1 histidine. It is possible that the heme may sit between a p22-phox dimer, or it may be shared between p22-phox and p91-phox(48) . Using radiation inactivation, the heme-containing polypeptide was predicted to have a molecular mass of 21 ± 5 kDa, suggesting that p22-phox carries the moiety(49) . Our data show that in the absence of heme there is an absence of stable expression of the p22-phox, whereas p91-phox is unaffected. This strongly suggests that p22-phox is involved in binding the heme but, however, does not exclude the possibility of assistance from gp91-phox. The resolution of this problem awaits further experimentation.
It has been widely reported that the neutrophils from CGD patients with a defect in either cytochrome b subunit lack the expression of both leading to suggestions that each was required for the stable expression of the other(50, 51) . The stable expression of p22-phox has been reported as requiring gp91-phox mRNA(45) . In contradiction to this, we have found that HL60 cells induced in the presence of succinyl acetone lack only the expression of p22-phox. We have as yet to determine the expression and stability of the mRNA for the various components under these conditions. We would, therefore, propose that regulation of expression at the transcription level, by interaction (direct or indirect) between p91-phox and p22-phox in CGD patients, as opposed to regulation of p22-phox at the protein translation level by the unavailability of heme in the case of the HL60 cells, as an explanation for the apparent contradiction in results. It should be pointed out that in EBV-transformed B lymphocytes the level of expression of other cytochrome b subunits in the cell lines derived from -91 and -22 CGD patients was lower but never completely absent. All the cells we have utilized in this paper, by the very nature of being immortal cell lines, are abnormal. The induction of HL60 cell takes 5-7 days; therefore, longer term instabilities in the expression of gp91-phox in the absence of p22-phox would not be apparent in our experimentation. These may also contribute to the discrepancy between our results and those published previously.
We have previously reported that, as with the
H conductance in snail neurons(52) ,
Zn
and Cd
(mM) inhibit the
H
channel (1, 2) . In patch clamp
studies of the HL60 and neutrophil H
channel, this
inhibition has been shown to be fully
reversible(40, 41, 42) . Therefore the use of
10 µM Cd
, a level very much lower than
that required to block the H
channel (1 mM),
for the induction of the expression of gp91-phox should not
interfere with the assay, as the cells are repeatedly washed before
being used for measurements.
By exploiting the properties of various
cell lines, we have been able to examine individually the involvement
of the components of the NADPH oxidase in the expression and/or
regulation of the H channel activity. We have
demonstrated that the expression of the gp91-phox correlates
with the expression of the NADPH oxidase-associated H
channel. Our data strongly suggest that this cytochrome b subunit is the AA-activable H
channel and that
its activity does not involve nor is regulated by the other oxidase
components. However we have not eliminated the possibility that the
H
channel is a separate protein who's expression
is tightly coupled to the expression of gp91-phox. The ability
of isolated p91-phox protein to function as the H
channel in an artificial phospholipid system is presently under
investigation. As well as functioning as the channel, gp91-phox has been reported previously to contain the FAD and NADPH binding
sites (53, 54) and may be involved in binding the heme
moiety, i.e. all the major functions of the oxidase. At
present, functions and roles for the other components of the oxidase,
especially the cytosolic factors, have not been identified.