©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Arachidonate-activable, NADPH Oxidase-associated H Channel
EVIDENCE THAT gp91-phox FUNCTIONS AS AN ESSENTIAL PART OF THE CHANNEL (*)

(Received for publication, October 18, 1994; and in revised form, December 16, 1994)

Lydia M. Henderson (§) George Banting J. Brian Chappell

From the Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human neutrophil NADPH oxidase-associated H channel acts as a charge compensator for the electrogenic generation of superoxide (O(2)). The expression of the channel activity was found to increase in parallel with that of the stimulatable generation of O(2) 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(2), 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.


INTRODUCTION

The activation of the NADPH oxidase is associated with a rapid depolarization of the membrane potential (Delta: -60 mV -15 mV), a slight fall in pH (0.1), and the generation of superoxide (O(2))(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(2) generation; either the H channel or O(2) 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(2) generation as a result of a stimulus. We have shown that the channel is not opened by depolarization of Delta, 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 (Deltap = Delta + 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.


EXPERIMENTAL PROCEDURES

Materials

The following were obtained from Sigma, Poole, Dorset, United Kingdom, and stock solutions were prepared as indicated: arachidonic acid, sodium salt (10 mM and 1 mM in 50% EtOH, stored under nitrogen), valinomycin (0.4 mM in EtOH), nigericin (1.5 mM in MeOH), CCCP (^1)(8 mM and 0.8 mM in EtOH, pH adjusted with NaOH), propidium iodide (10 mg/ml in H(2)O), succinyl acetone (1 mg/ml tissue culture medium, filter sterilized) and F(ab`)(2) fragment, fluorescein isothiocyanate-labeled antispecies antibodies were diluted as instructed by the supplier. BCECF-AM was obtained from Molecular Probes Inc., Eugene, OR and a stock solution of 1 mM prepared in Me(2)SO dried by freeze-thawing. The composition of the salt solutions was as follows: 150 mM KCl, 5 mM Hepes-Tris, 5.5 mM glucose pH = 7.3 (K medium) and 150 mM NaCl, 1 mM KCl, 5 mM Hepes-Tris, 5.5 mM glucose, pH = 7.3 (Na medium).

Tissue Culture

All manipulations of the cell cultures were performed in a Flow Laboratory's flow hood, and cultures were grown in a Heraeus incubator at 37 °C.

The human myeloid cell line (HL60) and the Epstein-Barr virus (EBV)-transformed B Lymphocyte cell lines were maintained in a CO(2)-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 times 10^5 cell/ml. The cells were harvested (day 5-7) by centrifugation at 800 times g for 10 min, washed in 1 volume of Na medium, and finally resuspended in Na medium at 2 times 10^7 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(2)-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 times 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.

Internal pH Measurements

All cell types were loaded with BCECF by incubation with 5 µM BCECF-AM for 5 min at 37 °C in Na medium, centrifuged (800 times g for 10 min) and resuspended in the appropriate assay buffer (either K or Na medium). Little leakage of dye was observed over a 2-h storage on ice.

Fluxes of H were monitored as changes in pH(i) determined by ratioing the fluorescence intensity after alternative excitation at 490 and 455 nm, at 37 °C as described previously(2) . An inward Deltap was imposed on the cells in a Na medium by the addition of valinomycin (2.7 µM) and Hepes (pH(o) = 6.7). Whereas an outward acting Deltap was imposed on cells in a K medium in the presence of valinomycin (2.7 µM) and Tris (pH(o) = 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(i) changes were calibrated in a K medium in the presence of 10 µM nigericin, as described previously (17) .

Construction of gp91-phox cDNA Plasmid

The gp91 construct (pMEP-4-91) was generated as follows. Nucleotides 19-1776 of the published gp91-phox cDNA sequence (18) (encoding amino acid 1 to the end of the protein coding sequence predicted by the published cDNA sequence(18, 19, 20) ) was excised as a KpnI/HindIII fragment, isolated from agarose gel, and ligated into KpnI/HindIII-digested pMEP-4 (Invitrogen Corp.) downstream of the inducible human metallothionein IIa promoter/enhancer (Fig. SII) using previously published procedures (21) . The construct was transfected, by electroporation (Bio-Rad gene pulser) into Escherichia coli, strain WM1100. Plasmid DNA was isolated using Qiagen columns as described by the manufacturer (Qiagen). The correct orientation of the gp91-phox cDNA insert was verified by diagnostic restriction enzyme digest using the BglII site within cDNA.


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.



Hygromycin b Sensitivity Assay

Hygromycin b is an inhibitor of eukaryotic protein synthesis. As susceptibility varies between cell lines a sensitivity assay was performed on both CHO and HL60 cells. The cells were cultured in the presence of 0, 25, 50, 75, 100, 150, 200, 300, and 400 µg/ml hygromycin b for 48-72 h. Cell survival was assessed as the ability of the cells to divide, to remain impermeant to trypan blue at 7 days and, in the case of CHO cells, remain adherent. The minimum concentration of hygromycin b required to lead to total cell death after 7 days was used to select for transfected cells.

Electroporation Survival Assay

The ability of both CHO and HL60 cells to recover following electroporation was assessed as the ability of cells to divide and in the case of CHO cells, remain adherent. The cells were electroporated at 200 V, 210 V, 230 V, 250 V, 270 V, 290 V, 300 V, and 950 µF and allowed to stand for 20 min before culturing as described above. The highest voltage giving good recover was used in the transfection experiments.

Transfection of CHO and HL60 Cells

The CHO (from two T75 flasks) and HL60 cells were harvested, as described above, and washed, twice, in filter-sterilized phosphate-buffered saline. The CHO cells were resuspended in 1 ml of a medium containing 20 mM Hepes, pH 7.0, 137 mM NaCl, 5 mM KCl, 0.7 mM Na(2)HPO(4), 250 mM sucrose in a 0.4-cm gene pulser cuvette (Bio-Rad) containing 10 µg of pMEP-4-91kDa or 10 µg of pMEP-4. The cells were electroporated (230 V, 960 µF) and allowed to stand at room temperature for 20 min before diluting with 20 ml Ham's-F-12 and split between two tissue culture flasks. Nonadherent/dead cells were removed 24 h later and the medium replaced. Hygromycin b (Sigma), 100 µg/ml, was added 48 h after electroporation to select for transfectants. The cells were passaged three times in the presence of hygromycin b before it was judged that stable transfectants were established.

The HL60 cells in 1 ml of CO(2)-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(2)-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.

Immunostaining

Immunocytochemical techniques in combination with confocal microscopy were used to examine the expression of gp91-phox, p67phox, p47-phox, and p22-phox. HL60 (±DMF), EBV-transformed B lymphocytes, CHO cells, and the transfected HL60 and CHO cell lines were immunostained using polyclonal anti-oxidase component antibodies by the method described previously(22) . The cells were fixed (4% formaldehyde, 10 min) and permeabilized (0.2% Triton X-100, 2 min) prior to staining. The CHO cell lines were cultured on round glass coverslips for 24 h prior to staining.

Arachidonate-induced Cell Lysis

Propidium iodide shows an increase in fluorescence upon binding to DNA but is cell impermeant and therefore can be used as an indicator of cell integrity. The susceptibility of transfected cell lines (±Cd) to lysis by AA was followed by excitation at 500 nm with emission at 540 nm, in the presence of 7.5 µg/ml propidium iodide in Na medium at 37 °C. A 1/100 dilution of Triton X-100 was used as a control to validate the assay.

Superoxide Assay

The generation of superoxide was monitored as the superoxide dismutase- or diphenylene iodonium-sensitive reduction (550 nm to 540 nm) of horse heart cytochrome c (100 µM) in Na medium at 37 °C as described previously(1) .

Isolation of White Blood Cells from Chinese Hamsters

Chinese hamster blood (0.5 ml) was obtained by cardiac puncture. The buffy coat layer (rich in white blood cells) was aspirated from the top of the cell pellet following centrifugation at 800 times g for 10 min. The remaining red blood cells were removed by hypotonic lysis as described previously(1) .

Western Blot

Two polyclonal anti-p22-phox protein antibodies and a anti-p47-phox were supplied by Prof A. W. Segal (University College, London). The polyclonal anti-gp91-phox was raised to the C-terminal 14-amino acid peptide coupled to keyhole limpet hemocyanin. The antibodies were verified by Western blot against isolated human neutrophils.


RESULTS AND DISCUSSION

The flux of H through the channel was monitored as the fall of pH(i) 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 Delta, negative inside. This together with a favorable pH gradient (pH(o) < pH(i)) 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(o) > pH(i). In both situations the counter movement of K provides charge compensation for H movements (Fig. SI). The measurements of pH(i) 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(2) generation and Na/H exchanger, respectively(11) .

Induction of H Channel in HL60 Cells

The myeloid cell line, HL60, grows continuously in culture but may be induced to differentiate into neutrophil like cells (5-7 days) by the addition of DMF, Me(2)SO, or retinoic acid(23) . An increase in the expression of oxidase components (gp91-phox, p67-phox, p47-phox, and p22-phox) and an increase in their ability to generate O(2) have been previously reported to be associated with this differentiation (24, 25, 26, 27) . As shown in Fig. 1the increase in O(2) generating ability (Fig. 1, a and b) is paralleled by an increase in the expression of the H channel activity (Fig. 1, c and d). Therefore the differentiation of HL60 cells is associated with an increased expression of the H channel.


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.

Contribution of p22-phox to H Channel Activity

Succinyl acetone inhibits -aminolevulinic acid dehydratase, an enzyme in the biosynthetic pathway for heme(30) . HL60 cells which have been induced to differentiate in the presence of 10 µg/ml succinyl acetone fail to show an increase in the ability to generate O(2) (Fig. 2, a-c). It has previously been reported that succinyl acetone-treated cells lack the characteristic cytochrome b difference spectrum(31) . This suggested that this failure to generate O(2) results from the absence of a heme moiety in the cytochrome b, rendering it nonfunctional.


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(2) also demonstrates that the channel is functionally independent of the activity of the oxidase.

H Channel in EBV-transformed B Lymphocytes

It has previously been reported the human B lymphocytes immortalized by EBV transformation generate low levels of O(2) in response to an appropriate stimulus(32) . This ability is constitutively expressed, and it shares some characteristics with the neutrophil NADPH oxidase (33) .

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(2) 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(i) change was only observed following the addition of an uncoupler. Thus the failure to observe an AA induced pH(i) 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.



Transfection of CHO and HL60 Cell with gp91-phox cDNA

To distinguish between these two roles for gp91-phox in the H channel, we investigated the ability of the protein, expressed in eukaryotic cell lines, to conduct H ions in response to AA. gp91-phox cDNA was inserted into the eukaryotic expression vector pMEP-4 (Fig. SII) and transfected, by electroporation, into CHO and HL60 cells. This vector allows the regulated expression of appropriately inserted cDNAs as expression is driven by the CdCl(2)-inducible human metallothionein IIa promoter; the increase in level of protein expression correlates with addition of increasing and nontoxic amounts of CdCl(2) to the tissue culture medium of transfected cells (36) . As anticipated, cells transfected with pMEP-4-91(CHO-91,HL60-91) express gp91-phox in a CdCl(2)-inducible manner (Fig. 4a; CHO-91,HL60-91, not shown). As the confocal microscope performs noninvasive optical sectioning the image is obtained from a discreet cross-section through the cell(37, 38) . The annular staining observed (Fig. 4a, i-iii) is indicative of plasma membrane location for the expressed protein.


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 pHunder 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(i) 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(i) change in nonexpressing CHO-91 cells argues against AAinduced cell lysis as the cause of the observed pH(i) 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(i) 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.




CONCLUSION

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 Delta, 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.


FOOTNOTES

*
This work was supported by a grant from the Arthritis and Rheumatism Council, United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 117-9287437; Fax: 117-9288274; L.M.Henderson{at}bristol.ac.uk.

(^1)
The abbreviations used are: CCCP, carbonyl cyanide m-chlorophenyl-hydrazone; AA, arachidonate; BCECF-AM, biscarboxyl 5 (and 6)-carboxyfluorescence acetomethyl ester; CHO, Chinese hamster ovary; CGD, chronic grandulomatous disease; DMF, dimethylformamide; EBV, Epstein-Barr virus; FCS, fetal calf serum; HL60, human leukemic 60 cell line; SA, succinyl acetone-4,6-dioxoheptanoic acid.


ACKNOWLEDGEMENTS

We thank Prof. A. W. Segal (University College, London) for his generous gifts of polyclonal antibodies, EBV-transformed B lymphocyte cell lines and gp91-phox cDNA and Elizabeth Hammond for the gift of the original CHO cells. We also thank Bush & Bush, Solicitors for their support and assistance.


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