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J Biol Chem, Vol. 274, Issue 31, 21603-21608, July 30, 1999

Essential Requirement of Cytosolic Phospholipase A₂ for Activation of the H⁺ Channel in Phagocyte-like Cells^*

Alexander Lowenthal and Rachel Levy

From the Laboratory of Infectious Diseases, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer-Sheva 84105, Israel

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We previously established a model of cytosolic phospholipase A₂ (cPLA₂)-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA₂-generated arachidonic acid (AA) is essential for NADPH oxidase activation (Dana, R., Leto, T., Malech, H., and Levy, R. (1998) J. Biol. Chem. 273, 441-445). In the present study, we used this model to determine the physiological role of cPLA₂ in the regulation of both the H⁺ channel and the Na⁺/H⁺ antiporter and to study whether NADPH oxidase activation is regulated by either of these transporters. PLB-D cells and two controls: parent PLB-985 cells and PLB-985 cells transfected with the vector only (PLB cells) were differentiated using 1.25% Me₂SO or 5 × 10⁸ M 1,25-dihydroxyvitamin D₃. Activation of differentiated PLB cells resulted in a Zn²⁺-sensitive alkalization, indicating H⁺ channel activity. In contrast, differentiated PLB-D cells failed to activate the H⁺ channel, but the addition of exogenous AA fully restored this activity, indicating the role of cPLA₂ in H⁺ channel activation. The presence of the H⁺ channel inhibitor Zn²⁺ caused significant inhibition of NADPH oxidase activity, suggesting a role of the H⁺ channel in regulating oxidase activity. Na⁺/H⁺ antiporter activity was stimulated in differentiated PLB-D cells, indicating that cPLA₂ does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA₂-generated AA for activation of the H⁺ channel and suggest the participation of this channel in the regulation of NADPH oxidase activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The phagocyte NADPH oxidase is a multicomponent transport chain that transfers electrons from NADPH to molecular oxygen and generates superoxide, a precursor of microbicidal oxidants important to host defense. NADPH oxidase subunits include three cytoplasmic components, p47^phox, p67^phox, and Rac2, and a membrane flavocytochrome b₅₅₈ composed of gp91^phox and p22^phox (1-9). In phagocytes, stimulation results in translocation of the cytosolic NADPH oxidase components to the membrane, where they interact with the flavocytochrome to form the activated oxidase. Oxidation of NADPH to NADP⁺ results in an increase in intracellular H⁺, a concomitant drop in pH_i (10, 11), and a significant membrane depolarization (12). As a result, protons are transported across the plasma membrane via three different pathways: the Na⁺/H⁺ antiporter (10, 13), the vacuolar H⁺ ATPase (14), and the heavy metal-sensitive H⁺ channel (15-17).

It has been demonstrated that the single electron transfer from internal NADPH, through the oxidase complex, to external oxygen is an electrogenic process and that the efflux of H⁺ through the H⁺ channel is necessary for charge compensation (17-20). The channel was found to be singularly H⁺-selective, voltage-gated outwardly rectifying, regulated by both external and internal protons and sensitive to heavy metals (Cd²⁺ and Zn²⁺). A series of recent studies suggest that AA¹ regulates H⁺ channel activity in neutrophils (21, 22) and in macrophages (23). Transfection studies demonstrating heterologous expression of gp91^phox have indicated that gp91^phox or its N-terminal membrane-spanning domain (residues 1-230) also constitutes an AA-activated H⁺ channel (24, 25). The histidine 115 was found to be an amino acid important to the ability of gp91^phox to function as the NADPH oxidase-associated H⁺ channel (26). All of these in vitro studies suggest a role for AA in the regulation of H⁺ channel activity. However, the role of AA in a physiological system and the type of phospholipase A₂ responsible for its release have not as yet been defined. Furthermore, the effect of AA in these studies is not specific, since, apart from AA, a variety of unsaturated fatty acids has been shown to significantly activate a proton conductance in both macrophages (23) and neutrophil cytoplasts (21).

We previously established a model of cytosolic phospholipase A₂-deficient differentiated PLB-985 cells and demonstrated that cPLA₂-generated arachidonic acid is essential, by an unknown mechanism, for the activation of NADPH oxidase (27). The model of differentiated PLB-D cells offers a unique tool to determine the physiological role of cPLA₂ and of its metabolite AA in regulation of the various phagocytic functions. In the present study, this model was used to determine whether the H⁺ channel is regulated by cPLA₂ and whether this channel is involved in the regulation of NADPH oxidase activity. In addition, the role of cPLA₂ in the regulation of the Na⁺/H⁺ exchanger was investigated.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cell Culture and Induction of Differentiation

PLB-985 cells and selected PLB-D clones were grown in a stationary suspension culture in RPMI 1640 medium as described earlier (27). The results presented were found in seven individual clones.

Optimal concentrations of 1.25% Me₂SO or 5 × 10⁸ M 1,25(OH)₂D₃ were added to PLB cells or PLB-D cells (2 × 10⁵ cells/ml) at their logarithmic growth phase to induce differentiation toward granulocyte- or monocyte-like cells, respectively. Differentiation was induced for 4 days and determined by Mac-1 antigen expression detected by indirect immunofluorescence as described previously (28). Me₂SO was chosen in the present study instead of retinoic acid to induce differentiation toward the granulocyte lineage (27). PLB cells or PLB-D cells treated for 4 days with Me₂SO exhibited 85 ± 7 and 86 ± 8% differentiation, respectively. The expression of the cytosolic and membrane oxidase components was similar in both types of differentiated cells. Differentiated PLB cells produced 14.53 ± 2.8 nmol of O₂/10⁶ cells/min upon the addition of 50 ng/ml PMA. In contrast, superoxide production could not be stimulated in differentiated PLB-D cells with the addition of 50 ng/ml PMA. The addition of 10 µM AA to PMA-stimulated differentiated PLB-D cells restored this activity as shown in our previous study in PLB-D differentiated with 1,25(OH)₂D₃ (27).

Neutrophil Isolation

Granulocytes were purified by Ficoll/Hypaque centrifugation, dextran sedimentation, and hypotonic lysis of erythrocytes (29).

pH_i Measurements

pH_i Determinations-- Cells (5 × 10⁷) were loaded with 2',7'-bis(carboxyethyl)-5-(6)- carboxyfluorescein (BCECF) (3 µg/ml) for 10 min at 37 °C. 1 × 10⁷ cells were suspended in 3 ml of Na⁺ medium (140 mM NaCl, 5 mM KCl, 10 mM glucose, and 10 mM HEPES, pH 7.4). Cells were stimulated with 50 ng/ml PMA, and the changes in pH_i were recorded.

H⁺ Channel Activity-- H⁺ channel activity was monitored by recording changes in pH_i under conditions where the contribution of other H⁺ translocating systems and major acidifying mechanisms were shown to be eliminated (11, 19). Briefly, pH_i was monitored in cells (1 × 10⁷) suspended in 3 ml of KCl-based Na⁺ and HCO₃-free medium (145 mM KCl, 10 mM glucose, and 10 mM HEPES, pH 7.5), thus excluding the involvement of Na⁺/H⁺ and Cl/HCO₃ antiporters. Bafilomycin (100 µM) was applied to inhibit the vacuolar type H⁺-ATPase, and diphenylene iodonium (3 µM) to block H⁺ generation by the NADPH oxidase (30). Formation of a diffusion potential that might limit the free permeation of protons through the channel was prevented by the addition of the K⁺ ionophore valinomycin (1.5 µM). In order to generate an outward proton motive force, the K⁺ medium was titrated to pH 7.5 by the addition of KOH.

For acid loading, cells were preincubated in Na⁺ buffer containing 50 mM NH₄Cl for 10 min (NH₄Cl prepulse technique (31)). After removal of NH₄Cl, cells were suspended in N-methyl-D-glucammonium⁺-rich solution; the resultant pH_i was ~6.2. Upon the addition of 20 mM Na⁺ to the medium, recovery of pH_i occurred rapidly, ostensibly through Na⁺/H⁺ exchange that was shown to be significantly inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a specific Na⁺/H⁺ antiporter inhibitor (results not shown).

Calibration of the BCECF fluorescence was done as described previously (28, 32). Fluorescence, indicating changes in internal pH (pH_i), was monitored by a Perkin-Elmer LS50 B luminescence spectrometer with wavelengths of 485 and 540 nm for excitation and emission, respectively.

Superoxide Anion Measurements

The production of superoxide anion (O₂) by intact cells was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome c as described earlier (27).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

pH_i Changes Stimulated with PMA-- This set of experiments was performed in order to determine whether PMA induces a change in pH_i in differentiated PLB-985 cells similar to that observed in human neutrophils. In all of the experiments, both the parent PLB-985 cell line and the G418-resistant clones transfected with the empty pcDNA₃ vector were used as controls. Since no changes were obtained between the two controls, for simplicity we present the results of the clones transfected with the empty pcDNA₃ vector only (PLB cells). PLB cells were differentiated toward granulocyte- or monocyte-like cells by 1.25% Me₂SO or 5 × 10⁸ M 1,25(OH)₂D₃, respectively. The cells loaded with BCECF were suspended in a Na⁺ medium and then stimulated with PMA (50 ng/ml). In agreement with earlier observations (10, 33), peripheral blood neutrophils activated with PMA exhibited a triphasic change in pH_i (Fig. 1A). An initial rapid acidification of 0.1 pH units due to the activation of NADPH oxidase was followed by a recovery toward normal pH_i mediated by both the H⁺ channel and the Na⁺/H⁺ antiporter. This was followed by a slow and variable acidification phase. Both in granulocyte-like PLB cells (Fig. 1B) and in monocyte-like PLB cells (Fig. 1C), the addition of PMA induced a pattern of pH_i changes similar to that observed in neutrophils. There were slight differences, first, in the magnitude of the rapid acidification phase, which is more significant in neutrophils than in differentiated PLB cells, and second, differentiated PLB cells exhibited a higher alkalization than that induced in neutrophils. These two phenomena reflect the significantly higher PMA-stimulated NADPH oxidase activity in peripheral blood neutrophils generating 32 ± 4.5 nmol of O₂/10⁶ cells/min compared with granulocyte-like PLB cells or monocyte-like PLB cells producing 14.53 ± 2.8 and 10.85 ± 3.3 nmol of O₂/10⁶ cells/min, respectively (the results are the mean ± S.E. from 10 different experiments). These changes in pH_i induced by PMA in differentiated PLB cells indicate that these cells are an appropriate model for studying the role of cPLA₂ in the regulation of proton transport and whether these transporters affect oxidase activity in phagocytes.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   pHi changes stimulated with PMA. Stimulated pHi changes were detected in neutrophils (A), granulocyte-like PLB cells (B), monocyte-like PLB cells (C), granulocyte-like PLB-D cells (D), and monocyte-like PLB-D cells (E) suspended in a Na+ medium (as described under "Experimental Procedures"). Stimulation was obtained with PMA (50 ng/ml). The restoration of pHi changes with the addition of 10 or 25 µM exogenous AA in granulocyte-like PLB-D cells (F) or monocyte-like PLB-D cells (G) stimulated with PMA are shown.

pH_i changes were then studied in differentiated PLB-D cells. These cells fail to activate NADPH oxidase due to the absence of cPLA₂, as described previously (27). Thus, no metabolic acidification is expected to trigger the alkalization phase. Both the H⁺ channel and the Na⁺/H⁺ antiporter were shown to be either indirectly stimulated by metabolic cytoplasmic acidification or directly activated by the phorbol ester in neutrophils (34); thus, the direct effect of PMA on these transporters was studied in differentiated PLB-D cells. The addition of 50 ng/ml PMA to PLB-D cells differentiated with Me₂SO (Fig. 1D) or with 1,25(OH)₂D₃ (Fig. 1E) induced a pH_i alkalization that was significantly lower (p < 0.001) than that observed in differentiated control PLB cells. In addition, the metabolic acidification phase prior to alkalization was not observed in PMA-stimulated differentiated PLB-D cells as expected due to the absence of oxidase activation. The typical changes in pH_i detected in differentiated PLB cells, both the acidification phase and a higher alkalization, could be restored in PMA-stimulated differentiated PLB-D cells by the addition of exogenous AA in a dose-dependent manner (Fig. 1, F and G). The restoration process by exogenous AA is identical to the dose-dependent effect of AA for restoring NADPH oxidase activity, as shown in our previous study (27). The acidification induced by AA in the PMA-stimulated cells is probably due to oxidase activity. The lower alkalization phase in differentiated PLB-D cells stimulated with PMA suggests that one of the H⁺ efflux transporters does not operate in the absence of cPLA₂ and that its activity is restored by addition of AA. Since cells differentiated to the granulocyte lineage by Me₂SO and cells differentiated to the monocyte lineage by 1,25(OH)₂D₃ showed similar results, the results from here onward are shown only for cells differentiated with Me₂SO.

The Role of cPLA₂ in Regulation of the H⁺ Channel-- The gp91^phox, which constitutes an AA-activated H⁺ channel (24), is found in minute quantities in undifferentiated HL-60 cells or PLB cells and is induced during differentiation (35-37). In order to determine whether cPLA₂ has a role in regulating H⁺ channel activity, the normal development of the H⁺ channel during differentiation of PLB-D cells first had to be established. An outward acting pH gradient was imposed on cells in K⁺ medium in the presence of valinomycin (1.5 µM) and Tris (pH_o = 8.2) (as described previously (24)). Fig. 2 demonstrates that the H⁺ channel is similarly activated by the addition of AA in differentiated PLB cells and in differentiated PLB-D cells, indicating that it develops and operates normally in differentiated PLB-D cells. H⁺ channel activity was not induced by the addition of AA in undifferentiated PLB cells (data not shown) as demonstrated for undifferentiated HL-60 cells (24).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Induction of H+ channel activity in differentiated cells. Differentiated PLB cells and differentiated PLB-D cells were suspended in a K+ medium as described under "Experimental Procedures." An outward acting proton motive force was imposed by the addition of Tris buffer (pHo = 8.2) and 1.5 µM valinomycin (Val). Exogenous AA (10 µM) induced an efflux of protons due to activation of the H+ channel in both differentiated PLB cells and differentiated PLB-D cells.

The next step was to study whether cPLA₂ is involved in the signaling leading to H⁺ channel activation. The addition of PMA triggered a substantial cytoplasmic alkalization in differentiated PLB cells (Fig. 3A) similar to that induced in peripheral blood neutrophils (Fig. 3B and Ref. 19), indicating a H⁺ efflux. Alkalization was prevented in the presence of the H⁺ channel inhibitor, Zn²⁺ (50 µM), further confirming that H⁺ movements take place through the heavy metal-sensitive H⁺ channel. In differentiated PLB-D cells, PMA did not induce any cytoplasmic alkalization (Fig. 3C), but H⁺ channel activity could be restored by the addition of exogenous AA to PMA-stimulated or -nonstimulated cells (Fig. 3D). These results clearly indicate that cPLA₂-generated AA is essential for the activation of the H⁺ channel in differentiated PLB-D cells. Previous publications, utilizing the PLA₂ inhibitor p-bromophenacyl bromide (38) and the newly developed selective blocker of cPLA₂, trifluoromethyl ketone analogue of arachidonic acid (AACOCF₃) (22), have proposed that cPLA₂ has a possible role in regulation of the electrogenic H⁺ channel in the plasma membrane of neutrophils.

View larger version (7K): [in this window] [in a new window]   Fig. 3.   The role of cPLA2 in stimulation of the H+ channel activity. H+ channel activity was monitored in differentiated PLB cells (A), neutrophils (B), and differentiated PLB-D cells (C) suspended in a K+ medium in the presence of diphenylene iodonium, valinomycin, and bafilomycin (as described under "Experimental Procedures"). Stimulation was obtained with PMA (50 ng/ml). Restoration of H+ channel activity with the addition of 10 µM exogenous AA in differentiated PLB-D cells stimulated with PMA is shown (D). The H+ channel inhibitor, Zn2+, at a final concentration of 50 µM was added where noted.

Involvement of the H⁺ Channel in Regulating NADPH Oxidase Activity-- Differentiated PLB-D cells fail to activate both the NADPH oxidase (27) and the H⁺ channel (Fig. 3C) upon stimulation. As demonstrated in Fig. 3D, the addition of AA restored H⁺ channel activity in differentiated PLB-D cells in the presence of the oxidase inhibitor, diphenylene iodonium, indicating that oxidase activity is not a prerequisite for activation of this channel. In order to determine the potential role of the H⁺ channel in promoting or maintaining NADPH oxidase activity, the effect of the H⁺ channel inhibitor, Zn²⁺, was studied. As shown in Fig. 4A, the presence of 50 µM Zn²⁺ prevented alkalization induced by PMA in granulocyte-like PLB cells suspended in a Na⁺ medium. The absolute inhibition of H⁺ channel activity by 50 µM Zn²⁺ in stimulated granulocyte-like PLB cells suspended in a K⁺ medium is demonstrated in Fig. 3A. The presence of 50 µM Zn²⁺ significantly inhibited the production of superoxide stimulated with PMA in either Na⁺ or K⁺ medium, retaining about 40% of the activity (Fig. 4B). Since H⁺ channel activity was assayed in the presence of valinomycin (Fig. 3), the effect of Zn²⁺ on superoxide production was studied also in its presence. The addition of valinomycin accelerated the inhibition of superoxide production in differentiated PLB cells, reaching about 75% inhibition (Fig. 4B) in agreement with earlier data on neutrophil cytoplasts (39). These results suggest the involvement of the H⁺ channel in the regulation of NADPH oxidase activity, similar to earlier studies in neutrophil cytoplasts (39). Thus, the inability of stimulated differentiated PLB-D cells to activate NADPH oxidase may account, in part, for the failure in activating the H⁺ channel in these cells. The partial inhibition of superoxide production under conditions in which absolute inhibition of the H⁺ channel was induced suggests that the AA-activable H⁺ channel is not the sole mechanism regulating oxidase activity. AA, which caused complete activation of oxidase activity, probably acts at other sites on the oxidase in addition to its effect on the H⁺ channel.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   The effect of the H+ channel inhibitor on stimulated pHi changes and on superoxide production in differentiated PLB cells. A, the effect of Zn2+ (50 µM) on PMA (50 ng/ml) stimulated pHi changes monitored in differentiated PLB cells suspended in a Na+ medium. B, the effect of Zn2+ (50 µM) on PMA (50 ng/ml) stimulated superoxide generation in differentiated PLB cells suspended in a Na+ or K+ medium. Valinomycin (Val) was added at a final concentration of (1.5 µM).

Does cPLA₂ Regulate the Na⁺/H⁺ Antiporter?-- The Na⁺/H⁺ exchanger has been shown to be expressed and active in human neutrophils, HL-60 cells, and PLB cells (37). To determine the normal development of the Na⁺/H⁺ antiporter during differentiation of PLB-D cells, Na⁺/H⁺ exchange activity was determined by their ability to recover from an acid load. Cells were acid-loaded using the NH₄Cl prepulse technique (40). As demonstrated in Fig. 5, Na⁺/H⁺ exchange activity was similar in control differentiated PLB cells and differentiated PLB-D cells, implying normal development and activity of the Na⁺/H⁺ antiporter during differentiation of PLB-D cells.

View larger version (8K): [in this window] [in a new window]   Fig. 5.   Na+/H+ antiporter activity determined by recovery from acid load. Differentiated PLB and differentiated PLB-D cells were acid-loaded by preincubation in a Na+ buffer containing 50 mM NH4Cl for 10 min (see "Experimental Procedures") and then sedimented and resuspended in an N-methyl-D- glucammonium+-rich media. NaCl was added (final concentration 20 mM), and pHi changes were monitored. No difference was observed between PLB and PLB-D cells in the activation pattern of the Na+/H+ antiporter.

We then studied the involvement of cPLA₂ in the signaling leading to activation of the Na⁺/H⁺ antiporter. Stimulation of this antiporter is performed in a Na⁺ medium as described in Fig. 1. As shown in this figure, alkalization induced by PMA in differentiated PLB-D cells (Fig. 1D) is significantly lower than that observed in differentiated PLB cells (Fig. 1, B and C). Since the H⁺ channel does not operate in differentiated PLB-D cells as demonstrated in Fig. 3, it is likely that the alkalization stimulated by PMA in these cells is mediated by the Na⁺/H⁺ antiporter only. To further demonstrate this point, we studied the effect of the specific inhibitors for both H⁺ transporters. As shown in Fig. 6, stimulation of differentiated PLB-D cells in the presence of the H⁺ channel inhibitor, Zn²⁺, resulted in an alkalization identical to that observed in the absence of Zn²⁺, while the presence of the Na⁺/H⁺ antiporter inhibitor, EIPA, totally inhibited the alkalization. These results indicate that alkalization induced in stimulated differentiated PLB-D cells is mediated by the Na⁺/H⁺ antiporter and, furthermore, that cPLA₂ does not participate in regulation of Na⁺/H⁺ exchange activity. Alkalization following PMA stimulation under conditions in which the NADPH oxidase does not function was observed in neutrophils of patients with chronic granulomatous disease (20, 34). However, in contrast to the differentiated PLB-D cell model, in chronic granulomatous disease neutrophils, both Na⁺/H⁺ antiporter and H⁺ channel activities operate and contribute to the efflux of H⁺, resulting in higher alkalization induced by PMA.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   The effect of Na+/H+ antiporter and H+ channel inhibitors on pHi changes in differentiated PLB-D cells. pHi changes were monitored in differentiated PLB-D cells suspended in a Na+ medium as described under "Experimental Procedures." Stimulation was induced with PMA (50 ng/ml). Zn2+ (50 µM) or EIPA (10 µM) was added where noted.

Is the Na⁺/H⁺ Antiporter Involved in Regulating NADPH Oxidase Activity?-- To determine whether the Na⁺/H⁺ antiporter has a role in regulating the NADPH oxidase, by a mechanism which does not involve cPLA₂, the effect of EIPA on NADPH oxidase activity was studied. Stimulation of granulocyte-like PLB cells with PMA in the presence of EIPA resulted in a dramatic drop in pH_i due to an uncompensated accumulation of protons within the cytoplasm (Fig. 7A). These data, similar to previously published data (34, 41), indicates that the Na⁺/H⁺ antiporter has a major role in regulating pH_i. However, the presence of EIPA in PMA-stimulated granulocyte-like PLB cells does not have any effect on NADPH oxidase superoxide generation (Fig. 7B), indicating that the Na⁺/H⁺ antiporter has no role in the regulation of the NADPH oxidase. This is in agreement with previous results demonstrating that EIPA and other amiloride derivatives do not affect superoxide generation in neutrophils (42), murine peritoneal macrophages (43), and rat alveolar macrophages (44).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   The effect of Na+/H+ antiporter inhibitor on both pHi changes and superoxide production in differentiated PLB cells. A, differentiated PLB cells were suspended in a Na+ medium as described under "Experimental Procedures." The addition of PMA (50 ng/ml) to differentiated PLB cells pretreated with EIPA (10 µM) caused an uncompensated cytoplasmic acidification. B, the presence of EIPA in a range of 1-20 µM had no effect on PMA (50 ng/ml)-stimulated superoxide generation in differentiated PLB cells.

In conclusion, the development of a differentiated PLB-D cell model that lacks any cPLA₂ expression offers a unique tool for determining the physiological role of cPLA₂ in the signal transduction pathway leading to H⁺ channel activation. The present study demonstrates that cPLA₂ and its enzymatic production of AA are an essential requirement for H⁺ channel activation. Stimulation of differentiated PLB-D cells did not induce activation of H⁺ channel, but the addition of exogenous AA fully restored this activity. The present study clearly demonstrates that cPLA₂ specifically regulates the H⁺ channel and is not involved in the activation of the Na⁺/H⁺ exchanger. The similar restoration patterns of H⁺ channel activity (Fig. 2D) and of NADPH oxidase activity (27) by AA in differentiated PLB-D cells supports the presence of a tight coupling between these two processes. The significant inhibition of NADPH oxidase activity by the H⁺ channel inhibitor, Zn²⁺, suggests that the H⁺ channel participates in the regulation of NADPH oxidase activity. The exact mechanism by which AA activates the H⁺ channel and the NADPH oxidase and its precise site of action still remain to be elucidated.

	FOOTNOTES

^* This work was supported by United States-Israel Binational Science Foundation (Jerusalem, Israel) Grant 97-00178.

To whom correspondence should be addressed: Dept. of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Tel.: 972-7-6403186; Fax: 972-7-6467477; E-mail: ral@bgumail.bgu.ac.il.

	ABBREVIATIONS

The abbreviations used are: AA, arachidonic acid; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; PMA, phorbol 12-myristate 13-acetate; BCECF, 2',7'-bis(carboxyethyl)-5-(6)- carboxyfluorescein; EIPA, 5-(N-ethyl-N-isopropyl) amiloride.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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				T. E. DeCoursey During the Respiratory Burst, Do Phagocytes Need Proton Channels or Potassium Channels, or Both? Sci. Signal., May 18, 2004; 2004(233): pe21 - pe21. [Abstract] [Full Text] [PDF]

				Z. Shmelzer, N. Haddad, E. Admon, I. Pessach, T. L. Leto, Z. Eitan-Hazan, M. Hershfinkel, and R. Levy Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes J. Cell Biol., August 18, 2003; 162(4): 683 - 692. [Abstract] [Full Text] [PDF]

				T. E. Decoursey Voltage-Gated Proton Channels and Other Proton Transfer Pathways Physiol Rev, April 1, 2003; 83(2): 475 - 579. [Abstract] [Full Text] [PDF]

				L. M. Henderson and R. W. Meech Proton Conduction through gp91phox J. Gen. Physiol., November 25, 2002; 120(6): 759 - 765. [Full Text] [PDF]

				N. Touret and S. Grinstein Voltage-gated Proton "Channels": a Spectator's Viewpoint J. Gen. Physiol., November 25, 2002; 120(6): 767 - 771. [Full Text] [PDF]

				X. Zhao, E. A. Bey, F. B. Wientjes, and M. K. Cathcart Cytosolic Phospholipase A2 (cPLA2) Regulation of Human Monocyte NADPH Oxidase Activity. cPLA2 AFFECTS TRANSLOCATION BUT NOT PHOSPHORYLATION OF p67phox AND p47phox J. Biol. Chem., July 5, 2002; 277(28): 25385 - 25392. [Abstract] [Full Text] [PDF]

				D. Morgan, V. V. Cherny, M. O. Price, M. C. Dinauer, and T. E. DeCoursey Absence of Proton Channels in COS-7 Cells Expressing Functional NADPH Oxidase Components J. Gen. Physiol., May 28, 2002; 119(6): 571 - 580. [Abstract] [Full Text] [PDF]

				E. Mollapour, D. C. Linch, and P. J. Roberts Activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate oxidase and phospholipase A2 are dissociated by inhibitors of the kinases p42ERK2 and p38SAPK and by methyl arachidonyl fluorophosphonate, the dual inhibitor of cytosolic and calcium-independent phospholipase A2 Blood, April 15, 2001; 97(8): 2469 - 2477. [Abstract] [Full Text] [PDF]

				A. Maturana, S. Arnaudeau, S. Ryser, B. Banfi, J. P. Hossle, W. Schlegel, K.-H. Krause, and N. Demaurex Heme Histidine Ligands within gp91phox Modulate Proton Conduction by the Phagocyte NADPH Oxidase J. Biol. Chem., August 3, 2001; 276(32): 30277 - 30284. [Abstract] [Full Text] [PDF]

I. Pessach, T. L. Leto, H. L. Malech, and R. Levy Essential Requirement of Cytosolic Phospholipase A2 for Stimulation of NADPH Oxidase-associated Diaphorase Activity in Granulocyte-like Cells J. Biol. Chem., August 31, 2001; 276(36): 33495 - 33503. [Abstract]