Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2α activity

  1. Deri Morgan1,
  2. Vladimir V. Cherny1,
  3. Alison Finnegan2,
  4. James Bollinger3,
  5. Michael H. Gelb3 and
  6. Thomas E. DeCoursey1
  1. 1Department of Molecular Biophysics & Physiology and 2Department of Immunology/Microbiology, Rush University Medical Center, Chicago, IL 60612, USA3Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA
  1. Corresponding author T. E. DeCoursey: Department of Molecular Biophysics and Physiology, Rush University Medical Center, 1750 West Harrison, Chicago, IL 60612 USA.  Email: tdecours{at}rush.edu
 
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Abstract

The prevailing hypothesis that a signalling pathway involving cPLA2α is required to enhance the gating of the voltage-gated proton channel associated with NADPH oxidase was tested in human eosinophils and murine granulocytes. This hypothesis invokes arachidonic acid (AA) liberated by cPLA2α as a final activator of proton channels. In human eosinophils studied in the perforated-patch configuration, phorbol myristate acetate (PMA) stimulation elicited NADPH oxidase-generated electron current (Ie) and enhanced proton channel gating identically in the presence or absence of three specific cPLA2α inhibitors, Wyeth-1, pyrrolidine-2 and AACOCF3 (arachidonyl trifluoromethyl ketone). In contrast, PKC inhibitors GFX (GF109203X) or staurosporine prevented the activation of either proton channels or NADPH oxidase. PKC inhibition during the respiratory burst reversed the activation of both molecules, suggesting that ongoing phosphorylation is required. This effect of GFX was inhibited by okadaic acid, implicating phosphatases in proton channel deactivation. Proton channel activation by AA was partially reversed by GFX or staurosporine, indicating that AA effects are due in part to activation of PKC. In granulocytes from mice with the cPLA2α gene disrupted (knockout mice), PMA or fMetLeuPhe activated NADPH oxidase and proton channels in a manner indistinguishable from the responses of control cells. Thus, cPLA2α is not essential to activate the proton conductance or for a normal respiratory burst. Instead, phosphorylation of the proton channel or an activating molecule converts the channel to its activated gating mode. The existing paradigm for regulation of the concerted activity of proton channels and NADPH oxidase must be revised.

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Crucial to innate immunity, the NADPH oxidase complex produces the superoxide anion (O2·−), a precursor to many other reactive oxygen species. Because NADPH oxidase is electrogenic (Henderson et al. 1987; Schrenzel et al. 1998), sustained activity requires activation of voltage-gated proton channels to compensate charge (Henderson et al. 1988; DeCoursey et al. 2003). For over a decade, it seemed fairly well established that phospholipase A2 (PLA2) contributed to activation of NADPH oxidase (Maridonneau-Parini & Tauber, 1986; Henderson et al. 1989), presumably by liberating arachidonic acid (AA). Abundant evidence (summarized by DeCoursey & Cherny, 1993; DeCoursey, 2003) established that AA is released by phagocytes during the respiratory burst and that AA itself is a powerful stimulus for O2.− generation, both in intact cells and in cell-free systems (the NADPH oxidase complex reconstructed from its component parts). The cell-free system requires AA or another amphiphile to activate NADPH oxidase (McPhail et al. 1985; Bromberg & Pick, 1985). Despite the evidence that AA can activate NADPH oxidase, the question whether it does so under physiological conditions has been controversial. For example, although a variety of PLA2 inhibitors inhibit O2.− release (Henderson et al. 1989; Dana et al. 1994; Daniels et al. 1998), others do not (Tsunawaki & Nathan, 1986; Suszták et al. 1997; Daniels et al. 1998; Mollapour et al. 2001). In some studies, PLA2 inhibitors, now known to be of questionable potency and specificity, prevented the respiratory burst stimulated by AA or fMetLeuPhe, but not that by PMA (Maridonneau-Parini & Tauber, 1986; O'Dowd et al. 2004). Several PLA2 inhibitors were shown to act in macrophages by inhibiting glucose uptake rather than NADPH oxidase activity (Tsunawaki & Nathan, 1986). In eosinophils, PLA2 inhibitors prevented O2.− production, but the response was not restored by AA (White et al. 1993), in contrast with neutrophils in which AA restored the burst after PLA2 inhibition (Henderson et al. 1989; Dana et al. 1994). In the context of confusing and contradictory pharmacological studies, the use of antisense to create a cPLA2α deficient PLB-985 cell line (Dana et al. 1998) promised to clarify the situation. The cPLA2α deficient cells failed to produce O2.− upon stimulation, and the response was fully restored by exogenous AA (Dana et al. 1998), apparently confirming a requirement for cPLA2α and AA to activate the respiratory burst. However, in a recent study, a completely normal respiratory burst was observed in the presence of potent and selective cPLA2α inhibitors that demonstrably prevented arachidonic acid release (Rubin et al. 2005). Similarly, PLA2 inhibitors did not affect O2.− production in rac2 knockout mouse neutrophils stimulated with PMA or AA (Kim & Dinauer, 2006). Finally, macrophages and neutrophils from cPLA2α knockout mice have a normal respiratory burst despite reduced AA release (Gijón et al. 2000; Rubin et al. 2005). The present results extend this conclusion, showing that NADPH oxidase activation does not require cPLA2α in human eosinophils stimulated by PMA or in murine granulocytes stimulated by PMA or fMetLeuPhe.

A similar, but less complete and thus far, less controversial, story exists for voltage-gated proton channels in phagocytes, the main focus of the present study. These channels mediate the proton efflux that balances the electronic charge translocated by NADPH oxidase (Henderson et al. 1987; Murphy & DeCoursey, 2006), preventing extreme depolarization that would otherwise abolish NADPH oxidase activity (DeCoursey et al. 2003). Henderson & Chappell (1992) proposed that AA was the final, necessary activator of the proton conductance, gH, during the respiratory burst. For PMA stimulation, the proposed pathway is (Chappell & Henderson, 1991): Formulawhere H* indicates the activated gating mode of proton channels (Bánfi et al. 1999; DeCoursey et al. 2000b). In a number of cells, PKC is immediately upstream of cPLA2 (Qiu & Leslie, 1994; Xia et al. 1995; Han et al. 2004).

Substantial indirect evidence supports this hypothesis. AA enhances proton currents in whole-cell voltage-clamp studies of neutrophils (DeCoursey & Cherny, 1993), macrophages (Kapus et al. 1993b; Suszták et al. 1997), and eosinophils (Gordienko et al. 1996; Schrenzel et al. 1996). Using pH changes to measure proton fluxes, Kapus et al. (1993b) found that the PLA2 inhibitor bromophenacyl bromide prevented the PMA-induced cytoplasmic alkalinization believed to be mediated by proton channels. Suszták et al. (1997) found that the cPLA2α inhibitor AAOCOCF3 prevented PMA- or fMetLeuPhe-induced pH changes, but did not inhibit NADPH oxidase, nor did it directly affect proton currents in whole-cell studies. Schrenzel et al. (1996) reported that proton currents were larger in cells with high [Ca2+]i and that 10 μm bromophenacyl bromide inhibited proton currents, suggesting that proton current enhancement was mediated by PLA2. Proton currents also were enhanced by AA, even after bromophenacyl bromide, leading to the conclusion that activation of the gH during the respiratory burst is controlled by elevation of [Ca2+]i, which activates PLA2 (Schrenzel et al. 1996). The final evidence that seemed to confirm this hypothesis was again the cPLA2α deficient cell line. Levy and colleagues (Lowenthal & Levy, 1999; Levy et al. 2000) showed that cPLA2α deficient PLB-985 cells lacked the Zn2+ sensitive alkalinization that is seen in normal cells after stimulation by PMA. AA restored this response. Finally, PMA stimulated proton efflux in PLB-985 cells transfected with a fragment of gp91phox, but not in identical cells with cPLA2α knocked out (Mankelow et al. 2003). Thus, in contrast to the NADPH oxidase story, the implication of cPLA2α and its product AA as the ‘final’ necessary physiological activators of proton channels seemed to be clearly established.

Despite the unanimity in the literature on the requirement for cPLA2α to activate the proton conductance, the revision of the NADPH oxidase story (Rubin et al. 2005) stimulated us to re-examine the idea that proton channels are obligatorily activated during the respiratory burst by AA generated by cPLA2α. The previously discussed electrophysiological studies of AA effects in phagocytes were all done using the whole-cell configuration, in which the pipette solution replaces the cytoplasm and abolishes many signalling pathways, including the activation of NADPH oxidase and H+ channels by PMA (DeCoursey et al. 2000b). In contrast, in the perforated-patch configuration, which preserves the cytoplasm, AA activates electron currents that reflect NADPH oxidase activity, and also dramatically alters the properties of proton channels to promote their opening, which we consider a manifestation of ‘activation’ of these channels (Cherny et al. 2001). The effects of AA on H+ currents in perforated-patch configuration were more profound than in whole-cell studies, and, except for tail current kinetics, closely resembled the constellation of effects seen with PMA stimulation (DeCoursey et al. 2000b, 2001a) or in cells that are spontaneously activated (perhaps by adherence) (DeCoursey et al. 2001a). Here we explore the potent and selective cPLA2α inhibitors pyrrolidine-2, also known as pyrrophenone (Seno et al. 2001; Ono et al. 2002; Ni et al. 2006; Vandal et al. 2006), Wyeth-1 (Ni et al. 2006), and AACOCF3 (Street et al. 1993; Bartoli et al. 1994; Riendeau et al. 1994), in human eosinophils studied in the perforated-patch configuration. Surprisingly, we found no evidence that cPLA2α is necessary for activation of proton channels. This conclusion was tested independently by examining the activation of granulocytes from cPLA2α knockout mice. The phenomenology of proton channel activation in these mice was indistinguishable from that in control mice. We conclude that AA generated by cPLA2α is not a necessary component of the signalling pathway that leads to activation of voltage-gated proton channels. In contrast, we show that PKC inhibition prevents the proton channel response and can also reverse the response. Thus, the activation of proton channels is stimulated and sustained by PKC via a pathway that does not require cPLA2α.

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Methods

Isolation of human neutrophils and eosinophils

Venous blood was drawn from healthy adult donors under informed written consent in accordance with the procedures outlined by the Rush University Institutional Review Board, Federal regulations, and the Declaration of Helsinki. Neutrophils were isolated by density gradient centrifugation (Boyum, 1968). Blood was mixed into a sterile 0.9% sodium chloride solution with 3 mm EDTA and layered onto Lymphocyte Separation Medium (LSM, Cambrex Bioscience, Walkersville, MD, USA). The blood was then separated by centrifugation at 400 g at room temperature (21–24°C) for 30 min. Mononuclear cells and plasma were aspirated off and the pellet resuspended in ice-cold distilled water for 10–20 s to lyse the red blood cells. The water–cell mixture was diluted with ice-cold 2 × Hanks' balanced salt solution (HBSS) with 5 mm Hepes (adjusted to pH 7.4) and centrifuged at 350 g for 10 min at 4°C, and then this process was repeated. The pellet was then resuspended in 1 × HBSS with 2.5 mm Hepes.

Eosinophils were isolated from the neutrophil preparation by negative selection. After counting, 1 × 107 cells were seeded in 10 μl PBS with 2 mm EDTA and 0.5% bovine serum albumin (BSA, Sigma Chemical Co., St Louis, MO, USA) and mixed with an equal volume (10 μl) of MACS CD16 MicroBeads (Miltenyi Biotec, Auburn, CA, USA). The resulting mixture was incubated at 4°C for 45 min, mixing by pipette every 10 min. The beads–cells mixture was diluted with 0.5 ml PBS–EDTA–BSA (with EDTA and BSA as described above), and then added to a magnetized column. The column was washed twice with PBS–EDTA–BSA. Cells were kept on ice in this solution or resuspended in RPMI medium with granulocyte–macrophage colony-stimulating factor (GM-CSF) (at a working concentration of 1 ng ml−1) and stored in an incubator. The eosinophils or neutrophils were suspended in PBS with 2 mm EDTA and 0.5% BSA. Neutrophil purity was routinely 95% or greater. Eosinophil purity was typically > 98% as determined by counting Wright-stained cytospin preparations.

Murine granulocytes

C57BL/6 mice with the cPLA2α gene disrupted (cPLA2α knockout mice) were obtained from Professor T. Shimizu (University of Tokyo, Japan). The cPLA2α transgene is in a C57BL/6 background and is a targeted vector using a PGK-neo expression cassette to disrupt the endogenous cPLA2α gene. The genotype of the mice studied was verified by using PCR to amplify the gene, as follows. One forward primer, cPLAF (5′-TTC TCT GGT GTG ATG AAG GC-3′), was designed against an exonic sequence and a reverse primer, cPLAR (5′-AAA CTG ACT GTA GCA TCA CAC-3′), was designed to recognize a downstream intron. A third primer was useful in keeping the diagnostic product bands within a range compatible with the selected polymerase. The third primer, NeoF2 (5′-ATC GCC TTC TTG ACG AGT TC-3′), was designed to recognize a sequence in the 3-prime side of the poly-A region of the PGK-neo cassette. In a multiplex format (all three primers in one PCR reaction), the expected PCR products are 570 bp for the knockout allele and 224 bp for the wild-type allele. PCR conditions described are for the Qiagen Hot Start TAQ Polymerase Kit (Qiagen, Valencia, CA, USA) with 2.5 mm MgCl2 final concentration. After an initial denaturation step at 94°C for 15 min, 35 cycles of PCR were performed (94°C for 45 s, 55°C for 1 min, and 72°C for 1 min), with a final elongation cycle of 72°C for 10 min. PCR products were resolved on a 2% agarose gel with ethidium bromide. Further details are described elsewhere (Bonventre et al. 1997).

We used three strains of control mice: BALB-c, SCID mice (in BALB-c background) obtained from the National Canter Institute, and C57BL/6 mice that were littermates of the KO mice. The bulk of the control data were collected on C57BL/6 mice. All animal protocols were approved by the Rush University Medical Center Institutional Animal Care and Use Committee, and are in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Surgical procedures were performed in mice anaesthetized by ketamine (100 mg kg−1) and xylazine (5 mg kg−1) administered intraperitoneally. Granulocytes were isolated from blood obtained either by cardiac puncture or from the supraorbital plexus of anaesthetized mice. The mice were killed while under anaesthesia, by cervical dislocation. Granulocytes were isolated using Lympholyte-M or Lympholyte-Mammal (Cedarlane Laboratories Ltd, Burlington, NC, USA). A small volume of blood (< 0.5 ml) was diluted into 3 ml EDTA–saline solution (PBS with 2 mm EDTA and 0.5% BSA), layered onto 3 ml Lympholyte in a 15 ml tube and centrifuged at 800 g for 20 min at room temperature. The layer above the Lympholyte (containing lymphocytes and monocytes) was discarded and the pellet (containing granulocytes and red cells) was resuspended in ice-cold sterile water for 10 s (to lyse the red cells), then diluted with an equal volume of 2 × HBSS (with 5 mm Hepes at pH 7.4). After mixing, the result was centrifuged at 4°C for 10 min at 400 g. This step was repeated and then the pellet was resuspended in PBS with 2 mm EDTA and 0.5% BSA. Cells were kept on ice until use. We selected non-adherent cells for recording.

H2O2 measurement

H2O2 release (next paragraph) was measured using an Amplex red kit purchased from Molecular Probes (Eugene, OR, USA) according to the instructions. Fluorometric measurements were done in a volume of 120 μl, with typically 2 × 104 cells per well. Absolute concentrations of H2O2 were determined from a calibration curve. AACOCF3 produced a baseline signal that was subtracted from data at subsequent time points.

cPLA2α inhibitors and other materials

Pyrrolidine-2 and Wyeth-1 were prepared as described (Ni et al. 2006), and used at concentrations shown previously to abolish cPLA2α activity (Seno et al. 2001; Ono et al. 2002; Ni et al. 2006; Vandal et al. 2006). Aliquots of AACOCF3 (Calbiochem, San Diego, CA, USA) were kept at −20°C in argon-filled vials. The AA analogue, AACOCF3, reportedly prevents activation of proton flux by PMA or fMetLeuPhe (Suszták et al. 1997). The IC50 of AACOCF3 for inhibiting AA release is 2–10 μm in platelets (Bartoli et al. 1994) and U937 cells (Riendeau et al. 1994). The IC50 of AACOCF3 reported to prevent proton channel activation is also 3–5 μm (Suszták et al. 1997). In preliminary experiments, we examined its effects on NADPH oxidase activity, assessed as H2O2 production. AACOCF3 alone (10–100 μm) did not elicit significant H2O2 production. AACOCF3 inhibited PMA-stimulated H2O2 release significantly only at high concentration (100 μm, n = 6, ANOVA, P < 0.01). Thus NADPH oxidase activity is not inhibited at concentrations that inhibit cPLA2α activity as assessed directly by AA release (Street et al. 1993; Bartoli et al. 1994; Riendeau et al. 1994) or proton channel activation assessed by pH changes (Suszták et al. 1997). Concentrations of AACOCF3 used here (up to 20 μm) were sufficient to inhibit cPLA2α activity (Street et al. 1993; Bartoli et al. 1994; Riendeau et al. 1994), but lower than those that inhibit H2O2 release.

Stock solutions (5 mm) of arachidonic acid (AA) and oleic acid (both from Sigma) were prepared in 50% ethanol (‘vodka’) and aliquots were stored in argon or nitrogen atmosphere at −20°C. PMA, DPI (diphenylene iodonium) and staurosporine were obtained from Sigma, GFX (GF109203x) was from Calbiochem, and okadaic acid was from LC Laboratories (Woburn, MA, USA)

Electrophysiology

The data collection setups and analysis software have been previously described (Morgan et al. 2003). Pipettes were made from 8250 glass (Garner Glass Co., Claremont, CA, USA). Seals were formed with Ringer solution (mm: 160 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5 Hepes, pH 7.4) in the bath, and the potential zeroed after the pipette was in contact with the cell. The pipette solution for perforated patch recording contained 128 mm KCH3SO3 or 130 mm tetramethylammonium methanesulphonate (TMAMeSO3), 50 mm NH4+ in the form of 25 mm (NH4)2SO4, 2 mm MgCl2, 10 mm BES buffer and 1 mm EGTA, and was titrated to pH 7.0 with KOH or tetramethylammonium hydroxide (TMAOH). Then ∼500 μg ml−1 solubilized amphotericin B (∼45% purity) (Sigma) was added. The pipette was backfilled after first dipping the pipette tip in amphotericin-free solution. The bath solution contained (mm): 130 TMAMeSO3, 25 (NH4)2SO4, 2 MgCl2, 1.5 CaCl2, 1 EGTA, and 10 BES at pH 7.0. All solutions were roughly 300 mosmol l−1. Studies were done at room temperature (20–25°C).

Most data were acquired with a sampling rate of 50–200 Hz and lowpass filtering at 10–100 Hz. For the purpose of displaying long records (e.g. Fig. 7) the data were digitally re-filtered at 2 Hz in order to show the amplitude of the H+ current, without its being obscured by capacity transients. This filtering attenuated the capacity current spikes with little effect on the proton current.

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Results

cPLA2α inhibition does not prevent the response of NADPH oxidase or proton channels to PMA in human eosinophils

The activation of NADPH oxidase can be detected in individual granulocytes studied in the perforated-patch configuration as an inward electron current (Ie) (DeCoursey et al. 2000b). Figure 1A illustrates the response of a human eosinophil stimulated with 60 nm PMA. Robust Ie was also activated in the presence of two specific cPLA2α inhibitors, 10 μm Wyeth-1 (Fig. 1B) and 5 μm pyrrolidine-2 (Fig. 1C). The mean Ie was the same in cells stimulated by PMA in the absence or presence of cPLA2α inhibitors (Table 1). Although the kinetics of Ie turn-on differs in these cells, this variability occurs normally, and the time course did not noticeably depend on the cPLA2α inhibitor used. That the inward current activated by PMA is Ie generated by NADPH oxidase was confirmed by its sensitivity to inhibition by 10 μm diphenylene iodonium (DPI) (e.g. Fig. 1B and C). Of 14 cells pretreated with cPLA2α inhibitors, inward current that was clearly inhibited by DPI was seen in 12, with two being equivocal. Evidently, NADPH oxidase in human eosinophils can be activated by PMA without the involvement of cPLA2α. This result extends similar conclusions reached in human neutrophils stimulated by PMA or opsonized zymosan (Rubin et al. 2005).

In contrast, pretreatment with 1 μm GFX (GF109203X), a PKC inhibitor, prevented any Ie response to PMA (Fig. 1D) in all 13 cells tested (P < 0.001 by a χ2 test versus PMA stimulation after pretreatment with cPLA2α inhibitors). Eosinophils studied in the perforated patch configuration occasionally convert to the whole-cell configuration via spontaneous patch rupture (Morgan et al. 2003). No response to PMA occurs in the whole-cell configuration (DeCoursey et al. 2000b); thus we confirmed the configuration by including Lucifer yellow in the pipette solution in some experiments. Lucifer yellow rapidly enters the cell upon patch rupture (Morgan et al. 2003). Absence of Lucifer yellow fluorescence in the cell confirmed that the patch was intact in three cells pretreated with 1 μm GFX that failed to respond to PMA. Pretreatment with staurosporine, another PKC inhibitor, also prevented activation by PMA in three cells. In two of these cells, after washout of staurosporine, PMA elicited a normal response (both Ie and enhanced proton current, IH) that could then be inhibited by staurosporine (vide infra). The reversibility of staurosporine allows confirmation that the configuration is perforated patch and that the cell is responsive.

In addition to reliably activating NADPH oxidase, PMA profoundly alters the gating kinetics of voltage-gated proton channels, greatly enhancing their probability of opening (DeCoursey et al. 2000b, 2001a). Proton currents measured at various times (indicated by lower case letters) during the experiments in Fig. 1 are superimposed to the right of the Ie records. Characteristically drastic changes in proton channel gating (increased gH, faster activation, slower deactivation) are evident in the response to PMA of the untreated human eosinophil (Fig. 1A) and the cells pretreated with Wyeth-1 (Fig. 1B) or pyrrolidine-2 (Fig. 1C). Evidently, the response of voltage-gated proton channels to PMA in human eosinophils does not require functional cPLA2α. Pretreatment with 1 μm GFX prevented the proton channel response to PMA (Fig. 1D), consistent with a previous report (Bankers-Fulbright et al. 2001). We found previously that a lower concentration of GFX (200 nm) reduced, but did not abolish, the PMA response in human neutrophils (DeCoursey et al. 2000a). GFX, staurosporine, and cPLA2α inhibitors had no detectable effect on proton currents in unstimulated, resting cells.

The several responses of proton channels to stimulation occur at different rates (DeCoursey et al. 2000b, 2001b) and exhibit differential sensitivity to DPI (DeCoursey et al. 2000b, 2001a), which might indicate that the responses are not monolithic. To evaluate the changes in proton channel gating in more detail, we compared families of H+ currents recorded during an identical series of depolarizing pulses in each cell before and after PMA stimulation (Fig. 2A). Analogous measurements in an eosinophil pretreated with pyrrolidine-2 (Fig. 2B) produced responses similar to those in the control cell in Fig. 2A. Figure 3A summarizes these results together with data from other experiments in which cells were pretreated with AACOCF3, Wyeth-1, or GFX. Average gH–V (gHversus voltage) relationships before and after PMA stimulation are plotted in Fig. 3A (▪ and ▴, respectively), revealing a roughly 40 mV hyperpolarizing shift and an increase in the maximum gH (gH,max) that are characteristic of the PMA response (DeCoursey et al. 2000b, 2001a). The average gH–V relationships of PMA-stimulated cells were identical whether they were pretreated with cPLA2α inhibitors or not. In contrast, cells pretreated with the PKC inhibitor GFX (○) did not respond to PMA.

In Fig. 3B, the faster activation of PMA-stimulated proton currents is compared in the presence or absence of the signalling inhibitors. Currents were fitted by a single rising exponential to obtain the activation time constant (τact). Confirming the impression of the IH responses in Figs 1 and 2, Fig. 3B shows that PMA decreased τact to a similar extent at all voltages in the presence or absence of the cPLA2α inhibitors pyrrolidine-2 or Wyeth-1. Furthermore, cPLA2α inhibitors did not prevent the slowing of tail current deactivation (τtail; Table 1). The responses of human eosinophils to PMA that are summarized in Table 1 indicate no effect of pretreatment with cPLA2α inhibitors. Responses of all cells are similar to those previously reported in human neutrophils and eosinophils (DeCoursey et al. 2000b, 2001a).

GFX reverses the activation of NADPH oxidase and proton channels

Pretreatment with GFX prevented activation of NADPH oxidase and proton currents, as shown in Fig. 1D. GFX presumably prevents the phosphorylation of the cytosolic oxidase components, especially p47phox, and thereby prevents assembly of the NADPH oxidase complex (Babior, 1999). In the absence of a known mechanism, it might be speculated that GFX prevents the phosphorylation of the proton channel itself or an activating molecule. Given these mechanisms, it was not clear whether the activation proton currents would be reversible. However, as shown in Fig. 4, addition of 3 μm GFX at the peak of the PMA response reversed the activation of both molecules. The time course of these GFX actions exhibited marked variability and differed even in the same cell. In the example in Fig. 4, there was a pronounced delay before Ie began to decline. In other cells, this delay ranged from 0 to 3 min and appeared to be independent of GFX concentration (100 nm to 3 μm). In contrast, the proton current, IH, usually responded almost immediately. For example, in Fig. 4, Ie had hardly changed by time point ‘c’, but already IH was distinctly attenuated. Once Ie began to decay, however, it decreased marginally faster (τ = 99 ± 49 s, mean ± s.d., n = 8) than did IH (τ = 140 ± 59 s, n = 9) (P = 0.07, but P = 0.01 for a paired t test with n = 7). In a minority of cells, GFX even at high concentration did not produce effects within a few minutes. Although we detected no concentration dependence of the rate of action of GFX on either molecule, low concentrations of GFX (50–200 nm) inhibited Ie only partially, whereas 2–3 μm GFX effectively abolished Ie in most cells. Proton currents appeared never to be completely restored to their preactivated state. However, the extent of deactivation is difficult to quantify, because there is a tendency for H+ currents to increase gradually during experiments. In four cells activated by PMA, addition of 100 nm staurosporine attenuated IH and Ie, closely resembling the effects of GFX. Overall, the effects of GFX are consistent with independent regulation of proton channels and NADPH oxidase. Bankers-Fulbright et al. (2001) reached a generally similar conclusion, because rottlerin inhibited NADPH oxidase activity but not the activation of the proton conductance. Taken together, the kinetics of these responses suggests that the signalling pathways immediately proximal to the activation of the two molecules may be distinct, and the regulatory mechanisms that govern the level of activation also may be distinct.

Because PMA activates PKC, it is of interest whether PKC inhibitors also inhibit the responses when the respiratory burst is triggered by other kinds of stimuli. Effects of PKC inhibition on activation by AA or oleic acid are described below. Some eosinophils become activated spontaneously, possibly in response to contact with the glass microelectrode. Figure 5 illustrates currents recorded in a cell that is representative of four spontaneously activated cells tested. Addition of 100 nm staurosporine reversed the activation of both Ie and IH. The proton current began to decrease immediately, but Ie decreased after a delay, a time course reminiscent of that for addition of GFX to PMA-stimulated cells.

Okadaic acid preserves the activated state of proton channels during GFX exposure

The failure of cPLA2α inhibitors to prevent activation of proton channels rules out Scheme 1. However, proton channels might also be activated by an alternative signalling pathway, as follows: FormulaHere PMA can activate proton channels either via cPLA2α and AA or by phosphorylation of the channel or an intermediary.

If activation of the proton channel by PMA is due to phosphorylation, the return toward the resting state that is induced by GFX may reflect ongoing phosphatase activity. Figure 6 illustrates the average time course of deactivation of proton currents in eosinophils that were activated with PMA, then treated with GFX in the presence (□) or absence (▴) of okadaic acid, a phosphatase inhibitor (Bialojan & Takai, 1988). After a transient increase due to temperature, IH decreased by > 60% in the presence of GFX alone. Okadaic acid greatly reduced the extent of deactivation. To the extent that okadaic acid acted by inhibiting phosphatases, this result supports the idea that the proton channel is activated by phosphorylation of either the channel itself or a regulatory intermediary. Usually okadaic acid was added simultaneously with PMA. Pretreatment of two cells with okadaic acid for 5 min did not noticeably activate either IH or Ie, although both were activated by subsequent addition of PMA.

Activation of proton channels by AA is also sensitive to PKC inhibitors

The observation that AA has substantially greater effects on proton currents in perforated-patch than in the whole-cell configuration (Cherny et al. 2001) suggests that diffusible second messengers contribute to its effects in perforated-patch studies. The possibility that AA might activate PKC was explored using GFX or staurosporine. Figure 7A illustrates the response to AA of an eosinophil in perforated patch configuration. After addition of 5 μm AA, IH increased progressively for several minutes. The inward current at the holding potential, − 60 mV, also increased progressively. We interpret the increase in inward current as electron current, Ie. Addition of 100 nm staurosporine in the continued presence of the same AA concentration (followed by stirring) inhibited Ie and progressively reduced IH, although not to the level before stimulation with AA. GFX (1.5–3.0 μm, n = 4) and staurosporine (100 nm, n = 3) had similar effects, reducing proton currents and Ie after activation with AA. Evidently, part of the effect of AA on proton currents is mediated by PKC, and is reversed by PKC inhibition. In addition, PKC inhibitors inhibited Ie despite the continued presence of AA in the bath. Evidently, both NADPH oxidase and the enhanced gating mode of proton channels are sustained by continuous phosphorylation, the prevention of which results in dephosphorylation and deactivation of both molecules.

Families of H+ currents during the experiment in Fig. 7A are illustrated in Fig. 7B–D. AA greatly increased IH, accelerated activation of the H+ current during voltage pulses, and shifted the threshold for activating the conductance (Vthreshold) toward more negative values. In addition, the inward current at −60 mV increased, reflecting Ie generated by NADPH oxidase activity. Each of these effects was reversed partially by 100 nm staurosporine, added to the bath in the continued presence of AA (Fig. 7D). The AA induced inward current cannot be ascribed to leak current because it was abolished by staurosporine in the continued presence of AA. Later, AA was washed out of the bath, and IH decreased gradually (not shown). The effects of AA that are sensitive to PKC inhibitors presumably reflect the consequences of phosphorylation, whereas the residual effects of AA in the presence of PKC inhibitors reflect direct effects on proton channels (see Discussion).

After a response to AA and subsequent partial reversal by staurosporine (e.g. Fig. 7A), the bath could be washed and the entire process repeated, with similar results. Cells that had responded to AA (n = 3) or PMA (n = 2) and then were inhibited by staurosporine, responded to subsequent exposure to AA with increased IH and activated Ie, and both responses were again partially reversed by addition of staurosporine to the bath (in the continued presence of AA).

Other unsaturated fatty acids, such as oleic acid, also activate NADPH oxidase (Badwey et al. 1984) and enhance proton currents (Kapus et al. 1994). In 12 eosinophils in the perforated patch configuration, 2.5–10 μm oleic acid elicited presumed electron current and profound, progressive increases in proton current reminiscent of AA effects (not shown). However, these responses did not stabilize, and without further treatment, all cells rapidly became leaky or spontaneously went to the whole-cell configuration, precluding further tests. In two cells, however, we added 100–300 nm staurosporine shortly after the progressive increase in IH began. After a delay during which both IH and Ie continued to increase progressively, both IH and Ie stabilized and then began to decrease. Despite the harshness of oleic acid, some of its effects appear to be reversed by PKC inhibition. A minimum estimate of the magnitude of IH enhancement by fatty acids was obtained by taking the ratio of gH during the test pulses (IH is defined here as the time-dependent increase in current during the pulse) in the presence of fatty acid to gH,max determined from the gH–V relationship and Vrev measured before addition. The cells did not survive oleic acid long enough to obtain a full gH–V relationship in its presence. By this somewhat crude measure, the mean increase in gH,max was 5.0 ± 1.5-fold by AA (mean ± s.d., n = 10) and 5.0 ± 2.5-fold by oleic acid (n = 12).

The responses of NADPH oxidase and proton channels are identical in normal and cPLA2α knockout mice

The existence of mice with the cPLA2α gene disrupted (cPLA2α ‘knockout’ mice) enables a direct test of the proposed requirement for cPLA2α in activating proton current during the respiratory burst. It was shown previously that NADPH oxidase activity (assessed as cytochrome c reduction) stimulated by PMA or zymosan was similar in normal and cPLA2α knockout mouse macrophages (Gijón et al. 2000) and neutrophils (Rubin et al. 2005). Figure 8 illustrates experiments in which we examined this question in murine peripheral granulocytes, assessing NADPH oxidase activity directly in individual cells as Ie. We could detect no difference between the responses of control and knockout murine cells, whether they were stimulated with fMetLeuPhe (vide infra) or PMA. In both the normal mouse cell (Fig. 8A) and the cPLA2α KO cell (Fig. 8B), addition of 10 μm fMetLeuPhe had no effect, but subsequent introduction of PMA produced a vigorous Ie that was inhibited by DPI. Proton currents in both normal and cPLA2α KO cells were enhanced profoundly by PMA (current records on the right in Fig. 8).

No previous studies of proton currents in murine granulocytes exist, although proton currents in murine macrophages have been studied extensively (Kapus et al. 1993a, 1994; Grinstein et al. 1994; Suszták et al. 1997). That the time- and voltage-dependent outward currents in murine granulocytes were proton selective was confirmed by appropriate shifts of Vrev and Vthreshold (the threshold voltage at which distinct IH is first elicited) when pHo was changed directly or when pHi was changed indirectly by varying the NH4+ concentration (data not shown). Proton currents in murine granulocytes were generally similar to those in human granulocytes in terms of gating kinetics, amplitude, and the voltage dependence and pH dependence of gating. More important for the present purposes, proton channels in murine cells responded to PMA (or to fMetLeuPhe) in a manner reminiscent of, but not identical to, the responses of human cells. Figure 9A and B, respectively, illustrate families of proton currents in a murine granulocyte before and after stimulation with PMA. All of the changes produced by PMA in human cells also occur in murine cells, but in most cases, the magnitude of the effect is smaller. It is evident in Fig. 9 that after PMA stimulation, the H+ current is larger, activates at more negative voltages, turns on faster, and less obviously, the tail current decays more slowly. Table 2 summarizes some of the effects of PMA on proton currents in murine granulocytes. The amplitude of IH at the end of the test pulse increased about 4-fold. Activation of proton current (τact) was faster after PMA stimulation, but only by about 2-fold. Channel closing, measured as the time constant of tail current decay (τtail), was 2- to 3-fold slower. All of these parameters were changed significantly by PMA. There was no significant difference between control and cPLA2α KO cells for any parameter, either before or after stimulation.

Figure 10 shows that PMA shifted the gH–V relationship negatively, but only by ∼20 mV, as opposed to the 40 mV shift in human cells (Table 1). All of these effects of PMA on proton channel gating in cPLA2α KO murine cells were indistinguishable from those in control cells.

Stimulation of murine granulocytes with fMetLeuPhe

Although cPLA2α is evidently not required for the response of murine granulocytes to PMA, it was possible that cPLA2α might be required in a more physiological signalling pathway such as that triggered by fMetLeuPhe. Therefore, we examined the effects of 10 μm fMetLeuPhe in normal and cPLA2α KO mice. The effect of fMetLeuPhe on murine proton currents was similar to that of PMA, although the extent of the response was more variable. Intriguingly, proton currents responded to fMetLeuPhe in all cells in which Ie was elicited, including one cPLA2α KO cell in which Ie was very small. In the cells illustrated in Fig. 11B and E, the IH in the presence of 10 μm fMetLeuPhe was approximately doubled and Vthreshold shifted about 20 mV more negative. In both cells, the inward current elicited by fMetLeuPhe was confirmed to be Ie because it was inhibited by 20 μm DPI (not shown). Subsequent addition of 100 nm PMA produced a response that was greater than that elicited by fMetLeuPhe (Fig. 11C and F).

The Ie response of murine granulocytes to fMetLeuPhe was highly variable. About half of the cells did not respond detectably (Fig. 8A and B), others exhibited a weak response (compared with the response to PMA added subsequently to the same cell), and in some cells there was a dramatic response comparable with the PMA response (e.g. Fig. 11E and F). This variability is consistent with a previous study in which only about half of murine neutrophils responded to fMetLeuPhe in an nitro blue tetrazolium (NBT) test that reflects O2.− production by individual cells (Kim & Dinauer, 2001). We excluded cells that did not respond to fMetLeuPhe or subsequently to PMA, because they might have spontaneously gone to the whole-cell configuration, which precludes any response (Morgan et al. 2003). The peak Ie elicited by fMetLeuPhe was −1.5, −7.2 and −8.0 pA in 3/5 control cells that responded, and −4.0, −1.5, −0.9 and possibly −0.2 pA in 4/7 cPLA2α KO mouse cells. The small numbers of responding cells preclude quantitative comparison, but it is clear that some granulocytes from cPLA2α KO mice respond to fMetLeuPhe with both Ie and enhanced proton currents.

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Discussion

cPLA2α and increased AA levels are not required for activation of proton channels

The cPLA2α isoform has been proposed to be required for agonist-induced AA release, NADPH oxidase activity, and proton fluxes associated with the respiratory burst (Dana et al. 1998; Lowenthal & Levy, 1999). This study demonstrates that the transformation of voltage-gated proton channels into their ‘activated’ state or ‘enhanced gating mode’ occurs completely normally in the absence of cPLA2α activity. Furthermore, we show that cPLA2α activity is not required for a normal respiratory burst stimulated by PMA in human eosinophils or stimulated by PMA or fMetLeuPhe in murine granulocytes, extending similar findings in other cells (Gijón et al. 2000; Rubin et al. 2005). The cPLA2α inhibitors used here, especially pyrrolidine-2 and Wyeth-1, appear to be more selective for this isoform than are previously used inhibitors (Street et al. 1993; Ni et al. 2006). For the purpose of this study, however, perfect selectivity is not required – what is required is that the inhibitors prevent cPLA2α activity at the concentrations used. The results do not categorically rule out any involvement of cPLA2α in responses to agonists that were not studied. However, the studies that provided the evidence for the cPLA2α hypothesis tested here all used PMA as a stimulus (Henderson et al. 1989; Chappell & Henderson, 1991; Kapus et al. 1993b; Suszták et al. 1997; Lowenthal & Levy, 1999; Levy et al. 2000; Mankelow et al. 2003).

The results in cPLA2α KO mice do not in themselves rule out a role for AA in the activation of either protein. Because AA release (spontaneous or stimulated by PMA and A23187) is reduced but not abolished in cPLA2α KO macrophages (Bonventre et al. 1997; Gijón et al. 2000), it could be speculated that other phospholipases might be up-regulated in the KO mice. In PLB-985 cells, reduction of cPLA2α activity by antisense RNA abolished both PMA-stimulated AA production and O2.− release (Dana et al. 1998), suggesting that no compensatory up-regulation of other isoforms occurred in this system. Furthermore, acute application of specific cPLA2α inhibitors abolished agonist-stimulated AA release from human phagocytes (Rubin et al. 2005). Consequently, the lack of effect of specific cPLA2α inhibitors indicates that proton channel activation does not require newly generated AA. It remains conceivable that AA already present before stimulation may play some role or interact in some way with the proton channel, perhaps analogous to the role of AA on NADPH oxidase activity in a cell-free system. However, it seems clear that a different mechanism, most likely phosphorylation, mediates the activation of the proton channel per se.

Phosphorylation of proton channels and NADPH oxidase

The activation of proton efflux by PMA (Henderson et al. 1987; Nanda & Grinstein, 1991; Kapus et al. 1992) immediately suggested the possibility that proton channels might be activated directly by PKC (rather than indirectly via cPLA2α and AA). Because most early studies inferred proton channel activity from pH changes, their interpretation is complicated because PMA also activates proton extrusion by Na+/H+-antiport and a V-type H+-ATPase (Nanda et al. 1992), as well as NADPH oxidase, which creates a large driving force for proton extrusion. The idea that phosphorylation is a key process is supported by the prevention of activation of either NADPH oxidase or proton channels by the PKC inhibitors, GFX (Fig. 1D; Bankers-Fulbright et al. 2001) and staurosporine. GFX exhibits high selectivity for PKC compare with other protein kinases (Toullec et al. 1991; Davies et al. 2000), and inhibits most conventional and novel PKC isoforms (Martiny-Baron et al. 1993). In the case of NADPH oxidase, phosphorylation of its components precedes assembly of the oxidase complex (Babior, 1999). The ‘activation’ of proton channels may also be less straightforward than direct phosphorylation of the channel molecule by PKC. Our results do not distinguish whether the proton channel itself or an intermediate channel-activating molecule is phosphorylated.

Might the PKC inhibitors act by inhibiting cPLA2α? In many cells, PKC is upstream of cPLA2 and PKC activation enhances cPLA2 activity. However, in several systems, GFX inhibited only the PKC-dependent cPLA2 activity and did not inhibit basal activity (Qiu & Leslie, 1994; Xia et al. 1995; Han et al. 2004). Similarly, staurosporine does not inhibit AA release from macrophages (Beppu et al. 2002). Because selective inhibitors of cPLA2α do not inhibit activation of IH or Ie, there is no reason to expect that PKC inhibitors might act in this way. The effects of the PKC inhibitors clearly are not mediated by incidental effects on cPLA2α.

The active states of both proton channels and NADPH oxidase, whether resulting from PMA or AA stimulation or spontaneous activation, were reversed by GFX or staurosporine. This reversibility links phosphorylation by PKC not only to the initial activation, but also to the sustained activity of both proteins. Several types of studies support the idea that NADPH oxidase activity is regulated by a balance between phosphorylation and dephosphorylation (Heyworth & Badwey, 1990), although a number of other explanations for deactivation exist (DeCoursey & Ligeti, 2005). Electron currents generated by NADPH oxidase reportedly run down more slowly with time in excised membrane patches upon addition of cytosolic ATP and GTPγs (Petheö et al. 2003). Although ATP has effects on proton currents that are unrelated to phosphorylation (pp. 533–534 in DeCoursey, 2003, and DeCoursey, 2006), the possibility remains that the channel may be phosphorylated in intact cells. The time constants of inhibition of Ie and IH were not obviously dependent on GFX concentration, suggesting that when phosphorylation is interrupted, deactivation proceeds by another mechanism that becomes rate determining.

The hypothesis that the activated gating mode of proton channels reflects phosphorylation of the channel or a closely related regulatory element was supported by the observation (Fig. 6) that okadaic acid largely prevented deactivation by GFX. Because GFX simply prevents further phosphorylation, but would not be expected to affect already phosphorylated channels, the diminution of H+ currents toward their resting condition must reflect another process, such as the activity of phosphatases. This interpretation is supported by the independence of the rate of the GFX effect on concentration. Thus, okadaic acid must interfere with the process that causes deactivation, presumably phosphatase activity. Neither staurosporine nor GFX had detectable effects on IH in unstimulated eosinophils, suggesting that the resting level of phosphorylation of proton channels must be minimal. Similar to the lack of activation of NADPH oxidase by okadaic acid alone (Ding & Badwey, 1992; Lu et al. 1992; DeCoursey & Ligeti, 2005; present results), okadaic acid alone did not activate IH significantly. Thus, neither NADPH oxidase nor proton channels are kept in their resting state by ongoing serine–threonine phosphatase activity.

Role of AA in proton channel activation

Consistent with an earlier study (Cherny et al. 2001), we found that AA profoundly activated both IH and Ie in human eosinophils. Oleic acid appeared to be equally effective. Unlike AA, oleic acid is not metabolized by the lipoxygenase or cyclooxygenase pathways. Hence, the efficacy of oleic acid in activating IH (and Ie) indicates that AA itself is active, not a metabolite. The proton channel response to AA was graded, in contrast with the PMA response that appears to be all-or-nothing. Nevertheless, it is possible to distinguish two types of AA responses, direct and indirect. The direct response is seen in whole cell configuration and consists of a modest hyperpolarizing shift in the gH–V relationship (≤ 20 mV), faster H+ current activation (≤ 2-fold smaller τact), faster deactivation (2-fold smaller τtail), and a (≤ 2-fold) larger gH,max (DeCoursey & Cherny, 1993; Kapus et al. 1994; Gordienko et al. 1996). The AA response in perforated patch studies can be more profound and in some respects qualitatively different, strongly implicating a diffusible second messenger in the response. The perforated patch configuration preserves signalling pathways by retaining intracellular constituents that rapidly diffuse into the pipette in whole-cell configuration. The additional effects of AA that occur in perforated-patch studies thus reflect an indirect response. Even in perforated patch studies, low [AA] (1 μm) increased IH without shifting the gH–V relationship and hastened both τact and τtail, without activating detectable Ie (Cherny et al. 2001), which can be taken as the ‘direct’ response. However, higher [AA] (3–10 μm) induced a profound negative shift of the gH–V relationship (resulting in distinct inward H+ current negative to EH), 4-fold faster τact, 4.6-fold larger IH (measured during pulses to 40 or 60 mV), and also activated Ie (Cherny et al. 2001), all of which indicate an indirect response, analogous to that seen with PMA stimulation. The main difference between the responses to AA and PMA is that τtail was slowed at least 3- to 5-fold by PMA (DeCoursey et al. 2000b, 2001a; Table 1), but was not consistently changed by AA (Cherny et al. 2001). This difference can be explained by a combination of the direct effect of speeding τtail and the indirect of slowing τtail, as follows: FormulaHere we assume that phosphorylation of the proton channel or an intermediary after PMA or AA stimulation results in slowing of τtail. Because AA acts via both pathways, it simultaneously speeds and slows τtail, and the net result is little or no change. The possibility that the indirect effects of AA are mediated by PKC was suggested by the partial reversal of proton channel activation by GFX or staurosporine (Fig. 7).

Oleic acid induced profound activation of both IH and Ie in human eosinophils studied in the perforated patch configuration. Although oleic acid increased IH in a whole-cell study (Kapus et al. 1994), this effect was much weaker than that for AA in the same study (a 1.3-fold increase in IH by oleic acid, versus a 3.7-fold increase by AA). In contrast, in the present measurements, oleic acid appeared to activate IH as profoundly as did AA. Taken together, these observations suggest that AA has greater direct effects on proton channels, but oleic acid and AA are comparably effective in activating IH by the indirect pathway (presumably via PKC activation).

One of the earliest proposed mechanisms of activation of NADPH oxidase by AA was through PKC activation (McPhail et al. 1984). Unsaturated fatty acids like AA and oleic acid activate PKC directly (McPhail et al. 1984; Murakami & Routtenberg, 1985). Subsequent studies questioned whether this mechanism occurred at physiologically relevant levels of AA (Ely et al. 1995), which is difficult to evaluate. The PKC inhibitor H-7 partially inhibited O2.− production stimulated by PMA but not by 25 μm AA in intact neutrophils (Maridonneau-Parini & Tauber, 1986). A recent study demonstrates translocation (but not activation) of PKC by 10–100 nm AA, with activation occurring at higher concentrations (O'Flaherty et al. 2001). Effects of AA on translocation of PKC were partially inhibited by GFX (Hii et al. 1998). We conclude that at least part of the effect of AA on proton currents in intact phagocytes is due to activation of PKC. This conclusion for proton channels may parallel the phenomenon that NADPH oxidase can be fully activated by high [AA] alone, or by lower [AA] in synergism with PKC (Shiose & Sumimoto, 2000). In murine neutrophils, activation of NADPH oxidase by AA requires PKCζ (Kim & Dinauer, 2006). PKC inhibitors reduced or abolished AA-induced Ie despite the continued presence of AA in the bath. Evidently, both NADPH oxidase activity and the enhanced gating mode of proton channels are sustained by continuous phosphorylation, the prevention of which results in dephosphorylation and deactivation of both molecules.

Proton and electron currents in murine granulocytes

This is the first study of proton and electron currents in murine granulocytes and their responses to stimulation. In most respects, the properties of proton currents in murine cells resembled those in their human counterparts. Stimulation with either PMA or fMetLeuPhe produced changes in proton channel gating (enhanced gating mode) that were qualitatively like those in human cells. However, most of the changes in proton channel gating were distinctly less profound. The speeding of H+ current turn-on (τact), the slowing of deactivation (τtail), and the hyperpolarizing shift of the voltage dependence of gating were all only about half of their magnitude in human cells. In this sense, the mouse is a more difficult model system because the effects of stimulation are simply less obvious; its utility derives from the possibility of generating knockouts.

Responses to fMetLeuPhe

To confirm that the lack of a requirement for cPLA2α was not restricted to activation of cells by PMA, we used fMetLeuPhe in murine granulocytes. The response of both Ie and IH in individual cells ranged from no response to a full PMA-like response, consistent with a previous study in which only half of murine neutrophils responded to fMetLeuPhe in an NBT test that reflects O2.− production by individual cells (Kim & Dinauer, 2001). A response to fMetLeuPhe may require priming (DeCoursey & Ligeti, 2005), and spontaneous priming may occur in only a subpopulation of cells. In the cells in Fig. 8, there was no response to fMetLeuPhe, but subsequent introduction of PMA produced a large Ie that was inhibited by DPI. These results clearly demonstrate that the lack of fMetLeuPhe response was not due to the cells being non-viable or having already exhausted their capacity to assemble NADPH oxidase. All murine granulocytes that responded to fMetLeuPhe with Ie also exhibited enhanced proton channel gating. This result suggests that the ‘activation’ of H+ channels and NADPH oxidase are mediated by common or overlapping pathways that diverge relatively late.

In this study, we found no evidence to support the prevailing hypothesis regarding the mechanism of activation of the proton conductance during the respiratory burst in human eosinophils and murine granulocytes. Despite extensive circumstantial data showing (1) production of AA during the respiratory burst, (2) stimulation of NADPH oxidase and proton channels by AA, (3) inhibition of responses by broad-spectrum PLA2 antagonists, and (4) a requirement for AA or another amphiphile in the cell-free NADPH oxidase system, neither direct, specific inhibition of cPLA2α (which generates AA during the respiratory burst) nor genetic knockout of cPLA2α detectably impaired the enhanced gating of proton channels or NADPH oxidase activity. Instead, a role for PKC was demonstrated in activation of proton channels as well as NAPDH oxidase. Both molecules were deactivated within a few minutes of GFX or staurosporine addition, demonstrating that the signalling pathways downstream from PKC are rapidly reversible upon removal of the stimulus. Okadaic acid prevented the deactivation of proton current, implicating phosphatases in this process. Stimulatory effects of AA and oleic acid on proton currents were separated into direct and indirect components, and the indirect effects on both proton channels and NADPH oxidase are due at least in part to activation of PKC by AA.

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Acknowledgements

This work was supported in part by the Heart, Lung and Blood Institute of the National Institutes of Health (research grants HL52671 and HL61437 to T.D. and HL50040 to M.G.). The authors thank Prof T. Shimizu (University of Tokyo) for providing cPLA2α KO mice, Prof Linda C. McPhail (Wake Forest University) for a helpful critique of the manuscript, and Dr Tatiana Iastrebova for excellent technical assistance.

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Footnotes

  • (Resubmitted 3 November 2006; accepted after revision 18 December 2006; first published online 21 December 2006)

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Figure 1. cPLA2α inhibitors have no effect on the activation of NADPH oxidase or H+ current by PMA Records on the left show the current at −60 mV in human eosinophils in the perforated patch configuration. The traces on the right show proton currents in response to pulses to +60 mV applied at the time points indicated by lower case letters. Currents after PMA stimulation are shown as darker lines. Bath and pipette solutions consisted of TMA MeSO3 at pH 7. Cells were pretreated with nothing (A), 10 μm Wyeth-1 (B), 5 μm pyrrolidine-2 (C), or 2 μm G109203FX (D) and then stimulated with 60 nm PMA.

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Figure 2. Proton channel ‘activation’ occurs despite cPLA2α inhibition Proton currents elicited by identical families of 8 s voltage pulses in 20 mV steps from −40 mV to 60 mV (inset) in human eosinophils before (left) or after (right) stimulation with 60 nm PMA. The eosinophil in A was untreated, that in B was pretreated with 10 μm pyrrolidine-2.

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Figure 3. Enhanced proton channel gating occurs despite cPLA2α inhibition A, summary of gH–V (mean ± s.e.m) relationships before (▪, n = 9) or after stimulation with PMA alone (▴, n = 9) or following pretreatment with 10–20 μm pyrrolidine-2 (▾, n = 5), 1–10 μm Wyeth-1 (♦, n = 5), 10–20 μm AAOCOF3 (▪, n = 5), or 0.2–3 μm GFX (○, n = 8). Data are fitted with Boltzmann curves constrained to limit to 0. Conductance was calculated from IH measured at the end of the pulse, with leak subtracted. In most cases, Vrev was measured, or was assumed to be 0 mV. B, the time constant of activation (τact) of proton currents in human eosinophils before (▪, n = 20) or after stimulation with 60 nm PMA (▴, n = 9), in the presence of 10–20 μm pyrrolidine-2 (▾, n = 6), 10–20 μm Wyeth-1 (♦, n = 5) and 1 μm GFX (○, n = 6). The turn-on of proton current during depolarizing pulses was fitted by a single exponential, whose time constant is plotted here (mean ± s.e.m).

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Figure 4. GFX reverses activation of both NADPH oxidase and proton channels in human eosinophils Current at −60 mV (left records) in a human eosinophil in the perforated patch configuration stimulated with PMA and then treated with 3 μm GFX. The upper record shows currents during steps to +60 mV applied every 30 s, as well as the current at the holding potential, −60 mV. The lower record shows the same holding current at higher gain, with the currents during the test pulses blanked. On the right are proton currents during pulses to +60 mV recorded in this experiment at the times indicated by lower case letters.

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Figure 5. Staurosporine reverses spontaneous activation of both NADPH oxidase and proton channels in human eosinophils Current recorded shortly after establishing perforated-patch recording. Voltage pulses to +20 mV were applied from a holding potential of −60 mV every 15 s. At the arrow, 100 nm staurosporine was introduced into the bath. The time-dependent outward currents are proton currents; the inward current is presumably largely Ie. The horizontal line indicates zero current. It was evident that this cell was activated because a pulse to −20 mV elicited inward proton current (not shown), something never seen in resting cells. In addition, superposition of the inward current, presumed to be Ie, and the proton current during the test pulses results in a net inward current throughout the pulses to +20 mV, despite the activation of several picoamperes of outward proton current. Capacity transients have been truncated for clarity.

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Figure 6. Okadaic acid inhibits the deactivation of proton current induced by GFX Eosinophils were stimulated with PMA and then 2–3 μm GFX was introduced in the absence (▴) or presence of 100 nm okadaic acid (□) (n = 5 cells for both). Test pulses to +60 mV (+40 mV in one cell) were applied every 30 s. The mean (± s.e.m.) leak-corrected IH (the time-dependent rising current) at the end of the 4 s test pulse is plotted, normalized to its value during the final test pulse before addition of GFX, which is indicated as a horizontal line. All IH values obtained > 1.5 min after addition GFX in the presence of okadaic acid are significantly greater than those in its absence (P < 0.05, by Student's two-tailed t test). The IH during the first few pulses after the bath change increased presumably due to a transient temperature increase. (Evaporation of the bath reduces its temperature below ambient.)

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Figure 7. Staurosporine partially reverses the activation of proton currents by AA A, a continuous record of currents during test pulses to +20 mV applied every 15 s from a holding potential of −60 mV. At the first arrow, 5 μm AA was added. Ie and H+ currents increased progressively. The interruptions (boxes) are due to recording a family of currents (shown in C) and then addition of 100 nm staurosporine (STR) directly into the bath followed by stirring. The horizontal line indicates zero current. Digitally filtered at 2 Hz to reduce capacity transients. B–D, currents in the same cell before AA (B), in the presence of 5 mm AA (C) (the blanked family in A), and after addition of 100 nm staurosporine to the bath, in the continued presence of AA (D). Currents in B–D are from −20 mV to +30 mV in 10 mV increments, from a holding potential of −60 mV.

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Figure 8. Similarity of the PMA responses of NADPH oxidase and H+ channels in granulocytes from control or cPLA2α KO mice Currents at −60 mV (left traces) in granulocytes in the perforated patch configuration from a control mouse (A) and a cPLA2α KO mouse (B). Arrows indicate addition of 10 μm fMetLeuPhe, 60 nm PMA, or simple bath exchanges (‘wash’). Currents during 4 s depolarizing pulses to +60 mV (A) or +40 mV (B) (right traces) before (a) and after (b) addition of 60 nm PMA at the times indicated by lower case letters.

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Figure 9. PMA enhances proton channel gating in murine granulocytes Families of currents in a normal mouse granulocyte during identical pulses to −40 through +80 mV in 20 mV increments, before (left) and after stimulation with PMA (right). The dotted line shows zero current: PMA elicited inward electron current.

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Figure 10. The gH–V relationships before and after stimulation are similar in granulocytes from control and cPLA2α KO mice Mean ± s.e.m. values of gH calculated from IH at the end of 8 s pulses and measured Vrev values (in most cases) in control granulocytes (▪) and cPLA2α KO (•) mice. The open symbols indicate data after stimulation with 60 nm PMA in control (□) or cPLA2α KO cells (○). All data are from 10 to 12 cells.

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Figure 11. Responses to fMetLeuPhe are similar in granulocytes from control and cPLA2α KO mice Families of currents before (A and D) and after stimulation with 10 μm fMetLeuPhe (B and E) or 100 nm PMA (C and F) in a normal mouse cell (A–C) and a cPLA2α KO cell (D–F). All families illustrated are from −20 mV through +60 mV in 20 mV increments. The horizontal line indicates zero current. Calibration bars apply to all parts.

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Table 1. Comparison of PMA responses of human eosinophils in the absence or presence of cPLA2α inhibitors

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Table 2. Comparison of PMA responses of granulocytes from control or cPLA2α knockout mice

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  1. Published online before print December 21, 2006, doi: 10.1113/jphysiol.2006.124248 March 1, 2007 The Journal of Physiology, 579, 327-344.
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