©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of NADPH-Oxidase and Its Associated Whole-cell H Current in Human Neutrophils by Recombinant Human Tumor Necrosis Factor and Formyl-Methionyl-Leucyl-Phenylalanine (*)

Muhammad A. Schumann (§) , Chi Chiu Leung , Thomas A. Raffin

From the (1) Division of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, California 94305-5236

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Proton accumulation and efflux associated specifically with NADPH oxidation in neutrophils remains to be elucidated. Using confocal fluorescence and patch-clamp recordings from single human neutrophils, in the presence of protein kinase C inhibitors, we studied the transient cytosolic acidification and whole-cell H current induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and recombinant human tumor necrosis factor (rhTNF). Intracellular pH changes were monitored utilizing the ratiometric imaging of the dual emission fluoroprobe, carboxyseminaphthorhodafluor-1, AM acetate. Bath application of 1000 units/ml rhTNF or 0.1 µM fMLP changed the fluorescence of fluoroprobe-loaded cells, indicating generation of cytosolic H ions. In the absence of Ca in the pipette solution, exposure of cells to rhTNF or fMLP for 10 s activated voltage-dependent H currents. From tail current analysis, the threshold voltage for H current activation was -50 mV. These fMLP- or rhTNF-activated voltage-dependent H currents were augmented further in the presence of 0.1 mM of NADPH in the pipette solution, and they were inhibited by bath application of 50 µM of apocynin, an NADPH oxidase inhibitor. These results indicate that rhTNF- or fMLP-induced NADPH oxidase in human neutrophils gives rise to the activation of voltage-dependent H currents.


INTRODUCTION

Human neutrophils are the principal phagocytic cells during the acute phase of inflammation. During phagocytic stimulation, activated neutrophils utilize molecular oxygen for the killing of microbial pathogens (1) . The immense rise in oxygen consumption and the associated production of oxygen-free radicals are designated ``respiratory burst'' (2, 3, 4) . Oxygen molecules undergo one-electron reduction catalyzed by an oxidase whose substrate is the pyridine nucleotide, NADPH (5) . Once assembled from its components (see Ref. 6 for review), the activated NADPH oxidase is in effect an electron transport chain bound to the plasma membrane. With the action of enzymes released from cytoplasmic granules, the initially produced superoxide anion (O) and its dismutated product HO are subjected to a complex series of reactions, taking place in the phagosome, leading to the formation of reduced oxygen species. The oxidase is normally inactive but can be readily activated by various stimuli (7, 8) including tumor necrosis factor (9) , N-formyl-methionyl-leucyl-phenylalanine (fMLP)()(8) , and phorbol 12-myristate 13-acetate (PMA)(10, 11) . Coupled with the reduction generating the O is the accumulation of protons (H ions) in the cytoplasm of neutrophils. The cell immediately rids itself from the ensuing acidification by extruding H ions in the external milieu (12, 13) . This homeostatic response is necessary for protecting metabolic reactions in general and maintaining NADPH as a steady electron donor in particular. The mechanism involved in the proton efflux associated with phagocytosis remains to be elucidated.

A recent body of evidence supports the existence of putative H-conducting channels in the plasma membrane of human neutrophils (14, 15, 16, 17, 18) . The evidence is based on the following observations in PMA-stimulated cells: 1) the mode of action of NADPH oxidase has been shown to be electrogenic; 2) the subsequent efflux of produced H ions, through a proposed Zn- and Cd-inhibitable channel (known as ``H-channel''), provides the necessary charge compensation; and 3) the efflux of H ions (repolarization) initially lagging behind the generation of O (depolarization) may explain the membrane depolarization-repolarization sequence. In these studies, correlative changes in pH to membrane potential have been determined using the cytosolic pH indicator, 2`,7`-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein and a membrane potential-sensitive probe. However, the characterization of an accumulation of cytosolic H ions or of a current carried by H ions in human neutrophils as a direct function of NADPH oxidase activation, without protein kinase C (PKC) influence, remained to be elucidated. Functional regulation of cytosolic pH is essential to phagocytosis (19) . Generation of H ions can take place in many of cytoplasmic reactions and in response to various stimuli (e.g. PMA) that produce a respiratory burst even in the presence of an inhibited NADPH-oxidase (14). Furthermore, there is a strong evidence that receptor-mediated stimulation of the NADPH oxidase can occur by pathways not involving PKC (9, 10, 20) . With this knowledge, the goal of the present study was to monitor transient changes in cytosolic [H] or induced H currents in response to the activation of NADPH oxidase, separate from the activation of PKC. Thus, we used the whole-cell patch-clamp technique and confocal fluorescence microscopy to characterize the activated H current and to monitor the associated change in cytoplasmic pH, respectively, after stimulation of NADPH oxidase by fMLP or rhTNF, in the presence of PKC inhibitors. Our results indicate that, separate from the role of PKC, activated NADPH oxidase induces a voltage-dependent H current in human neutrophils.


EXPERIMENTAL PROCEDURES

Reagents

We purchased rhTNF having specific activity of 5 10 units/mg from BioSource International, Camarillo, CA. Purity was >98%, as determined by electrophoresis and amino acid amino terminus sequencing analysis; endotoxin content was <0.1 ng/1 g, by limulus amebocyte lysis assay. Cytochrome C (type iii), fetal bovine serum, superoxide dismutase, and Ficoll-Hypaque were purchased from Sigma; minimum essential medium with Hanks' salts and L-glutamine, and phenol red-free Hanks' balanced salt solutions from Life Technologies, Inc.; PMA and 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (H-7) from Calbiochem Corp.; the PKC inhibitory peptide, PKC (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) , the PKC control peptide, [Glu]PKC (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) , and fMLP from Peninsula Laboratories, Belmont, CA; carboxysemi-naphthorhodafluor-1 acetate in the acetoxymethyl acetate ester form (C-SNARF-1/AM) and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) from Molecular Probes, Eugene, OR; and 4-hydroxy-3-methoxyacetophenone (apocynin) from Carl Roth GmbH, Karlsruhe, Federal Republic of Germany.

Neutrophil Separation

Cells were isolated from blood of healthy donors using the method (21) of density gradient centrifugation with Ficoll-Hypaque. We visualized the cells by phase-contrast inverted microscopy, confirmed their nuclear morphology by staining (with Wright's stain), and checked their viability (by trypan blue dye exclusion), yield, and purity. We washed the cells twice and resuspended them (0.5 10 cell/ml) in a filtered recording solution. Isolation of cells and all recordings were performed at room temperature (20-22 °C).

Superoxide Production Assay

This assay is an indirect estimation of NADPH oxidase activity during respiratory bursts. The amount of O generated was determined by the reduction of cytochrome c as described previously (22) . All incubations were done in 16-mm tissue culture wells coated with fetal bovine serum; wells were precoated with 300 µl of serum, in 5% CO atmosphere at 37 °C for 1 h, and then washed three times with normal saline. A suspension of 0.25-1 10 cells in 100 µl, pH 7.4, Hanks' balanced salt solutions was added to 800 µl of a reaction mixture containing 85 µM cytochrome c, with or without an activator or inhibitor. The control contained 300 units of superoxide dismutase. Wells were incubated at 37 °C for 1 h before stopping the reaction with the addition of 300 µl of solution of superoxide dismutase in Hanks' balanced salt solutions. The reaction mixture was harvested and centrifuged at 500 g for 5 min. The optical density of the supernatant was read at a wavelength of 550 nm using a spectrophotometer (DU-64, Beckman Instruments, Inc., Fullerton, CA). Production of superoxide was obtained by dividing the difference in absorbency value between control and sample by an extinction coefficient of 29.5 mm(23) .

Electrophysiological Recordings

We used the whole-cell configuration of the patch-clamp technique (24) to record from single neutrophils of nearly equal sizes (10-12 µm). Pipettes were pulled using the Flaming/Brown programmable micropipette puller, P-87 (Sutter Instrument Co., San Rafael, CA) to provide electrode resistance of 4-6 M in whole-cell recording. We electronically compensated series resistance, which was obtained after capacitance compensation by direct readout from the patch amplifier. Nonetheless, base-line H currents were small enough to minimize voltage errors. Families of whole-cell currents were elicited from a holding potential (HP) of -60 mV by voltage pulses delivered in 20 or 25 mV steps, from -100 to +120 or +150 mV at a frequency of 0.1 Hz. Recording was performed by an Axopatch-1C (Axon Instruments, Foster City, CA) patch-clamp amplifier with a 10 G feedback resistor and active low-pass filter. Records were digitized at 1 and 10 kHz and were filtered at 5 kHz. We performed data acquisition with pClamp Clampex software (Axon Instruments) running on a computer (IBM PC/AT) that interfaced with the amplifier by means of an analog-to-digital converter. Measurements and plotting were carried out by pClamp Clampfit (Axon Instruments) and Sigma Plot software (Jandel Scientific, Corte Madera, CA).

Recording Solutions and Drugs

To record H-selective currents from neutrophils, we used solutions devoid of the cations Na, K, and Ca, and of Cl as a major anion. We also used the following buffers (Sigma): MES (pK= 6.1); PIPES (pK= 6.8); HEPES (pK= 7.5); and TAPS (pK= 8.4). We adjusted the pH of solutions with either methanesulfonic acid or choline base. We utilized N-methyl-D-glucamine (NMDG) as the major cation (in a hydroxyl form). The bath solution contained 170 mM NMDG-OH, 2 mM MgCl, 10 mM buffer, and 15 mM glucose (pH values: 6.1, 6.8, 7.4, and 8.4; osmolality: 320 mosm kg). The pipette solution included 140 mM NMDG-OH, 0.5 mM EGTA, 2 mM MgCl, 2 mM ATP (Mg salt), and 5 mM HEPES (pH 7.3 or 7.4, 295 mosm kg). The minor content of Cl- in both solutions is symmetrical. We made the bath solution slightly hypertonic to avoid cell swelling. To alter H gradients in recording solutions to levels close to those found in the physiological milieu, we used the appropriate buffer so that we can achieve bath-to-pipette pH ratios (reflecting those of extracellular pH, pH, to intracellular pH, pH ) of 6.1:7.4, 6.8:7.4, 7.4:7.4, 8.4:7.4. Using a ratio higher than the latter ratio or lower than the former ratio resulted in unstable recording, which is perhaps ascribable to the resultant deviation from enzyme pH optima. Neither Cs nor Tris was used as a major cation in pipette or bath solutions since Cs and Tris have been shown to exhibit some cationic permeability through nonspecific cationic channels in neutrophil membranes (25) . Stock solutions (0.1 M) of apocynin, H-7, W7, fMLP, and PMA were made in MeSO; stock solutions of rhTNF (1000 units/ml) and NADPH (0.1 M) were made in distilled water. All stocks were kept in vials at 20 °C and added freshly to the bath solution (or pipette solution in the case of NADPH and PKC-related peptides). There was no significant effect for MeSO on H currents.

Loading of C-SNARF-2/AM into Neutrophils

Forty µl of cell suspension (1 10 cell/ml) were placed in the center of one well or two wells (for a duplicate experiment) of a two-well coverglass chamber whose bottom is a glass coverslip (Nunc Inc., Naperville, IL). One-hundred µl of minimal essential medium with Hanks' salts and L-glutamine were mixed carefully with the cell suspension, and the mixture was allowed to stand in a moist compartment for 10-15 min so that cells can attach. Cells were then washed with 1 ml of medium to remove dead or unattached cells and, subsequently, incubated in a fresh 1 ml of medium. We measured cytosolic [H] changes by using the single-excitation, dual-emission wavelength pH fluoroprobe C-SNARF-1/AM (26, 27). Although C-SNARF-1/AM can also be utilized in the dual-excitation ratioing mode, it was used throughout the present study in the dual-emission ratioing mode.

Measurements of Cytosolic pH Changes by Laser Confocal Fluorescence Microscopy

A scanning confocal microscope (developed by Dr. Stephen Smith, Stanford University, CA) was utilized for imaging measurements of C-SNARF-1 emission fluorescence after excitation at 514-nm line of the argon laser. For emission ratioing, two filters were employed: 590 nm band-pass (bp) filter (30 nm bandwidth) and 600 nm long-pass (lp) filter. Thus, derived emission ratios are referred to as ratios of 600 lp/590 bp. Fluorescence ratio measurements were conducted to circumvent variations in the extent of dye loading, cell thickness, photobleaching, and dye leakage. Upon binding with H ions, C-SNARF-1 displays diminishing fluorescence at the longer wavelength and increasing fluorescence at the shorter wavelength (28, 29) . To attain a loading concentration of 5 µM of C-SNARF-1/AM, 5 µl of 1-mm stock solution (stored at -70 °C in 50-µl aliquots of MeSO) were added, in the dark, to the incubating medium. To facilitate cell loading with the fluoroprobe, the latter and 5 µl of 25% (weight/weight) solution of the surfactant Pluronic F-127 (Molecular Probes) were added concurrently to the incubating medium. To complete loading, cells were incubated in the moist compartment for 45 min and then washed with fresh medium. Fluorescent pictures in this work are only representative examples of at least five replicates recorded from different cell preparations. In all assays, control samples of cell suspension that had been treated similarly but not loaded with the fluoroprobe were set aside to measure autofluorescence.

Adjustment of Intracellular pH and Calibration

To eliminate pH gradient across the cell membrane, the method of Thomas et al.(30) was used as follows. At the end of the experiment, cells were equilibrated with 10 µg/ml nigericin (Sigma)/high potassium calibration solutions of pH 5.5-9.5. Under these conditions, nigericin acts as both K and H ionophore. By raising extracellular [K] to 140 mm, the membrane potential should depolarize to 0 mV and pH should equal pH. The pH may then be controlled simply by changing pH.

Data Analysis

Leak current, which was small, was subtracted before determining current amplitudes. We derived the leak current from ohmic sweeps, at -80 and -100 mV steps and then digitally subtracted a linearly scaled inverted pulse from all test potential steps. Current amplitudes were measured 1 ms before the termination of the test pulse. Neutrophil cell capacitance in our experiments was about 4 pF. Data were expressed as mean ± standard error of the mean with n indicating the number of cells contributing to the mean. When appropriate, we performed comparisons of groups using paired or independent Student's t tests. Measurements of brightness, ratio imaging, and subtracting background fluorescence were done by Image-1 software (Universal Imaging Corp., West Chester, PA) in the absence and presence of fMLP or rhTNF.


RESULTS

Overview

Experimental data, subsequently shown, were derived using enzymatic, spectrofluorimetric, and electrophysiologic investigations designed to shed light on whether the activation of NADPH-oxidase alone (separate from that of PKC) could be associated with the activation of membrane H conductance in human neutrophils during respiratory burst activity. Specifically, we studied the effect of activating human neutrophils' NADPH oxidase by PMA, fMLP, or rhTNF on O production, cytoplasmic pH changes, and the associated generation of whole-cell H currents (in the presence of PKC inhibitors), using spectrophotometry, confocal fluorescence microscopy, and patch-clamp techniques, respectively.

Activation of NADPH Oxidase in Human Neutrophils by PMA, fMLP, and rhTNF

Agents such as PMA, fMLP, or rhTNF are known to enhance respiratory burst activity, and effects of PMA, fMLP, or rhTNF on neutrophil NADPH oxidase have been investigated (31, 32, 33) . It was necessary, however, to begin our investigations with a quantitative comparison between the effects of these agents on respiratory burst activity under our experimental conditions. Although chemiluminescence could have been used as an indirect sensitive measurement of NADPH oxidase activity in human neutrophils, we used a more quantitative method utilizing a cytochrome reduction to investigate the production of O by neutrophils exposed to PMA, fMLP, and rhTNF. To bioassay O levels induced by rhTNF, neutrophils had to be adherent prior to incubation with this cytokine (33). As neutrophils were incubated with appropriate concentrations of each of the three agents, there were significant enhancements of O production, compared to the basal level as shown in Fig. 1. As indicated by the level of O, the activation of NADPH oxidase induced by PMA (0.1 µM) was more significant than that induced by either rhTNF (1000 units/ml) or fMLP (0.1 µM). In the presence of 15 µM H-7, a potent inhibitor of PKC (34, 35) , the PMA-induced O generation was blocked, while fMLP- or rhTNF-induced O generation was partially reduced but remained significant.


Figure 1: Stimulation of O production by rhTNF, fMLP, and PMA in human neutrophils. *p < 0.01, compared to base-line. NADPH oxidase activity was determined by measuring O level before (base-line) or after stimulation of neutrophils with rhTNF, fMLP, or PMA at the indicated concentrations. The incubations lasted 1 h.



Cytosolic Acidification of Stimulated Neutrophils and the in Vitro Calibration of C-SNARF-1

To monitor transient acidifications as they occurred in the presence of fMLP or rhTNF, we used pH indicator fluorimetry. The emission intensities of the fluoroprobe C-SNARF-1 at two different wavelengths were simultaneously collected for ratio measurements (see ``Experimental Procedures''). Fluorescence was monitored continuously before, during, and after bath exposure to either rhTNF (1000 units/ml) or fMLP (0.1 µM). Before exposure, the fluorescence intensity of C-SNARF-1-loaded cell was quite pronounced; the emitted fluorescence associated with the unprotonated dye was 4-fold greater than that of protonated form. Both fMLP and rhTNF produced a significant time- and dose-dependent decline in fluorescence intensity. This decline indicated transient cytosolic acidification. To dissect the role of PKC or other kinases in transient cytosolic acidification of stimulated neutrophils, measurements were also performed in the presence or absence of 15 µM of H-7 and 10 µM of W7, an inhibitor of Ca


Figure 2: Effect of fMLP on cytosolic acidification in human neutrophils. Fluorescent photomicrographs representing the change in intracellular pH from 7.3 before (upper panel) to 6.4 after (lower panel) bath administration of 0.1 µM fMLP for 5 s. The scale of gray (indicative of fluorescence arbitrary units) is shown in the upper micrograph. Corresponding gray scale values for pH 7.4 and 6.3 were 116.5 and 28.7 arbitrary units, respectively. The experiment was done in the presence of 15 µM H-7 and 10 µM W7.




Figure 3: Calibration of C-SNARF-1. Effect of pH on calculated emission ratio (600 lp/590 bp) of C-SNARF-1 fluorescence. Ratio is plotted against intracellular pH that has been controlled extracellularly in single cells (n = 10-18) made permeant by nigericin 10 µg/ml nigericin (Sigma)/high-potassium (140 mM) calibration solutions of pH 5.5-9.5 made by MES, PIPES, HEPES, and TAPS buffers. Under these conditions, nigericin acts as both K and H ionophore. Fitted curve matches the Henderson-Hasselbalch equation.



Dependence of Whole Cell H Currents on pH Gradients across the Plasma Membrane

To separate whole-cell H currents in the absence of major permeant ions (Na, K, Cl, and Ca), we performed recording experiments using salts whose cationic and anionic radicals were relatively impermeant through various ionic channels in the neutrophil membrane. Leak current, which was small (0-3%, n = 18), was subtracted before determining current amplitudes. In unstimulated neutrophils, recorded base-line H currents were small, outwardly rectifying, and showing voltage-dependent activation. We observed some cell-to-cell variability in expressing voltage-dependent base-line currents; out of 23 unstimulated cells, 17 cells expressed similar voltage-dependent H currents. In addition to being elicited by depolarizing voltage steps, amplitudes of these base-line H currents were dependent on pH gradients imposed across the plasma membrane of human neutrophils in the presence or absence of the specific PKC inhibitory peptide PKC()(39, 40, 41) . The PKC inhibitory peptide (PKC) was included in the pipette solution at 10 µM. Fig. 4illustrates the pH dependence of H currents as observed in one representative experiment (n = 6) in which the peptide inhibitor was included. In this experiment, different pH /pH ratio (bath to pipette) of 6.1:7.4, 6.8:7.4, 7.4:7.4 (1:1), and 8.4:7.4 were imposed by recording solutions. Recorded current amplitudes were found to be proportional to the applied pH gradient, being larger with higher ratios. As the bath solution was made more alkaline than the pipette solution, the voltage-dependent outwardly rectifying current became more evident.


Figure 4: Dependence of whole-cell H currents on pH gradient across the plasma membrane of unstimulated human neutrophils. Families of base-line H currents monitored at different pH ratios (pH/pH), each recorded from a different cell, reveal voltage-dependent responses to 25-mV steps (from -100 mV to +150 mV) applied from a holding potential of -60 mV (left bars). Leak was subtracted from records. Recording solution: bath, 170 mM NMDG-OH, 1 mM MgCl, and 10 mM buffer; pipette, 140 mM NMDG-Cl, 0.5 mM EGTA, 2 mM MgCl, and 5 mM HEPES, pH 7.4. Buffers (pK): MES (6.1), PIPES (6.8), HEPES (7.5), and TAPS (8.4). pH/pH ratio (indicated above traces) are 6.1/7.4, 6.8/7.4, 7.4/7.4 (1:1), and 8.4/7.4. The PKC inhibitory peptide (PKC) was included in the pipette solution at 10 µM. Calibration is indicated at the bottom.



Effect of fMLP and rhTNF on Whole Cell H Currents

Both fMLP and rhTNF trigger a respiratory burst activity in human neutrophils. Thus, we examined the effects of the both agents on the induction of H currents in these cells. In all experiments, the PKC inhibitory peptide PKC was included in the pipette solution at 10 µM. Fig. 5A illustrates a family of H current traces recorded in a cell before and after exposure to 0.1 µM fMLP for 10 s. In contrast to the small base-line H current, fMLP-activated H currents were substantially augmented, prominently voltage-dependent, and outwardly rectifying. Time-dependent current responses were evident at higher depolarizing voltages. Tail currents were also enhanced noticeably. Both the amplitude and activation rate of fMLP-activated H currents were increased markedly with membrane depolarization. The H current was augmented in all tested cells (875 ± 15.3 pA, n = 6). To confirm that the recorded currents were carried by H cations, we plotted the recorded current amplitudes versus their corresponding voltages at two different pH gradients. In the case of fMLP-activated currents after leak subtraction, only the net activated current (the current obtained by subtracting the base-line current from the activated current) was considered for plotting. The current-voltage relationships for apparently voltage-dependent base-line and fMLP-activated H currents at two different pH gradients, respectively, are depicted in Fig. 5B. Illustrated relationships demonstrate the outward rectification of currents at more depolarized potentials. As indicated in Fig. 5B for both base-line and activated currents, the current reversal potential (the potential at which there is no current) shifts from -52 mV in a pH /pH (bath to pipette) ratio of 8.4/7.4 to +36 mV in a pH /pH ratio of 6.8/7.4. As predicted by the Nernst equation for a predominantly H-selective channel, reversal potential values are -58 and +35 mV, respectively. Thus, in our experiments, there was an approximate 88-mV shift in the reversal potential to the right of the current-voltage relationship, corresponding to a pH gradient (between bath and pipette solutions) change equivalent to 1.6 pH units. Currents were also activated by rhTNF; however, the effect of stimulating neutrophils by rhTNF on the current was dependent on cell adherence. Using the same voltage protocol, we registered activated H currents 30 s after bath exposure to 1000 units/ml rhTNF from cells adherent to the recording dish (n = 19). In contrast, there was slight or no activation of currents recorded from nonadherent neutrophils treated with rhTNF (n = 13). Current amplitudes were significantly larger in adherent cells (3.7 ± 0.2 folds, n = 10). Activated currents exhibited marked outwardly rectification with voltage- and time-dependent properties. A representative comparison between two families of rhTNF-induced H currents recorded from an adherent and a nonadherent cell, respectively, is illustrated in Fig. 6. There was no difference in the corresponding base-line H currents (recorded prior to exposure to rhTNF) between adherent and nonadherent neutrophils. Data subsequently shown for rhTNF were derived from adherent cells only.


Figure 5: Activation and voltage properties of whole-cell H currents in fMLP-stimulated human neutrophils. A, base-line H currents (upperpanel) elicited by 25-mV steps delivered from a holding potential of -60 mV were activated (lowerpanel) by bath application of 0.1 µM fMLP for 10 s. The pH/pH ratio was 7.4/7.3 for this experiment. B, current-voltage relationships for base-line currents and fMLP-activated currents recorded at two indicated pH/pH ratios. Amplitude were measured 1 ms prior to the termination of the 250-ms voltage pulse. Observed values of reversal potential are -52 mV (167, n = 3; , n = 5) and +36 mV (, n = 3; , n = 6). Points are connected by lines. The PKC inhibitory peptide (PKC) was included in the pipette solution at 10 µM.




Figure 6: Activated whole-cell H currents in rhTNF-stimulated human neutrophils. Two families of H currents elicited by 25-mV steps delivered from a holding potential of -60 mV (left bars) were recorded from an adherent cell (upper panel) and a nonadherent cell (lower panel) after bath application of 1000 units/ml rhTNF for 30 s (n = 19 and 13, respectively). Corresponding base-line currents are not shown. The pH/pH ratio was 7.4/7.3.



Voltage Dependence of H Currents Activated by fMLP or rhTNF in Human Neutrophils

Activated H currents induced by 10-20-s exposure to 1000 units/ml rhTNF or 0.1 µM fMLP exhibited signs of voltage dependence. To determine the conductive and voltage properties of the channel involved separate from its activation pathway, we performed experiments to analyze activated H tail currents after activation of the neutrophil by bath application of 1000 units/ml rhTNF or of 0.1 µM fMLP 10 s. We utilized pHo/pH ratios of 7.4:7.3 and pipette solutions containing no Ca


Figure 7: Voltage properties of tail H currents in fMLP-stimulated human neutrophils. A, tail currents were elicited in a cell stimulated by 0.1 µM fMLP for 10 s by stepping from a holding potential of +90 mV (applied for 400 ms) to test voltages of +30, +10, -10, -30, -50, -70, -90, -110, and -130 mV. Insets show the voltage protocol and calibration. The pH/pH ratio was 7.4:7.3. B, current-voltage relationship viewing tail current amplitudes (recorded in fMLP-stimulated cells) as a function of respective test voltages (n = 6). The tail current amplitude was measured 1 ms before the termination of the current trace. Ratios of pH/pH were 7.4:7.3 for all experiments. The PKC inhibitory peptide (PKC) was included in the pipette solution at 10 µM.



As indicated by Fig. 5B, onsets of membrane current activation were noticeable at more negative potentials with higher pH /pH ratios. This early activation was studied in a greater detail by analyzing activated tail H currents under near symmetrical conditions with respect to [H] across cell membrane. Thus, we determined the threshold of voltage activation of tail H currents after stimulation of neutrophils with 10-s bath administration of 1000 units/ml rhTNF or 0.1 µM fMLP. We performed the following protocol using pH /pH ratios of 7.4:7.3 and pipette solutions containing 10 µM PKC inhibitory peptide PKC, 0.5 mM EGTA, and no Ca


Figure 8: Voltage dependence of tail H currents in fMLP-stimulated human neutrophils. A, tail currents were elicited in a cell stimulated with 0.1 µM fMLP for 10 s by stepping from holding potentials of +75, +50, +25, 0, -25, -50, and -75 mV (applied for 400 ms) to -90 mV. Insets show the voltage protocol and calibration. B, amplitudes of tail currents recorded in fMLP-stimulated cells and measured (n = 4) at -90 mV are plotted against respective holding voltages. The tail current amplitude was measured 1 ms before the termination of the current trace. Ratios of pH/pH were 7.4:7.3 for all experiments. The PKC inhibitory peptide (PKC) was included in the pipette solution at 10 µM.



Involvement of NADPH Oxidase in the Activation of H Currents

The involvement of NADPH oxidase in the activation of H currents was investigated by conducting two separate approaches. The first approach involved inhibition of the NADPH oxidase; the second approach involved activation of the oxidase. For the inhibition study we utilized apocynin, a potent cell permeant NADPH oxidase inhibitor (42) . We used apocynin, alone or concurrently applied with a mixture of 15 µM H-7 and 10 µM W7, to study its blocking effect on rhTNF- and fMLP-activated voltage-dependent H currents in human neutrophils. After incubation of the cells with 50 µg/ml apocynin alone or in combination with other inhibitors, no activation of H currents by any of the tested agents was observed. As shown in Fig. 9A for a representative experiment, already activated H currents with 10-s exposure to 0.1 µM fMLP were significantly blocked by 7-min exposure to 50 µg/ml apocynin. Comparable results were obtained in the case of 1000 units/ml rhTNF as subsequently indicated. For the activation study, we did not activate NADPH oxidase directly, but we promoted its forward reaction by supplying with extra NADPH molecules. Thus, we tested the effect of the coenzyme NADPH on the activation of H currents induced by rhTNF and fMLP. NADPH (1 mM) was included in the pipette solution for a 5-min intracellular dialysis. After obtaining a stable whole cell base-line currents, we applied 1000 units/ml rhTNF or 0.1 µM fMLP. In the presence of NADPH in the pipette solution, either agent significantly activated voltage-dependent outwardly rectifying H currents. Cumulative results derived from experiments with apocynin and NADPH can be seen in Fig. 9B. Peak outward H currents (base-line, fMLP-, or rhTNF-induced) recorded at +100 mV were plotted in the absence or presence of apocynin or NADPH. Data were obtained from different experiments and different cell populations. It is interesting to note that apocynin and NADPH significantly inhibited and activated, respectively, only rhTNF- or fMLP-induced voltage-dependent H currents but had no significant effect on voltage-dependent base-line H currents.


Figure 9: Inhibition and enhancement of fMLP-activated H currents in human neutrophils. A, activated H currents (upper panel) elicited by 25-mV steps delivered from a holding potential of -60 mV after bath exposure to 0.1 µM fMLP for 10 s were inhibited by 7-min bath application of 50 µg/ml apocynin (lower panel). The pH/pH ratio was 7.4:7.3 for this experiment. B, maximal outward base-line, rhTNF (1 µM, 10 s)-activated, or fMLP (0.1 µM, 10 s)-activated currents recorded at +100 mV in the absence and presence of 1 mM NADPH (in the pipette) or 50 µg/ml apocynin (in the bath), respectively. (n = 4-8 for each treatment pair; base-line responses are combined; and *p < 0.01, compared to respective base-line responses.)




DISCUSSION

We demonstrated that activated human neutrophils exhibited voltage-dependent H currents in response to fMLP and rhTNF, both of which are known to elicit respiratory bursts in these cells (32, 33) . In particular, we characterized a portion of activated currents, carried by a pool of H ions, that was generated by the differential activation of neutrophil NADPH oxidase (Fig. 10). Characterization of this pool of currents is arguably an essential step in correlating the transport of H ions with the activity of NADPH oxidase. Establishing such a correlation is necessary considering four important facts linking NADPH oxidase and H transport: 1) the specialized protein components of NADPH oxidase are either membrane bound (flavocytochrome b) or translocated (phosphoproteins and GTP-binding proteins) to the membrane upon activation of the oxidase (43) ; 2) NADPH oxidase-mediated phagocytosis and the efflux of H ions driven by the resultant acidification are membrane-coupled (44) ; 3) killing of pathogens is more effective the more acidic the cytosol becomes (compared to the phagosome or external milieu) (6, 19) ; 4) evidence has been mounting in support of proposing operation of ``H-selective channels'' in the plasma membrane of human neutrophils having an activated NADPH oxidase (see Introduction); and 5) there is an abnormal activation of H conductance in NADPH oxidase-defective neutrophils of chronic granulomatous disease patients (45) . Metabolic processes giving rise to H ion generation, uptake, and homeostasis are numerous (e.g. a significant contribution to the transient acidification of the cytoplasm during phagocytosis can also be provided by the activation of PKC (46) ); many of these processes are not related to the NADPH oxidase-mediated pathway nor to the hexose monophosphate shunt, the supplier of NADPH molecules in the pathway.


Figure 10: Diagrammatic model of phagocytosis in a human neutrophil. After receptor-mediated endocytosis of a bacterium, the NADPH oxidase is activated in the membrane surrounding the phagosome, generating O and hydrogen peroxide in the phagosome. There is a subsequent massive acidification of the cytosol. The pH in the phagosome increases and activates the proteinases (6). Both proteinases and reactive oxygen species participate in the killing of the bacterium.



We studied H currents in human neutrophils, and we were aware of only one simultaneous work (47) describing H conductance in human neutrophils. However, this work does not address any direct correlation between H conductance and NADPH oxidase activity in the absence of PKC activity in these cells. It is also difficult to rule out any contamination of H conductance with Ca or Cl conductance. In contrast, we used recording solutions free from all permeant monovalent cations and containing only H ions whose gradient across the cell membrane was controlled by imposed alterations of pH. Any minor traces of Cl were kept strictly symmetrical. Ca ions were also excluded from recording solutions since their presence would lead to recording of Ca currents (25) which may mask H currents. In other than human neutrophils, a pH- and voltage-dependent H conductance has been characterized in murine peritoneal macrophages (48) . A Ca role in superoxide production does not seem to be required (49, 50) . Under these condition, most base-line H currents (in unstimulated neutrophils) showed pH and voltage dependence. These base-line currents were not inhibited by the NADPH oxidase inhibitor or activated by NADPH (Fig. 8), indicating that they were not induced by NADPH oxidase. When stimulated by fMLP or rhTNF, however, all neutrophils expressed activated voltage-dependent H currents which could have been produced by PKC or NADPH oxidase. Since our main concern was to characterize only those H currents that were associated with the activation of NADPH oxidase, we eliminated any possible role for PKC by recording in the presence of a specific PKC inhibitor. In doing so, we dissected the effect of PKC from the function of the NADPH oxidase in H ion generation. It has been reported that stimulation of PKC by PMA induces the activation of an electrogenic H-conducting pathway in the plasma membrane of human neutrophils. The rate of H extrusion (as a function of the conductance) can increase 2.5-fold even when the NADPH oxidase is blocked by the thiol reagent p-chloromercuribenzene (17) . On the other hand, an active PKC is not necessary to the activation of NADPH oxidase in human neutrophils (20, 51) . Having dissociated the role of PKC, we were able to manipulate the activity of NADPH oxidase by fMLP or rhTNF and evaluate the concomitant H conductance.

We characterized the conductive and voltage properties of H currents and determined their linkage to NADPH activation. Several properties of the whole cell currents before and after their induction by the activated NADPH oxidase were consistent with time- and voltage-dependent macroscopic H currents. Currents were: 1) both outwardly rectifying and depolarization sensitive; 2) contributing to the ionic basis of transient depolarization of membrane potential during activation of neutrophils; 3) displaying characteristic current-voltage relationships with observed shifts in current reversal potentials corresponding to imposed pH gradients across the cell membrane according to the Nernst equation; 4) exhibiting activated voltage-sensitive tail currents with a threshold of voltage activation at -50 mV which differs from that (-69 mV) of voltage-dependent Cl currents (52) ; and 5) showing a magnitude of conductance directly proportional to pH /pH ratio, being larger when pH< pH. The following criteria also indicate that all activated H currents were induced by and associated with the selective activation of NADPH oxidase: 1) the activated H currents were recorded with dialyzing pipettes containing a specific PKC inhibitor (PKC, a pseudosubstrate peptide reported to inhibit PKC-dependent currents in neutrophils (41, 52) ); 2) activated H currents did not persist in the presence of apocynin, an NADPH oxidase inhibitor; 3) they were also induced by fMLP or rhTNF in the presence of H-7, an inhibitor of PKC, and W7, an inhibitor of Ca/calmodulin-dependent kinases; 4) the activated H currents were further augmented by introducing NADPH in dialyzing pipettes; 5) increases in fMLP- or rhTNF-induced H current amplitudes paralleled increases in fMLP- or rhTNF-induced O production levels in the presence of H-7 and W7 (i.e. correlated with NADPH oxidase activities); and 6) our fluorescent data indicated the occurrence of cytosolic transient acidification in human neutrophils undergoing respiratory burst activity in the presence of PKC and calmodulin inhibitors.

The obvious significance of NADPH oxidase to phagocytosis is underscored by patients suffering from chronic granulomatous disease (53). It is responsible for generation of superoxide and subsequent microbicidal effects. However, this multisubunit complex may also be involved in other pathways for neutrophil activation. It has been observed that reduction of the cytoplasmic [Cl] concentration can result in spontaneous activation (independent of Ca) of NADPH oxidase (54) . Changes in the cytoplasmic [Cl] concentration can be brought about by the activation of Ca-activated Cl channels (41, 55) and/or Ca-independent voltage-dependent Cl channels (52) .

In summary, we characterized H waves across the plasma membrane or in the cytosol of human neutrophils, both electrophysiologically (H current changes) and fluorimetrically (pH alterations), respectively. To prevent overlapping effects from different transduction pathways, our characterization was achieved under conditions that separate the role of PKC from that of NADPH oxidase in respiratory burst. Our results indicate that there are voltage-dependent H currents associated with resting and activated (undergoing respiratory burst activity) human neutrophils. These currents flow as effluxes of H ions resulting from the accumulated cytosolic acidity during NADPH-mediated phagocytosis. It is known that activation of either PKC or NADPH oxidase results in a cytosolic transient acidification and subsequent extrusion of H ions. According to our results, the main observation of our report is that there are ``H channels'' that can be activated following stimulation of NADPH oxidase in the absence of Ca ions or of an active PKC. Nonetheless, both Ca and PKC may modulate superoxide production (56) . The significance of this finding is that it sheds light on exploring various strategies necessary for successful pharmacological interventions in some neutrophil-mediated pulmonary injuries (57, 58, 59, 60) . These strategies could be devised to design effective drugs that manipulate either NADPH oxidase or PKC, or both, as dictated by various clinical cases. Whether there is a cross-talk between mechanism pathways initiated by the activation of both PKC or NADPH oxidase also remains to be investigated.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL455330. Preliminary data of this work were presented in part in an abstract form ((1993) Biophys. J. 64, 98 (abstr.)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed. Tel.: 415-725-0421; Fax: 415-725-5489.

The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; TNF, tumor necrosis factor; rhTNF, recombinant human tumor necrosis factor ; HP, holding membrane potential; NMDG, N-methyl-D-glucamine; C-SNARF-1, carboxyseminaphthorhodafluor-1; MES, 2-(N-morpholino)ethanesulfonic acid; PIPES, piperazine-N,N`-bis(2-ethane-sulfonic acid); TAPS, N-tris(hydroxymethyl)methyl-3-aminopropane-sulfonic acid; PKC, protein kinase C; H-7, 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine; W7, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide.

This peptide sequence which is based on the sequence of the autoinhibitory, regulatory region of PKC serves as a pseudosubstrate competitive inhibitor, presumably by binding to the catalytic domain. Thus, we used the peptide inhibitor to dissect away the role of PKC. The substituted version, [Glu]PKC (19-36), serves as control and had no effect on H currents throughout.


ACKNOWLEDGEMENTS

We thank Stephen Smith for his valuable suggestions and Judie Schumann for excellent technical assistance and dedication.


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