From the
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
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
A recent body of evidence supports the existence of putative
H
We demonstrated that activated human neutrophils exhibited
voltage-dependent H
We characterized the conductive and voltage
properties of H
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
In summary, we characterized
H
We thank Stephen Smith for his valuable suggestions
and Judie Schumann for excellent technical assistance and dedication.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
) and its dismutated product
H
O
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.
-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.
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 Me
SO; 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 Me
SO 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
Me
SO) 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.
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
To separate whole-cell
H Currents on
pH Gradients across the Plasma Membrane
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
Both fMLP and rhTNF on Whole Cell H
Currents
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
Activated
H Currents
Activated by fMLP or rhTNF
in Human Neutrophils
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
pH
o
/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
The involvement of NADPH oxidase in
the activation of H Currents
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.)
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.
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.
] 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) .
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.
,
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.
]PKC (19-36), serves as control and
had no effect on H
currents throughout.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.