1 Division of Clinical Biochemistry, University of Geneva Medical School, 1211
Geneva 14, Switzerland
2 Division of Infectious Diseases, University Hospital Geneva, 1211 Geneva 14,
Switzerland
3 Immunology Laboratory, Faculty of Sciences, University Nancy 1, 54506
Vandoeuvre-les-Nancy, France
* Author for correspondence (e-mail: Sten_Theander{at}hotmail.com )
Accepted 6 May 2002
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Summary |
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Key words: Exocytosis, ATP affinity, Granule populations, Ca2+, Neutrophils
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Introduction |
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The current model of the exocytotic mechanism is mainly based on work in
excitable cells. Numerous cells of the immune system, such as mast cells,
neutrophils or cytotoxic T-lymphocytes, also use regulated granule exocytosis
to fulfil their physiological role. Taking the example of the neutrophil, some
proteins belonging to the SNARE family have been identified, whereas others,
among them NSF, have not been found in neutrophils (reviewed in
Ligeti and Mocsai, 1999).
Furthermore, neutrophils lack voltage-gated Ca2+ channels
(Krause and Welsh, 1989
), and
docked vesicles are not found in unstimulated neutrophils
(Borregaard et al., 1993
).
In view of these differences between excitable cells and neutrophils we
wanted to assess the role of ATP in Ca2+-induced exocytosis of
neutrophils and to determine the dose-response curve between intracellular ATP
and exocytosis. Previous investigations on permeabilized mast cells and
neutrophils revealed both ATP-dependent and -independent pathways of
exocytosis (Churcher and Gomperts,
1990; Cockcroft,
1991
; Rosales and Ernst,
1997
).
Here, we used chemical depletion of intracellular ATP and determined the
resulting time-course of ATP depletion by using the luciferase assay
(Stanley and Williams, 1969).
We studied Ca2+-induced exocytosis, using both patch-clamp
capacitance measurements (Lollike and
Lindau, 1999
) and the ß-glucuronidase release assay
(Nüße et al.,
1997
), over a wide range of intracellular ATP concentrations.
Since different granule populations in neutrophils have different
Ca2+ sensitivities for exocytosis
(Borregaard et al., 1993
;
Lew et al., 1986
;
Nüße et al., 1998
),
it was, furthermore, possible to address the ATP dependence of these granule
populations. This approach gave for the first time an estimated Km
for ATP dependence of exocytosis of different granules in the same cell. Our
results show different ATP requirements for the release of two main granule
population and substantial release of apparently undocked granules in the
absence of ATP.
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Materials and Methods |
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Measurements of intracellular ATP Cell extract preparation
A neutrophil suspension of 106 cells per ml of ES was kept on
ice. Just before further preparations, the cell suspension was warmed to room
temperature. For incubations, 0.5 ml of cell suspension was centrifuged for 3
minutes at 224 g. The supernatant was taken off, replaced with
ATP depletion medium and left to incubate for various times. Aliquots were
then centrifuged again for 3 minutes at 224 g (for incubation
times less than 2 minutes the time of centrifugation had to be shortened
appropriately), and the supernatant was quickly removed and replaced with ice
cold ES + 0.01% Triton X-100 for lysis of cells. After lysis for 5 minutes the
aliquots were centrifuged for 3 minutes at 2012 g. The
supernatant was taken and quickly stored at -20°C for later analysis. For
control samples only the lysis step of the procedure was applied.
ATP measurements
For measurements of intracellular ATP, the luciferase bioluminescence assay
(Stanley and Williams, 1969)
was used. Firefly luciferase extract (FLE50 Sigma) was dissolved in water (10
mg/ml) and 1 ml of the dissolved extract was added to 200 ml arsenate buffer
(100 mM Na2HAsO4, 20 mM MgSO4, pH 7.4 with
H2SO4) and mixed in the dark at 4°C for 90 minutes.
An ATP standard was constructed by adding various amounts of MgATP salt to a
glycine buffer (glycine 50 mM, pH 11 with NaOH). For the bioluminescent assay
of ATP, 100 µl cell extract or ATP standard was added to 5 ml enzymatic
mixture, and light emission was measured with a LS 8000 beta counter
(Beckman). After calibration against the ATP standard, the ATP content of the
cell extract was determined. For the calculation of intracellular ATP
concentrations, the cell count (106 per ml) and an estimate of the
neutrophil volume (Nüße and
Lindau, 1988
) to 250 µm3 (=250 fl) was used to
calculate the dilution factor for the cell extract.
Capacitance measurement of secretion
For patch-clamp recordings, the neutrophils were placed on glass
coverslips. After 3-5 minutes, non-adherent cells were washed away with ES or
ES lacking glucose for ATP depletion experiments. The adherent cells were
incubated with ES containing 5 µg/ml cytochalasin B or, for ATP depletion
experiments, with ES without glucose supplemented with 6 mM 2-deoxyglucose, 5
µM antimycin and 5 µg/ml cytochalasin B. Treatment with cytochalasin B,
an inhibitor of actin polymerization, enhances Ca2+-induced
exocytosis of primary, secondary and tertiary granules
(Lew et al., 1986) and is
required for fMLP-stimulation of primary granule exocytosis from human
neutrophils. All experiments were done at room temperature. For ATP depletion
experiments, the time between incubation and patch rupture was carefully noted
and registered as the incubation time. Experiments were performed with an
inverted microscope (Nikon, Diaphot) with a 40x oil immersion objective.
Patch pipettes were pulled from borosilicate glass capillaries (GC150F-10,
Clark Instruments, Reading, UK) on a Model P-97 puller from Sutter Instruments
(Novato, CA). Pipette resistance was between 4 and 10 MOhm. Patch-clamp
recordings were performed with a List EPC 9 amplifier (HEKA, Darmstadt,
Germany) in voltage-clamp mode. Capacitance measurements were performed after
applying a 1-kHz, 50-mV peak to peak sinusoid stimulus from a dc holding
potential of -10 mV. The `sine+dc' mode of the software lock-in extension of
the PULSE software was used to calculate the equivalent circuit parameters
Cm (membrane capacitance), membrane conductance (Gm) and
access resistance (Ra) from the current recordings.
Measurement of exocytosis in suspension
1.25x106 cells were suspended in 500 µl of ice-cold
medium. Aliquots were then warmed to 22°C and either glucose (controls) or
6 mM deoxyglucose plus 5 µM antimycin were added. The aliquots were then
left to incubate for various times (controls for 30 minutes). 5 minutes prior
to stimulation, 5 µg/ml cytochalasin B was added to the aliquots to enhance
exocytosis (see above). Stimulation of exocytosis was elicited by adding 1
µM ionomycin, 1 µM fMLP or the DMSO vehicle only. Incubation was
terminated by rapid cooling with an equal volume of ice-cold buffer and
further cooled on ice before centrifugation (800 g for 10
minutes). ß-glucuronidase in the supernatant was measured
fluorimetrically with 4-methylumbelliferyl-glucuronide as described previously
for ß-glucosaminidase
(Nüße et al.,
1997). The results were calculated as a percentage of initial
total cellular content. Background exocytosis (5 to 10% of total cellular
content) was subtracted. This background was independent of any
pre-incubation. The total enzyme content was determined from an aliquot of the
same cell suspension treated with 0.025% Triton X-100 for 5 minutes at
37°C.
Measurement of intracellular Ca2+ after ATP depletion
The intracellular free Ca2+ concentration
([Ca2+]i) was measured using the fluorescent
Ca2+ indicator fura-2/AM. Cells (2x107 per ml)
suspended in ES containing 0.1% BSA were loaded for 45 minutes at room
temperature with 2 µM fura-2/AM. Just before measurement, 0.5 ml of the
cell suspension was taken and centrifuged for 3 minutes at 224
g. The supernatant was taken off, and the cells were
resuspended in 2.5 ml glucose free ES and transferred to a cuvette for
measurements. The excitation wavelength was alternating between 340 and 380
nm, and emission was recorded at 509 nm at 0.26 Hz in a Perkin Elmer LS-50B
spectrofluorimeter. 5 µg/ml cytochalasin B was added at time zero when the
recordings started. For the ATP depletion protocol it was found that antimycin
displayed too much autoflouresence to be used with fura-2. Hence, it was
replaced by 2 µM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone),
another blocker of the mitochondrial ATP production. 6 mM of deoxyglucose and
2 µM FCCP were added to the cuvette at 10 and 20 minutes, respectively,
after which measurements proceeded for another hour. Calibration was performed
for each cuvette by sequential addition of 1 µM ionomycin to measure
Ca2+-saturated fura-2 signal (Rmax), followed by the
simultaneous addition of 8 mM EGTA and 3 mM Tris (pH 9.3). Finally, 0.1%
Triton X-100 was added to measure the Ca2+-free fura-2 signal
(Rmin). [Ca2+]i was calculated from the
340/380 nm ratio (R) according to the formula
(Grynkiewicz et al., 1985):
[Ca2+]i=KD(F2min/F2max)((R-Rmin)/(Rmax-R))
where F2min and F2max are the fluorescence at 380 nm at
times corresponding to Rmin and Rmax, respectively, and
KD=224 nM.
Analysis
The plot of intracellular ATP concentration, [ATP], over time of ATP
depletion (t) was fitted to a sum of two exponential functions: [ATP]=2111
µMxe-t/3.5+250 µMxe-t/27. The value of
[ATP] has three potential sources of error, the measurement of ATP, the
fitting and the estimation of the cytoplasmic volume, which was based on
electron microscopy micrographs. The absolute value of [ATP] may have a
significant error margin; however, the relative decline of [ATP] over time is
well represented by the fit-function. From capacitance recordings, the
amplitude of exocytosis, Cm, was calculated as final
capacitance minus initial capacitance immediately after patch rupture. Cells
with the same intracellular solution and similar times of ATP depletion prior
to patching were grouped in 20 minutes intervals. All cells with more than 60
minutes of ATP depletion were pooled in one group. Choosing smaller time
intervals for the groups of cells did not change the overall shape of the dose
response curves (data not shown). The mean ATP depletion time and the mean
Cm were calculated. For each group, the mean [ATP] was
calculated from the mean ATP depletion time. The plots of
Cm
over [ATP] were fitted with a logistic equation:
Cm=
C0/[1-(Km-1/[ATP])n]
where
C0 was the capacitance change at [ATP]=0,
Km was the apparent affinity, and n was the cooperativity factor.
Data are presented as mean±s.e.m.
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Results |
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ATP requirements of different granule populations
We used two complementary techniques to stimulate neutrophil exocytosis by
Ca2+, the Ca2+ ionophore ionomycin and the perfusion of
Ca2+ buffers through the patch pipette. Patch-clamp capacitance
measurements monitor the increase in plasma membrane surface during
exocytosis. The patch pipette provides a direct control of the cytosolic
Ca2+ concentration independently of cellular Ca2+
regulation. Furthermore, the amplitude and the kinetics of exocytosis are
recorded, and two granule populations are released by quite different
Ca2+ concentrations
(Nüße et al.,
1998). Infusion of a 10 µM Ca2+ pipette solution
elicited the release of peroxidase-negative granules (secondary and tertiary
granules), whereas 300 µM elicited exocytosis also of primary granules
(Nüße et al.,
1998
). Under these experimental conditions, almost all neutrophil
granules are released. The differences in Ca2+ sensitivities make
it possible to investigate the ATP dependence of different granule populations
by infusion of the appropriate Ca2+ buffers.
In Fig. 2, representative
examples of capacitance traces from cells in control conditions (0 minutes
ATP-depletion) or after various times of incubation in an ATP-depleting medium
and stimulated with either 10 µM (A) or 300 µM (B) Ca2+ in
the pipette are displayed. Under control conditions, cells were in
glucose-containing medium without metabolic inhibitors, and the patch-pipette
contained 1 mM ATP. For cells in ATP-depleting medium, the pipette solution
did not contain ATP. 10 µM Ca2+ in the pipette stimulated an
essentially monophasic increase in capacitance. The traces (control and 10
minutes ATP depletion) obtained using 300 µM Ca2+ in the pipette
displayed typically biphasic secretion. ATP depletion for more than 15
minutes resulted in a progressive loss of secretion. For both Ca2+
concentrations, however, the cells display robust exocytosis also after very
long (>60 minutes) incubations in ATP-depleting media. The initial
capacitance of the neutrophils was unaffected by incubation in the
ATP-depleting medium (Fig. 2C),
demonstrating that the ATP depletion itself did not induce exocytosis.
|
Unlike the situation in many other cell types
(Henkel and Almers, 1996),
exocytosis in patch-clamped neutrophils does not appear to be accompanied by
concomitant endocytosis, and the capacitance traces usually reach a stable
plateau value. The difference between the capacitance value at the plateau and
the initial capacitance,
Cm, therefore, directly reflects
the amplitude of exocytosis
(Nüße et al., 1998
;
Nüße and Lindau,
1988
). Fig. 3 shows
Cm as a function of time of incubation in ATP-depleting
medium for cells stimulated with 10 µM Ca2+ or with 300 µM
Ca2+. In both cases, exocytosis was reduced by ATP depletion but
reached similar stable amplitudes after more than 40 minutes. The decline of
exocytosis was delayed with higher Ca2+ stimulation, suggesting
different ATP requirements for different granule populations. By calculating
the intracellular ATP concentration at the time of patch rupture for each
experiment (see Materials and Methods), a dose-response curve for exocytosis
versus intracellular ATP could be constructed
(Fig. 3B). The
Cm obtained with 10 µM Ca2+ could be well
fitted by a logistic function with an apparent Km of 328 µM ATP
and a cooperativity factor of 2.0. Furthermore
56% of the granules were
released in an ATP-independent manner. Secondary and tertiary granules have an
average capacitance of 1.1 fF (Lollike et
al., 1995
), thus some 1250 granules were released at very low
intracellular ATP concentrations. The dose-response curve for stimulation with
300 µM Ca2+ shows that exocytosis remained unaltered down to
concentrations of
200 µM ATP. At lower values, however, exocytosis
dropped steeply down (Km of 95 µM, cooperativity 3.7) to a level
that was, interestingly, similar to that obtained with 10 µM
Ca2+. This suggests that the secretion of granules that require
more than 10 µM Ca2+ was completely abolished at low ATP
concentrations.
To exclude the possibility that ATP depletion caused irreversible damage to the exocytotic machinery of the cells, we re-introduced ATP, using the same pipette solutions as for the controls (1 mM ATP), after prolonged depletion. On the basis of Fig. 3A, cells were treated with metabolic inhibitors for at least 25 and 40 minutes before stimulation with 10 µM Ca2+ and 300 µM Ca2+, respectively, to lower the intracellular ATP concentration to levels that affect granule exocytosis. Reperfusion of these cells with 1 mM ATP in the pipette solution completely reversed the effect of ATP depletion with both Ca2+ concentrations (Fig. 3C).
ATP may not only influence the amplitude of exocytosis but also its speed.
We measured the maximal rate of secretion by taking the maximum derivative of
the capacitance traces. The results, plotted as a function of intracellular
ATP at the time of patch rupture, are given in
Fig. 4. The maximum rates of
capacitance increase displayed a strikingly similar ATP dependence, as did the
measurements of total increase in capacitance. The kinetic analysis confirmed
the different Km values and the cooperativity for stimulation with 10 µM
and 300 µM Ca2+. In an ATP-consuming enzymatic process, the
concentration of ATP should affect the rate of secretion. It is unlikely that
ATP was completely used up during secretion. Therefore, the amplitude of
secretion should only be affected if the ATP concentration had an influence on
the number of granules that are capable of fusion. This phenomenon, called
submaximal secretion in response to submaximal stimulation
(Nüße et al.,
1998), resembles the effect of different Ca2+
concentrations on the kinetics and amplitude of exocytosis in neutrophils.
Like Ca2+, the ATP concentration appears to influence the size of
the granule pool that is available for exocytosis. For each granule in this
pool of available granules, the probability of fusion may be the same.
Therefore, the rate of exocytosis would be directly linked to the number of
fusing granules.
|
Primary granule exocytosis following ATP depletion
In order to have an independent assessment of the ATP dependence of primary
granule exocytosis we used the ß-glucuronidase release assay for the
detection of exocytosis in conditions of ATP depletion.
Fig. 5A shows the results of
ionomycin-induced secretion as a function of intracellular ATP at the
beginning of stimulation, as calculated from the time-course of ATP depletion.
Also shown is the response when exocytosis was elicited by receptor
stimulation with 1 µM fMLP. All values were normalised to their respective
value in the control situation. Ionomycin-induced primary granule release had
an ATP affinity of 55 µM and an apparent cooperativity of four. The result
obtained by receptor stimulation, however, displayed a very high sensitivity
to decreases in intracellular ATP. The high ATP dependence of receptor
stimulation may reflect the need for phosphorylated compounds, which are
essential in the stimulus response coupling. The direct stimulation with
Ca2+ ionophores or Ca2+ buffers in the patch pipette
probably bypassed these requirements.
|
As shown previously (Nüße et
al., 1998), 10 µM Ca2+ in the patch pipette
stimulates mainly the complete release of secondary and tertiary granules, and
300 µM Ca2+ stimulates the release of primary as well as
secondary and tertiary granules. The difference between the amplitude of
secretion with 300 µM Ca2+ and 10 µM Ca2+ should,
therefore, reflect primary granule release alone. Thus we could obtain the ATP
dependence of primary granule release under patch-clamp conditions by
subtracting the curve fitted to the ATP dose-response relationship (from
Fig. 3B) obtained for 10 µM
Ca2+ from the one obtained for 300 µM Ca2+.
The direct comparison of the ATP dependence of primary granule exocytosis induced by ionomycin or by Ca2+ buffers in the patch pipette is shown in Fig. 5B. Both methods revealed a sharp fall in exocytosis with a midpoint around 60-80 µM ATP. It is interesting that two so different approaches to measuring primary granule release give, essentially, the same result.
The capacitance measurements suggested that essentially no primary granule
exocytosis is possible in the absence of ATP, whereas the ß-glucuronidase
release assay indicated some ATP-independent secretion. Possibly, this
discrepancy arose as a consequence of some overlap in vesicle contents such
that a small amount of ß-glucuronidase may have been derived from
vesicles that are less sensitive to ATP depletion than the primary granules.
In fact, secondary or tertiary granules may contain low amounts of
ß-glucuronidase (Edwards,
1994). In control cells, ionomycin released about 45% of the total
cellular ß-glucuronidase content. The fraction of ATP-independent
granules (0.2) therefore represented less than 10% of the total. We conclude
that no or few primary granules were released in the absence of ATP.
The ß-glucuronidase release assay gave values that are somewhat left-shifted with respect to those obtained using capacitance measurements. This may reflect ATP washout into the patch pipette that we have neglected in the calculation of the intracellular ATP concentration. Consequently we slightly overestimated the true ATP concentration sensed by the exocytotic machinery. Nevertheless, the two rather different techniques provided very similar results.
Effects of ATP depletion on intracellular Ca2+
concentrations
In the patch-clamp situation, the intracellular solute composition is
clamped by the pipette solution, whereas in the ß-glucuronidase release
assay, changes secondary to ATP depletion such as alterations of ionic
composition and, in particular, intracellular Ca2+, may well occur.
In order to investigate this we measured [Ca2+]i after
depletion of intracellular ATP. Fig.
5C shows that [Ca2+]i increased slowly after
ATP depletion and that most of the increase occurs after 40 minutes of ATP
depletion. After 1 hour of ATP depletion, [Ca2+]i rose
to 800 nM, a level that stimulates very little exocytosis of secondary
and tertiary granules in patch-clamp experiments
(Nüße et al., 1998
)
and is well below the EC50 for release of primary granules in
non-permeabilised cells (Lew et al.,
1986
). These results are therefore in good agreement with the
absence of any increase in initial capacitances
(Fig. 2C) and with the stable
baseline during ATP depletion in the ß-glucuronidase release assay.
Addition of ionomycin even after 1 hour of ATP depletion caused a rapid and
sharp rise in [Ca2+]i, which is known to induce
exocytosis in human neutrophils (Lew et
al., 1986
). We conclude that the slow rise in
[Ca2+]i during ATP depletion did not cause significant
exocytosis and did not interfere with the stimulation with ionomycin.
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Discussion |
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ATP-independent exocytosis without docking?
It has been claimed that `it is dogma that neutrophil granules are not
docked' (Lollike et al.,
1999), and it is indeed a puzzling circumstance that granules of
resting neutrophils do not appear to be docked to the plasma membrane when
seen in electron microscopy micrographs (e.g.
Borregaard et al., 1993
).
Docking could be very brief and immediately precede fusion. However, our
measurements detected over 1000 granules that were released in the absence of
ATP. A pool of this size should not go unnoticed if they were accumulated just
beneath the plasma membrane. We have to conclude that these granules can dock
and fuse with the plasma membrane without using ATP. A similar phenomenon has
indeed been described for the `reserve' granules in sea urchin eggs
(Chestkov et al., 1998
). The
granules of unstimulated neutrophils may be already primed. However, priming
is reversible (Klenchin and Martin,
2000
) with a half-life of
4 minutes in permeabilized
chromaffin cells (Holz et al.,
1989
). Most probaby, primed granules would be unprimed during the
prolonged ATP depletion in our experiments. Alternatively, only a few granules
may fuse directly with the plasma membrane. Then, more granules fuse with the
membrane of granules that are already inserted into the plasma membrane. Such
cumulative fusion occurs in other cells such as eosinophils
(Scepek and Lindau, 1993
) and
pancreatic ß-cells (Bokvist et al.,
2000
). However, there is no evidence, that neutrophil granules are
chained up or otherwise associated prior to exocytosis. Thus the problem of
how the granules attach (dock) to and fuse with the target membrane without
ATP remains to be solved.
Localisation of the ATP-sensitive element to the granules?
Our results show that primary granule release has a strong dependence on
ATP, with a apparent Km in the range of 80 µM and a high degree
of cooperativity (n=4). A similarly high degree of cooperativity was
also found for the Ca2+ dependence of primary granules
(Nüße et al.,
1998). The high cooperativity for ATP dependence as well as
Ca2+ dependence may indicate that complexes of several primary
granules have to be formed prior to or in the process of exocytosis.
The granules released at lower Ca2+ concentration probably
represent a mixture of secondary, tertiary granules and a small contribution
from secretory vesicles (Nüße
et al., 1998). This heterogeneous granule population showed a more
shallow ATP dependence than the primary granules, perhaps reflecting several
underlying processes. The apparent Km for release of this
population was higher than for primary granules. In contrast to the primary
granules, the release process is not absolutely dependent on ATP as more than
half of this population can be released also in the absence of ATP. Our
experimental approach does not permit us to further distinguish the ATP
dependence of the different subpopulations. It is possible that those
vesicles, which are released in an ATP-independent manner, represent a single
subpopulation. The observation that different granule populations display
different ATP affinities suggests that the ATP-dependent mechanism is granule
specific, at least in part. This is obviously in contradiction to the idea of
a universal ATP-dependent step in granule exocytosis. Our data do not exclude
a common ATP-dependent mechanism, but this mechanism is not rate limiting in
neutrophils. They do, however, indicate that the ATP-sensitive elements are
granule specific and may, therefore, be localised to the granules.
The nature of the ATP-requiring steps
Which ATP-binding proteins could explain the apparent Km for ATP
of neutrophil exocytosis? Multiple ATP-dependent steps may be involved. The
Km of 80 µM ATP for the primary granule release is much lower
than has been reported for NSF and for other cells. Holz et al. found a
half-maximal activation of exocytosis in chromaffin cells at 400 to 600 µM
ATP (Holz et al., 1989),
compatible with the Km of 650 µM that was determined for NSF in
vitro (Tagaya et al., 1993
).
It therefore appears unlikely that NSF is causing the ATP dependence of
primary granule exocytosis. The apparent Km for release of
secondary and tertiary granules (330 µM) is closer to the reported
Km of NSF and might be compatible with NSF participating in the
release machinery for one or both granule types.
NSF was not found when screening a cDNA library from HL-60 cells, a cell
line derived from neutrophil precursors
(Ligeti and Mocsai, 1999). A
possible explanation for the absence of NSF in neutrophils is that the
hydrolysis of ATP by the NSF serves mainly to dissolve the fusion complex and
thereby prepare for a second round of fusion events
(Burgoyne and Morgan, 1998
;
Robinson and Martin, 1998
).
Neutrophils are terminally differentiated cells with a very short life span
and may not be capable of repeated rounds of fusion events
(Ligeti and Mocsai, 1999
),
possibly making the NSF unnecessary.
Numerous other ATP-binding proteins may be involved in exocytosis. The
ATPase p97 is a homolog of NSF and could take up some of its function
(Robinson and Martin, 1998).
Many protein serine/threonine kinases have a Km below 100 µM ATP
(Edelman et al., 1987
). Protein
kinase C requires only 6 µM ATP for half-maximal activity. Protein kinase A
(Km of 40 µM) has recently been implicated in insulin secretion
from ß-cells (Takahashi et al.,
1999
). Phosphoinositides play an important role in exocytosis
(Klenchin and Martin, 2000
;
Pinxteren et al., 2001
). The
phosphoinositide kinases, phosphoinositide 3-kinase (Km<10
µM), phosphoinositide 4-kinase [Km 18 µM
(Porter et al., 1988
)] and
phosphoinositide 5-kinase [0.5 mM (Singhal
et al., 1994
)] generate important signalling molecules.
Proteins with a Km for ATP significantly higher than 80 µM and 330 µM are unlikely to represent the critical steps in primary and secondary/tertiary granule exocytosis, respectively. Furthermore, the reversibility by reperfusion with ATP (Fig. 3C) suggests that the ATP-sensitive element acts rapidly and is either constitutively active or is activated by Ca2+ infusion through the patch pipette.
Why neutrophils may be different
Immune cells appear to have their own way of regulating exocytosis, namely
stimulation by either Ca2+ or guanine nucleotides alone
(Pinxteren et al., 2000),
absence of voltagegated Ca2+ channels, lysosome-like granules that
undergo regulated secretion (Griffiths,
1996
), little evidence for docked vesicles and low numbers or
absence of SNARE proteins (Ligeti and
Mocsai, 1999
). The neutrophil has the additional feature of
several populations of releasable granules with distinct requirements for
exocytosis.
Infection often occurs in poorly irrigated regions of the body. Neutrophils
are designed to combat such infections. They have few mitochondria and
therefore use mainly oxygen-independent glycolysis to derive energy
(Edwards, 1994). However,
glucose supply may be low at sites of infection
(Chavalittamrong et al., 1979
).
A low Km for ATP dependence of the secretory machinery may be
needed to keep the cells functional in such situations. Docking and priming of
granules before reaching the site of infection could solve the energy
requirement for exocytosis. However, it must be remembered that those cells in
which docking has been observed are part of a fixed geometric arrangement
within the tissue, whereas the neutrophil is a highly mobile cell for which
the release target(s) are not defined by any tissue geometry. Apparently,
stimulation of neutrophils with chemoattractants does not cause docking
(Lollike et al., 1999
).
Furthermore, it would be rather hazardous to have primary granules docked and
ready to fuse in large numbers given their toxic contents.
In conclusion, the ATP requirement for neutrophil exocytosis appears to be
a feature of individual granules instead of being a general mechanism for all
granules within the cell. This suggests that the rate limiting ATP-dependent
step in exocytosis involves granule-specific elements, such as protein or
lipid kinases or their substrates, located on the granules. In addition, about
50% of all secondary and tertiary granules are secreted at very low ATP
concentrations despite the fact that they do not appear to be docked to the
plasma membrane. For comparison, only 4% of the vesicles in chromaffin cells
undergo exocytosis in the absence of ATP
(Holz et al., 1989). Thus, a
reversible priming step either before or after docking is not obligatory for a
substantial number of neutrophil granules. Our results strengthen the idea
that immune cells have adapted the mechanism of exocytosis to their particular
physiological needs.
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Acknowledgments |
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References |
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