Bioscience Reports, Vol. 9, No. I, 1989
ATP Inhibits Onset of Exocytosis in
Permeabilised Mast Cells
P. E. R. Tatham and B. D. Gomperts
Received October 20, 1988
ATP is not required for exocytosis from permeabilised mast cells, and therefore there is no direct role
for protein phosphorylation in the late stages of the activation pathway. We have measured the
timecourse of exocytosis from permeabilised cells triggered to release hexosaminidase following
addition of C a 2+ to cells equilibrated for 2 min with GTP-u
If ATP is included at the time of
permeabilisation, then exocytosis commences after a delay, the duration of which depends on the
square root of the product [Ca2+][GTP-7-S], and which may extend to beyond 3 min. When ATP is
excluded then the maximal rate of exocytosis is established within 3 secs of completing the effector
combination. These results suggest that the achievement of a new steady-state, induced by Ca2+ and
GTP-7-S , and required for exocytosis is inhibited by ATP. From this we conclude that dephosphorylation of an unknown regulator protein may comprise a step in the exocytotic pathway.
KEY WORDS: mast cells; exocytosis; G-protein; GE; calcium; ATP.
INTRODUCTION
T h e key event in stimulus-secretion coupling is an elevation in the level of cytosol
Ca 2§ a l t h o u g h there are exceptions (the most notable being the p a r a t h y r o i d
( B r o w n et al., 1987)). A r e q u i r e m e n t for an intact cellular m e t a b o l i s m is almost
certainly universal, p r o b a b l y reflecting a role for A T P as a d o n o r in p h o s p h o r y l a tion reactions. Using permeabilised secretory cells (Knight and Scrutton, 1986;
B a k e r et al., 1985; G o m p e r t s and F e r n a n d e z , 1985) we can n o w pose direct
questions regarding b o t h the identity and effective c o n c e n t r a t i o n s of all soluble
cytosolic effectors. F o r exocytosis o f histamine and lysosomal e n z y m e s f r o m
permeabilised mast cells an effector c o m b i n a t i o n comprising b o t h C a 2§ and a
guanine nucleotide m u s t be supplied (Howell et al., 1987). G u a n i n e nucleotides
regulate late events in exocytosis in m a n y secretory cells ( B a r r o w m a n e t a l . , 1986;
Cockcroft et al., 1987; Ullrich and W o l l h e i m , 1988; Vallar et al., 1987;
G o m p e r t s , 1989) a n d it has b e e n inferred that this action is m e d i a t e d by a
G T P - b i n d i n g p r o t e i n , (GE). A T P is n o t required, t h o u g h w h e n p r o v i d e d it
enhances the effective affinity of b o t h essential effectors t h r o u g h reactions
Department of Experimental Pathology, University College London, University Street, London
WC1E 6JJ, UK.
99
0144-8463/89/0200-0099506.00/0 (~ 1989 Plenum Publishing Corporation
100
Tatham and Gomperts
catalysed by protein kinase C (Howell et al., 1987; Cockcroft et al., 1987; Howell
1988). To learn about the sequence of the intracellular events it is
necessary to undertake kinetic experiments. Here we show that the onset of
exocytosis from permeabilised mast cells triggered with Ca 2§ and GTP-y-S ( a
non-hydrolysable analogue of GTP) is retarded when ATP is provided. This
suggests that the mechanism of triggering by Ca 2+ and GTP may involve a protein
dephosphorylation reaction.
et al.,
MATERIALS A N D METHODS
Mast cells obtained from the peritoneal cavity of a single rat were isolated
and purified by centrifugation through a 2 ml cushion of 25% Ficoll as previously
described (Howell and Gomperts, 1987). They were then washed and resuspended in a buffered salt solution (pH6.8) which comprised 137 mM NaCI,
2.7mM KC1, 20mM piperazine-N,N'-bis(2-ethanesulphonate) and lmgm1-1
bovine serum albumin. In those experiments in which the cells were to be
preincubated (e.g. with phorbol ester (Fig. 4), forskolin, dibutyrl cyclic AMP,
etc.), 5.6 mM glucose, 2 mM CaC12 and i mM MgCI2 were also included at this
stage. At the end of such preincubations the cells were transferred by washing to
a divalent cation- and glucose-free solution.
To measure the timecourse of exocytosis, ceils ( - 0 . 5 • 106) were incubated
for 5 min in 3 ml of buffer containing 6 mM 2-deoxyglucose, 5/~M antimycin A,
and 0.2 mM Ca.EGTA, pCa7 (in some experiments, pCa8). The cells were then
permeabilised by transferring 0.5 ml aliquots at 3 second intervals to six tubes
containing streptolysin 0 (SL-0; Howell and Gomperts, 1987) (0.2-0.4 IU m1-1
final) and ATP as indicated, in 0.5 ml, together with GTP-y-S (final concentrations as indicated). After 1.5 min a zero time aliquot (70/~1) was withdrawn from
each tube and quenched in ice cold 0.15 M NaCI/5 mM E D T A (0.5 ml, buffered
at p H 7 with 10raM K phosphate). At 2rain, the exocytotic reaction was
triggered by addition of 0.1 ml of 30 mM Ca.EGTA (pCa as indicated). This was
done in sequence at 3s intervals such that the fastest responding cells (i.e. those
loaded with the highest concentrations of Ca z+ or GTP-y-S) were triggered last
and then sampled and quenched first in a reverse sequence, starting 6 seconds
later. Thereafter, the six tubes were sequentially sampled and quenched as
described above at 3 s intervals (i.e. a cycle time of 18 s), timed with a clockwork
metronome (setting allegretto, 80 beats min -1, 4/4 time). Finally, the cells were
sedimented by centrifugation at 4~ (1800g, 5 min) and samples (typically 50/~1)
of supernatant were removed for determination of secreted /3-N-acetylgtucosaminidase (hexosaminidase) as previously described (Cockcroft and Gomperts,
1979a; Howell and Gomperts, 1987).
Ca 2+ was buffered at concentrations between 10-7M and 10-SM (pCa7 to
pCa5), and Mg z+ was set at 2 mM by the use of appropriate E G T A buffers which
were prepared as previously described (Barrowman et al., 1987).
ATP Inhibits Onset of Exocytosis
101
RESULTS
In these experiments, mast cells were pretreated with metabolic inhibitors to
the point at which they fail to respond to activating ligands such as compound
48/80. They were then permeabilised with streptolysin-O (Howell and Gomperts,
1987) in the presence of GTP-7-S, with or without ATP. After a further 2 min the
secretory reaction was initiated by addition of Ca ~+ and timed samples were
removed and quenched. Figure 1A illustrates the results of such an experiment in
(3TP-S it0gMi
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Fig. 1. Timecourse of GTP-)'-S- and Ca2+-dependent hexosaminidase secretion from permeabilised
rat mast cells. (A) Mast cells were treated with metabolic inhibitors for 2 min and then permeabilised
by treatment with SL-O in the presence of Ca.EGTA (0.1 mM, pCa7), 1 mM MgATP and GTP-y-S
as indicated. After a further 2 min Ca.EGTA (3 mM, pCa5) was added and timed samples were
removed, quenched in ice-cold phosphate-buffered saline and processed for measurement of secreted
hexosaminidase (GTP-7-S , 21.6 #M, ~ ; 10 #M, Q; 4.64/~M, i ; 2.15 #M, II; 1 #M, O; 0.464 #M,
T.) The inset illustrates the relationship between Iog[GTP-~,-S] and the reciprocal of the delay
(plotted on a logarithmic scale) before the commencement of exocytosis. (B) Mast cells were
permeabilised in the presence of Ca.EGTA (0.1 mM, pCa7), 1 mM Mg.ATP and 21.6 #M GTP-7-S.
After 2 min they were transferred to tubes containing 3 mM Ca.EGTA (final concentration) buffered
to regulate [Ca 2 + ] as indicated and processed as above (pCa5, Q; pCa5.25, &;_pCa5.5, II; pCa5.75,
I1'; pCa6, 9 pCa6.167, [].) The inset illustrates the relationship between log[Ca ~+] and the reciprocal
of the delay (plotted on a logarithmic scale) before the commencement of exocytosis.
102
Tatham and Gomperts
which the cells were first exposed to a range of concentrations of GTP-y-S
together with 1 mM MgATP and then triggered to secrete by elevating [Ca2+]
from pCa7 to pCa5. It can be seen that while the three higher concentrations of
the guanine nucleotide all induce the same final extent of secretion (approximately 70% in this experiment), the onset of release is delayed for periods which
become more prolonged as its concentration is reduced. The rate of release of
secretory product also declines as the concentration of guanine nucleotide is
reduced. At concentrations of GTP-y-S below 5.6/tM (i.e. 10 -5.33 M) the final
extent of release declines. The extension of the delays preceding the onset of
secretion becomes even more evident under these conditions.
The timecourses illustrated in Fig. 1B were obtained from the same cell
preparation as used in the experiment of Fig. la. Here, the cells were first loaded
with GTP-y-S at 21.6/~M and then stimulated to secrete by transferring the cells
to solutions containing Ca 2+ in the range pCa6.167-5. In this experiment the two
highest concentrations of Ca 2+ both induced a maximal level of secretion
(approximately 75%); this declined to 20% at pCa6.167. As with the previous
experiment, secretion is seen to occur only after a delay following completion of
the pair of essential effectors. As the concentration of Ca 2+ is reduced the delays
become more prolonged and the maximal rates of (normalised) secretion also
decline. Similar delays occur when the order of addition of the two effectors is
reversed (data not shown).
The above results are summarised in the insets to Fig. 1 in which we attempt
to discern the relationships between the effector concentrations and kinetic
parameters obtained from the exocytosis progress curves. We have defined the
delay (tau) as the time intercept of the tangent to the progress curves at the point
of maximal rate of secretion. The insets illustrate the relationships of log(1/tau)
vs Iog[GTP-y-S] and log[Ca2+] derived from the data of the experiment shown in
Fig. 1. Expressed in this format it is apparent that there exists a systematic
relationship between the concentrations of the two effectors and the factors which
determine the duration of the delay. In 5 separate experiments the average slope
of the lines relating log(1/tau) to log[Ca2+] was 0.489 + 0.054 (r > 0.975), and in
11 experiments the average slope of the lines relating log(1/tau) to log[GTP-?-S]
was 0.522 + 0.041 (r > 0.98). Thus
1/tau = k- {[Ca2+][GTP-7-S]} 1/2
and 1/k has the dimensions of a second order rate constant; in other words a
100-fold increase in concentration of either effector reduces the delay preceding
the onset of exocytosis to 10% of its original duration. In this series of
experiments the maximal extents of secretion varied widely (between 40 and
80%) as also did the onset times for secretion measured under comparable
conditions. However, the linearity and the slope of the log-log relationships are
remarkably consistent features, showing that the duration of the delays preceding
exocytosis depends on the square root of the concentrations of both effectors.
The average maximal rates from many experiments are presented in Fig. 2
and, in contrast to the delay parameter these are seen to be much less dependent
on effector concentration. These maximal rates were extracted from normalised
ATP Inhibits Onset of Exocytosis
103
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r
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effecfor concentration (togM)
Dependence of maximal rates of secretion on effector concentrations. The average
normalised maximal rates of secretion of hexosaminidase (i.e. the slopes of the normalised progress
curves which are generally linear over the range 25-75% of the maximal extent of secretion) are
plotted against the logarithm of the concentrations of Ca2+ and GTP-7-S. The slopes are:
(Caz+) = 1.09 (open circles, r=0.989, n=5 independent experiments), GTP-~,-S=0.96 (closed
circles, r = 0.98, n, the number of independent experiments at each concentration of GTP-~,-S is
indicated above each point).
Fig. 2.
I
progress curves in order to be able to c o m p a r e m a n y experiments. While this
procedure tends to reduce the spread of the measured rates, in any given
experiment the variation in the actual maximal rates is still small in comparison
with the variation in delay times.
The purpose of the experiment illustrated in Fig. 3 was to test the effect of
3mitting A T P . In this experiment we t o o k repeated samples f r o m single
incubations of cells in order to improve the time resolution, and in a g r e e m e n t
with previous results (Howell et al., 1987) we find that higher concentrations of
GTP-7-S are required to cause secretion when A T P is absent. H o w e v e r , under
these conditions the delays which precede secretion when A T P is provided (Fig.
3B), are effectively abolished (Fig. 3A). Release of hexosaminidase is clearly
apparent within 3 secs of completing the effector pair. In experiments (not shown)
in which we tested the effect of varying the concentration of A T P in the range
0 - 1 raM, we found that full expression of the delays is manifest at 30/~M A T P
and on some occasions as low as 10/uM. The non-metabolisable analogue of
A T P , A p p N H p (1 m M ) was without effect in this system, secretion occurring
abruptly in the presence of this c o m p o u n d (1 mM) as it does in the absence of
ATP. On the other hand A T P - v - S which can act as a thiophosphoryl donor
induces delays, producing traces closely resembling those (i.e. Fig. 3B) obtained
with A T P (not shown).
104
Tatham and Gomperts
B
(~
GTP-S
(logM)
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0
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(LogM)
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60
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Fig. 3. Effectof ATP on the onset kinetics of exocytosis.In this experiment the progress of secretion
of hexosaminidaseunder each set of conditionswas followedindividuallyin order to permit finer time
resolution, especially at the early time points. (A) Mast cells were treated with metabolic inhibitors
for 2min and then permeabilised by treatment with SL-O in the presence of Ca.EGTA (0.1 mM,
pCa7), GTP-y-S as indicated. After a further 2 min, Ca.EGTA (3 raM, pCa5) was added and timed
samples were removed, quenched in ice-cold phosphate-bufferedsaline and processed for measurement of secreted hexosaminidase.(B) As (A) except that MgATP (1 mM) was included at the time of
permeabilisation.
In view of our observation that the onset of secretion is delayed by ATP, we
tested the effect of pretreating the cells (i.e. before permeabilisation) with
compounds known to activate cyclic AMP-dependent kinase (protein kinase A)
and protein kinase C. Preincubation of the cells with dibutyrylcyclic AMP
(10 mM, 30 min in the presence of 1 mM isobutylmethyl xanthine) was without
effect on either the time-course or the final extent of secretion from
permeabilised cells triggered in the absence of ATP. Nor was there any effect of
preincubation with forskolin (5/~M, 5 min) even when cells were subsequently
permeabilised and triggered to secrete in the presence of ATP and cyclic AMP
(100/~M) (not illustrated). By contrast, preincubation with phorbol myristate
acetate (100 nM, 5 min) sharply reduced the delays such that they were now only
discernible at and below 10-TM GTP-%-S (Fig. 4). There was also an increase in
the rate of the already immediate exocytosis which occurs when A T P is not
provided (data not shown). We are thus unable to ascribe the ATP-induced
delays to phosphorylations catalysed by protein kinases A or C. Phorbol ester
pretreatment has the predicted effect of enhancing the affinity for the two
effectors and this most likely accounts for the accelerated onset in secretion which
we have observed. Since the cells were washed after treatment with the phorbol
ester, these results indicate that the reactions induced by protein kinase C and
which result in acceleration of exocytosis, had occurred before the permeabilisation and administration of the effector combination, Ca2+-plus-GTP-%-S.
ATP Inhibits Onset of Exocytosis
105
A
100I
GTP-S ItogM]
0
~ sc
.E_
o
~e-- 25
6
minutes after pCa5
10C
7E
B
[iTP-S [togM]
-5"67
-6.33
-6"67_7
5c
r-
GA
r-
minutes after pCa5
Fig. 4. Effect of pretreating cells with PMA on the timecourse of secretion of hexosaminidase. Cells
were incubated for 5 min at 37~ in the absence (A) or presence (B) of PMA (0.1/~M). They were
then washed once and permeabilised in the presence (i.e. without pretreatment) of metabolic
inhibitors, 1 mM ATP and GTP-7-S as indicated. After 2 min, CaEGTA (3 mM, pCa5) was added
and samples taken and processed as described.
DISCUSSION
O u r key o b s e r v a t i o n c o n c e r n s the inhibitory effect of A T P on the reactions
preceding the onset of exocytosis. In its absence secretion achieves its maximal
rate within 3 secs of completing the dual effector combination. W h e n A T P is
provided, exocytosis is p r e c e d e d by a delay, the d u r a t i o n o f which is inversely
p r o p o r t i o n a l to the square r o o t of the p r o d u c t of the concentrations of the two
effectors.
Since delays preceding exocytosis are i n d u c e d by A T P , but not by its
n o n - p h o s p h o r y l a t i n g a n a l o g u e A p p N H p , it is m o s t likely that they are due to
maintained p h o s p h o r y l a t i o n of an inhibitory regulator protein. Since exocytosis
106
Tatham and Gomperts
can occur when ATP is excluded it is clear that phosphorylation does not
constitute an essential step in the sequence of reactions leading to exocytosis, and
under these conditions secretion commences rather abruptly following completion
of the dual effector system. When ATP is-provided there is a tendency to
maintain the putative regulator in its inhibitory form and secretion then ensues
only after attainment of a new permissive steady-state. This implies the existence
of a rate-limiting step in the sequence of reactions initiated by GTP and Ca 2§
which is opposed by ATP. The non-phosphorylating analogue AppNHp is
without effect. There are of course mechanisms other than phosphorylation by
which ATP could modulate a cellular process. These would include catalytic
activation by adenylation, and through allosteric modification of target proteins
(both of cell surface receptors and intracellular proteins). In the case of the mast
cell, application of exogenous ATP leads to membrane permeabilisation (Cockcroft and Gomperts, 1979b), but this is an effect which is insignificant in
streptolysin-O treated cells. No other effects of ATP at the surface of these cells
have been recorded. Until proved otherwise, a mechanism involving phosphorylation must certainly be considered the most likely.
Since the duration of the delay in our experiments depends on the
concentrations of both the guanine nucleotide and Ca 2+, we suggest that
activation of a CaZ+-dependent protein phosphatase might be under the control of
the GTP-binding protein, GE. However, the kinetic experiments provide no clues
as to the identity of the catalytic entities (kinase and phosphatase) which mediate
these reactions.
There is a clear precedent for protein dephosphorylation as an essential step
in exocytosis in the trichocyst discharge process of Paramecium tetraurelia
(Momayezi et al., 1987; Gilligan and Satir, 1982). In this system also, ATP
appears to behave as an inhibitor (Wilmart-Seuwen et al., 1986). A 65 kDa
phosphoprotein has been identified which undergoes dephosphorylation within
one second of stimulation of wild-type, but not in those mutants (nd) which are
devoid of secretory organelles or which fail to undergo trichocyst discharge in
response to stimulation (Zieseniss and Plattner, 1985; Gilligan and Satir, 1982).
Injection of exogenous phosphatases such as calcineurin or alkaline phosphatase
induces exocytotic discharge and conversely, microinjection of anticatcineurin
antibody is inhibitory.
The different kinetic patterns of secretion generated by preincubation of the
mast cells with phorbol ester, and by provision of a phosphorylating environment
to permeabilised cells, have some resemblance to those obtained by stimulation
of intact cells with compound 48/80 (delay <5 secs, secretion complete within
20 secs) and with IgE-directed agonists (delay - 3 0 secs, secretion extending over
1-2min) (Gomperts and Fewtrell, 1985). Delays in the onset of exocytosis
following introduction of GTP-y-S into the cytosol of single mast cells have
recently been reported in experiments involving the use of patch pipettes to
monitor exocytotic degranulation (Fernandez et al., 1987; Zimmerberg et al.,
1987). Under the same conditions the reaction due to compound 48/80 was
prompt.
It is pertinent to ask why, since ATP appears to inhibit exocytosis, secretion
due to stimulation of intact cells with receptor directed agonists and ionophores is
ATP Inhibits Onset of Exocytosis
107
so sensitive to application of metabolic inhibitors? In intact cells ATP is required
to maintain phosphatidylinositol-4,5-bisphosphate, the substrate for formation of
inositol-l,4,5-trisphosphate and diglyceride. ATP is also needed in the transphosphorylation reaction to maintain GTP which is required both at the level of
receptor activation (Gp) and again in the later stages of the pathway (GE). In
mast cells it is also required as the donor in reactions catalysed by protein kinase
C, by which effector affinity is regulated and maintained (Howell et al., 1988). In
permeabilised cells none of these constraints apply.
From the kinetic data presented in this paper, which demonstrate that ATP
retards the onset of exocytosis, we propose that protein dephosphorylation may
be a rate limiting step in the stimulus-secretion pathway of rat mast cells and that
the enzyme controlling this event, a protein phosphatase, could be the target of
the GTP-binding protein GE. This proposal is illustrated schematically in
Fig. 5.
m
cQ |
EXOCYTOSIS
Fig. 5. Schematic representation of the relationships between the early events in stimulus-secretion
coupling and late events mediated by the G-protein G E. Two GTP-binding proteins are understood to
be involved in the complete stimulus-secretion pathway. The first of these to be involved, Gp
transduces receptor (R) mediated signals and activates inositide-specific phosphodiesterase (PPI-pde)
to generate inositol trisphosphate (IP3) and diacylglycerol (dag). In the permeabilised cells IP 3 leaks
out and control of Ca 2§ is by the use of CaEGTA buffers. Diacylglycerol, which is retained in the
plasma membrane, can activate protein kinase C if ATP is provided and this enhances the effective
alfinity for both Ca 2+ and guanine nucleotide in the exocytotic reaction. Both of these effectors are
essential but elevation of the one reduces the requirement for the o t h e r , indicating communication
between the Ca2§
protein and the G-protein G E. ATP exerts an inhibitory action by
preserving a control protein in a phosphorylated form, so retarding the onset of exocytosis. This can
be accelerated by increasing the concentrations of Ca 2+ and/or GTP-y-S. In the absence of this
nucleotide the protein is rapidly dephosphorylated so initiating prompt exocytosis. The scheme is a
hypothesis based on previously published data (Gomperts et al., 1987; Howell et al., 1987; Cockcroft
et al., 1987; Howell et al., 1988) as well as the kinetic experiments described here.
108
Tatham and Gomperts
ACKNOWLEDGEMENTS
W e are grateful for s u p p o r t f r o m the W e l l c o m e T r u s t , the A r t h r i t i s a n d
R h e u m a t i s m R e s e a r c h C o u n c i l a n d the V a n d e r v e l l F o u n d a t i o n . W e also t h a n k
D r J. E a s t e r b y for his help a n d advice.
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