Rat mast cells degranulate in response to microinjection of guanine
nucleotide
PETER E. R. TATHAM and BASTIEN D. GOMPERTS
Department of Physiology, University College London, University Street, London WC1E 6JJ, UK
Summary
Investigation of regulated exocytosis has frequently
required the use of permeabilised cell preparations.
This has provided evidence that Ca2+-binding and
guanine nucleotide-binding proteins can mediate
secretion. Since the manner and extent of membrane
permeabilisation affect the requirements for Ca2+
and guanine nucleotide, we have introduced such
effectors directly into intact, rat peritoneal mast cells
by microinjection. During this brief procedure (~1 s)
a glass needle forms a seal with the plasma membrane. Following injection and withdrawal of the
needle the membrane reseals without apparent loss
of cell contents. Using fluorescent dye, we estimate
that the volume injected is ~5fl and that the dilution
of injected solutes is —100-fold. Injection of the
nucleotides inosine triphosphate, guanosine 5'-[ythio]triphosphate (GTPyS) and guanosine 5'-[/Jyimido]triphosphate causes degranulation. The ECso
for GTPyS is ~10/iM (concentration in the needle)
equivalent to an intracellular concentration of
Introduction
The mechanism of regulated exocytosis has been investigated in many cell types by manipulating the composition
of the cytosol following permeabilisation of the plasma
membrane. Mast cells in suspension may be permeabilised
by a variety of agents, including bacterial cytolysins such
as streptolysin O (Howell and Gomperts, 1987). Streptolysin O generates lesions that allow the rapid leakage of the
soluble contents of the cytosol, including lactate dehydrogenase (Howell and Gomperts, 1987) and actin (Koffer
and Gomperts, 1989). At the same time, this treatment
provides access to the cytoplasm, so that if the cells are
permeabilised in the presence of guanine nucleotide
analogues such as guanosine 5'-[y-thio]triphosphate
(GTPyS), and if Ca 2+ is buffered in the micromolar range,
then exocytotic secretion occurs (Howell et al. 1987;
Gomperts et al. 1987).
The use of patch-clamp techniques has also led to many
new insights. By the use of the patch-pipette in the wholecell mode, the cytoplasm is brought into contact with an
effectively infinite volume of fluid within the pipette,
causing rapid dilution of the cytosol and permitting
control over its subsequent composition. The patch-pipette
can be used to measure the capacitance of the cell
membrane and thus to monitor exocytosis in real time. In
Journal of Cell Science 98, 217-224 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
~100nM. However, the effect of GTPyS is dependent
This
on the presence of Ca2+ in the external medium.
may be explained by a transitory influx of Ca2+ that
occurs during impalement, since the seal between
needle and membrane will not prevent the movement
of small ions. Thus an increase in cytoplasmic Ca2+
appears to be necessary for secretion induced by
GTPyS. Using metabolic inhibitors we have investigated the requirement for ATP. Under conditions
where [ATP]j has fallen to 60±18/iM (S.E.M., n=3) the
mast cell agonist, compound 48/80, is unable to
induce degranulation, yet injection of GTPyS still
activates the cells. Thus the ATP-dependent processes that mediate 48/80-induced secretion are not
part
of the pathway that is activated by GTPyS and
Ca2+.
Key words: mast cells, exocytosis, microinjection, calcium,
guanine nucleotides.
this way it has been shown that non-hydrolysable
analogues of GTP can serve as intracellular effectors
while, under these conditions, Ca 2+ acts as a modulator,
serving only to accelerate the process (Fernandez et al.
1984; Fernandez et al. 1987; Lindau and Niisse, 1987;
Neher, 1988; Penner, 1988).
These approaches have led to the identification of at
least two sites of action of guanine nucleotide, termed Gp
and GE (Gomperts et al. 1986; Fernandez et al. 1987;
Penner, 1988; Cockcroft et al. 1987; Gomperts and Tatham,
1988). However, the manner in which these influence or
control the activation of intact, as opposed to permeabilised, cells can only be inferred. In this paper we describe
the use of the microinjection technique to introduce
nucleotides into intact rat mast cells. The injection
procedure involves the introduction of a small volume of
fluid directly into the cytoplasm of a cell by means of a fine
glass needle. As the needle enters it forms a seal with the
plasma membrane and after injection, as it is withdrawn
from the cell, the membrane reseals without apparent loss
of cell contents. The process takes about a second and,
unlike the methods described above, inflicts minimal
damage.
Exocytosis in mast cells is accompanied by a substantial
morphological change as the numerous intracellular
granules fuse with the plasma membrane and release
217
their contents. We have used this 'degranulation' event,
which is readily detected in the light microscope, to assess
individual cell responses following injection. We define
here the conditions for the microinjection of soluble
substances into individual rat mast cells and describe the
effects of injecting nucleoside triphosphates in terms of
exocytotic degranulation. We find that the pattern of
responses to different nucleotides and nucleotide analogues is similar but not identical to that observed in
permeabilised cells. However, the microinjected cells
exhibit a Ca 2+ requirement for activation and so do not
resemble patch-clamped mast cells in the whole-cell
configuration (Neher, 1988), but are more similar to
streptolysin O-permeabilised cells, which require both
Ca 2+ and guanine nucleotide for full activation (Howell et
al. 1987). Activation by nucleotide injection is inhibited
when Mg2"1" is included in the injected solution.
Materials and methods
Microinjection of mast cells
Mast cells were obtained from adult male Sprague Dawley rats
and purified by sedimentation through Percoll as described
elsewhere (Tatham and Gomperts, 1990). The purified cells were
resuspended at ~ 5 x l 0 B m l - 1 in a buffered salt solution containing 137mM NaCl, 2.7 mw KC1, 1.8mM CaCl2> lmM MgCl2,
5.6mM glucose, lmgml" 1 bovine serum albumin and 20 mM
NaHepes, pH7.2; 200/d samples of this suspension were then
placed in 35 mm diameter plastic Petri dishes (Falcon). These had
previously been thermally impressed with a lettered grid (BBForm, Mecanex, Vuarpilliere, Switzerland) to enable the subsequent location of individual cells. These were allowed to adhere
to the covered dishes at room temperature for 20-40 min before
the addition of a further 2 ml of medium. After 1—2h, adherence
was usually complete. Before injections were commenced, the
buffer was aspirated from the Petri dish and replaced with fresh
medium. For most experiments this consisted of the standard
buffer solution described above. Ca2+-free medium was prepared
by omitting CaCl2 from the standard buffer solution and
including 1 mM NaEGTA, while low Na medium was obtained by
replacing NaCl in the standard buffer with potassium gluconate
(145 mM). Transfer to a different medium was always preceded by
two washes.
Cell microinjection was carried out using glass needles
prepared from thin-walled filament capillaries (1 mm diameter,
Kwik-fil, Clarke Electromedical Instruments, Pangbourne, UK)
drawn to a fine tip with a vertical needle puller (Kopf 720,
Tujunga, CA USA) and then back-filled with injection solution
(1-2^1), using a fine plastic tube connected to a micrometer
syringe. Filled needles were mounted in the holder of a Leitz
micromanipulator (Leitz Wetzlar, FRG) and a hand-held syringe
provided continuous gentle positive pressure, which was maintained as the needle was moved from cell to cell. Each injection
took approximately 1 s.
Assessment of cell responses
In order to evaluate the effect of injecting the nucleotide solutions
each was introduced into a group of at least 60 cells, usually
occupying one grid square in the Petri dish. Before treatment, a
video image of the field was printed. After injection (10-15 min
later) the cells within the field that had not been injected were
marked on the picture and after a further 10-20 min the
responses of the remainder were evaluated. Mast cell degranulation is a dynamic transformation that may be observed in the
light microscope as a characteristic morphological change (Bloom
and Chakravarty, 1970; Rolich et al. 1971). Under phase-contrast
illumination, it is manifest as a sequence of events in which the
cells successively lose their sharp circular contours and take on a
diffuse, grey appearance. This is frequently accompanied by the
emergence of free granule matrices undergoing Brownian motion.
Fig. 1 shows a phase-contrast image of mast cells some of which
218
P. E. R. Tatham and B. D. Gomperts
Fig. 1. Phase-contrast image of microinjected rat mast cells.
Adherent mast cells were microinjected with 1 mM GTP)S
(arrows) or a similar volume of water (asterisks). The
remaining cells were not injected.
were previously microinjected. The five cells indicated by arrows
were injected with an aqueous. GTPyS solution (1 mM in the
needle). Typical degranulation morphology is apparent and these
cells are readily distinguishable from control cells (asterisks)
injected with a similar volume of water. These are indistinguishable from the remaining uninjected cells. In this paper responses
are reported as the percentage of injected cells that degranulate.
Only full degranulations in which the refractive changes
described above were observed were scored as positive responses.
ATP permeabilisation and ethidium bromide loading
Medium was removed by aspiration from Petri dishes, each of
which contained approximately 106 adherent mast cells, and was
replaced by buffered saline lacking divalent cations and containing 500 /JM ATP, 1 mM EDTA and a range of concentrations of
ethidium bromide. Since high concentrations of the dye caused
intact mast cells to degranulate, neomycin (2.5 mM), which
inhibits phospholipase C (Cockcroft and Gomperts, 1985; Cockcroft et al. 1987), was also added to this medium. Alternatively,
the cells were pretreated in glucose-free medium with metabolic
inhibitors (5 JJM antimycin A and 6 mM deoxyglucose) to prevent
activation. After incubation of the dishes for 40 min at 37 °C in a
humidified atmosphere, the cells were rinsed twice with buffered
saline lacking CaCl2 but containing MgClj (2mM) and either
NaEGTA (3 mM) or EGTA/CaEGTA (3 mM, pCa>8) to reseal the
membranes and trap the dye. Under these conditions, cell
fluorescence could be measured reproducibly over a period of at
least 45 min.
Fluorescence measurements
The fluorescence of ethidium-stained cells was measured with a
modified Leitz Fluovert microscope fitted with a fixed stage and
an MPV photometer, the output of which was fed to a pen
recorder. Fura2 fluorescence measurements in individual cells
were performed using an apparatus previously described (Swann
and Whitaker, 1986). Estimations of [Ca 2+ ]| were calculated from
the ratio of the fluorescence signals obtained by exciting
alternately at 350 and 380 nm (Grynkiewicz et al. 1985).
Measurement of intracellular ATP
Intracellular ATP was measured by an adaptation of a method
described by Johansen (1987). Briefly, samples of cell suspension
were added to equal volumes of perchloric acid (final concentration 0.33 M) and kept on ice for at least 15 min. The precipitated
protein was then removed by centrifugation (1800 #). The
supernatanta were neutralised by adding Tris/KOH and were
kept on ice for a further 15 min before another spin to remove the
precipitated potassium perchlorate. The final supematants were
mixed with luciferin/luciferase reagent solution (ATP Bioluminescence CLS, Boehringer-Mannheim) and the constant bioluminescence light signal was measured using an LKB Luminometer.
Estimates of the concentration of cytosolic ATP were made
assuming a mean cytosol volume of 540 fl (see Results).
Materials
GTP)fi was supplied by Boehringer-Mannheim as a solution of its
tetralithium salt (100 mM). This stock solution was diluted
appropriately in water before injection. No significant difference
was observed between the responses of cells injected with 1 mM
GTP>6 obtained by diluting the stock solution in either water,
potassium glutamate (150mM, pH7.2) or KC1 (150mM, pH6.8).
All the remaining nucleotides were obtained as their sodium salts
from Boehringer-Mannheim, except for GTP and guanosine 5'[/iy-imido]triphosphate (GppNHp), which were supplied as lithium salts, and xanthosine triphosphate (XTP), which was
provided as a sodium salt by Sigma Chemical Co.; 100mM stock
solutions of each of these were obtained by dissolving them in
water with sufficient Tris to take the pH to 7. GTP was made up in
a similar way to 50 mM.
Bthidium bromide, compound 48/80, potassium gluconate,
deoxyglucose and antimycin A were obtained from Sigma. The
pentapotassium salt of fura2 was supplied by Molecular Probes
Inc.
Results
Determination of the final concentration of injected
solutions
Since mast cells are terminally differentiated and are
therefore (predominantly) diploid, the nuclear DNA can be
used, in an inverse fashion, as a probe for measuring the
concentration of ethidium as its fluorescent DNA adduct.
Calibration of cell fluorescence in terms of cytosol
ethidium concentration can be achieved by equilibrating
permeabilised cells with known concentrations of ethidium bromide. At increasing levels of intracellular dye,
binding to DNA increases until saturation is achieved,
and so by comparing either the saturating concentrations
or the concentrations for 50 % saturation for injected and
permeabilised cells, it is possible to evaluate the dilution
factor.
To make accurate measurements of fluorescence from
individual cells, it is necessary to remove any external
dye. Unfortunately, changing the medium to one that is
dye-free causes the nuclei of permeabilised cells to destain.
This problem can be overcome, however, by adopting a
method of permeabilisation that is reversible, so that the
dye is trapped in the cells. For mast cells this can be
achieved by exposing them to ATP4" and after dyeequilibration removing the free ATP by adding MgCl2
(Gomperts, 1983; Tatham and Gomperts, 1990). The
details of the procedure for plated cells are given in
Materials and methods.
Fig. 2 shows the relationship between ethidium concentration and cell fluorescence for both injected and ATPpermeabilised cells. The pattern of binding, saturation
and apparent quenching observed in the permeabilised
cells is repeated at much higher concentrations (in the
needle) for the dye-injected cells. The shift between the
two curves indicates the dilution of the injected dye as it
entered the cytosol. The concentration of dye giving 50 %
saturation of the fluorescence signal when admitted to the
cells through permeability lesions was 20 (JM. For injected
dye the needle concentration giving 50% saturation was
2.5mM and the ratio of these is 1:125. Taking into account
0.001
0.01
0.1
1
10
[Ethidium bromide] (mM)
100
Fig. 2. Relationship between cellfluorescenceintensity and
ethidium bromide concentration for permeabilised and
microinjected cells. Mast cells in dishes were permeabilised in
the presence of ethidium bromide at4 the indicated
concentrations by exposure to ATP ". After equilibration the
membranes were allowed to reseal by adding Mg2"1" to trap the
dye (neomycin or metabolic inhibitors were also present to
prevent degranulation; see Materials and methods for details).
After rinsing away external dye, thefluorescenceof groups of
5-10 cells were then measured. The data are plotted as single
cellfluorescenceintensity versus ethidium concentration (•).
Alternatively, mast cells in dishes were injected with ethidium
bromide solutions and after rinsing, the single cell fluorescence
intensity was measured. The data (•) refer to the
concentration of ethidium in the needle.
the fact that variations in the quantity injected will have
most effect at the half-saturation points and allowing for
other inaccuracies (see Discussion), we estimate that the
dilution is between 60- and 160-fold. Henceforth, as a rule
of thumb we assume a factor of 100.
The mean cell diameter of rat mast cells has been
estimated to be 13.5 /xm (Kruger et al. 1974), and the fluid
space within the cytoplasm of these cells occupies 42 % of
the cell volume (Helander and Bloom, 1974), so we deduce
a mean cytosol volume of 540 fl. On this basis the volume
injected is approximately 5fl.
Injection of nucleotides
The effects of injecting various nucleoside triphosphates
and nucleotide analogues into mast cells are summarised
in Fig. 3. GTP (50 mM, needle concentration) and XTP
(100 mM) failed to bring about degranulation of a significant proportion of the cells, while CTP and UTP at 100 mM
activated only a very limited number. Inosine triphosphate (TIP) and GppNHp were considerably more effective, while GTPyS was the most potent. Fig. 4 illustrates
the concentration dependence of degranulation in response to injections of the poorly hydrolysable guanine
nucleotide analogues, GTPyS and GppNHp. At concentrations above 100 ftM, corresponding to an intracellular
concentration of ~1 JIM, GTPyS caused the majority of cells
to degranulate within 1-5 min. The EC50 is 10 /JM,
equivalent to —100 nM inside the cell. GppNHp was less
effective and somewhat slower, degranulating no more
than about 20 % of the cells with an EC50 of 3 mM (—30 JCM,
intracellular). Since GTP, GTPyS and GppNHp were
provided as lithium salts, we tested the effects of injecting
LiCl on its own and together with GTPyS; 400 J/M LiCl did
not cause degranulation when introduced into cells nor did
it cause inhibition when provided at this concentration
together with GTPyS (100/*M).
Microinjection of mast cells
219
100
I
Ca 2 + (1.8 mM)
£ 80
V
r
o
^^•.
«
1
7
•a
2 60
/
1
£40
a
s? 20
No Ca 2 +
20
40
% Degranulated cells
60
Fig. 3. Response of mast cells to injections of nucleotides. Cells
were injected with different nucleoside triphosphates or
guanine nucleotide analogues dissolved in water. The
concentration of each in the needle was 100 /IM, except for
GTP, which was 50/iM. The data represent the mean and S.E.M.
of 3 experiments. In each experiment 60 cells were injected for
each nucleotide.
0.001
0.01
[Guanine
0.1
10
10
^ ^ (mM)
Fig. 4. Concentration dependence of the response of mast cells
to injections of guanine nucleotide analogues. Either GTPyS
(•) or GppNHp (A) was injected into mast cells at the
indicated concentrations (in the needle). Data represent mean
and S.E.M. of 3 experiments; 60 cells were injected for each
measurement.
Ca2+ requirement for degranulation
When GTPyS was injected into cells maintained in Ca 2+ free medium (lmM EGTA, lmM MgCl2; pCa>8), the
response was greatly reduced at all concentrations
(Fig. 5). This dependence on external Ca 2+ is unlikely to
be due to depletion of the ion from the cells, since they
were not transferred to the Ca2+-free medium until just
before they were injected. Under these conditions, plated
cells kept at room temperature remained responsive to the
mast cell agonist, compound 48/80 (10 ^gml" 1 ) for over 2 h
(2=95% degranulation). Although the time between penetration and withdrawal of the injection needle is brief and
occurs without apparent loss of cell contents, the seal
between the needle and plasma membrane is likely to
possess an impedance similar to that observed when cells
are impaled by conventional glass microelectrodes, i.e. in
the megohm range. In the presence of a steep Ca 2+
concentration gradient across the plasma membrane, it is
likely that each injection event will be accompanied by a
brief inward flux of Ca 2+ . This will then contribute a
transitory signal component that, together with the
injected guanine nucleotide, becomes sufficient momen220
P. E. R. Tatham and B. D. Gomperts
*~— o
0.001
0.01
0.1
1.0
[GTPySJnKdk. (mM)
10
o
100
Fig. 5. Effect of the presence of external Ca 2+ on the cell
responses to injections of GTPyS. Experiments were performed
in medium containing 1.8 mM CaCl2 ((•) same data as Fig. 4)
or 1 mM EGTA ((O) Ca 2+ omitted, see text for details). Data
represent mean and S.E.M. of 3 experiments; 60 cells were
injected for each measurement.
tarily to trigger exocytosis. Restoring Ca 2+ (1.8 HIM) to
dishes of cells previously injected with GTPyS (1 mM) in
the absence of Ca 2+ ( < 1 0 ~ 8 M ) had virtually no effect on
the injected cells, increasing the proportion of degranulated cells from 4 ± 1 % to 6±2% (S.E., n=3). This suggests
that an increase in cytoplasmic Ca 2+ during the injection
is necessary for secretion in these experiments.
In an attempt to detect an influx of Ca 2+ , cells were
loaded with the fluorescent Ca2+-indicator fura2. Since
mast cells incubated with the acetoxymethyl esters of
Ca2+-indicator dyes accumulate substantial amounts of
dye in their granules (Bibb et al. 1986; Aimers and Neher,
1985), it is more satisfactory to load the free acid form of
the dye directly by microinjection. During this process
considerable quantities of dye escape from the needle prior
to and after injection, preventing the monitoring of [Ca 2+ ];
during the operation. However, after allowing the external dye to disperse or after washing, it is possible to make
a subsequent estimation of [Ca 2+ ];. Thus, cells in the
standard buffer were injected with fura2 (1-3 mM as its
potassium salt in water) and 5 min or more later, when the
background was negligible, values of [Ca 2+ ]; were calculated. This treatment gave rise to two groups of cells in
which resting [Ca 2+ ], was 143±39nM (S.D., 9 cells) or
474±33nM (S.D., 4 cells).
Injection of divalent cations and chelators
Since our data implicate Ca 2+ in the activation of mast
cells by nucleotide injection, we have tested the effects of
including the ion, or the chelator EGTA in the injection
solution. However, in order to have a significant effect on
the ultimate [Ca ], by this means, it is necessary to
overcome the substantial Ca2+-buffering capacity of
cytoplasm. Injections of CaCl2 at needle concentrations
between 10/iM and lmM failed to induce degranulation
but did lead to cell injury. In response to the higher
concentrations of CaCl2 the cells immediately formed
blebs. These tended to be reabsorbed during the ensuing
minutes but the cells remained swollen and had a
damaged appearance. When 500 mM NaEGTA (which had
no effect when provided alone) was injected into cells
together with GTPyS (1 mM) the proportion of degranulating cells was still very high (90±2 %, data not shown). It is
likely that even when EGTA is introduced into cells at a
final level of 5 mM, a Ca 2+ -transient caused by leakage
100
JC
80
•o
a 60
l
Q
3? 20
+ Mg 2 +
GTPyS
+ Mg2
ITP
10
20
30
40
50
Time after addition of inhibitors (min)
60
Fig. 6. Inhibitory effect of Mg 2 + injected together with GTPyS
or ITP. Cells were injected with a solution containing MgCl2
(100 mM) and either GTPyS (IIDM) or ITP (100 nun) (hatched
bars). MgClj was omitted from the needle solution in the
control experiments (open bars). Data represent mean and
S.E.M. of 3 experiments; 60 cells were injected for each
measurement.
from the outside during impalement is not eliminated.
(This leakage will commence as soon as the needle enters
the cell and before the chelator has been delivered.)
We also tested the effect of including MgCl2 in the
needle solutions of both GTPyS and ITP. These experiments are illustrated in Fig. 6, where it can be seen that
this ion has a substantial inhibitory effect on the actions of
the two nucleotides, when provided at 100 mM. We would
expect this to increase the intracellular concentration of
Mg2"*" by ~lmM.
Monovalent ions
Under the conditions of a leaky seal, other ions (Na + , K +
and Cl~) may also move into or out of the cell down their
respective concentration gradients. To determine if such
ion movements are necessary for the activation of cells by
guanine nucleotide, injections were performed in medium
containing potassium gluconate instead of NaCl, in order
to reduce or eliminate the normal concentration gradients
of the principal monovalent ions. Na + was not completely
absent from this solution, since it was buffered by
NaHepes. The EC50 of GTPyS and the maximum extent of
degranulation for cells maintained in this medium are not
significantly different from the values obtained from cells
in the standard buffer (data not shown), indicating that
leakage of Na + , K + or Cl~ is not essential for secretion in
our experiments.
Effect of metabolic inhibition
Work with permeabilised mast cells has shown that there
is no absolute requirement for ATP when exocytosis is
induced by an effector combination consisting of GTPyS
and Ca 2+ , although ATP does control the effective
affinities for these two effectors (Howell et al. 1987). In
order to gain some insight into the role of ATP in intact
cells, we have examined the effect of reducing intracellular ATP levels by metabolic inhibition prior to microinjection of GTPyS. In cells suspended in the standard buffer
solution (including glucose) the estimated intracellular
concentration of ATP is 3.4±0.3mM (S.E.M., n=5 experiments). Fig. 7 A shows the time-course of the fall in [ATP]!
following metabolic inhibition at room temperature.
Antimycin A (5/JM) and deoxyglucose (6mM) were added
at zero time to cells in medium lacking glucose. Also
0.001
0.01
0.1
1.0
[GTPyS] needl , (HIM)
10
Fig. 7. Effect of metabolic inhibitors. Mast cells in medium
lacking glucose were treated with antimycin A (5 /IM) and
2-deoxyglucose (0.6 mM) at room temperature. (A) The time
course of the effect of the inhibitors on cell responsiveness to
compound 48/80 (10/wgmr 1 ; (O)) and on cytosol [ATP] ((•);
see text for details). (B) The responses of cells inhibited as
described above for at least 45 min and then injected with
GTPyS at the indicated concentrations (O), compared with the
responses of normal cells in medium containing glucose (•)
(same data as Fig. 4). Data represent mean and S.E.M. of 3
experiments; 60 cells were injected for each measurement.
shown is the concomitant decline of responsiveness of cells
to compound 48/80 (lO^gml" 1 ). At 45 min, when the cells
had become refractory to the agonist, [ATP], was 60 ±18 /JM
(S.E.M., / I = 3 ) ; (note that the loss of ATP and the loss of
responsiveness to agonist stimulation is much more rapid
when metabolic inhibition is carried out at 37°C; P. E. R.
Tatham, unpublished results; Howell et al. 1987). Fig. 7B
shows the effect of injected GTPyS on mast cells that had
been subjected to antimycin A (5 /JM) and 2-deoxyglucose
(6mM) for at least 45 min in standard medium lacking
glucose. Although the EC50 of GTPyS for the inhibited
cells is —10 times that of untreated cells, the nucleotide is
fully effective at the higher concentrations. Thus, under
conditions where compound 48/80 is unable to bring about
degranulation, injection of GTPyS can still activate the
cells.
Discussion
Estimation of final concentrations
We have derived a relationship between the cytosolic level
of an injected solute and its concentration in the injection
needle by comparing the fluorescence of cells injected with
Microinjection of mast cells
221
ethidium bromide with that of cells permeabilised in the
presence of the dye and resealed after an equilibration
period. The emission from the DNA-bound dye depends on
the cytoplasmic dye level in each case. Factors that
contribute to the uncertainty in these measurements
include variations in the sizes of individual mast cells and
in the amount injected into each cell. Indeed, at intermediate and low dye concentrations, the observed fluorescence
was variable from cell to cell. Moreover, it is possible that
although the ATP-treated cells were equilibrated with the
dye for 40 min before resealing, it may not have reached
complete equilibrium across the membrane pores. This
would lead to an overestimate of the intracellular
ethidium level (i.e the left-hand curve of Fig. 2 should lie
further to the left) and hence an u^wferestimation of the
dilution. Conversely, ATP4"-treated mast cells can leak
out proteins such as actin (Koffer and Gomperts, 1989) and
this may allow the dye to permeate the cytoplasm more
extensively than it can in the injected cells. In this case,
even when the levels of cytoplasmic dye are equivalent,
the nuclear fluorescence would be brighter in the
permeabilised cells (effectively shifting the left-hand
curve of Fig. 2 to the right) leading to an ouerestimation of
the dilution. In view of these uncertainties we assume an
approximate dilution factor of 100.
The mobility and ultimate distribution of substances
injected into cells is determined by the submicroscopic
structure of the cell interior. Not only are the physical
characteristics of cytoplasm far from isotropic but local
structural elements may also undergo temporal changes,
and these properties will affect the way in which injected
solutes are accommodated within the cell. For example,
substances of high molecular mass may be restricted, at
least initially, to the site of injection and even low
molecular mass solutes may not be able to permeate the
entire space (Luby-Phelps et al. 1988). The notion of (final)
cytosolic concentration is therefore a simplification and
from the outset its estimation must be viewed as
approximate.
Nucleotide-induced degranulation and Ca2+ dependence
Of the nucleotides tested, only ITP, GTPyS and GppNHp
gave rise to substantial degranulation when injected into
cells bathed in medium containing Ca 2+ (Fig. 3). Table 1
compares these responses with the ability of the same
nucleotides to support secretion in permeabilised preparations of mast cells and neutrophils. In making these
comparisons, it is important to remember that MgATP
(present in intact cells at millimolar levels) has a number
of modulatory effects upon secretion in the permeabilised
preparations. In permeabilised neutrophils and HL60
cells, the presence of MgATP augments the extent of
lysosomal enzyme secretion. The rather low levels of
release (indicated in Table 1) from permeabilised neutrophils evoked by GTP, ITP, XTP, CTP and UTP reflect the
omission of MgATP in these particular experiments
(Barrowman et al. 1987). Thus, the nucleotides that
activate mast cells upon injection, appear to be a subset of
those that support secretion in the permeabilised cells.
The exceptions are XTP and GTP, which failed to activate
the cells when injected. XTP does support secretion in
permeabilised mast cells, neutrophils and HL60 cells, and
GTP is effective in permeabilised mast cells and neutrophils (Table 1). GTP is of course already present in intact
cells and the injection will have merely increased its
intracellular level by approximately 1 mM. XTP, like ITP,
is a potential substrate for intracellular nucleotidases, and
this may account for its failure to activate upon injection.
This may also explain why ITP is the most potent
nucleoside triphosphate when provided to permeabilised
mast cells under conditions where its supply is effectively
infinite (Howell et al. 1987), and yet is only partially
effective when it is injected.
The most effective activator of mast cells by injection is
GTPyS. This poorly hydrolysable analogue of GTP is well
established as an activator of G proteins in both cell-free
and permeabilised cell systems, and it has been used
extensively to investigate the involvement of G proteins in
intracellular processes. When it is injected into intact
cells, surface receptor events are bypassed and cell
responses are activated directly, presumably through
interactions with G proteins such as Gg, a hypothetical
mediator of exocytosis for which there is evidence in a wide
range of cell types including mast cells (Gomperts,
1990a,6). Direct activation can also be achieved using
permeabilised cells, but it must be recognised that these
are modified or depleted systems. Cells with small
permeability lesions exchange ions and small molecules
such as nucleotides with the surroundings, while substantially permeabilised cells also leak out soluble proteins.
Leakage of cytosol proteins is not necessarily inhibitory as
it has been found that mast cells permeabilised lightly (by
ATP4") require a concentration of GTPyS to elicit
secretion that is several hundred times that required by
streptolysin O-permeabilised cells at the same [Ca 2+ ]
(Koffer and Gomperts, 1989). Thus care must be
Table 1. Extent of secretion induced by nucleotides in permeabilised and microinjected cells
Nucleotide
GTP
Permeabilised mast cells (ATP4")*
Permeabilised mast cellB (SKD)t
Permeabilised neutrophils^
Permeabilised HL60 cells§
Microinjected mast cellsl
ITP
XTP
CTP
UTP
nd
nd
nd
nd
GTPyS
GppNHp
+
+ , extent of response; •, poor or no response; nd, not tested.
* Histamine secretion from ATP4"-permeabilised rat mast cellB, loaded with nucleotide and resealed. Response to subsequent challenge with Ca2+
(6inM external) (Gomperts, 1983).
tHistamine secretion from metabolically inhibited cells permeabilised by streptolysin 0 in the presence of Ca a+ (pCa5) and in the absence of
MgATP (Howell et al. 1987).
t /J-Glucuronidase Becretion from Sendai virus permeabilised rabbit neutrophils in the presence of cytochalasin B and Ca2+ (pCa5). MgATP was
provided only in the experiments with the analogues (**) (Barrowman et al. 1987; Barrowman et at. 1986).
§/3-Glucuronidase secretion from streptolysin O-permeabilised, undifferentiated cells, in the presence of MgATP and Ca 2+ (pCa5) (Stutchfield and
Cockcroa, 1988).
H Degranulation of rat mast cells (this work)
222
P. E. R. Tatham and B. D. Gomperts
exercised when interpreting the results of experiments on
permeabilised cells that are likely to have lost modulatory
factors. During the microinjection of intact cells, the
positive pressure in the needle, the brevity of the injection
and the rapid resealing of the membrane, allow substances
to be introduced into the cytosol without significant
leakage. Thus there is an important complementarity
between the different methods of gaining access to the
cytosol. Streptolysin O-permeabilisation and the application of patch-pipettes produce substantially depleted
cells; ATP4" and agents such as o--toxin (see AhnertHilger and Gratzl, 1988) cause less damage and microinjected cells sustain the least damage.
In permeabilised cells GTPyS is always more potent
than GppNHp (Howell et al. 1987), and the microinjected
cells adhere to this pattern. The concentration dependence
of the response to GTPyS (Fig. 4) indicates an EC50 in the
region of 100 nM (final). This is similar to the level
required for streptolysin O-permeabilised cells buffered at
pCa5 but lower than that required (10 /JM) by ATP4"permeabilised cells also at pCa5 (Koffer and Gomperts,
1989). To compare our data with these results, it is
necessary to have some idea of [Ca 2+ ], in the injected cells.
Since the effect of injected GTPyS is almost completely
dependent on the presence of Ca 2+ in the external medium
and since late provision of Ca 2+ does not induce exocytosis
in pre-injected cells, a transitory Ca 2+ increase must
accompany each injection event. The extent of the influx
may be considerable. (Indeed, fura2 fluorescence revealed
that some of the cells retained high levels of Ca 2+ .)
Inclusion of EGTA in the GTPyS solution to a final
concentration of ~5 mM did not inhibit the degranulation
response. This level should be adequate to overcome
normal intracellular Ca2+-buffering but is not likely to be
sufficient to withstand a substantial inward leak of Ca 2+
down a concentration gradient from millimolar extracellular levels. Moreover, the leakage of Ca 2+ into the cell will
commence as soon as the needle penetrates the membrane
and will continue during the injection, so that [Ca2+]i may
increase before the EGTA has reached its final concentration. In contrast with this, the permeabilised cell data
were obtained under Ca 2+ clamp conditions (pCa5) and
this makes direct comparison of GTPyS requirements with
those of injected cells somewhat difficult. For further
clarification it will be necessary to define the Ca 2+
requirement in terms of both concentration and timing.
Mast cells can be activated by treatment with ionophores such as A23187 and ionomycin, which allow the
entry of external Ca 2+ , and can also release the ion from
intracellular storage sites (Foreman et al. 1973; Bennett et
al. 1980). In a similar vein, permeabilisation of mast cells
by ATP4" in the presence of Ca 2+ gives rise to secretion
(Cockcroft and Gomperts, 1980). In consequence, it might
be expected that microinjection of CaCl2 would also trigger
secretion. In agreement with an earlier report (Tasaka et
al. 1970), we find that injection of CaCl2 solution into cells
in Ca2+-containing medium does not lead to degranulation, but can cause cell damage. However, degranulation
has been reported to occur when CaCl2 is introduced by
iontophoresis into mast cells attached to mesentery
(Kanno et al. 1973). Since these are presumably gentler
conditions, it may be that pressure injection of high
concentrations of Ca 2+ in the presence of extracellular
Ca 2+ has a deleterious effect on the exocytotic apparatus,
perhaps through damage to structural elements such as
the filamentous network within the cytoplasm.
Although we have been unable to trigger cells by
injection of Ca 2+ , it is clear that under our experimental
conditions Ca 2+ is required for degranulation induced by
injection of GTPyS. In intact rat mast cells extracellular
Ca 2+ is required for activation through the IgE receptor
pathway (Foreman and Mongar, 1972), but not through
non-immunological polycationic stimuli such as compound
48/80 (Atkinson et al. 1979). On the other hand, when
GTPyS is introduced into cells through a patch-pipette,
degranulation can occur even when [Ca 2+ ]; is clamped at
pCa8 (Neher, 1988), although the response is slow and
occurs after a time-lag of minutes. Since cells both in the
whole-cell configuration of the patch-pipette and in
streptolysin O-permeabilised preparations are substantially depleted by leakage, it is important when making
comparisons to give consideration to the differences in
these systems. In both cases the cells lose soluble factors
from their cytosols and these may possess modulatory
activities. In metabolically inhibited mast cells permeabilised by ATP4" 10jiM GTPyS elicits 50% secretion at
pCa5. When the cells are more substantially permeabilised by streptolysin O at the same [Ca 2+ ], only 100 nM
GTPyS is required to produce the same level of secretion.
This may be explained by the loss from the streptolysin Opermeabilised cells of factors that can suppress the
sensitivity to Ca 2+ and GTP analogues (Koffer and
Gomperts, 1989). Since ATP4" permeabilisation causes
less membrane damage than permeabilisation by streptolysin O, it might appear that ATP*"-treated cells should
be more comparable to microinjected cells. However, as
stated above, a direct comparison here is not possible,
since [Ca2+] was held at pCa5 in the permeabilised cells
but could vary in the injected cells.
In permeabilised cells it is also necessary to provide a
suitable ionic environment. In patch-clamp experiments
the intracellular solution conventionally contains glutamate as the principal anion (Neher, 1988). In permeabilised mast cells glutamate stands out from other anions
(chloride, acetate, succinate etc.) in that it can support a
measure of Ca2+-independent secretion (Churcher and
Gomperts, 1990).
In assessing the dependence of GTPyS-induced secretion
on intracellular ATP levels, we are hampered by the fact
that, using metabolic inhibition, we are not able to
eliminate ATP completely from intact cells. We are
therefore limited to concluding that the ATP-dependent
processes that mediate compound 48/80-induced secretion
are not part of the pathway that is activated by GTPyS and
Ca 2+ .
It is difficult to explain the inhibitory effect of Mg2"1" in
our experiments, but we note that it also inhibits secretion
from electrically permeabilised, bovine adrenal chromaffin cells; both the extent of release and the apparent
affinity for Ca 2+ are reduced (Knight and Baker, 1982).
Mg2"1" also suppresses Ca2+-induced secretion from rabbit
neutrophils when admitted to the cells by ionophore (Di
Virgilio and Gomperts, 1983). Mg2"1" has been shown to be
necessary for the dissociation of activated, GTP-bound Gs
into its a-and /Jy subunits in rat liver, membranes (Iyengar
and Birnbaumer, 1982; Iyengar et al. 1988). Binding sites
for Mg2"1" have also been detected on G-protein-receptor
complexes in HL60 membrane preparations (Gierschik et
al. 1989), and these are associated with the modulation of
the number and affinity of formylmethionyl peptide
receptors. However, it is not clear how such sites could
affect the activation by injection of guanine nucleotide
when [Mg2"1"]; is elevated from approximately lmM to
2mM.
Microinjection of mast cells
223
This work was supported by grants from the Wellcome Trust,
the SmithKline (1982) Foundation and the Central Research
Fund of the University of London. Support was also provided
under NATO grant RG.0908/87 and in this regard we are
indebted to Dr Dafna Bar-Sagi for her advice and assistance in
setting up the microinjection facility, to Dr Michael Whitaker for
his assistance in the measurements of intracellular [Ca 2+ ], and to
Professor David Saggerson and Dr Jenny Fordham for their
cooperation in ATP measurement.
References
AHNEET-HILGKH, G. AND GRATZL, M. (1988). Controlled manipulation of
the cell interior by pore-forming proteins. Trends Pharmac. Sci. 9,
196-197.
ALMBRS, W. AND NEHER, E. (1985). The Ca-eignal from fura-2 loaded
maat cells depends strongly on the method of dye loading. FEBS Lett.
192, 13-18.
ATKINSON, G., ENNIS, M. AND PEARCE, F. L (1979). The effect of alkaline
earth cations on the release of histamine from rat peritoneal mast
cells treated with compound 48/80 and peptide 401 Br. J. Pharmac.
6fi, 395-402.
BARHOWMAN, M. M., COCKCROW, S. AND GOMPERTS, B D. (1986). Two
roles for guanine nucleotides in the stimulus secretion sequence of
neutrophils. Nature 319, 504-607.
BABROWMAN, M. M., COCKCEOFT, S. AND GOMPEBTS, B. D. (1987).
Differential control of azurophilic and specific granule exocytosis in
Sendai virus permeabilised rabbit neutrophils. J. Physiol. Land. 383,
115-124.
BENNETT, J. P , COCKCROFT, S. AND GOMPERTS, B. D. (1980) Ionomycin
stimulates mast cell histamine secretion by forming a lipid soluble
calcium complex. Nature 282, 851-853.
BIBB, P. C , COCHRANE, D. E. AND MOREL-LAURENS, N. (1986). Loss of
quin 2 accompanies degranulation of mast cells. FEBS Lett 209,
169-174.
BLOOM, G. D. AND CHAKRAVARTY, N. (1970). Time course of anaphylactic
histamine release and morphological changes in rat peritoneal mast
cells. Acta physiol. scand. 78, 410-419.
CHURCHER, Y. AND GOMPERTS, B. D. (1990). ATP dependent and ATP
independent pathways of exocytosis revealed by interchanging
glutamate and chloride as the major anion in permeabilised mast
cells. Cell Reguln 1, 337-346.
COCKCROFT, S. AND GOMPERTS, B. D. (1980). The ATP*~ receptor of rat
mast cells. Biochem. J. 188, 789-798.
COCKCROFT, S. AND GOMPERTS, B. D. (1985) Role of guanine nucleotide
binding protein in the activation of polyphosphoinositide
phosphodiesterase. Nature 314, 534-536.
COCKCROFT, S., HOWELL, T. W. AND GOMPERTS, B D. (1987) Two G-
proteins act in series to control stimulus-secretion coupling in mast
cells: Use of neomycin to distinguish between G-proteins controlling
polyphosphoinositide phosphodiesterase and exocytosis. J. Cell Bwl.
105, 2745-2760.
Di VIRGILJO, F. AND GOMPERTS, B. D. (1983) Cytosol Mg** modulates
Ca 2+ ionophore induced secretion from rabbit neutrophils. FEBS Lett.
163, 315-318.
FERNANDEZ, J. M., LINDAU, M. AND ECKSTEIN, F (1987). Intracellular
stimulation of mast cells with guanine nucleotides mimic antigenic
stimulation. FEBS Lett. 216, 89-93.
FERNANDEZ, J M., NEHER, E. AND GOMPKRTS, B. D. (1984) Capacitance
measurements reveal stepwise fusion events in degranulating mast
cells. Nature 312, 453-455.
FOREMAN, J. C. AND MONOAR, J. L. (1972) The role of the alkaline earth
ions in anaphylactic histamine secretion. J. Physiol. Land. 224,
753-769.
FOREMAN, J C, MONGAR, J L. AND GOMPERTS, B D (1973). Calcium
ionophores and movement of calcium ions following the physiological
stimulus to a secretory process. Nature 245, 249-251.
GlSRSCHIK, P , STEISSIJNGER, M , SmiDOPOULOS, D., HERRMAN, E . AND
JAKOBS, K. H. (1989). Dual Mg3"1" control of formyl peptide receptor G
protein interaction in HL60 cells Evidence that the low agonist
affinity receptor interacts with and activates the G protein. Eur. J.
Biochem. 183, 97-105
GOMPERTS, B. D (1983). Involvement of guanine nucleotide-binding
proteins in the gating of Ca a+ by receptors. Nature 306, 64-66.
GOMPERTS, B. D. (1990a). G^: A GTP-binding protein mediating
exocytosis. A. Rev. Physiol. 52, 591-606.
GOMPERTS, B. D. (19906). GTP-binding Proteins and Exocytotic
224
P. E. R. Tatham and B. D. Gomperts
Secretion, in G Proteins, pp. 601-637. Academic Press, New York and
London.
GOMPERTS, B. D , BARROWMAN, M. M. AND COCKCROFT, S. (1986). Dual
role for guanine nucleotides in stimulus-secretion coupling: an
investigation of mast cells and neutrophils. Fedn Proc. Fedn Am. Socs
exp. Biol. 45, 2156-2161.
GOMPERTS, B. D., COCKCROFT, S., HOWELL, T. W., NUSSE, O. AND
TATHAM, P. E. R. (1987). The dual effector system for exocytosis in
mast cells: Obligatory requirement for both Ca 2+ and GTP. Biosci.
Rep. 7, 369-381.
GOMPERTS, B. D. AND TATHAM, P. E. R. (1988). GTP-binding proteins in
the control of exocytosis. Cold Spring Harbor Svmp. quant Biol. 53,
983-992.
GRYNKIEWICZ, G., POENIK, M. AND TSIEN, R. Y. (1985). A new generation
of Ca 2+ indicators with greatly improved fluorescence properties
J. biol. Chem. 260, 3440-3450.
HELANDER, H. F. AND BLOOM, G. D. (1974). Quantitative analysis of
mast cell structure. J. Microsc. 100, 315-321.
HOWELL, T. W., COCKCROFT, S. AND GOMPERTS, B. D. (1987). Essential
synergy between Ca 2+ and guanine nucleotides in exocytotic secretion
from permeabilised mast cells. J. Cell Biol. 105, 191-197.
HOWELL, T. W. AND GOMPERTS, B. D. (1987). Rat mast cells
permeabilised with streptolysin-0 secrete histamine in response to
Ca 2+ at concentrations buffered in the micromolar range. Biochun.
bwphys. Acta 927, 177-183.
IYENGAR, R. AND BIRNBAUMER, L. (1982). Hormone receptor modulates
the regulatory component of adenylyl cyclase by reducing its
requirement for Mg 2+ and enhancing its extent of activation by
guanine nucleotides Pnx. natn. Acad Sci. U.S.A. 79, 5179-5183.
IYENOAR, R., RICH, K. A., HERBERG, J. T., PREMONT, R. T. AND CODINA,
J. (1988). Glucagon receptor-mediated activation of G s is accompanied
by subunit dissociation. J. bwl. Chem. 283, 15 348-15353.
JOHANSEN, T. (1987). Energy metabolism in rat mast cells in relation to
histamine secretion. Pharmac. Toxicol. 61, 1-20.
KANNO, T., COCHRANE, D. E. AND DOUGLAS, W. W. (1973). Exocytosis
(secretory granule extrusion) induced by injection of calcium into
mast cells. Can J. Physiol. Pharmac. 51, 1001-1004.
KNIGHT, D. E. AND BAKER, P. F. (1982). Calcium-dependence of
catecholamine release from bovine adrenal medullary cells after
exposure to intense electric fields. J. Membr. Biol. 68, 107-140.
KOFTER, A. AND GOMPERTS, B. D. (1989) Soluble proteins as modulators
of the exocytotic reaction of permeabilised rat mast cells. J. Cell Sci.
94, 585-591.
KRUGER, P. G., BLOOM, G. D. AND DIAMANT, B. (1974). Structural
aspects of histamine release in rat peritoneal mast cells. Int. Archs
Allergy appl. Immun. 47, 1-13.
LINDAU, M. AND NUSSK, O. (1987). Pertussis toxin does not affect the
time course of exocytosis in mast cells stimulated by intracellular
application of GTPyS. FEBS Lett. 122, 317-321.
LUBY-PHELPS, K., LANNI, F AND TAYLOR, D. L. (1988). The
submicroscopic properties of cytoplasm as a determinant of cellular
function. A. Rev. Biophys. biophys. Chem. 17, 369-396.
NEHER, E. (1988). The influence of intracellular calcium concentration
on degranulation of dialysed mast cells from rat peritoneum. J.
Physiol. Lond. 395, 193-214.
PENNER, R. (1988). Multiple signaling pathways control
stimulus-secretion coupling in rat peritoneal mast cells. Proc. natn.
Acad. Sci. U.S.A 85, 9856-9860.
ROUCH, P., ANDERSON, P. AND UVNAS, B. (1971). Electron microscope
observations of compound 48/80-induced degranulation in rat mast
cells. J. Cell Biol. 51, 465-483.
STUTCHFIELD, J. AND COCKCROFT, S. (1988). Guanine nucleotides
stimulate polyphosphoinositide phosphodiesterase and exocytotic
secretion from HL-60 cells permeabilised with streptolysin O.
Biochem. J. 250, 375-382.
SWANN, K. AND WHITAKER, M. (1986) The part played by inositol
trisphosphate and calcium in the propagation of the fertilization wave
in sea urchin eggs. J. Cell Biol. 103, 2333-2342.
TASAKA, K., SUQIYAMA, K., KOMOTO, S. AND YAMASAKI, H. (1970).
Degranulation of isolated rat mast cellB induced by adenosine
triphosphate in the presence of calcium ions. Proc. Japan Acad. 46,
317-321.
TATHAM, P. E. R. AND GOMPERTS, B. D. (1990). Cell permeabilisation In
Peptide Hormones - A Practical Approach, vol. 2, pp. 255-267. IRL
Press, Oxford.
(Received 25 October 1990 - Accepted 20 November 1990)