(Received for publication, January 12, 1996; and in revised form, March 15, 1996)
From the
We developed a unidirectional Ca
efflux technique in which 60 cumulative doses of inositol
1,4,5-trisphosphate (InsP
), each lasting 6 s, were
subsequently added to permeabilized A7r5 cells. This technique allowed
an accurate determination of the threshold for InsP
action,
which was around 32 nM InsP
under control
conditions. The InsP
-induced Ca
release
was characterized by an initial rapid phase, after which the normalized
rate progressively decreased. The slowing of the release was associated
with a shift of the threshold to higher InsP
concentrations. Stimulatory concentrations of thimerosal (10
µM) shifted the threshold to 4.5 nM InsP
and increased both the cooperativity and the maximal normalized
rate of Ca
release. This low threshold was maintained
when the thimerosal concentration was increased to inhibitory levels
(100 µM) but then the effects on the cooperativity and on
the normalized rate of Ca
release disappeared.
Oxidized glutathione (5 mM) was much less effective in
stimulating the release and did not have an effect on the threshold or
on the cooperativity. ATP (5 mM) stimulated the release
despite a shift in threshold toward higher InsP
concentrations. Luminal Ca
did not affect the
threshold for InsP
action but stimulated the normalized
release at each InsP
concentration. The inhibitory effect
of 10 µM free cytosolic Ca
was
associated with a shift in threshold to higher InsP
concentrations and a decreased cooperativity of the release
process. We conclude that this novel technique of accurately measuring
the threshold for InsP
action under various experimental
conditions has allowed us to refine the analysis of the kinetic
parameters involved in the regulation of the InsP
receptor.
Many hormones, neurotransmitters, and growth factors induce the
hydrolysis of phosphatidylinositol 4,5-bisphosphate and thereby produce
inositol 1,4,5-trisphosphate (InsP) (
)as an
intracellular messenger(1) . Once threshold concentrations of
InsP
are reached and conditions for the regenerative
release of Ca
are created(2) , InsP
mobilizes Ca
from the nonmitochondrial stores
through interaction with the InsP
R. The precise kinetics of
the [InsP
] rise in an intact cell are unknown,
but it is possible that there is a gradual increase in the
[InsP
] before the first Ca
response(3, 4) .
It is not clear how the
Ca stores in cells respond to such progressively
increasing [InsP
]. On the one hand, there is the
technical difficulty in accurately measuring the Ca
release induced by very low doses of InsP
above the
background Ca
leak. On the other hand, it is not
clear whether classical dose-response relationships, obtained by
acutely introducing one concentration of InsP
, can predict
the response when the [InsP
] is slowly
increasing, especially if inactivation of the InsP
R (5) would occur. We have now developed a
Ca
efflux technique in which cumulative
doses of InsP
are subsequently added. This technique, which
mimics the gradual accumulation of InsP
at the onset of a
Ca
response, allows an accurate measurement of the
threshold [InsP
] for InsP
-induced
Ca
release. With this technique, we have investigated
how the InsP
thresholds are affected by thimerosal, GSSG,
ATP, luminal Ca
, and cytosolic Ca
and how a repeated application of InsP
acts on the
threshold value. This novel technique of measuring thresholds for
InsP
action allowed a refined analysis of the kinetic
parameters controlling the mechanisms of action of several regulators
of the InsP
R.
A7r5 cells, an established cell line derived from embryonic
rat aorta, were used between the 7th and the 17th passage after receipt
from the American Type Culture Collection (Bethesda, MD) and
subcultured weekly by trypsinization. The cells were cultured at 37
°C in a 9% CO incubator in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, 3.8 mML-glutamine, 0.9% (v/v) nonessential amino acids, 85 IU
ml
penicillin, and 85 µg ml
streptomycin. The cells were seeded in 12-well dishes (4
cm
, Costar, MA) at a density of approximately 10
cells cm
.
Ca
fluxes on permeabilized cells were done on a thermostated plate
at 25 °C. The culture medium was aspirated and replaced by 1 ml of
permeabilization medium containing 120 mM KCl, 30 mM imidazole HCl (pH 6.8), 2 mM MgCl
, 1 mM ATP, 1 mM EGTA, and 20 µg ml
saponin. The saponin-containing solution was removed after 10
min, and the cells were washed once with a similar saponin-free
solution.
Ca
uptake into the
nonmitochondrial Ca
stores was accomplished by
incubation for 60 min in 2 ml of loading medium containing 120 mM KCl, 30 mM imidazole HCl (pH 6.8), 5 mM MgCl
, 5 mM ATP, 0.44 mM EGTA, 10
mM NaN
, and 100 nM free
Ca
. After this phase of
Ca
accumulation, the monolayers were
incubated in 1 ml of efflux medium containing 120 mM KCl, 30
mM imidazole HCl (pH 6.8), 2 mM MgCl
, 1
mM ATP, 1 mM EGTA, 100 nM free
Ca
, and 2 µM thapsigargin.
The inclusion of thapsigargin was necessary to inhibit any further
Ca
-ATPase activity during the efflux. The main
difference with our previous
work(6, 7, 8, 9, 10, 11, 12, 13) is that the technique was modified to allow a sampling
resolution of 6 s during the efflux. This was performed by
automatically transferring the efflux medium from the cells into
scintillation vials using a high speed P-50 Pump (Pharmacia Biotech,
Inc.) connected to a Pharmacia LKB 158 SuperFrac fraction collector.
The pumping rate was 1 ml/6 s. The advantage of this modification is
that the rate of Ca
release can be measured, and not
only the extent of Ca
release as in our previous
work. The first 5 min of efflux were not monitored. InsP
was included in the efflux solutions during the time periods and
at the concentrations indicated in the figures. At the end of the
experiment the
Ca
remaining in the
stores was released by incubation with 1 ml of a 2% sodium dodecyl
sulfate solution for 30 min.
Figure 1:
Kinetics of the
InsP-induced Ca
release from
permeabilized A7r5 cells. The nonmitochondrial stores were loaded for
60 min at 100 nM free
Ca
and
then incubated in an efflux medium containing 2 µM thapsigargin. The first 5 min of efflux were not monitored. A shows, on a logarithmic scale, the decrease of the Ca
content of the stores during further incubation in efflux medium
and its modification by 2 additions of 0.5 µM InsP
, added as indicated by the bars above the trace. B shows, on a linear scale, the fractional
loss (the amount of Ca
leaving the stores in 6 s,
divided by the total store Ca
content at that time)
as a function of time. The inset shows the effect of shortly
removing 0.5 µM InsP
at the maximum of the
InsP
response. Typical for four
experiments.
The release
was rather slow, since it was still significant at the end of the first
stimulation period, i.e. after 3.5 min (Fig. 1B). This observation contrasts with the much
faster kinetics observed in some other experimental systems (e.g. rat brain synaptosomes(15) ). These differences are
unlikely to be due to a slow diffusion of Ca out of
our permeabilized cell preparation, since the release dropped to
control values only upon removing the InsP
and again
increased upon restimulation (Fig. 1B). The long time
period that is needed to empty the stores in A7r5 cells might be
related to the larger amount of Ca
ions that have to
pass through the channels: more than 95% of the accumulated
Ca
is releasable by a maximal
[InsP
] in A7r5 cells(7) , while the
figure for rat brain synaptosomes is only 6%(15) .
The
effect of the [InsP] on the kinetics of the
release is shown in Fig. 2. Increasing the
[InsP
] from 0.2 to 1 µM increased
the extent of Ca
release, as deduced from the lower
Ca
content at the end of the efflux (Fig. 2A), confirming the ``quantal'' nature
of the release in A7r5 cells(6) . Fig. 2B illustrates that the normalized maximal rate of Ca
release was higher at 1 than at 0.2 µM InsP
. The normalized rates of release at both
concentrations of InsP
then gradually decreased and
eventually became identical, i.e. they became independent of
the [InsP
]. At these later time points, the
efflux rates were still significantly higher than the basal efflux rate
in the absence of InsP
. These data obtained with the
technique of
Ca
fluxes are in agreement
with similar findings made with protocols in which the release was
monitored with fluorescent Ca
indicators in the
cytosol (16, 17, 18) or in the
store(19) . The arrows in Fig. 2B indicate the time at which the fractional loss has decreased to
half of its maximal value. This phenomenon occurred earlier at 1
µM than at 0.2 µM InsP
. This
progressively decreasing normalized rate of Ca
release also occurred in the presence of 0.1 µM InsP
, although it took a much longer time for the
release to reach half of its maximal rate (data not shown). A
progressive decrease in the normalized rate of Ca
release therefore occurred at all InsP
concentrations. The rate constant of this decline was, however,
dependent on the [InsP
]. In contrast, Sugiyama
and Goldman (19) concluded from very similar experiments that
low and high concentrations of InsP
induced different
patterns of Ca
release, 0.1 µM InsP
only induced a monoexponential decline of the
store Ca
content, while higher concentrations induced
a biphasic release. In contrast, we could only observe one pattern of
Ca
release with a slowly decreasing rate constant.
Figure 2:
Effect of the [InsP]
on the kinetics of the InsP
-induced Ca
release. A shows, on a logarithmic scale, the decrease
of the Ca
content of the stores during incubation in
efflux medium and its modification by 0.2 (
) and 1 µM (
) InsP
, added as indicated by the bar above the traces. B shows, on a linear scale, the fractional
loss for the two experimental conditions as a function of time. The arrows indicate the time at which the fractional loss has
decreased to half of its maximal value. Typical for four
experiments.
There is evidence that the InsPR might be stimulated by
Ca
inside the
store(6, 7, 8, 11, 13, 19, 20, 21, 22, 23, 24, 25) .
Part of these effects could be exerted at the cytosolic side of the
receptor(26, 27) , but other authors (8, 11, 24) have brought forward arguments
against the latter hypothesis. Since the release process is associated
with a decline in the luminal [Ca
], we have
compared the kinetics of the release induced by 1 µM InsP
in stores that were allowed to deplete by
blocking their pumps with 2 µM thapsigargin either for 6
min (Fig. 3, A and B, closed circles)
or for 21 min (Fig. 3, A and B, open
circles) before the InsP
challenge. The maximal
normalized rate of Ca
release as well as the
fractional loss at the later time points appreciably decreased when the
InsP
challenge was given to the 4 times less filled stores (Fig. 3B). The effect of luminal Ca
was also apparent when the increase in fractional loss was
normalized to that in the presence of 10 µM A23187 given
at the same time in parallel experiments (data not shown). These
findings therefore confirm that the decreasing rate of Ca
release in the continuous presence of InsP
(Fig. 1) is, at least partly, explained by the decreasing
Ca
content of the store. This interpretation of the
data can explain why the normalized release rate decreased faster at
high InsP
concentrations (Fig. 2B), because
high concentrations induce a more rapid decrease in the store
Ca
content.
Figure 3:
Effect of the level of store loading on
the kinetics of InsP-induced Ca
release.
The nonmitochondrial stores were loaded for 60 min at 100 nM free
Ca
and then incubated in
efflux medium containing 2 µM thapsigargin. The efflux was
allowed to proceed either for 6 min (more filled stores,
) or for
21 min (less filled stores,
) before the InsP
challenge. A shows, on a logarithmic scale, the decline
of the Ca
content of the stores during the incubation
in the efflux medium and its modification by the addition of 1
µM InsP
as indicated by the bar above the trace. B shows, on a linear scale, the corresponding
fractional loss as a function of time. Typical for four
experiments.
Figure 4:
Effect of gradually increasing the
[InsP] on the Ca
release. A shows, on a logarithmic scale, the decrease of the store
Ca
content. B shows, on a linear scale, the
corresponding fractional loss during incubation in efflux medium and
its modification by a gradual increase of the
[InsP
] from 10 nM to 10 µM in 60 individual steps each lasting 6 s. The
[InsP
] was increased in a logarithmic way, as
shown by the full line in B. The inset in A shows, on a linear scale, the extent of Ca
release, i.e. the difference between the Ca
content in the absence and presence of InsP
, as a
function of the cumulative [InsP
]. The release at
10 µM InsP
was taken as 100%. Typical for four
experiments.
Figure 5:
Effect of 10 µM thimerosal on
the InsP-induced Ca
release. A shows, on a logarithmic scale, the decline of the Ca
content of the stores during incubation in efflux medium
containing 10 µM thimerosal in 0.1% Me
SO
(
) or containing 0.1% Me
SO only (
) and its
modification by a gradual logarithmic increase of the
[InsP
] from 3.2 nM to 3.2 µM in 60 individual steps each lasting 6 s. B shows, on a
linear scale, the fractional loss for the above-mentioned experimental
conditions as a function of time. Typical for four
experiments.
Higher concentrations of thimerosal become
inhibitory(10, 32, 34) . In agreement with
these findings, we observed that 316 µM thimerosal
completely prevented any effect of InsP (data not shown).
The nature of this inhibition was further characterized using a lower,
but still inhibitory, thimerosal concentration (100 µM, Fig. 6, A and B). This concentration of
thimerosal still allowed InsP
to act at a lower threshold.
However, the increased cooperativity was lost and the maximal
normalized rate of Ca
release became smaller (Fig. 6B). The impaired InsP
-induced
Ca
release in the presence of high concentrations of
thimerosal was not the consequence of an increased passive
Ca
leak and, therefore, the lower store
Ca
content at the time of InsP
addition(10) .
Figure 6:
Effect of 100 µM thimerosal
on the InsP-induced Ca
release. A shows, on a logarithmic scale, the decline of the Ca
content of the stores during incubation in efflux medium
containing 100 µM thimerosal in 0.1% Me
SO
(
) or containing 0.1% Me
SO only (
) and its
modification by a gradual logarithmic increase of the
[InsP
] from 3.2 nM to 3.2 µM in 60 individual steps each lasting 6 s. B shows, on a
linear scale, the fractional loss as a function of time. Typical for
three experiments.
From these findings, we propose that
thimerosal might act on at least two different sites on the
InsPR or, alternatively, on some proteins associated with
the InsP
R. Interaction with one site results in the
increased sensitivity for InsP
, the increase in
cooperativity and of the maximal normalized rate of Ca
release. This interaction may also explain the decreased K
for InsP
binding(32, 34, 35, 36) . The
other interaction site for thimerosal could be responsible for the
inhibition by high thimerosal concentrations.
Oxidized glutathione
(GSSG) can also stimulate the InsPR, but this stimulation
has so far only been reported in
hepatocytes(20, 21, 35, 36, 37) .
The effect of 5 mM GSSG in A7r5 cells was further
characterized. A very modest stimulation of the release could be
observed, but its overall effect was much smaller than that of 10
µM thimerosal (data not shown). GSSG had no effect on the
threshold for InsP
-induced Ca
release nor
on the cooperativity. In contrast, the normalized rate of
Ca
release at each [InsP
]
increased by about 20% (data not shown).
These functional data
indicate that the two sulfhydryl reagents that we have tested
(thimerosal and GSSG) exerted different effects on the
InsPR. Our findings are in agreement with
InsP
-binding studies in permeabilized rat hepatocytes
indicating that GSSG only increased the number of binding sites for
InsP
without changing the K
, whereas
thimerosal both increased the number of binding sites and decreased the K
(36) . The effect of GSSG on
Ca
release in A7r5 cells was much smaller than
reported in rat hepatocytes (20, 21, 36, 37) . This finding
reinforces previous conclusions that InsP
Rs of different
sources can be differently affected by sulfhydryl
reagents(33) .
Figure 7:
Effect of 5 mM ATP on the
InsP-induced Ca
release. A shows, on a logarithmic scale, the decline of the Ca
content of the stores during incubation in efflux medium
containing 5 mM ATP (
) or in control medium without ATP
(
) and its modification by a gradual logarithmic increase of the
[InsP
] from 3.2 nM to 3.2 µM in 60 individual steps each lasting 6 s. The efflux medium did not
contain MgCl
. B shows, on a linear scale, the
fractional loss as a function of time. Typical for four
experiments.
Figure 8:
Effect of the level of store loading on
the InsP-induced Ca
release. The
nonmitochondrial stores were loaded for 60 min at 100 nM free
Ca
and then incubated in efflux medium
containing 2 µM thapsigargin. The efflux was allowed to
proceed for 6 min (
) or 21 min (
) before the InsP
challenge. A shows, on a logarithmic scale, the change
of the Ca
content of the stores elicited by a gradual
logarithmic increase of the [InsP
] from 3.2
nM to 3.2 µM in 60 individual steps each lasting
6 s. B shows, on a linear scale, the fractional loss as a
function of time. Typical for four
experiments.
Figure 9:
Effect of 10 µM free
Ca on the InsP
-induced
Ca
release. A shows, on a logarithmic scale,
the Ca
content of the stores during incubation in
efflux medium buffered at 10 µM (
) or 100 nM (
) free Ca
. In the experiment at 10
µM Ca
, the [InsP
]
was gradually increased from 3.2 nM to 32 µM in
80 individual steps each lasting 6 s and in that at 100 nM Ca
from 3.2 nM to 3.2 µM in 60 individual steps each lasting 6 s. B shows, on a
linear scale, the fractional loss as a function of time. Typical for
four experiments.
Lower concentrations of cytosolic Ca stimulate the InsP
-induced Ca
release(8, 15, 42, 43, 44) .
However, these effects were small in permeabilized A7r5 cells with
relatively filled stores(8) . An analysis with the present
Ca
efflux technique in strongly depleted
stores was technically impossible, since not enough radioactivity is
then left to sample every 6 s.
Figure 10:
InsP-induced
Ca
release during two consecutive stimulations. A shows, on a logarithmic scale, the change of the Ca
content of the stores during incubation in efflux medium and its
modification by two consecutive stimulation periods in which the
[InsP
] was gradually increased on a logarithmic
scale from 3.2 nM to 500 nM in 40 individual steps
each lasting 6 s and then kept at 500 nM for another 2 min.
The two periods of the InsP
challenge were interrupted by a
2.5-min incubation in efflux medium without InsP
. B shows, on a linear scale, the superimposed curves of the
fractional loss as a function of time for the first (
) and the
second (
) stimulation. The pattern of the InsP
addition was the same for both traces. Typical for five
experiments.