(Received for publication, October 31, 1994)
From the
In many cell types, depletion of Ca stores
causes activation of Ca
influx by a mechanism whose
molecular basis remains unclear. We recently described a new messenger
that is released by empty Ca
stores and that
activates Ca
influx in heterologous cells
(Randriamampita, C. & Tsien, R. Y.(1993) Nature 364,
809-814). This factor, provisionally named CIF (for
Ca
influx factor), seems to be a small nonprotein
factor possessing a phosphate group. Meanwhile Parekh et al. reported that okadaic acid, an inhibitor of protein phosphatases 1
and 2A, potentiates Ca
influx in Xenopus oocytes (Parekh, A. B., Terlau, H. &
Stühmer, W.(1993) Nature 364,
814-818). A link between these two observations is presented in
this paper. We show that in astrocytoma cells, okadaic acid and
cyclosporin A (an inhibitor of calcineurin) both potentiate the
Ca
elevations due to low doses of CIF, thapsigargin,
or carbachol. In lymphocytes, okadaic acid potentiates the
Ca
elevations due to low doses of phytohemagglutinin
and increases the amount of extractable CIF. CIF degradation can be
observed in cell-free homogenates of lymphocytes and is prevented by
the above phosphatase inhibitors, an effect that can at least partly
explain their potentiation of Ca
influx. CIF
degradation is also prevented by lowering free Ca
concentrations, which could be a feedback mechanism to enhance
Ca
influx when cells are depleted of
Ca
.
In many cell types, depletion of intracellular Ca stores causes compensatory influx of Ca
across
the plasma membrane. The mechanisms that link these two events have
been the subject of widespread speculation and intensive
investigation(1, 2, 3, 4, 5) .
Recently, two very different lines of experiments have suggested that
empty Ca
stores may release a diffusible messenger
that transmits a signal to the plasma membrane to activate influx
pathways. We recently showed that Jurkat lymphocytes contain a soluble
factor of low molecular weight that can induce Ca
influx when applied extracellularly to various other mammalian
cell types(6) . This factor does not match any of a wide
variety of known messengers, but seems to contain phosphate and a
periodate-sensitive group. Until its chemical structure is completely
defined, it is provisionally referred to as CIF (
)(for
Ca
influx factor). CIF is stored in
digitonin-resistant organelles of resting cells and is released into
the cytoplasm upon stimulation, although pathways for synthesis and
breakdown of CIF presumably also exist. Meanwhile, Parekh et al.(7) reported that in Xenopus oocytes, depletion
of intracellular Ca
stores caused Ca
influx, measured indirectly as a Ca
-activated
Cl
current. If the patch of plasma membrane was
ripped off the oocyte, the current immediately disappeared, but could
be reactivated if the patch was reinserted into the oocyte cytoplasm,
as if the cytoplasm contained a soluble ligand that activated the
influx. The current could be enhanced by okadaic acid (OA), an
inhibitor usually considered specific for protein phosphatases 1 and
2A(8) .
The enhancement found by Parekh et al.(7) might be attributed to an effect of OA on various
stages between the emptying of the Ca stores and the
activation of the Ca
-activated Cl
channels. To decide whether something like CIF is involved in
this effect, an obvious experiment is to test in intact mammalian cells
whether OA and other phosphatase inhibitors can enhance Ca
entry, activated either by depletion of endogenous Ca
stores or by application of exogenous CIF. If so, then one could
test more directly in vitro the tempting hypothesis that cells
possess enzymes that degrade CIF, but are directly or indirectly
inhibited by OA. Inhibition of such enzymes could then lead to an
increase in CIF concentration, which would provide a mechanism for
potentiating Ca
influx, but would not exclude other
possible effects of OA. Attempts to prevent breakdown could give
valuable hints about the metabolism of CIF even while its detailed
structure remains the subject of separate investigations.
Figure 1:
Protein phosphatase inhibitors
potentiate the [Ca]
response to cell extract. A-C, Jurkat cell
extract was added (1:20 dilution in A, 1:2 in B, and
1:12 in C) to fura-2-loaded astrocytoma cells where indicated
by the dashedlines marked CE. 50 nM OA (A), 100 nM OA (B), or 100 nM CsA (C) was added a few minutes later (second dashedlines). D, astrocytoma cells were stimulated
with submaximal doses of Jurkat extract (diluted 4-8 times)
either under control conditions (CTL; no pretreatment) or
after 3 min of incubation with 500 nM 1-norokadaone, 100
nM OA, or 1 µM CsA, each normalized to its
respective control. For each condition, values (integrated from 0 to
6-8 min after cell extract addition) correspond to the mean of
six to eight individual dishes resulting from three independent
experiments.
For in vitro degradation
experiments, Jurkat lymphocytes (0.3-ml packed volume) were stimulated
with PHA (20 µg/ml) for 10 min in 1 ml of the following medium: 145
mM NaCl, 5 mM KCl, 5 mM HEPES, 1 mM MgCl, and no added Ca
. The cells
were then sonicated for 2.5 min (Deltasonic 011C) and finally
centrifuged. The pellet was discarded, and the supernatant was
isolated, aliquoted, and kept at room temperature (22-25 °C)
for different periods of time to allow degradation of CIF activity.
Incubations were terminated by rapid freezing. In some runs, okadaic
acid (500 nM), cyclosporin A (CsA; 1 µM), or EGTA
(1 mM) was added to the samples at the beginning of the
incubation period. (The OA and CsA concentrations were set higher than
in the intact cell experiments because the sonicated homogenates were
derived from much more concentrated cell suspensions.) The same
inhibitor concentrations were then added to other aliquots at the end
of the incubation period, just before freezing, so that each pair of
samples would have the same final composition of additives. For
[Ca
]
measurements, 150 µl
of extract were rapidly thawed and added to astrocytoma cells in 450
µl of standard medium with 1 mM Ca
.
OA has been described as a specific
inhibitor of protein phosphatases 1 (EC = 20
nM) and 2A (EC
= 0.2 nM), the
EC
for protein phosphatase 2C being much higher (5
µM)(8) . Several structurally unrelated
phosphatase inhibitors with different specificities were tested on the
response to threshold concentrations of CIF. CsA (100 nM),
whose complex with cyclophilin specifically inhibits calcineurin
(protein phosphatase 2B)(10) , was comparable to OA (Fig. 1C) at potentiating
[Ca
]
increases. Calyculin A (50
nM), which inhibits protein phosphatase 1 as well as protein
phosphatase 2A(11) , was also as effective as OA (data not
shown). However, this concentration of calyculin A seemed to be highly
toxic because the cells developed blebs within a few minutes. For this
reason, calyculin A was not further studied.
The potentiating
effects of these inhibitors were equally evident if OA (100
nM) or CsA (1 µM) was added 3 min before the
submaximal dose of Jurkat extract. 1-Norokadaone, a structural analogue
of OA unable to inhibit protein phosphatases(12) , was also
tested as a control for nonspecific effects. Fig. 1D shows that OA and CsA amplified the
[Ca]
response by a factor of
3. Both were statistically different from the control without
inhibitors. 1-Norokadaone even at 500 nM did not produce a
statistically significant potentiation.
Figure 2:
Protein phosphatase inhibitors potentiate
the [Ca]
responses to
low doses of carbachol and thapsigargin. Fura-2-loaded astrocytoma
cells were stimulated with 1 nM thapsigargin (TG; A and C) or 1 µM carbachol (CCh; B) either under control conditions (CTL; no pretreatment) or after 3 min of incubation with 100
nM OA or 1 µM CsA. An example of such experiment
is presented in A. Pooled results resulting from three
independent experiments are shown in B and C.
Individual response magnitudes have been normalized to the control. For
each condition, values have been integrated from 0 to 6-8 min
after cell extract addition and correspond to the mean of 10-12
individual dishes.
Such
potentiations of threshold stimuli are not restricted to astrocytoma
cells. As shown in Fig. 3A, responses of Jurkat
lymphocytes to a low dose of phytohemagglutinin (5 µg/ml), a
classic phosphoinositide-mobilizing agonist, are greatly potentiated by
subsequent addition of 100 nM OA. This dose of OA by itself
did not elevate [Ca]
above
resting levels (data not shown).
Figure 3:
Okadaic acid increases CIF concentration
in PHA-stimulated Jurkat T cells. A, fura-2-loaded Jurkat
cells were stimulated with 5 µg/ml PHA (firstdashedline). OA (100 nM each) was added twice (second and third dashedlines). B,
acidic extract was prepared from Jurkat T cells stimulated with 5
µg/ml PHA in the absence (control (CTL)) or presence of
500 nM OA for 15 min. The same amount of OA was added to the
control cells just before centrifugation to equalize OA carryover.
[Ca]
elevations
measured in fura-2-loaded astrocytoma cells were normalized to the mean
of the control. Each point corresponds to the mean of seven dishes from
two independent experiments. The difference is statistically
significant to p < 0.05 (Student's t test).
These results show that the
response to threshold concentrations of CIF, either exogenously applied
or endogenously released from the Ca stores, is
synergistically potentiated by protein phosphatase inhibitors. Two main
explanations can be proposed, which are not mutually exclusive. Either
OA and CsA enhance the sensitivity to CIF of its targets, for example
the Ca
channels, or they directly increase CIF
concentration, for example by inhibiting its degradation in the
cytoplasm. To begin to discriminate between these two possibilities,
experiments were designed to test whether OA affected CIF
concentrations in intact cells and in cell-free homogenates. Such
experiments were performed with Jurkat lymphocytes because such
nonadherent cells were much easier than astrocytoma cells to culture in
the necessary large quantity and high density.
Figure 4:
Time course and pharmacology of CIF
degradation in vitro. A, cell extract was prepared
from sonicated Jurkat lymphocytes and kept for variable times (shown on
the abscissa) at room temperature. Degradation was stopped by
freezing. Each ordinate is the mean of 12-27
[Ca]
increases in
astrocytoma cells in response to equal dilutions of the incubated
extract from three to six independent experiments. Values have been
normalized to those obtained with no added incubation time before
freezing. B, cell extract activity was measured for 0 or 5 min
of incubation under control conditions (CTL) or for 5 min with
500 nM OA, 1 µM CsA, or 1 mM EGTA.
Values have been normalized as described for A. Each bar
corresponds to the mean of 7-18 dishes from two to three
independent experiments.
The
pharmacology of the degradation (Fig. 4B) was then
investigated using the above protocol, but with drugs added just before versus after room temperature incubation for 5 min. This mode
of comparison ensured that the same amount of drug was carried over
into the assay on the responder astrocytes. As in Fig. 4A, 50% of the initial activity remained
after 5 min at room temperature with no inhibitors added. However, the
presence of OA (500 nM) or CsA (1 µM) during
incubation completely prevented net degradation. Because calcineurin
has been reported to be sensitive to Ca
and
calmodulin(8) , the effect of Ca
deprivation
on CIF degradation was also tested. For all the in vitro experiments presented above, Ca
was not buffered
and was probably several µM. The addition of EGTA (1
mM) at the beginning of the incubation also completely
abolished the run-down of CIF activity. The activities left after 5 min
of incubation in the presence of OA, CsA, or EGTA were all
statistically different from the incubation without additives, but
similar to the initial activity before incubation.
The aim of this study was to determine whether phosphatases
control Ca influx in mammalian cells and are
implicated in metabolic inactivation of a putative messenger mediating
Ca
influx. Our results show that protein phosphatase
inhibitors potentiate Ca
influx during submaximal
stimulation via conventional receptors in the plasma membrane, by
thapsigargin blockade of endoplasmic reticulum pumps, or by direct
application of CIF. CIF assays examining the effects of phosphatase
inhibitors in intact cells or sonicated homogenates suggest that this
potentiation can be at least partly explained as an increase in CIF
concentration due to prevention of CIF degradation, which is also
Ca
-dependent. We doubt that phosphatase inhibitors
act mainly by stimulating CIF synthesis because it would be surprising
if such stimulation so exactly canceled the degradation observed under
control conditions. However, we cannot exclude that mechanisms other
than protection of CIF contribute in intact cells to the potentiation
of the [Ca
]
response.
Furthermore, because CIF activity was assayed in a crude cell extract
rather than as a purified single species, the present results cannot
yet exclude the possibility that these modulations involve more than
one interacting molecular species in the extract.
Several points
still remain unclear. First, the molecular targets of OA and CsA are
not identified. These drugs have been reported to be specific
inhibitors of protein phosphatases(8, 14) . However,
current evidence indicates that CIF is a small nonprotein factor.
Several possibilities can be proposed to reconcile those two points. 1)
Protein phosphatases could dephosphorylate and activate a CIF-degrading
enzyme, which might or might not itself be a phosphatase. Because OA
and CsA are not supposed to act on the same protein phosphatase, one
would presume that the CIF-degrading enzyme is sensitive to protein
phosphatase 1 (or protein phosphatase 2A) and to protein
phosphatase 2B. A more complex cascade can also be proposed where this
enzyme would be dephosphorylated only by protein phosphatase 1 and
where protein phosphatase 2B would modulate the activity of protein
phosphatase 1 by dephosphorylating inhibitor-1. 2) Dephosphorylations
of nonprotein substrates by protein phosphatases 1 and 2B have been
reported(15) . However, these observations result from in
vitro experiments, not yet in intact cells. 3) Finally, it is
possible that OA as well as CsA are able to inhibit enzymes or
phosphatases other than protein phosphatases. Although other defined
phosphatases so far tested are insensitive to these agents, one cannot
exclude that such a phosphatase could exist. Whatever are the relevant
immediate targets of OA and CsA, our results in intact cells suggest
that enzymes involved in CIF degradation are active in unstimulated
cells or at least at resting [Ca]
levels.
CIF degradation can be demonstrated in a cell-free
homogenate, always appearing within the first 3 min after extraction
and never proceeding beyond 60% reduction of the initial activity.
Because the degradation can be blocked by OA, CsA, or EGTA, it probably
results from an enzymatic process and not from a spontaneous breakdown
of CIF, which is stable in the absence of proteins(6) . The
inability to degrade more than 60% of the initial activity could be due
to run-down of the enzyme(s) involved after cell disruption, so that
CIF molecules that have not been metabolized in the first few minutes
will never be. Because no reducing agents were added to the extract,
one possible inactivation mechanism could be oxidation. Another
possibility is that some fresh synthesis of CIF takes place in parallel
to CIF degradation. Two observations argue in favor of this latter
hypothesis. In the presence of EGTA, the activity after 5 min of
incubation seemed to be even greater than at time 0, as if the
concentration of CIF had increased. Even without inhibitors, CIF
activity seems to increase slightly after its initial decline. This
augmentation varied from one experiment to another and may reflect a
delayed appearance of fresh CIF in the extract. The pharmacology of the
synthesis has not been yet established. In intact stimulated cells,
Ca influx can last for minutes or hours, suggesting
that if degradation events take place, they have to be balanced by the
appearance of CIF in the cytoplasm. Under our cell-free conditions,
with enough Ca
to allow microsomal stores to refill,
degradation is presumably somewhat favored compared with synthesis.
In these in vitro experiments, 2-3 min were
required for a 50% loss of activity. This value appears to be high
compared with shorter values that have been reported in the literature
for the decline of the Ca
influx after Ca
store refilling (e.g. see (16) ). Perhaps our in vitro conditions are not optimal for enzymatic degradation.
Simple dilution in preparing the homogenate would tend to slow
enzymatic reactions. However, CIF degradation by phosphatases might not
be the only mechanism to remove CIF from the cytoplasm when
Ca
stores are allowed to refill. Reuptake of CIF into
its organellar storage sites (perhaps the endoplasmic reticulum) may
also contribute. Phosphatase action may be more important during cell
stimulation for adjusting the concentration of CIF and the amplitude of
Ca
influx especially for submaximal conditions of
stimulation. Indeed, with saturating doses of carbachol, thapsigargin,
or cell extract, the Ca
response was not modified by
protein phosphatase inhibitors. Furthermore, because CIF degradation
seems to be very Ca
-dependent, phosphatases may play
a role in negative feedback induced by
[Ca
]
on Ca
influx, which has been observed in many cell types with a time
course compatible to the degradation observed here (17, 18, 19, 20) .