(Received for publication, December 22, 1994; and in revised form, January 24, 1995)
From the
Randriamampita and Tsien (Randriamampita, C., and Tsien, R.
Y.(1993) Nature 364, 809-814) suggested that an
acid-extracted fraction from a Jurkat cell line contains a messenger
responsible for the coupling of calcium entry to the depletion of
intracellular stores, i.e. capacitative calcium entry. We
found that the extract, prepared as described by Randriamampita and
Tsien, caused Ca entry in 1321N1 astrocytoma cells
which was not blocked by the D-myo-1,4,5-trisphosphate-receptor antagonist,
heparin. In contrast to astrocytoma cells, when applied to mouse
lacrimal acinar cells and rat hepatocytes the Jurkat extract always
caused the release of intracellular Ca
, followed by
Ca
entry across the plasma membrane. This activity of
the extract on lacrimal cells was blocked by either intracellular
injection of heparin or extracellular atropine. Similarly prepared
lacrimal cell extracts gave Ca
responses when applied
to astrocytoma cells or lacrimal cells which were similar to those for
Jurkat-derived extract. However, extracts from hepatocytes had no
effect. In most Xenopus oocytes, the Jurkat extract had no
effect, while in a few oocytes, the extract gave a
[Ca
]
response similar
to that seen in lacrimal cells, that is, release of Ca
followed by Ca
entry. We conclude that the
actions of the Jurkat cell extract are not consistent with its
containing the long sought messenger for capacitative calcium entry. It
is likely that this fraction contains a number of factors that mediate
Ca
response in different cell types, possibly through
receptor-mediated mechanisms.
Activation of surface membrane receptors in many cell types
results in a complex, biphasic Ca response composed
of an initial mobilization of internally stored Ca
,
followed by entry of extracellular Ca
. An early event
following receptor activation is the well characterized generation of
the putative second messenger, D-myo-inositol
1,4,5-trisphosphate ((1,4,5)IP)
, (
)which is
responsible for the mobilization of internally stored Ca
(1) . Considerable evidence suggests that depletion of
the agonist- and IP
-sensitive intracellular
Ca
-pool then leads to the activation of the secondary
Ca
entry phase of the response, a process which has
been termed capacitative calcium
entry(2, 3) . Although the nature of the linkage
between the depletion of internal Ca
pools and the
entry of Ca
is unclear, recent reports have suggested
that internal pool depletion leads to the generation of a soluble
messenger that signals the activation of the entry
process(4, 5) . In addition, there is evidence for a
GTP-dependent step in regulating this entry
process(6, 7) . Recently, Randriamampita and Tsien (5) reported the isolation of an acid extracted fraction from
Jurkat T-cells that contained a factor that could specifically activate
capacitative Ca
entry, a factor which they termed CIF
(calcium influx factor).
In the studies reported here, we have
isolated an acid-extracted fraction from Jurkat T-cells and other
mammalian cell types, and examined the effects of these extracts on
calcium signaling in various mammalian cells, including astrocytoma
cells, and in Xenopus laevis oocytes. The activity of the
Jurkat extract on astrocytoma cells was similar to that described for
CIF, confirming observations of Randriamampita and Tsien. However, the
effects of the Jurkat extract on other cell types and the effects of
extracts isolated from cells other than Jurkat cells are not consistent
with its purported role as a mediator of capacitative calcium entry.
Rather, our findings suggest that such crude extracts contain a number
of factors that mediate Ca response in different cell
types, possibly through receptor-mediated mechanisms.
Following the procedures described by Randriamampita and
Tsien(5) , we isolated an acid-extracted fraction from the
Jurkat cells. The cells, 0.4-0.6 ml packed cell volume, were
washed in nominally Ca-free HPSS. After
centrifugation (180
g, 4 min), the cell pellet was
resuspended in 700 µl of the same HPSS buffer, acidified to pH 1
with approximately 150 µl of 1 M HCl, and kept on ice for
20 min. The suspension was then centrifuged (180
g for
10 min), and the supernatant was retained and restored to pH 7.3 with 1 M NaOH. The final volume of the extract at this stage was
approximately 1 ml. When necessary, the extract was further incubated
for 20 min with 2 units/ml hexokinase (Sigma) to remove ATP, thereby
avoiding any possible effects through activation of purinergic
receptors. In the experiments described below, 200 µl of the Jurkat
extract plus 400 µl of HPSS (final [Ca
]
was either 1.8 mM or nominally Ca
-free) were
added to the cells tested.
One modification to this procedure was in
the preparation of an extract for oocytes. The procedure was
essentially as described above, except that the initial cell pellet was
brought up in water to minimize the osmolality of the extract. With
this procedure, the osmolality of the extract was approximately 275
mOsM and retained Ca-mobilizing activity
when assayed on astrocytoma cells. The extract was diluted to an
osmolality of 200 mOsM before being applied to oocytes.
The
distribution of calcium signaling activity between soluble and
particulate fractions of stimulated and nonstimulated Jurkat cells was
determined as described by Randriamampita and Tsien(5) . Packed
cells (0.4-0.6 ml) were resuspended in 60 mls of nominally
Ca-free HPSS supplemented with 0.5 mM EGTA.
The cells were then treated with or without phytohemagglutinin (PHA; 20
µg/ml) for 10 min. After stimulation, the suspension was
centrifuged (180
g, 4 min), and the pellet was
resuspended in 700 µl of nominally Ca
-free HPSS
containing saponin (2000 µg/ml) and incubated for 5 min to
permeabilize the plasma membrane. Permeabilization was monitored by
trypan blue dye exclusion. The suspension was centrifuged (180
g for 10 min) and the supernatant retained, while the pellet
was resuspended in an equal volume of nominally
Ca
-free HPSS. Both fractions were acidified to pH 1
with 1 M HCl and kept on ice for 20 min. Each fraction was
then centrifuged, and the supernatants were passed through a
reverse-phase octadecyl silica cartridge (SPICE C18 cartridge, Rainin)
to remove saponin. The extracts were neutralized, and ATP was removed
with hexokinase treatment, as described above. The fraction derived
from the supernatant after saponin treatment was considered the soluble
fraction, and the fraction from the resulting pellet was the
particulate fraction.
For
[Ca]
measurements, the oocytes
were injected with 50 nl of OIM containing 2 mM fura-2 free
acid (Molecular Probes) 30 min prior to the beginning of the
experiment. The fluorescence of the fura-2-loaded cells was monitored
as described except that a Nikon 20
Neofluor objective was used.
For membrane current measurements, two electrodes (filled with 3 M KCl, resistance 1 megaohms) were inserted into oocytes
and connected to a voltage-clamp amplifier (GeneClamp 500, Axon
Instruments, Foster City, CA). The membrane potential was held at
-60 mV, and recordings were sampled at 5 Hz and low pass filtered
at 100 Hz.
All figures show either means ± S.E. or representative results from at least three independent experiments.
Figure 1:
Ca responses in
1321N1-astrocytoma cells. Single, fura-2-loaded astrocytoma cells were
incubated in the absence of extracellular Ca
, and
then treated with a, 100 µM MeCh; b, 2
mM thapsigargin; or c, Jurkat extract. Extracellular
Ca
(1.8 mM) was restored to the cell at the
time indicated. Typically, astrocytoma cells responded to both MeCh (15
cells) and thapsigargin (11 cells) with mobilization of intracellular
Ca
and a sustained elevation of cytoplasmic
Ca
on restoring extracellular Ca
to
1.8 mM. The Jurkat extract mainly caused Ca
entry in 1321N1 astrocytoma cells when extracellular
Ca
was restored (29/34 cells), but 5/34 of the cells
responded with an initial release of Ca
from internal
stores in the absence of extracellular Ca
(not
shown).
Figure 2:
The(1, 4, 5) IP-receptor
antagonist, heparin, blocks MeCh but not Jurkat extract responses in
1321N1 astrocytoma cells. Single, fura-2 loaded astrocytoma cells were
microinjected with heparin (200 mg/ml, pipette concentration) as
described under ``Materials and Methods.'' The astrocytoma
cell was first exposed to a, 100 mM MeCh, and then to b, Jurkat extract. Whereas the actions of the muscarinic
agonist MeCh were blocked, Jurkat extract-activated Ca
entry in astrocytoma cells was not affected by intracellular
application of
the(1, 4, 5) IP
-receptor
antagonist, heparin. The data are representative traces from three
similar experiments.
By using the responsiveness of astrocytoma cells as an assay, we evaluated the subcellular distribution in Jurkat cells of this calcium entry stimulating activity. Following the method described by Randriamampita and Tsien(5) , the soluble fraction was derived from the supernatant following the centrifugation of saponin treated Jurkat cells (see ``Materials and Methods''), and the remaining pellet yielded the particulate fraction. As shown in Fig. 3, in nonstimulated Jurkat cells, the calcium entry stimulating activity appeared exclusively in the particulate fraction. Upon stimulating the cells with PHA (20 µg/ml), activity appeared in the soluble fraction (Fig. 3), although with little apparent loss of activity from the organelle fraction. Although this redistribution of activity is qualitatively consistent with the data of Randriamampita and Tsien (5) , these authors reported a quantitative redistribution of activity from a particulate to a soluble fraction which we were never able to achieve. Thus, the findings to this point largely reproduce those of Randriamampita and Tsien (5) and indicate that the extract we have prepared contains the active principle which Randriamampita and Tsien designated as CIF.
Figure 3:
Subcellular distribution of calcium
signaling activity isolated from either resting or PHA-stimulated
Jurkat cells. Cytoplasmic and particulate fractions of Jurkat cells
were prepared, as described under ``Materials and Methods.''
The traces on the left demonstrate the effect on astrocytoma cells of a
soluble and particulate fraction of non-stimulated Jurkat cells. The
traces on the right demonstrate the effect on astrocytoma cells of the
same fractions prepared from PHA-stimulated Jurkat cells. The activity,
assayed on astrocytoma cells, was entirely in the particulate fraction
of resting Jurkat cells, and stimulation of the cells with PHA resulted
in the appearance of activity in the soluble fraction. Note that
dilution of the extracts yielded appreciably less Ca signaling activity, indicating that the material was assayed at a
less than supramaximal concentration. The data are representative
traces from five independent preparations.
Figure 4:
Jurkat extract effects on mouse lacrimal
acinar cells. A single, fura-2-loaded mouse lacrimal cell was incubated
in the absence of extracellular Ca, and then treated
with the Jurkat extract. When
[Ca
]
returned to basal
levels, extracellular Ca
(1.8 mM) was
restored to the cell. The same protocol was also followed on a lacrimal
cell injected with the (1, 4, 5) IP
-receptor antagonist,
heparin (200 mg/ml, pipette concentration). Under these conditions, the
response to the Jurkat extract was completely blocked. The data shown
are representative traces from three similar
experiments.
We considered that the failure of hepatocytes and lacrimal cells to respond to CIF might reflect the absence of the principle in these cell types. We thus applied the isolation procedure described by Randriamampita and Tsien (5) to these cell types, assaying the subsequent activities on astrocytoma cells and lacrimal cells. An acid extracted fraction from rat hepatocytes displayed no activity when applied to either astrocytoma cells or lacrimal cells (three experiments, data not shown). However, an extract isolated from mouse lacrimal cells induced a response in astrocytoma cells similar to that induced by the Jurkat extract (Fig. 5a). As found for the Jurkat extract, the lacrimal extract induced a muscarinic-like activity when applied to mouse lacrimal cells (Fig. 5b).
Figure 5:
A lacrimal cell derived extract exhibited
effects similar to those produced by the Jurkat extract on
1321N1-astrocytoma cells and lacrimal cells. The extraction procedure
for Jurkat cells was also performed on lacrimal acinar cells, and the
activity of the resulting extract was assayed on astrocytoma cells (a) and lacrimal cells (b). The response to the
lacrimal extract on both cell types was essentially the same as that
observed for the Jurkat extract. The extract was added to astrocytoma
cells in the continued presence of external Ca, while
for the lacrimal cells, the extract was added in the absence of
external Ca
, and external Ca
was
then restored as indicated. The experiments shown are representative of
similar findings in three independent
experiments.
We next investigated
the actions of the Jurkat extract in oocytes of X. laevis.
Previous studies with this cell type have provided some of the most
compelling evidence for the existence of a diffusible messenger for
capacitative calcium entry(4) . (1, 4, 5) IP, or metabolically
stable analogues of (1, 4, 5) IP
,
are known to activate Ca
entry in this cell
type(11, 12) . In confirmation of these findings, Fig. 6shows that intracellular injection of the
nonmetabolizable(1, 4, 5) IP
analogue(2, 4, 5) IP
(9) , caused, in all oocytes, an intracellular
Ca
release, followed by a sustained elevation of
[Ca
]
on restoring extracellular
Ca
. By contrast, most of the oocytes tested (9/14)
were insensitive to the Jurkat extract (Fig. 6a). The
remainder responded to the extract with a
[Ca
]
response involving both
the release of internal Ca
as well as entry of
extracellular Ca
(Fig. 6b). In
addition, a small percentage of oocytes (5/19) responded to MeCh with a
typical biphasic mobilization of Ca
. Some oocytes
were tested for their responsiveness to both MeCh and the Jurkat
extract, and only MeCh-responsive oocytes were responsive to the
extract (three cells responsive to both; three cells responsive to
neither) (Fig. 6b). However, in the presence of 10
µM atropine, no oocytes responded to either MeCh or the
extract. Thus, the [Ca
]
responses to extracellularly applied Jurkat cell extract seen in
a few oocytes apparently represents activation of muscarinic receptors
as seen in lacrimal acinar cells. Intracellular injection of the Jurkat
extract in oocytes perifused with a medium containing 5 mM Ca
gave no
[Ca
]
response (n = 6).
Figure 6:
Effects of extracellular application of
the Jurkat extract on
[Ca]
in Xenopus oocytes. Fura-2-injected oocytes were initially bathed
in a Ca
-free medium. After testing their response to
10 µM MeCh, they were perifused with a medium containing 5
mM Ca
. A small transient rise in
[Ca
]
was sometimes
seen on addition of Ca
to unstimulated oocytes.
Thereafter, the Jurkat extract (JE) was applied in a medium
lacking Ca
and then in the presence of 5 mM Ca
. At the end of the experiment, intracellular
release and entry of Ca
was activated by injection of (2, 4, 5) IP
. As illustrated by
these two representative traces, the small percentage of oocytes
sensitive to the Jurkat extract was also sensitive to MeCh. The data
shown are representative traces from three experiments (for a and b) carried out with this protocol; however, the
majority of oocytes were not responsive to MeCh (see
text).
In the course of studying the effects of the
Jurkat extract on Ca signaling in Xenopus oocytes, we also examined the effects of the extract on membrane
currents because in this cell type, an endogenous
Ca
-dependent chloride current is often used as an
indicator of [Ca
]
changes(13, 14) . As shown in Fig. 7,
intracellular injection of(2, 4, 5) IP
produced a large transient chloride current in a
Ca
-free medium which was reactivated on restoring
extracellular Ca
. Application of the extract in a
Ca
-free medium produced a large and sustained inward
current (n = 32; Fig. 7, a and b), which occurred in oocytes insensitive to 10 µM MeCh and which was resistant to 10 µM atropine. This
current did not result from Ca
mobilization since it
also developed after the intracellular Ca
pools were
depleted by (2, 4, 5) IP
injection (Fig. 7b). Furthermore, the extract
current was totally insensitive to 500 µM niflumic acid, a
potent blocker of the endogenous Ca
-activated
chloride current(15) . Voltage ramps between -80 and
+80 mV (2-s duration) indicated that the current was relatively
linear with slight inward rectification at negative potentials and with
a reversal potential of -16.5 ± 0.3 mV (n = 3; Fig. 8). The extract-induced current was
largely reduced in a medium containing 1 mM Ca
(n = 3) and completely abolished in a medium
containing 5 mM Ca
(n = 13; Fig. 7a). Intracellular injection of the extract
(approximately diluted 10-fold after injection in the oocyte) did not
affect the current (n = 23), whereas the same oocyte
displayed a large current response to extracellular application of the
extract diluted to a similar extent (n = 4; Fig. 7b). Thus the Jurkat extract activated, in all
oocytes, a current response that was independent of any change in
[Ca
]
, and that was blocked by
extracellular Ca
.
Figure 7:
Effects of intracellular and extracellular
application of the Jurkat cell extract on the voltage-clamped current
in Xenopus oocytes. The oocytes were initially bathed in a
Ca-free medium. Thereafter, they were perifused with
a medium containing 5 mM Ca
, 10 µM MeCh or Jurkat extract (JE) where indicated, and they
were injected with(2, 4, 5) IP
or
the extract as indicated. Intracellular application of (2, 4, 5) IP
always induced a
large Ca
entry on restoring Ca
which was reflected by an increase in the
Ca
-dependent chloride current. By contrast, the
extract was without effect in the presence of 5 mM Ca
, whereas it induced a large sustained current
in a Ca
-free medium (a). This current could
not be fully reversed by washing the extract with a
Ca
-free medium, whereas it returned quickly to basal
level upon readdition of 5 mM Ca
to the
medium (a). Intracellular injection of the extract (final
dilution 10-fold) did not activate any current response, whereas
extracellular application of the extract diluted to a similar extent
produced a large current in a Ca
-free medium, even
when intracellular Ca
pools were emptied
following(2, 4, 5) IP
injection (b). The data shown are representative traces from 13 (a) and 4 (b) similar
experiments.
Figure 8:
Current-voltage relationship of the
responses activated by the Jurkat extract () and
Ca
injection (
) in Xenopus oocytes
bathed in a Ca
-free medium. The currents of the two
traces were normalized by dividing all points by the amplitude of the
current at +80 mV. The current activated by the Jurkat extract at
+80 mV averaged 6.22 ± 0.19 µA and that activated by
Ca
injection was 2.25 ± 0.4 µA. Each trace
was generated from voltage ramps (2-s duration) obtained in three
oocytes.
In this study, we have followed on the work of Randriamampita
and Tsien (5) who reported the presence of a Ca entry-stimulating principle in acidic extracts of Jurkat cells,
and who concluded that this principle may be the long sought messenger
for capacitative calcium entry. We confirmed some of the observations
of Randriamampita and Tsien in that extracellular application of an
acid extract of Jurkat cells to astrocytoma cells induced a calcium
entry and, at least in the majority of cells, this was not accompanied
by a release of intracellular Ca
. We also confirmed
that stimulation of the Jurkat cells with PHA caused a release of this
Ca
entry-stimulating principle into the soluble
fraction of Jurkat cells. Finally, we found that the actions of the
Jurkat extract on astrocytoma cells were not blocked by the
intracellular application of
the(1, 4, 5) IP
receptor
antagonist, heparin. All of these observations indicate that we have
prepared an extract containing the active principle which
Randriamampita and Tsien (5) called CIF. Further, they are
consistent with the interpretation given by Randriamampita and Tsien
that the calcium entry stimulating principle in the Jurkat extract is
the second messenger for capacitative calcium entry.
However, when
we investigated further the properties of this extract in astrocytoma
cells and other cell types, our findings cast doubt on this
interpretation. In three other cell types known to utilize the
capacitative Ca mechanism, lacrimal acinar cells,
hepatocytes, and X. laevis oocytes (only a minority responded
at all), Jurkat extracts did not induce Ca
entry
alone, but rather activated a biphasic Ca
mobilization pattern which appeared to result from activation of
surface membrane receptors; when these receptor responses were blocked,
the Jurkat extract was inactive in these cell types. This despite the
fact that lacrimal cells appeared to contain the same active
principle(s) as found in Jurkat cells. Further, in at least one cell
type in which capacitative Ca
entry is known to
occur, the rat hepatocyte(16, 17) , no Ca
signaling activity could be extracted. In a previous
communication(18) , we have pointed out that the temporal
pattern of [Ca
]
signaling
induced by Jurkat extracts in astrocytoma cells is quite different from
that induced by MeCh or thapsigargin, agents presumably acting through
the true messenger for capacitative Ca
entry. This
difference is also apparent in the experiments reported here, wherein
MeCh and thapsigargin induced sustained
[Ca
]
signals, while, as also
reported by Randriamampita and Tsien(5) , the extract tended to
induce irregular oscillations. In addition, we found that the
Ca
entry-stimulating activity in the Jurkat extract
was difficult to remove by washing astrocytoma cells; removal of MeCh,
which presumably acts through the true messenger for calcium entry,
resulted in rapid reversal of Ca
entry(18) .
It is important to note that the Jurkat cell extract was found to
contain not a single Ca signaling principle, but a
number of activities which affected not only Ca
movements across the cell membrane, but also surface membrane
receptors, as well as membrane channels for ions other than
Ca
. Further, these activities were expressed in a
cell type-specific manner. Given the complex signaling and paracrine
functions of the T-lymphocytes from which the Jurkat line is derived,
it is perhaps not surprising that these cells contain many substances
with diverse biological activities. However, we conclude from our data
that it is probably incorrect to attribute to any of these the role of
intracellular messenger for capacitative Ca
entry.