(Received for publication, December 6, 1994; and in revised form, January 23, 1995)
From the
Acid extracts of thapsigargin-activated Jurkat cells have been shown to have intracellular activity in inducing a dose-dependent rapid chloride current upon microinjection in Xenopus laevis oocytes. The extracts act by elevation of calcium through calcium entry. The factor(s) responsible for this activity have been termed calcium influx factor (CIF) and have been found to be small, relatively polar molecules (<1000 daltons) whose activity is abolished by alkaline phosphatase treatment and potentiated by co-injection of okadaic acid (a protein phosphatase inhibitor). CIF is produced in a time-dependent manner following thapsigargin treatment of Jurkat cells, being first elevated above basal levels by 2 min. Intracellular CIF activity is completely absent from NG115-401L neuronal cells, which lack capacitative entry. On this basis, it appears that Jurkat cells, activated by stimuli that deplete internal calcium stores, produce one or more CIF activities acting intracellularly, and Xenopus oocytes may be a powerful tool to purify and characterize CIFs.
Receptors that mobilize calcium through production of InsP(
)frequently exhibit two phases in the elevation of
cytosolic [Ca
]
, an
initial transient peak due to release from stores and a sustained entry
through surface channels(1) . The latter response has been
termed ``capacitative entry,'' and its underlying mechanism
is unknown(1, 2) . However, the trigger for activation
of capacitative calcium entry is widely held to be depletion of
intracellular calcium stores(3) . Recently, it was proposed
that coupling between the stores and the surface might be via a novel
diffusible messenger, which was identified in acid extracts of Jurkat
T-lymphocytes(4) . However, this activity was exclusively
characterized by extracellular application. The intracellular activity,
if any, of such extracts has not been evaluated. Here, we provide
evidence that activated, but not resting, Jurkat cells have a calcium
influx activity acting intracellularly when assayed in Xenopus oocytes.
In order to test the intracellular calcium-elevating
activity of Jurkat cell extracts, which have previously been shown to
have extracellular activity on mammalian cells(4) , we
have exploited unique advantages of the Xenopus oocyte. First, Xenopus oocytes are large cells that can be readily and
rapidly microinjected with small amounts of cell extracts. Second, the
well characterized Ca-activated chloride current is a
rapid electrogenic measure of calcium elevation. Third, the powerful
calcium stores-depleting reagent, TG, has no direct calcium-elevating
activity in Xenopus oocytes (Fig. 1A; also see (7) ), thus permitting its use for stimulating target mammalian
cells.
Figure 1:
Extracts from thapsigargin-stimulated
Jurkat cells, but not thapsigargin itself, activate large
Ca-dependent chloride currents by an
InsP
-independent mechanism in Xenopus oocytes. A, responses to InsP
(IP
) injection (1 pmol, dottedtrace), InsP
co-injected with thapsigargin (1
µM), and thapsigargin alone are shown. Direct responses to
InsP
were 520 nA ± 115 nA (n = 10),
whereas injecting 1 µM thapsigargin into oocytes always
failed to induce responses (n = 10). Including
thapsigargin in the InsP
injection pipette had no
significant effect on InsP
-induced responses (610 nA
± 100, n = 5). B, microinjection of 10
nl of extract prepared as described under ``Experimental
Procedures'' consistently induced large chloride currents (2120
± 445 nA, n = 35). Removal of extracellular
Ca
(inclusion of 1 mM EGTA to nominally
Ca
-free OR2 medium) completely abolished the response (n = 5). C, microinjection of diluted extracts
demonstrates a dose dependence for oocyte responses with the activity
becoming undetectable at a 1:10 dilution. D, injection of
approximately 500 µg/ml heparin into oocytes (dottedtrace) failed to block responses induced by extract
injection (n = 4). Jurkat T cells were maintained in
suspension in RPMI 1640 supplemented with 10% fetal bovine serum, 2
mML-glutamine, and penicillin (100
units)/streptomycin (100 µg/ml). Jurkat cells were passaged by 1:10
dilution every 4 days.
Acid extracts prepared from TG-stimulated Jurkat lymphocytes
rapidly induced large current responses (2 µA) when injected
into Xenopus oocytes (Fig. 1B). Removal of
extracellular calcium abolished current responses, suggesting absolute
dependence on calcium influx (Fig. 1B). On this basis,
the extracts contain a ``calcium influx factor'' (CIF),
acting from the inside. The activity in the extract exhibited dose
sensitivity as shown by serial dilution (Fig. 1C).
Extract responses could not be blocked by prior heparin injection (Fig. 1D), demonstrating that extracts induce
Ca
elevation in a non-InsP
-dependent
manner.
To obtain additional evidence that the Jurkat extract was
not releasing Ca from internal stores,
extract-induced current responses were tested for cross-desensitization
with receptor-induced current responses elicited by endogenous LPA
receptors(5) . Extract responses did not desensitize oocyte
responses to subsequent exposure to LPA. Similarly, oocytes first
exposed to LPA did not lose their ability to respond to the
microinjected Jurkat extract (data not shown). This clearly suggests
that Ca
was not released from internal stores by
Jurkat cell extracts, because both calcium-mobilizing
receptor-activated current responses and direct InsP
injections exhibited complete, long lived, and reciprocal
desensitization under these conditions in the Xenopus oocyte(8) .
Using the magnitude of the current response as a measure of the level of production of CIF activity, the time course for CIF appearance following TG stimulation was determined. Extracts prepared at intervals following TG stimulation showed a 4-fold enhancement of activity that was first observed between 1 and 2 min, which was then maintained (Fig. 2). This suggests a latency for production of CIF following TG treatment, which correlates exactly with the time to activation of the capacitative entry channel following TG treatment in the same cell type, Jurkat lymphocytes(9) . These experiments also detected intracellular CIF activity in resting cells, in agreement with the previous report(4) , although the levels were greatly enhanced by TG treatment, unlike earlier results.
Figure 2: Time-dependent appearance of extract CIF activity. Extracts from thapsigargin-stimulated Jurkat lymphocytes were taken at 0, 30, 60, 120, and 600 s. Thapsigargin stimulations were quenched at the indicated times by rapid immersion in liquid nitrogen, and extracts from each time point were prepared as described under ``Experimental Procedures.'' Responses were measured by microinjection into Xenopus oocytes as described under ``Experimental Procedures.'' Results are representative of three similar determinations.
To
rule out that the activity observed was due to TG itself, rather than
calcium depletion, Jurkat cells were activated by other
calcium-depleting treatments. The mitogenic lectin, PHA, increased
intracellular CIF activity but to a lower level than that elicited by
TG (Fig. 3A). Treating Jurkat cells with the structurally
distinct Ca-ATPase inhibitor cyclopiazonic acid (10) also generated extracts with activity comparable with the
level produced by TG stimulation (Fig. 3A, dottedtrace).
Figure 3: Extract activity is stimulus-specific, abolished by alkaline phosphatase treatment and potentiated by the protein phosphatase inhibitor okadaic acid. A, Jurkat lymphocytes were stimulated as described under ``Experimental Procedures'' with PHA, CPA, or TG. Acid extracts were prepared and tested for activity by oocyte injection. PHA-induced current was 676 ± 221 nA, CPA (dottedtrace) was 1786 ± 340 nA, and TG-induced current was 1876 ± 435 nA. PHA- and CPA-stimulated extracts were duplicated and gave similar current responses in different oocyte batches. B, extracts from TG-stimulated Jurkat lymphocytes were incubated with 10 units of alkaline phosphatase (10 units/100 µl of extract) for 20 min at 37 °C. Alkaline phosphatase-treated extracts were then microinjected into oocytes to test for activity. No attempt was made to remove the alkaline phosphatase because injecting equivalent doses of the enzyme caused no response in oocytes. The response is representative of at least three alkaline phosphatase treatments of different extracts. C, extracts were diluted 1:8 with injection medium (10 mM HEPES pH 7.4) to achieve small reproducible responses (111 ± 34 nA; note the different scale for C). Okadaic acid was included in the injection pipette (final estimated concentration, 5 nM), where it potentiated the diluted extract response (826 ± 122 nA, n = 3). Injection of okadaic acid alone at these levels and up to 1 µM (estimated final concentration in oocyte cytoplasm) did not induce a response.
As a preliminary chemical characterization,
the intracellular CIF activity was heat-stable (70-95 °C, 20
min), did not bind to fatty acid-free bovine serum albumin, and
migrated as a small molecule of less than 1000 daltons on gel
filtration. ()The intracellular CIF activity of Jurkat
extracts was completely abolished by alkaline phosphatase treatment (Fig. 3B). These properties are similar to those
reported earlier, but intracellular CIF activity was not retained on
reverse-phase C18 columns, unlike extracellular activity(4) ,
indicating the possibility of a chemical distinction between
extracellular and intracellular CIF activities.
Previously, it was
reported that the protein phosphatase inhibitor okadaic acid prolonged
capacitative Ca influx in Xenopus oocytes(11) . Strikingly, co-injection of low doses of
okadaic acid with Jurkat cell extracts gave an immediate and dramatic
enhancement of the current response (Fig. 3C).
The
cell specificity of production of intracellular activity was evaluated
using other mammalian cell lines. U937 monocytes showed TG-induced
production of intracellular CIF activity (data not shown). In contrast,
a neuronal cell line, NG115-401L, when stimulated by TG did not produce
detectable CIF activity. This is important inasmuch as the NG115-401L
cell line has been shown to lack capacitative entry, because these
cells exhibit only a transient calcium elevation upon TG treatment,
arising exclusively from discharge of calcium stores(12) . This
difference in extracts from stimulated Jurkat and U937 cells or
NG115-401L cells suggests that the latter lack the capacity to produce
CIF acting intracellularly, which may correlate with the absence of a
capacitative Ca entry pathway in this cell type.
These results are summarized in Fig. 4. These experiments also
constitute crucial controls indicating that intracellular CIF activity
is not attributable to trivial consequences, such as pH changes or
stretch-activated currents, of cell extraction or oocyte microinjection
assay procedures.
Figure 4:
Cell specificity of extract CIF
production. A, extracts obtained from TG-stimulated Jurkat
lymphocytes elicit large (>2,000 nA) Ca-dependent
chloride currents (n = 35). Inset shows
typical response when TG (1 µM) is added to a population
of Jurkat lymphocytes loaded with the Ca
-sensitive
dye Fura-2/AM. B, extracts obtained from the NG115-401L cell
line fail to induce responses in oocytes. Acid extracts were prepared
as for stimulated Jurkat cells in cell number-matched preparations and
tested for their ability to induce oocyte responses by intracellular
injection. Results were confirmed by duplicate NG115-401L cell extract
preparations. Inset shows typical responses when TG (1
µM) is added to populations of NG115-401L cells loaded
with Fura-2/AM. NG115-401L neuronal cells were maintained in monolayer
culture in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum, 2 mML-glutamine and
penicillin (100 units)/streptomycin (100 µg/ml). NG115-401L cells
were passaged at 1:10 ratios every 3 days by trypsinization. Fura-2
loading and measurements followed standard
procedures(6) .
Receptors activating intracellular calcium discharge through
the production of InsP have been repeatedly correlated with
triggering a second sustained phase of calcium entry, which has been
termed ``capacitative entry.'' This calcium entry pathway is
distinct from known voltage-sensitive calcium channels in that it is
initiated by loss of calcium from stores(3, 13) ,
implying a coupling mechanism between calcium stores and the channel
itself. There are several proposals for the mechanism of capacitative
entry, including physical conformational coupling (14) or
biochemical pathways involving cytochrome P450(15) , guanylate
cyclase(16) , tyrosine kinases(17) , sphingolipid
metabolism(18) , GTP-binding proteins(19) , or a novel
diffusible messenger(4) . In arguing for a novel messenger,
Randriamampita and Tsien (4) presented evidence for a calcium
entry activity extracted from Jurkat cells, but the assay and
characterization of this activity were based upon extracellular
application. Here, we have tested similar Jurkat cell extracts for
intracellular activation of calcium entry in Xenopus oocytes.
Extracts are here shown to contain one or more factors acting
intracellularly with properties consistent with functions in
depletion-activated calcium entry.
This factor can be described
correctly as a CIF, using the term first applied to an extracellular
activity(4) , on the basis that activated
Ca-dependent chloride currents elicited by
microinjection are absolutely dependent on extracellular calcium.
Consequently, the activity of this extract is acting intracellularly to elicit an inward calcium flux. We have
tested extracellular activities of crude and fractionated cell
extracts on Xenopus oocytes as well, and a distinct factor
acting extracellularly has been identified that can be purified away
from the intracellular factor.
Thus, the simplest
interpretation is that two or more active components contribute to the
CIF activities of mammalian cell extracts. Indeed, it is unclear what
the relationship of the intracellular CIF activity is to the
extracellular CIF activity reported earlier. We would suggest that
``CIF'' should be considered an operational term of a
functional activity, by analogy with the conventional use of
Endothelium-Dependent Relaxation Factor (``EDRF'') for an
activity that may be attributable to nitric oxide in some, but not
necessarily all, cases. Accordingly, CIF should not be presumed to be
an explicit extract component at this stage, since several substances
may have this activity. Indeed, the CIF described here is not identical
to that described earlier on the basis of extracellular actions.
Intracellular CIF activity is abolished by alkaline phosphatase
treatment, suggesting it is a phosphomonoester, and its behavior on gel
filtration indicates it is a small molecule of M < 1000. These properties are shared with the extracellular CIF
activity described earlier, but the intracellular activity behaves on
reverse phase chromatography as a polar molecule (data not shown). On
this basis, the activity described here would not cross membranes and
be active by external application. Intracellular CIF is unlikely to be
cyclic ADP-ribose, sphingosine-1-P, lysophosphatidic acid, or an
inositol polyphosphate in that none of these substances mimic extract
properties and actions in oocytes.
Intracellular CIF shows
time-dependent production following TG treatment. Thus, it appears to
be synthesized in response to stores depletion. Intracellular CIF is
correlated with capacitative Ca entry in that it
cannot be measured, and may be completely absent, in extracts from a
cell type (NG115-401L cells) that lacks TG-stimulated capacitative
entry. These results imply an interesting possibility that
non-excitable cells may preferentially express a capacitative entry
pathway, whereas excitable cells may not.
Earlier experiments using Xenopus oocytes, which established the possibility of a diffusible messenger activating capacitative entry, demonstrated potentiation of calcium entry by the protein phosphatase inhibitor okadaic acid(11) . A comparable potentiation was observed here upon co-injection of low doses of okadaic acid with Jurkat extracts. The speed, potency, and selectivity of the potentiation are consistent with okadaic acid acting on its identified protein phosphatase targets. This implies a serine/threonine kinase/phosphatase system in CIF regulation of capacitative entry channels.
In summary, these results show that Jurkat cells contain one or more substances with intracellular CIF activity. Moreover, Xenopus oocytes can be used as a simple and flexible assay for purification of such factors.