From the
Acid extracts of thapsigargin-stimulated Jurkat cells revealed
both intracellular and extracellular activities stimulating
Ca
Cytosolic Ca
We
recently reported that activated Jurkat cells produce an
intracellular-acting CIF activity measured indirectly as a
Ca
We have shown previously that acidic extracts from
TG-stimulated Jurkat lymphocytes have intracellular activity in
activating a calcium influx pathway
(5) . We now report that
these extracts also exhibit extracellular activity on oocytes in
stimulating the Ca
The activities were not retained on reverse phase cartridges
or by a high molecular weight cut-off filter. The first separation of
the activities into distinct fractions was observed on gel filtration
(Fig. 2). Two active fractions were obtained. The first, called
Peak 1, with an estimated molecular weight of 600, exhibited both
extracellular and intracellular activities. The second, called Peak 2,
with an estimated molecular weight of 400, exhibited exclusively
intracellular activity (Fig. 2 B).
In order to
compare the intracellular activity of Peak 1 to the previously reported
properties of crude acid extracts
(5) , the effect of external
calcium removal was examined. Using calcium-free medium (containing 1
mM EGTA) only 40% of the current could be blocked, indicating
the remaining response was due to calcium discharge. This incomplete
external calcium dependence of the current response was unlike that of
crude acid extracts and was confirmed using extracellular medium
containing 0.5 mM Ni
One
HPTLC-purified band ( R
In previous work, we have reported that a novel CIF can be
identified by intracellular injection of oocytes with extracts from
TG-activated cells. However, the issue of the possible extracellular
activity of CIF remained unresolved. The results reported here
demonstrate that extracellular activity can be detected by external
application of CIF-containing extracts to oocytes, but that it is
attributable to a factor distinct from an authentic CIF. Moreover, the
acid extracts are surprisingly complex in the diversity of distinct
components active on calcium elevation.
We have identified two size
fractions on gel filtration which are active. The smaller size
fraction, of approximately 400, is an intriguing and unexpected
activity in that it induces calcium discharge by an
InsP
The larger size
fraction, of approximately 600 daltons, contains distinct and separable
extracellular and intracellular factors. Only the latter, resolved by
HPTLC, is a candidate for authentic CIF based on 1) its increased
levels in cell extracts following thapsigargin treatment and 2) its
distinctive profile of biological activity, eliciting calcium entry,
but not discharge, exclusively by intracellular application.
Thus,
extracts thought to contain a single active component have, by this
analysis, proven to have at least three distinct factors regulating
calcium-dependent currents from external or intracellular sites of
action in Xenopus oocytes. This introduces a significant
complication in efforts to identify CIF activities in crude extracts.
We thank Dr. Brett Premack (Department of Molecular
Pharmacology, Stanford University School of Medicine) for helpful
discussions and his critical advice on the experiments described in
this manuscript. We also gratefully acknowledge Dr. Rich Nuccitelli
(Section of Molecular and Cellular Biology, University of California,
Davis) for usage of the X. laevis oocyte colony.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dependent Cl
currents on
Xenopus laevis oocytes. Chromatographic fractionation of these
extracts on gel filtration separated two active fractions of
M
approximately 600 and 400. Moreover, the
M
600 fraction exhibited both intracellular and
extracellular activities. However, the intracellular activity was
absent from extracts of unstimulated Jurkat cells, suggesting its
production was stimulated by thapsigargin. The further purification of
this fraction by high performance thin layer chromatography resolved a
single fraction which was active only on microinjection and which
required calcium entry for activation of current responses. These
results suggest that a single authentic calcium influx factor can be
resolved by purification from confounding activities detected in crude
acid extracts.
is elevated through multiple
mechanisms, including release from stores mediated by
InsP
(
)
or ryanodine receptor channels
(1) . Following release, the depletion of intracellular
Ca
stores induces Ca
influx across
the plasma membrane. This calcium influx pathway has been termed
``capacitative calcium entry''
(2) . However, the
mechanism which couples the depletion of the stores to Ca
influx across the plasma membrane is unclear. Recently, it was
proposed that depletion of Ca
stores might release a
novel diffusible messenger (calcium influx factor (CIF)) which opens a
specific class of calcium channels
(3, 4) .
-dependent Cl
current in
Xenopus oocytes
(5) . Here we establish that there are
different factors which act extracellularly or
intracellularly. The partially purified CIF reported here acts
exclusively intracellularly to induce the characteristic indicator of
elevated cytosolic calcium changes in oocytes, the
Ca
-dependent Cl
current. These
results suggest that acid extracts from Jurkat cells are a complex
mixture of agents active on calcium metabolism, but an authentic CIF
regulating the capacitative Ca
entry pathway can be
selectively purified away from both intracellular discharge and
extracellular calcium-elevating activities.
Materials
Thapsigargin (TG) was purchased from
LC Services (Woburn, MA). Hanks' balanced salt solution (HBSS)
and L-15 medium were from Life Technologies, Inc. RPMI 1640 was from
BioWhitaker (Walkersville, MD). Bio-Gel P-2 gel was from Bio-Rad.
Microcon-30 was from Amicon (Beverly, MA). Sep-Pak cartridge
(C) was from Millipore (Milford, MA). HPTLC plate
(200-µm layer) was from Whatman.
Cell Culture
Jurkat T lymphocytes 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.
Extract Preparation
Crude extracts were prepared
from resting and stimulated Jurkat cells as described previously with
minor modification
(5) . Briefly, cells were washed three times
with HBSS containing 20 mM HEPES. Jurkat cells were stimulated
with 1 µM TG for 15 min at 25 °C. Cells were
centrifuged for 5 min at 200 g, and the pellet was
resuspended in 0.85 ml of HBSS containing 20 mM HEPES. The
suspension was extracted with 0.15 M HCl and was incubated for
30 min at 25 °C. The extract was clarified by centrifugation for 10
min at 400
g, and the supernatant was neutralized by
addition of 10 M NaOH. After neutralization, 10 mM
BaCl
was added to the extract for precipitation of vicinal
phosphate compounds, including inositol polyphosphates. Insoluble
material was removed from extracts by centrifugation for 5 min at
12,000
g. The supernatant was lyophilized, and the
residue was extracted with methanol with continuous mixing for 15 min
at 25 °C. The methanol extract (0.8 ml) was loaded on the Sep-Pak
C
cartridge for removal of hydrophobic material and was
rinsed with methanol (0.8 ml, flow rate: 0.25 ml/min). The methanol
eluants were combined (1.6 ml), dried under N
gas (30
°C), and resuspended in 0.1 M acetic acid (0.25 ml). The
reconstituted extract was clarified by centrifugal ultrafiltration
through a Microcon-30 filter. The filtrate is enriched for materials of
M
< 30,000, whereas the retentate is material
M
> 30,000. The filtrate was loaded on the gel
filtration column for further purification.
Gel Filtration Chromatography
The filtrate was
loaded onto a Bio-Gel P-2 column (0.7 27 cm), equilibrated with
0.1 M acetic acid, and was eluted by the same buffer (7 ml/h),
collecting fractions of 0.5 ml. Activity was assayed by measuring
Ca
-dependent Cl
current in
Xenopus oocytes under voltage clamp as described previously
(5, 6) . Fraction 15 from TG-stimulated cell extracts
has both extracellular and intracellular activities and is termed
``Peak 1.'' Fractions 18 and 19 have only intracellular
activity, and were pooled to form ``Peak 2.''
HPTLC
Peak 1 was applied to a 200-µm HPTLC
plate and developed with chloroform/methanol/acetic acid/water
(20/15/8/4, v/v). Peak 1 was resolved into at least five bands using UV
detection. Each identified band was scraped from the plate, extracted
with 0.1 M acetic acid, and lyophilized. Each fraction was
then reconstituted in 50 µl of buffer (10 mM HEPES, pH
7.0) and activity tested extracellularly and intracellularly by
measuring Ca-dependent Cl
current
in Xenopus oocytes.
Oocyte Injections and Voltage Clamp Recording
Each
fraction was tested for activity by measuring
Ca-dependent Cl
currents under
voltage clamp conditions. Xenopus laevis oocytes were obtained
by ovarectomy. Follicular cells were removed from oocytes by treating
with collagenase (2 mg/ml) for 1 h and 45 min, followed by rolling
oocytes on plastic Petri dishes. Defolliculated oocytes were maintained
in modified L-15 medium (diluted 1:1 with 30 mM HEPES buffer,
pH 7.4, containing 0.25% chicken ovalbumin, 1 mML-glutamine, and 50 µg/ml gentamycin). Conventional
two-electrode voltage clamping was performed as described previously
(5, 6) . Oocyte injections were performed using the
Picospritzer (General Valve Corp, Fairfield, NJ) pressure injection
apparatus. Oocytes were voltage-clamped at
60 mV in OR2 medium
(82.5 mM NaCl, 2.5 mM KCl, 1 mM
CaCl
, 1 mM MgCl
, 1 mM
Na
HPO
, 5 mM HEPES, pH 7.4) and were
injected with 10 nl of each chromatographic fraction. Currents were
digitized using the Tl-1 A/D board (Axon Instruments Inc., Foster City,
CA) in combination with the current analysis program SCAN (Dagan Corp.,
Minneapolis, MN).
-dependent Cl
current. This extracellular response was, however, unlike the
intracellular activity, in that extracellular responses have no
dependence on extracellular calcium (Fig. 1). This is similar to
the earlier report of extracellular CIF activity
(3) , but the
dramatic difference in sensitivity to extracellular calcium removal
suggested that these intracellular and extracellular activities were
not the same and were therefore attributable to distinct and
independent factors.
Figure 1:
The extracellular response induced by
Jurkat extract does not require extracellular calcium. Extracts
prepared as described previously (5) from thapsigargin-stimulated
Jurkat lymphocytes were diluted 1:2 in Ca-free OR2
medium (82.5 mM NaCl, 2.5 mM KCl, 1 mM
MgCl
, 1 mM Na
HPO
, 5
mM HEPES, pH 7.4) supplemented with 1 mM EGTA, and 5
µl was applied to the oocyte bath (300 µl). To remove
extracellular Ca
( dotted trace) oocytes were
perfused for 2 min in nominally Ca
-free OR2
supplemented with 1 mM EGTA before application of extract. The
responses were representative of three similar experiments from
different batches of oocytes.
Accordingly, in order to purify intracellularly
acting CIF activity from Jurkat cell extracts, we have introduced a
sequence of purification steps: 1) Sep-Pak reverse-phase columns, 2)
Microcon-30 ultrafiltration, 3) Bio-Gel P-2 gel filtration, and 4)
HPTLC.
Figure 2:
Thapsigargin stimulation specifically
augments the intracellular activity of high molecular weight gel
filtration fraction. Fractionation of unstimulated ( A) and
TG-stimulated ( B) cell extracts on gel filtration
chromatography. Bio-Gel P-2 columns (0.7 27 cm) were
equilibrated and eluted with 0.1 M acetic acid. Fractions (0.5
ml) were collected at a flow rate of 7 ml/h. The extracellular
( open circles) and intracellular ( crosses) activities
(nA) are shown as Ca
-dependent Cl
current in Xenopus oocytes, as described under
``Experimental Procedures.''
The fractionation
of extracts from unstimulated Jurkat cells (Fig. 2 A) was
compared to that from TG-stimulated cells. Peak 2, which is only active
by intracellular injection, was unaffected by TG stimulation. Although
the extracellular activity of Peak 1 was also unaffected by TG
stimulation, the intracellular activity of Peak 1 was specifically
augmented (Fig. 2 A). These results suggest Peak 1 is
heterogenous and contains two distinct factors, the intracellular, but
not the extracellular, activity being produced by depletion of
Castores. Therefore, the intracellularly active
component in Peak 1 is a candidate for an authentic CIF.
as a Ca
entry channel blocker
(7) . Under these conditions, the
response was also blunted by about 40% (Fig. 3). These results
suggested that the intracellular activity of Peak 1 contained factors
influencing both calcium entry as well as calcium mobilization from
stores. The calcium discharge activity can be ascribed to a contaminant
in that it is completely lost by a 1:4 dilution. Using such diluted
extracts, the response to microinjection is not reduced, but shows
complete extracellular calcium dependence (data not shown).
Figure 3:
Gel filtration Peak 1 activates
Ca-dependent Cl
currents in
Xenopus oocytes by microinjection. Peak 1 (10 nl) prepared as
described under ``Experimental Procedures,'' induced chloride
current by intracellular injection (2300 ± 365 nA, n = 25). Removal of extracellular Ca
(inclusion of 1 mM EGTA to nominally Ca
free medium, dotted trace) blocked about 40% of maximum
current ( n = 4). Addition of 0.5 mM
Ni
to OR2 medium ( dashed trace) also blocked
about 40% of maximum current ( n = 4). The current shown
is carried by chloride ions as assessed by its appropriate reversal
potential (
25 mV) and blockade by the selective inhibitor
niflumic acid (1 mM, see Ref. 4). Injection of BAPTA (1
mM final concentration) eradicates all current activity,
indicating that the responses are completely
Ca
-dependent.
Thus, in
order to resolve authentic CIF from contaminating discharge activities,
Peak 1 was further fractionated using HPTLC. Peak 1 was indeed
heterogeneous and separated into at least five bands detectable under
UV light. Two bands exhibited significant intracellular activity, but
the extracellular activity of Peak 1 was resolved from both of these
bands (data not shown). Thus, the intracellular and extracellular
activities of Peak 1 are completely separated by this procedure.
= 0.57)
elicited current responses, exclusively on microinjection and not on
external application, that were eliminated by extracellular free
calcium removal or addition of extracellular NiCl
(Fig. 4). In unstimulated cells, this band is completely
absent. These results suggest that this HPTLC-enriched fraction
contains authentic CIF.
Figure 4:
Dependence on Ca entry
of CIF partially purified by HPTLC. Microinjection (10 nl) of
HPTLC-purified fraction ( R = 0.57), prepared as
described under ``Experimental Procedures'' induced current
(1776 ± 379 nA, n = 10). Removal of
extracellular Ca
(Ca
-free OR2
containing 1 mM EGTA) ( dotted trace, n = 8) or
addition of 0.5 mM Ni
( dashed trace, n = 9) inhibited current elicited by
microinjection.
-independent mechanism (data not shown). However, this
has not been characterized further, other than its activity is
unaffected by alkaline phosphatase treatment.
, Inositol 1,4,5-trisphosphate; TG,
thapsigargin; CIF, calcium influx factor; HBSS, Hanks' balanced
salt solution; BAPTA,
1,2-bis(2-aminophenoxy)ethane- N, N, N`, N`-tetraacetic
acid; HPTLC, high performance thin layer chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.