From the
Exposure of cells to the intracellular Ca
Intracellular Ca
Ca
In spite of the
profound influence of thapsigargin-induced Ca
The basis of the
current study was therefore to assess the intriguing question of how
DC-3F/TG2 cells survive in high levels of thapsigargin, and whether any
Ca
Considering these facts, it
was essential to examine Ca
The fact that there is pumping
activity within the DC-3F/TG2 cells grown in the presence of 2
µM thapsigargin was unexpected. Indeed, as shown in
, the maximal level of Ca
It has been suggested
that Ca
Pool function within the parent DC-3F
cells closely resembles that which we described in other cells, for
example, DDT
The ability of the channel to be deactivated and reduce its
conductance state may be an important factor in determining whether
cells can survive in the continuous presence of emptied pools which
activate capacitative Ca
Maximal
Ca
pump
blocker, thapsigargin (TG), results in emptying of Ca
pools and termination of cell proliferation (Short, A. D., Bian,
J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L.(1993)
Proc. Natl. Acad. Sci. U. S. A. 90, 4986-4990). DC-3F
Chinese hamster lung cells were made resistant to TG by long-term
stepwise exposure to increasing TG concentrations in culture (Gutheil,
J. C., Hart, S. R., Belani, C. P., Melera, P. W., and Hussain, A.(1994)
J. Biol. Chem. 269, 7976-7981). Since these cells
(DC-3F/TG2) grow in the presence of TG, it was important to ascertain
what Ca
pool function they retain. TG-resistant
DC-3F/TG2 cells cultured with 2 µM TG had a doubling time
(24 h) not significantly different from the parent DC-3F cells without
TG. Analysis of TG-induced inhibition of
Ca
uptake into permeabilized parent DC-3F cells revealed two
distinct Ca
pump activities with 20,000-fold
different sensitivities to TG; the IC
values for TG were
200 pM and 4 µM, representing 80% and 20% of
total pumping activity, respectively. Total pump activity in parent
DC-3F and resistant DC-3F/TG2 cells was similar (0.23 ± 0.10 and
0.18 ± 0.08 nmol of Ca
/10
cells,
respectively). In DC-3F/TG2 cells, up to 100 nM TG had no
effect on Ca
pumping; however, almost all pumping was
blocked at higher TG concentrations with an IC
of 5
µM. In both cell types, each Ca
pump
activity (regardless of TG sensitivity) had high Ca
affinity (K
values
0.1
µM) and similar ATP dependence and vanadate sensitivity.
In DC-3F cells, the TG-sensitive Ca
pool was
releasable with inositol 1,4,5-trisphosphate (InsP
) or GTP
and was oxalate-permeable; the TG-insensitive pool in these cells was
not InsP
-releasable. GTP-induced Ca
uptake in the presence of oxalate indicated Ca
transfer between distinct pools in the DC-3F cells. In resistant
DC-3F/TG2 cells, almost 50% of total TG-insensitive Ca
accumulation was releasable with InsP
; unlike the
parent cells, this pool was not oxalate-permeable, and GTP induced no
Ca
transfer between pools in the presence of oxalate.
Thus, whereas InsP
releases Ca
only from
the high TG sensitivity Ca
pumping pool in parent
DC-3F cells, in resistant DC-3F/TG2 cells the TG-resistant
Ca
pumping pool now contains functional InsP
receptors. It is concluded that the ability of TG-resistant
DC-3F/TG2 cells to grow in the presence of 2 µM TG results
from expression of a distinct TG-resistant Ca
pump
which serves to accumulate Ca
within an
InsP
-releasable Ca
pool; this provides
further evidence for the essential role of functional Ca
pools in progression of cells through the cell cycle and cell
division.
pools play an essential role
in signaling events within cells. Although the precise identity and
distribution of Ca
pools remain elusive, it is clear
that the endoplasmic reticulum (ER)
(
)
or
subdomains thereof constitute at least a major component of
Ca
pools
(1, 2, 3) . The
Ca
within ER not only provides an important source of
cytosolic Ca
signals, but also appears to be the
primary determinant in the opening of agonist-dependent
``capacitative'' Ca
entry channels
mediating sustained Ca
signaling
events
(4, 5) . However, generation and modulation of
Ca
signals is not the only role of Ca
accumulated within ER. Thus, intraluminal Ca
levels appear to exert control over essential ER functions
including folding, processing, and assembly of
proteins
(6, 7, 8) ; such control may be effected
through a range of luminal Ca
-binding and chaperone
proteins
(9, 10) . In addition, it has become clear that
the Ca
content of intracellular Ca
pools exerts profound control over cell proliferation and
progression of cells through the cell
cycle
(11, 12, 13, 14) . Therefore,
modification of Ca
within the ER exerts substantial
effects on the viability, growth, and function of cells.
is accumulated within pools via members of the
intracellular sarcoplasmic/endoplasmic reticulum
Ca
ATPase (SERCA) family of Ca
pump
proteins which appear widely distributed in the endoplasmic reticulum
of most cells
(15, 16) . These pumps have been shown to
be highly sensitive to blockade by the plant sesquiterpene lactone,
thapsigargin
(17, 18) . Thapsigargin binds with high
affinity to intracellular Ca
pumps resulting in
virtually irreversible inhibition of Ca
accumulation
within ER
(11, 18, 19) . The selective blockade
of Ca
accumulation within ER using thapsigargin has
made it possible to assess the significance of intraluminal
Ca
in cell function and growth. Thus, using cultured
DDT
MF-2 smooth muscle cells, brief treatment with nanomolar
thapsigargin levels causes a long-lasting emptying of Ca
pools and the entry of cells into a growth-arrested
G
-like state in which the cells remain stable for several
days
(11) . Treatment of these arrested cells with 20% serum
induces synthesis of new Ca
pump protein, the
refilling of Ca
pools, and the return of cells into
the cell cycle
(12, 13) . Not all cells undergo this
thapsigargin-induced cytostatic response; instead, other cells can
undergo cytotoxic responses to thapsigargin. For example, nanomolar
thapsigargin levels induce an irreversible cessation of proliferation
of prostatic cancer cells; in these cells, endonucleases are activated,
genomic DNA becomes fragmented, and cells undergo apoptosis resulting
in a generalized breakdown of cell morphology
(14) . Such a
cytotoxic response may be attributable to more substantial
thapsigargin-induced capacitative entry of Ca
in the
latter cells; in contrast, Ca
entry in
DDT
MF-2 cells due to thapsigargin is comparatively small
and short-lived
(11, 12, 13) .
pool
emptying on cell growth, it was recently shown that cells of the DC-3F
Chinese hamster lung fibroblast line could be made resistant to
thapsigargin
(20) . This was accomplished by gradual exposure of
DC-3F cells in culture to increasing concentrations of thapsigargin
over a period of 10 months. The resulting cell line, DC-3F/TG2,
exhibits neither the cytostatic effects of thapsigargin in
DDT
MF-2 cells nor the cytotoxicity observed in the parent
DC-3F cell line; indeed, cells retain normal morphology and continue to
divide while growing in 2 µM thapsigargin. The basis of
this remarkable resistance is an important question. Toxic hydrophobic
molecules can be efficiently transported out of cells by overexpression
of the multidrug resistance factor, P-glycoprotein (Pgp). However,
although overexpression and amplification of high levels of Pgp can
reduce TG sensitivity, the thapsigargin-resistant DC-3F/TG2 cells
express only modestly increased levels of Pgp (3.7-fold), enough to
only slightly decrease sensitivity to members of the multidrug
resistance class of compounds
(20) . Resistance to thapsigargin
might also have resulted from overexpression of Ca
pumps; however, the slight increase (3-fold) in SERCA pump
expression in DC-3F/TG2 cells
(20) was insufficient to explain
the massive decrease in thapsigargin sensitivity.
pools actually exist in these cells. To address
this, experiments sought to directly assess Ca
pool
function within permeabilized cells. The results reveal function of a
novel and remarkably thapsigargin-insensitive Ca
pumping activity. This pump accumulates Ca
within a pool that has characteristics similar to Ca
signaling pools in normal DC-3F cells but which also has certain
different properties. The results, in addition to providing evidence
for a novel Ca
pump, are consistent with the
hypothesis that intracellular Ca
pumping activity is
essential for cells to be able to grow and proliferate.
Culture of Parent DC-3F and Thapsigargin-resistant
DC-3F/TG2 Cells
Cells of the DC-3F Chinese hamster lung
fibroblast line were cultured in -modified Eagle's medium
(Life Technologies, Inc.) supplemented with 5% heat-inactivated fetal
bovine serum (Life Technologies, Inc.) as described
previously
(20) . Cells received a change of medium after 2 days
and were used in Ca
flux experiments the following
day. Selection of the thapsigargin-resistant DC-3F/TG2 cell line by
successive culturing in the presence of increasing thapsigargin
concentrations was as described previously (20). DC-3F/TG2 cells were
cultured in
-modified Eagle's medium with 5%
heat-inactivated fetal bovine serum together with 2 µM
thapsigargin; they received a change of medium (still containing 2
µM thapsigargin) on the 3rd day after passaging and were
used in Ca
flux experiments the following day. For
cell proliferation experiments, DC-3F or DC-3F/TG2 cells were passaged
directly into 4-well plates and counted over a period of 4 days. Both
cell types were also cultured on glass coverslips for use in
intracellular Ca
measurements as described below.
Permeabilization of Cells and Ca
Cells were permeabilized with 0.005%
saponin and used to measure CaFlux Measurements
fluxes into and out of
intracellular Ca
pools as described
previously
(21, 22) . Uptake was measured in the presence
of an intracellular-like medium (ICM: 140 mM KCl, 10
mM NaCl, 2.5 mM MgCl
, 10 mM
Hepes-KOH, pH 7.0) containing 1 mM ATP, 50 µM
CaCl
(with 150 Ci/mol
Ca
)
buffered to 0.1 µM with EGTA, 3% polyethylene glycol, and
10 µM ruthenium red, at 37 °C for the times shown. The
low free Ca
level together with the inclusion of
ruthenium red ensured no mitochondrial component of Ca
accumulation. Uptake was quenched with ICM containing 1
mM LaCl
, and cells were rapidly filtered on glass
fiber filters and accumulated
Ca
was
counted. Results are presented as ATP-dependent Ca
accumulation with that component of
Ca
associated with permeabilized cells in the absence of ATP
subtracted. For both cell lines, ATP-independent passive influx of
Ca
into pools was typically 1-2% of total
ATP-dependent Ca
pumping (between 2 and 5
pmol/10
cells/min).
Intracellular Free Ca
Procedures for measurement of
intracellular free CaMeasurements
within DC-3F and DC-3F/TG2
cells using fura-2 were as described
previously
(11, 12, 13, 23) . Cells were
grown on glass coverslips as described above and fura-2-loaded by
incubation with 2 µM fura-2/AM ester for 10 min at 20
°C in Hepes-buffered Krebs medium (107 mM NaCl, 6
mM KCl, 1.2 mM MgSO
, 1 mM
CaCl
, 1.2 mM KH
PO
, 11.5
mM glucose, 20 mM Hepes-KOH, pH 7.4, 0.1% bovine
serum albumin). Under these conditions, approximately 95% of dye was
restricted to the cytosol as judged by signal remaining after
permeabilization with saponin. Fluorescence emission at 505 nm was
monitored at 25 °C using a PTI dual wavelength spectrofluorometer
system, with excitation at 340 and 380 nm. Calculations of free
intracellular Ca
concentrations were as described by
Grynkiewicz et al.(24) , using a
K
of 135 nM. Dye was considered
saturated upon addition of 10 µM ionomycin, while minimum
fluorescence ratio was determined in the presence of both 10
µM ionomycin and 5 mM EGTA.
Materials and Miscellaneous Procedures
The DC-3F
and DC-3F/TG2 cell lines were provided by Dr. Arif Hussain (University
of Maryland Cancer Center). Thapsigargin was from LC Services (Woburn,
MA). Fura-2 was from Molecular Probes. Other materials and
miscellaneous procedures were as described previously (11-13).
RESULTS AND DISCUSSION
Effects of Thapsigargin on Growth Rates of DC-3F and
DC-3F/TG2 Cells
The ability of cells to grow and proliferate has
been found to closely correlate with their ability to express
functional intracellular Ca-pumping
pools
(11, 12, 13) . That Ca
pools are essential to cell proliferation has been shown by
blocking Ca
uptake into Ca
pools
using three different intracellular Ca
pump
inhibitors: thapsigargin, 2,5-di-tert-butylhydroquinone, and
cyclopiazonic acid
(11, 12) . The finding that cells
could be obtained, albeit through a lengthy and rigorous selection
procedure
(20) , that survive and grow in a supermaximally
effective concentration of thapsigargin (2 µM), was
therefore difficult to reconcile. Growth of normal DC-3F Chinese
hamster lung fibroblasts is blocked by thapsigargin with an EC
of less than 10 nM(20) . An initial question
concerning the selected thapsigargin-resistant DC-3F/TG2 cells was
whether the rate of cell division was appreciably different from that
of the parent cell line. The rates of cell division for both cell types
are compared in Fig. 1. In the absence of thapsigargin, both cell
types had doubling times of approximately 24 h. In the presence of 2
µM thapsigargin, the rate of DC-3F/TG2 cells was virtually
unaltered, whereas, for the parent DC-3F cells, 2 µM
thapsigargin totally prevented cell division. Indeed, after 3-4
days in culture with thapsigargin, cell viability decreased
considerably; by this time, the majority of cells were rounded,
detached from dishes, and nonviable, indicating a cytotoxic effect of
thapsigargin on the parent DC-3F cells. This contrasts with the
cytostatic effect of thapsigargin observed using the
DDT
MF-2 smooth muscle cell line in which cell viability is
maintained for up to 7 days even though cell division is completely
arrested
(11) . This is an interesting difference which may
relate to the coupling of Ca
pool emptying with
Ca
influx, as described below.
Figure 1:
Effects of
thapsigargin on growth rate of parent DC-3F and resistant DC-3F/TG2
cells. DC-3F cells (,
) or DC-3F/TG2 cells (
,
)
were cultured under identical conditions either in the absence of
thapsigargin (
,
) or in the presence of 2 µM
thapsigargin (
,
). Results are the means ± S.D. of
measurements from four wells, and are typical of three separate
experiments.
Distinct Intracellular Ca
A major question concerning cells
of the thapsigargin-resistant DC-3F/TG2 line was whether they contained
any functional intracellular CaPumps
within DC-3F and DC-3F/TG2 Cells Exhibit a 20,000-fold Difference in
Thapsigargin Sensitivity
pools. If they did
not, then our premise that Ca
pools are essential for
progression of cells through the cell cycle
(11, 12, 13) would be in question. As reported previously, the
possibility that thapsigargin resistance was arising from export of
thapsigargin mediated by Pgp overexpression, was not supported by
measurements on Pgp levels
(20) . Therefore, unless a different
resistance factor was being expressed, the levels of thapsigargin would
be sufficiently high within the cells to render any known intracellular
Ca
pumps nonfunctional. With regard to which pumps do
exist within these cells, previous Western analyses revealed that the
major pump protein of normal DC-3F cells was the SERCA-2 subtype; the
same pump was also expressed in the resistant DC-3F/TG2 cells at a
slightly increased level, but not sufficient to give a significant
change in thapsigargin sensitivity
(20) . It should be noted that
Western analysis also revealed SERCA-2 pump protein within
thapsigargin-treated quiescent DDT
MF-2 cells; however,
measurements of phosphorylated pump intermediate formation revealed
that none of this pump protein was functional due to irreversible
inhibition by thapsigargin
(13) .
pumping activity directly
within DC-3F/TG2 cells. This was undertaken using saponin-permeabilized
cells in which Ca
pool function could be measured
without the possibility of any interference from export of molecules
across the plasma membrane. The results of studies comparing the action
of thapsigargin on Ca
pump activity within
permeabilized normal DC-3F and resistant DC-3F/TG2 cells are shown in
Fig. 2
. In DC-3F cells grown under standard conditions, most
ATP-dependent Ca
pumping was highly sensitive to
thapsigargin (Fig. 2A). Thus, 1 nM thapsigargin
blocked almost 75% of uptake. The range of thapsigargin concentrations
from 3 nM up to 1 µM all induced virtually the
same effect, inhibiting approximately 80% of Ca
accumulation. A further reduction of the remaining 20% of
Ca
uptake was observed with 3 µM
thapsigargin, and 10 µM caused maximal inhibition.
Figure 2:
Inhibition of Ca
accumulation within DC-3F and DC-3F/TG2 cells by thapsigargin.
A, uptake was measured using permeabilized DC-3F cells (grown
under standard conditions without thapsigargin) in the presence of 0
(
), 1 (
), 3 (
), 10 (
), 30 (
), 1,000
(
), 3,000 (
), or 10,000 (
) nM thapsigargin.
B, uptake was measured using permeabilized DC-3F cells (grown
in the presence of 2 µM thapsigargin for 16 h before use)
in the presence of either 0 (
) or 3,000 (
) nM
thapsigargin. C, uptake was measured using permeabilized
DC-3F/TG2 cells (grown continuously in the presence of 2
µM thapsigargin) in the presence of either 0 (
), 300
(
), 1,000 (
), 3,000 (
), or 10,000 (
)
nM thapsigargin. Details of the Ca
uptake
conditions are given under ``Experimental Procedures.''
Results are typical of three similar
experiments.
If
DC-3F cells had been treated overnight with 2 µM
thapsigargin, there was essentially no thapsigargin-inhibitable
Ca accumulation (Fig. 2B).
Significantly, and in contrast to the parent DC-3F cells,
thapsigargin-resistant DC-3F/TG2 cells grown continuously in the
presence of 2 µM thapsigargin (the standard condition for
these cells), clearly displayed ATP-dependent Ca
pumping activity (Fig. 2C). This uptake could be
inhibited by thapsigargin. However, inhibition was observed only at the
higher concentrations of thapsigargin; that is, in the range of 300
nM to 10 µM.
uptake
attainable in these cells was not significantly less than in parent
DC-3F cells; however, substantial blockade of Ca
pumping using 100 nM thapsigargin could only be observed
with the latter. The differences in thapsigargin sensitivity of
Ca
pumping in the two cell lines suggested that
distinct Ca
pumps were operating. In order to
investigate this, a detailed analysis of the thapsigargin concentration
dependence in the two cell lines was undertaken, as shown in
Fig. 3
. For DC-3F cells, there were clearly two widely separated
regions of thapsigargin sensitivity. To more clearly illustrate this,
the effects of including thapsigargin in the 30 pM to 1
nM range are depicted separately from those resulting from
inclusion of thapsigargin in the 100 nM to 10 µM
range. The IC
values for thapsigargin in these regions
were 0.2 nM and 4 µM, respectively. The former
value is very close to that reported by Sagara and Inesi
(19) for inhibition of ATP-dependent Ca
pumping into sarcoplasmic reticulum vesicles. Interestingly, the
concentration dependence of the low sensitivity region for these cells
is almost superimposable upon that of the thapsigargin sensitivity of
Ca
pumping in the resistant DC-3F/TG2 cells
(Fig. 3), which in these cells comprises all of the pumping
activity. In the latter cells, the IC
was approximately 5
µM. Thus, it appears that the parent DC-3F cells contain a
mixture of two distinct Ca
pumps with widely
differing (20,000-fold) sensitivity to thapsigargin; in these cells,
the majority of Ca
accumulation occurs through the
high thapsigargin sensitivity Ca
pump. In DC-3F/TG2
cells, it appears that all of the Ca
pumping occurs
through the low thapsigargin sensitivity pump type (referred to
subsequently as the thapsigargin-insensitive pump) and that this is the
only Ca
pump activity functioning within these cells.
Selection of this type of Ca
pump likely represents
the major adaptation within this cell line that permits growth in
thapsigargin.
Figure 3:
Thapsigargin sensitivity of intracellular
Ca pump activity in DC-3F and DC-3F/TG2 cells. Uptake
was undertaken using either DC-3F cells (
) or DC-3F/TG2 cells
(
) under similar conditions as in Fig. 2 except accumulation
was for a standard 9-min period. For DC-3F cells, 100% of
Ca
uptake was defined as either that observed in the
absence of thapsigargin (left curve) or that observed in the
presence of 100 nM thapsigargin (right curve). For
DC-3F/TG2 cells, 100% uptake was defined as that observed in the
absence of added thapsigargin. Each point represents the mean
± S.D. of Ca
uptake measured in three separate
experiments.
Even though the thapsigargin concentration dependence
of both types of pumping activity differs widely, other parameters of
Ca pump function were remarkably similar. Thus the
K
for Ca
of pump
activity in DC-3F cells in the absence of thapsigargin and that for
Ca
pumping in DC-3F/TG2 cells were almost identical,
both measured as approximately 0.1 µM (data not shown)
using standard
Ca
flux procedures under
EGTA-buffered conditions, as described previously
(21) . This
value is very similar to the K
measured
for intracellular Ca
pumps in a variety of other cell
types
(2, 21, 25) . We also investigated the
sensitivity of thapsigargin-sensitive and -insensitive Ca
pumps in both cell types toward the other Ca
pump inhibitors, 2,5-di-tert-butylhydroquinone and
cyclopiazonic acid. In other cell types, the IC
for these
reagents is approximately 5 and 10 µM,
respectively
(11, 12) . Although resistant DC-3F/TG2
cells showed a slightly lower sensitivity to
2,5-di-tert-butylhydroquinone (approximately 30
µM), this difference was small; moreover, the nonspecific
effects of these agents at concentrations approaching or beyond 100
µM made determination of the absolute sensitivity of
pumping or growth to these reagents difficult to measure. Both
thapsigargin-sensitive and -insensitive Ca
pump
activities present in DC-3F and DC-3F/TG2 cells displayed similar
dependence on ATP, and both were completely inhibited by 1 mM
vanadate (data not shown). Therefore, it appears that the two pump
activities are functionally equivalent, each being capable of mediating
accumulation of Ca
into pools from normal resting
cytosolic free Ca
levels. Since measured
Ca
accumulation reflects both pumping and passive
leak rates, we cannot make assertions as to the actual or relative
amounts of Ca
pumping activity measured in these
cells and, hence, the relative sizes of the pools involved. However,
that intracellular Ca
pump activity exists within
DC-3F/TG2 cells continuously exposed to thapsigargin is clear;
therefore, these data do not challenge our previous assertion that
actively pumping intracellular Ca
pools are a
prerequisite for cell growth and
division
(11, 12, 13) .
accumulation into InsP
-sensitive
Ca
pools within pancreatic acinar cells may be
mediated by a mechanism distinct from direct ATP-dependent
Ca
pumping; this may involve a multistep process of
ATP-dependent proton transport and Ca
/H
exchange
(26, 27) . It was also shown that the
proton pump inhibitor, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, blocks
this Ca
accumulation within
InsP
-sensitive pools
(28) . However, experiments we
have undertaken reveal no effect of
7-chloro-4-nitrobenz-2-oxa-1,3-diazole on Ca
accumulation within either DC-3F or DC-3F/TG2 cells (data not
shown). This suggests that the thapsigargin-insensitive Ca
uptake activity observed in these cells is unlikely to involve
proton pumping and is instead more likely to reflect function of a pump
directly utilizing ATP hydrolysis to transport Ca
.
Thapsigargin-insensitive Ca
The important
question arising from discovery of a thapsigargin-insensitive
CaPumps
Fill an InsP
-sensitive Ca
Signaling Pool in DC-3F/TG2 Cells
pumping pool in the DC-3F/TG2 cells was whether
this pool is functionally significant, in particular, whether
Ca
could be released from the pool in response to
InsP
. We therefore examined the actions of InsP
on Ca
accumulated within both DC-3F and
DC-3F/TG2 cells as shown in Fig. 4. The data in Fig. 4A reveal that Ca
accumulated within
nonmitochondrial stores in saponin-permeabilized parent DC-3F cells
could be directly released by addition of InsP
or GTP. In
these cells, approximately 70% of ionophore A23187-releasable
Ca
was rapidly released upon addition of 10
µM InsP
. As with other cell types, addition of
20 µM GTP resulted in an apparently similar release of
Ca
; however, this action of GTP reflects function of
a very different mechanism believed to reflect G protein-mediated
translocation through junctions between closely apposed membranes of
distinct pools
(29, 30, 31) . Such junctions may
be important in maintaining and/or controlling luminal continuity of
Ca
pools in the ER
(32) . Although not observed
in all cells, GTP-mediated Ca
release into the medium
likely results from a small number of leaky or nonsealed pools through
which Ca
is lost to the medium after junctions are
formed between pools
(2, 33, 34) .
Figure 4:
InsP and GTP-mediated
Ca
release from Ca
pools within
DC-3F and DC-3F/TG2 cells. Ca
uptake experiments were
performed as described under ``Experimental Procedures''
using either permeabilized parent DC-3F cells (A) or
permeabilized DC-3F/TG2 cells (B). For DC-3F/TG2 cells only,
the entire uptake and release experiments were undertaken in the
presence of 100 nM thapsigargin. Release of Ca
was monitored after addition of either 10 µM
InsP
(
), 20 µM GTP (
), 10
µM InsP
together with 20 µM GTP
(
), 5 µM A23187 (
), or control buffer
(
), each added at the arrow. Arrows show the
time of reagent additions. Data are from single experiments
representative of at least three experiments with each cell type.
Details of uptake conditions are given under ``Experimental
Procedures.''
Most
importantly, InsP-mediated Ca
release was
also clearly observable using the thapsigargin-resistant DC-3F/TG2
cells (Fig. 4B). Although the extent of release was
somewhat less (approximately 45% of ionophore-releasable
Ca
), the actions of InsP
and GTP were
qualitatively similar. In this experiment, Ca
uptake
and Ca
release were undertaken throughout in the
presence of 100 nM thapsigargin. Using the parent DC-3F cells
under the same condition (that is, with 100 nM thapsigargin),
the small component of Ca
accumulation measurable
(see Fig. 2A) was entirely unaffected by addition of
InsP
or GTP (data not shown). These results demonstrate
therefore that Ca
pool function within DC-3F/TG2
cells has undergone a clearly defined modification in which the
thapsigargin-insensitive Ca
pump, which formerly
functioned to accumulate Ca
only within a pool
unresponsive to InsP
, now actively accumulates
Ca
within a functional, InsP
-releasable
Ca
pool.
MF-2 smooth muscle
cells
(2, 18, 29, 30, 31) . Thus,
we noted previously that InsP
-sensitive Ca
pools in DDT
MF-2 cells accumulate Ca
exclusively through thapsigargin-sensitive Ca
pumps
(18) . In those cells, although a distinct
thapsigargin-insensitive Ca
pool was measurable, that
pool was InsP
-insensitive and persisted within
thapsigargin-treated growth-arrested cells
(18) . These
observations contributed to the view that thapsigargin-insensitive
pumps lacked any physiological role in mediating signaling events.
Indeed, the inability of thapsigargin-treated cells to grow was
directly attributable to the lack of a functional
thapsigargin-sensitive, InsP
-releasable Ca
pool
(11, 12) ; moreover, the re-entry of
thapsigargin-arrested cells back into the growth cycle was dependent
upon expression of new SERCA pump protein and the return of a
functional InsP
-sensitive Ca
pool
(13) . With DC-3F/TG2 cells, we conclude that the
selection procedure has been successful in selecting cells in which
either (a) thapsigargin-insensitive Ca
pumps
have now been recruited to function within InsP
-releasable
pools or (b) InsP
receptors are now expressed
within the pool accumulating Ca
via
thapsigargin-insensitive Ca
pumps. Some limited
information on distinguishing between these possibilities is given
below. In addition, these results provide a substantial reinforcement
of our previous conclusion that functional InsP
-sensitive
Ca
pools are necessary for cells to be able to
proliferate
(2, 11, 12, 13) .
Distinctions in Ca
We have previously shown that a further
distinguishing characteristic of InsPPool Function
and Ca
Translocation between Pools in DC-3F and
DC-3F/TG2 Cells
-sensitive
Ca
pools within cells is their permeability to
anions, in particular,
oxalate
(29, 30, 35, 36) . The entry of
oxalate appears to be mediated by nonselective anion channels existing
within specific organelles including sarcoplasmic reticulum of muscle
and InsP
-releasable elements in
ER
(2, 36, 37) ; the role of such channels may be
to assist rapid Ca
release events by dissipation of
charge build-up
(36, 37) . In Ca
flux
experiments, entry of oxalate into Ca
pumping pools
results in greatly enhanced Ca
accumulation due to
formation of the insoluble Ca
-oxalate complex. In
previous studies with other cell types, we demonstrated that oxalate
permeability is a property specific to InsP
-sensitive
Ca
pools within
cells
(29, 30, 31, 33, 36) . As
shown in Fig. 5A using DC-3F cells, oxalate above 5
mM results in a large increase in Ca
accumulation; at 10 mM oxalate, Ca
uptake becomes approximately linear with time indicating
substantial entry of oxalate into Ca
-accumulating
pools within DC-3F cells. As was shown for other cell
types
(29) , this large increase in Ca
uptake
with oxalate was completely prevented by InsP
(data not
shown), indicating that oxalate was functioning exclusively upon the
InsP
-releasable pool of Ca
within these
cells. In contrast, with DC-3F/TG2 cells, addition of oxalate induced
almost no increase in Ca
accumulation
(Fig. 5B) indicating the absence of an oxalate-permeable
pool in these cells. It is concluded therefore that in DC-3F/TG2 cells,
the InsP
-sensitive Ca
pool, which
comprises at least a substantial fraction of the Ca
pumping pools present in these cells (see
Fig. 4B), is clearly different from that functioning in
parent DC-3F cells with respect to oxalate permeability.
Figure 5:
Oxalate dependence of Ca
uptake into DC-3F and DC-3F/TG2 cells. Ca
uptake was
measured in either permeabilized DC-3F cells (A) or DC-3F/TG2
cells (B) in the presence of either 0 (
), 3 (
), 5
(
), 8 (
), 10 (
), or 20 (
) mM oxalate,
as shown. Data are from a single experiment representative of four
experiments with each cell type. Details of Ca
uptake
conditions are given under ``Experimental
Procedures.''
The
difference in Ca pools within the two cell types is
further substantiated by examining the actions of GTP in the presence
of oxalate (Fig. 6). We noted previously that GTP can induce a
profound increase in Ca
accumulation in the presence
of submaximally effective oxalate concentrations due to GTP-activated
Ca
translocation between oxalate-permeable and
-impermeable compartments
(29, 33, 36) . Thus,
GTP appears to activate junctional processes between the two
compartments allowing Ca
pumped into
oxalate-impermeable pools to access oxalate-permeable pools,
effectively increasing the size of the oxalate-permeable
pool
(2, 29, 36) . It appears that
oxalate-precipitated Ca
cannot pass through the
GTP-activated junctions between pools, hence Ca
is
not released via connections with nonsealed pools and instead remains
trapped within the oxalate-permeable pool
(29, 36) . As
shown in Fig. 6A, whereas the presence of 5 mM
oxalate caused only a modest increase in Ca
accumulation in DC-3F cells, when 20 µM GTP was also
present, there was a very large increase in Ca
accumulation. In contrast, with DC-3F/TG2 cells, the slight
increase in Ca
accumulation in the presence of 5
mM oxalate was definitely not enhanced by GTP
(Fig. 6B); instead, in these cells GTP activates only
release of Ca
resulting in decreased Ca
accumulation just as in the absence of oxalate, as shown in
Fig. 4B. Thus, it is clear that whereas connections
between membranes can be activated by GTP in both cell types, DC-3F/TG2
cells contain only an oxalate-impermeable Ca
pool
within which Ca
can be accumulated. Thus, the
oxalate-permeable pool present in the parent line is clearly absent in
the resistant DC-3F/TG2 cells. These data may provide some information
to distinguish between the two possibilities described above, that is,
whether in DC-3F/TG2 cells it is InsP
receptors as opposed
to Ca
pumps that are being expressed in organelles
that are different from those in parent DC-3F cells. Thus, from the
oxalate data, it appears perhaps more likely that in these cells
InsP
receptors are being expressed within an enlarged
version of the TG-insensitive, InsP
-nonreleasable,
oxalate-impermeable pool that exists as a relatively minor component of
pools within parent DC-3F cells. Conversely, it appears less likely
that TG-insensitive Ca
pumps are being diverted to
pump Ca
into pre-existing InsP
-sensitive
Ca
pools.
Figure 6:
Measurement of GTP-mediated translocation
of Ca between pools in DC-3F cells (A) and
DC-3F/TG2 cells (B). Ca
uptake was measured
as described under ``Experimental Procedures'' under control
conditions (
) or in the presence of either 5 mM oxalate
(
) or 5 mM oxalate together with 20 µM GTP
(
). Data are from a single experiment representative of four
separate experiments with each cell type.
Although it is clear that the resistant
DC-3F/TG2 cells contain functional InsP-sensitive
Ca
pools, the oxalate permeability data indicate the
pool is not identical with that in normal DC-3F cells. A further
functional parameter of Ca
signaling pools is their
coupling to activation of Ca
entry across the plasma
membrane. Thus, in many cells, depletion of intracellular
Ca
pools results in activation of capacitative entry
of Ca
via an as yet undetermined coupling
process
(4, 5) ; this Ca
entry is
essential in the generation of cytosolic Ca
signals
and the refilling of depleted stores. We undertook experiments to
assess coupling between Ca
pool emptying and
Ca
entry in both normal DC-3F and resistant DC-3F/TG2
cells. The results
(
)
revealed that capacitative
Ca
entry is activated in normal DC-3F cells after
emptying pools with thapsigargin. Although influx becomes deactivated
with time, a small residual component appears to be maintained for at
least 60 min. During this time, a much larger component of
Ca
influx can be transiently reactivated by a
short-term (1 min) depletion of extracellular Ca
, as
has been shown in other cells
(38) . Interestingly, in resistant
DC-3F/TG2 cells, a similar small component of capacitative
Ca
entry appears to be constitutively activated,
presumably as a result of an empty Ca
pool. Moreover,
in these cells, a similar large transient activation of Ca
influx occurs upon short-term external Ca
depletion. This suggests that the resistant DC-3F/TG2 cells do
contain an emptied pool that is coupled to activation of capacitative
Ca
entry. Presumably, therefore, this is not the same
pool that accumulates Ca
via the
thapsigargin-resistant pump. Curiously, it would also appear to be
distinct from the InsP
-sensitive Ca
pool
which in these cells accumulates Ca
through the
thapsigargin-insensitive Ca
pump. Alternatively, the
pool activating Ca
entry may be nonexistent (as
opposed to merely empty) in these cells, and, instead, the downstream
pathway triggering Ca
entry constitutively activated.
entry. Thus, as mentioned
earlier, the level of cytosolic Ca
following pool
depletion may be critical to whether cells have a cytostatic as opposed
to a cytotoxic response to thapsigargin. In DDT
MF-2 smooth
muscle cells which have little Ca
entry in response
to pool emptying, cells respond to Ca
pump blockers
by entering a G
-like state from which they can be
rescued
(12, 13) ; in contrast, thapsigargin induces an
irreversible blockade of proliferation of prostatic cancer cells which
then undergo apoptosis (14).
Concluding Remarks
The major conclusion of the
studies presented here is that a novel thapsigargin-insensitive
Ca pump can be expressed within cells which have been
selected to grow in the presence of thapsigargin. This pump appears to
be endogenously expressed within these cells as well as other cells
(18); however, its function in normal cells does not appear to be
associated with Ca
signaling
events
(18, 29, 30, 31) . After cells are
induced to become resistant to thapsigargin, the
thapsigargin-insensitive Ca
pump accumulates
Ca
within a Ca
pool that functions
to release Ca
in response to InsP
. This
pool appears distinct from that which normally functions to generate
InsP
-induced Ca
signals with respect to
anion permeability and with respect to coupling to capacitative
Ca
entry. It may be that during the selection for
thapsigargin resistance, cells are selected that redirect functional
InsP
receptors from their normal thapsigargin-sensitive
Ca
pumping target pool to one that can still operate
in the presence of thapsigargin. Certainly, the presence of functional
Ca
pumping pools within the thapsigargin-resistant
DC-3F/TG2 cells is consistent with our previous assertion that cell
proliferation requires functional Ca
pools
(2, 11, 12, 13) . The
question of the molecular identity of the thapsigargin-insensitive
Ca
pump remains open. Although it has similar
Ca
affinity, ATP dependence, and vanadate sensitivity
to known SERCA pumps, the thapsigargin insensitivity of this pump
distinguishes it from most SERCA pumps that have so far been identified
(15, 16). Interestingly, evidence for a SERCA-like 97-kDa protein that
may function with low thapsigargin sensitivity has been presented in
platelets
(39, 40) ; its relationship to the activity in
resistant DC-3F/TG2 cells is unknown. From Western analysis, the
resistant DC-3F/TG2 cells have only slightly increased expression of
SERCA-2 type pump protein
(20) , all of which is presumed
nonfunctional as a consequence of irreversible thapsigargin binding.
The plasma membrane Ca
pump is also a possible
candidate for the pumping observed within DC-3F/TG2 cells, having a
greatly reduced sensitivity to thapsigargin that appears to be in the
low micromolar range
(41) . Obviously, this pump protein is
assembled in the ER; whether it can be retained within a subcompartment
inside cells and whether this process might be accentuated during the
selection of thapsigargin-resistant cells are intriguing questions.
Whatever the nature of the pump protein, the present study reveals that
thapsigargin-insensitive Ca
pumps can accumulate
Ca
within functional intracellular
Ca
pools.
Table:
Comparison of Ca uptake
in parent DC-3F and thapsigargin-resistant DC-3F/TG2 cells
accumulation was measured in both cell types in
the absence and presence of 100 nM thapsigargin in the
Ca
uptake assay. Total ATP-dependent intracellular
Ca
uptake was measured after 9 min of
incubation using either permeabilized DC-3F cells (grown without
thapsigargin) or DC-3F/TG2 cells (grown in the presence of 2 µM thapsigargin) as described under ``Experimental
Procedures.'' Results shown are in nanomoles of
Ca
/10
cells and are means ± S.D.
of 10 separate experiments.
, inositol
1,4,5-trisphosphate; fura-2/AM, fura-2 acetoxymethyl ester; Pgp,
P-glycoprotein; SERCA, sarcoplasmic/endoplasmic reticulum.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.