From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
Received for publication, January 12, 2001, and in revised form, March 22, 2001
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ABSTRACT |
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In nonexcitable cells, the predominant mechanism
for regulated entry of Ca2+ is capacitative calcium
entry, whereby depletion of intracellular Ca2+ stores
signals the activation of plasma membrane calcium channels. A number of
other regulated Ca2+ entry pathways occur in specific cell
types, however, and it is not know to what degree the different
pathways interact when present in the same cell. In this study, we have
examined the interaction between capacitative calcium entry and
arachidonic acid-activated calcium entry, which co-exist in HEK293
cells. These two pathways exhibit mutual antagonism. That is,
capacitative calcium entry is potently inhibited by arachidonic acid,
and arachidonic acid-activated entry is inhibited by the pre-activation
of capacitative calcium entry with thapsigargin. In the latter case,
the inhibition does not seem to result from a direct action of
thapsigargin, inhibition of endoplasmic reticulum
Ca2+ pumps, depletion of Ca2+ stores, or entry
of Ca2+ through capacitative calcium entry channels.
Rather, it seems that a discrete step in the pathway signaling
capacitative calcium entry interacts with and inhibits the arachidonic
acid pathway. The findings reveal a novel process of mutual antagonism
between two distinct calcium entry pathways. This mutual antagonism may provide an important protective mechanism for the cell, guarding against toxic Ca2+ overload.
In nonexcitable cells, the major mechanism for receptor-regulated
Ca2+ signaling involves the activation of
polyphosphoinositide-specific phospholipase C, formation of inositol
1,4,5-trisphosphate
(IP3),1 and
release of intracellular stored Ca2+ by the activation of
IP3 receptor/ion channels in the endoplasmic reticulum (1).
The decline in Ca2+ content of the intracellular stores
then secondarily signals the activation of plasma membrane calcium
channels, a process known as capacitative calcium entry (2,
3). Recent studies have indicated that the channels in the plasma
membrane may be members of the transient receptor
potential family of channel proteins (4-7). The
mechanism of coupling intracellular stores to the plasma membrane
channels may involve direct interactions between the channel molecules
and underlying IP3 receptors (8-11).
Although capacitative calcium entry seems to be present ubiquitously in
receptor-regulated nonexcitable cells, other mechanisms for signaling
entry have been found in specific cell types. Examples include
receptor-gated channels (12), channels activated by second messengers
such as cyclic nucleotides (13), phosphatidylinositol 3,4,5-trisphosphate (14), and arachidonic acid (15, 16) (for a review
see Refs. 17 and 18). In the HEK293 cell line, capacitative and
noncapacitative pathways co-exist (19, 20). In these cells, muscarinic
receptor activation can lead to baseline [Ca2+]i
spikes or [Ca2+]i oscillations. However, despite
the discharge of stored Ca2+ during these spikes, the
calcium entry necessary for maintaining continuous spiking seems to be
noncapacitative (16). Also, in this cell line arachidonic acid induces
a noncapacitative calcium entry; whether this response represents the
noncapacitative mechanism seen with low concentrations of muscarinic
agonists is controversial (21). Nonetheless, when exogenous arachidonic
acid is used to activate entry in HEK293 cells, a significant discharge
of stored calcium occurs, yet little if any capacitative entry is
activated (see below). Collectively, these observations suggest some
degree of interaction between the capacitative and noncapacitative
arachidonic acid-mediated pathways. Thus, in the current study we have
carried out experiments to examine the regulation of these two calcium entry mechanisms and specifically to determine in what ways the signaling pathways interact. Our findings indicate that although both
pathways involve the release of a common intracellular calcium pool,
the entry components of the two pathways operate in a mutually exclusive manner. This implies that the underlying signaling mechanisms may be able to interact in a way that prevents the occurrence of
supramaximal activation of calcium entry, which could have potentially
toxic effects on cell function.
Cell Culture--
Human embryonic kidney (HEK)-293 cells
obtained from the ATCC were grown at 37 °C in Dulbecco's modified
Eagle's medium supplemented with 10% heat-inactivated fetal bovine
serum and 2 mM glutamine in a humidified 95% air, 5%
CO2 incubator. For Ca2+ measurements, cells
were cultured to about 70% confluence, passaged onto glass coverslips,
and used 24-48 h after plating.
Fluorescence Measurements--
Fluorescence measurements were
made with Fura2-loaded single or groups of HEK293 cells as described
previously (22). In brief, the coverslips with attached cells were
mounted in a Teflon chamber and incubated in Dulbecco's modified
Eagle's medium with 1 µM acetoxymethyl ester of Fura2
(Fura2/AM, Molecular Probes) at 37 °C in the dark for 25 min. Before
[Ca2+]i measurements, the cells were washed three
times and incubated for 30 min at room temperature (25 °C) in
HEPES-buffered physiological saline solution (120 mM NaCl,
5.4 mM KCl, 0.8 mM Mg2SO4, 20 mM HEPES, 1.8 mM CaCl2, and 10 mM glucose, pH 7.4 (adjusted by NaOH)). Ca2+-free solutions contained
no added CaCl2 in the HEPES-buffered physiological saline solution.
Fluorescence was monitored by placing the Teflon chamber with
Fura2-loaded cells onto the stage of a Nikon Diaphot microscope (×40
Neofluor objective). The cells were excited alternatively by 340- and
380-nm wavelength light from a Deltascan D101 (Photon Technology
International, Ltd.) light source equipped with a light-path chopper
and dual excitation monochromators. Emission fluorescence intensity at
510 nm was recorded by a photomultiplier tube (Omega Optical). All
experiments were conducted at room temperature (25 °C) and carried
out within 2 h of loading for each coverslip. Changes in
[Ca2+]i are reported for one single cell in
oscillation experiments or a group of cells (6-10) in other protocols.
The data are expressed as the ratio of Fura2 fluorescence due to
excitation at 340 nm to that due to excitation at 380 nm.
In some experiments, [Ca2+]i was monitored on
attached populations of cells in 96-well plates using a fluorometric imaging plate reader (FLIPR384, Molecular Devices,
Sunnyvale, CA). When FLIPR384 was used, the cells (20,000 cells/well plated the day before use and cultured as above) were loaded
with the single visible wavelength indicator, Fluo-4 (2 µM Fluo-4/AM for 45 min at 37 °C), excited at 488 nm,
and emission-selected by a 510-570-nm bandpass filter. Detection was
performed with a cooled charge-coupled device camera.
Experiments were carried out at room temperature as described above.
Materials--
Arachidonic acid was obtained from BioMol.
Carbachol and thapsigargin were purchased from Calbiochem.
2-Aminoethyoxydiphenyl borane (2-APB) was synthesized as described
previously (23).
Statistics--
For some experiments, the average peak responses
(the ratio of Fura2 fluorescence due to excitation at 340 nm to Fura2
fluorescence due to excitation at 380 nm) were calculated and expressed
as mean ± S.E. for the indicated number (n) of
experiments. Statistical significance was determined with the
Student's t test (p < 0.05).
Thapsigargin and Arachidonic Acid Discharge the Same Intracellular
Ca2+ Pool--
Treatment of HEK293 cells with either 1 µM thapsigargin or 30 µM arachidonic acid
causes a release of intracellular stored Ca2+ as well as an
increase in Ca2+ entry across the plasma membrane (Fig.
1). Yet, 1 µM
Gd3+ completely blocks the thapsigargin-induced entry with
little effect on the entry caused by arachidonic acid (Fig. 1). Thus, arachidonic acid seems to release Ca2+ stores, but unlike
thapsigargin, arachidonic acid does not activate significant
capacitative calcium entry. We carried out the experiment illustrated
in Fig. 2 to determine whether
thapsigargin and arachidonic acid cause the release of Ca2+
from the same intracellular pool. HEK293 cells were treated with either
30 µM arachidonic acid or 1 µM thapsigargin
in the absence of extracellular Ca2+. In both cases a
transient increase in [Ca2+]i was observed,
indicative of the release of Ca2+ stores. When the
arachidonic acid-treated cells were subsequently treated with 1 µM thapsigargin or when the thapsigargin-treated cells
were subsequently treated with 30 µM arachidonic acid, no additional release of Ca2+ was observed. This indicates
that these two reagents release Ca2+ from the same
intracellular Ca2+ store, and the failure of arachidonic
acid to activate capacitative calcium entry cannot be explained by its
failure to release the pool of Ca2+ linked to calcium
entry. The addition of the Ca2+ ionophore, ionomycin, to
cells treated with both arachidonic acid and thapsigargin resulted in a
further release of Ca2+, presumably from mitochondria or
other cellular sites.
Mutual Antagonism of the Capacitative and Arachidonic
Acid-activated Calcium Entry Pathways--
One possible explanation
for the failure of arachidonic acid to activate capacitative calcium
entry is that arachidonic acid may act as an inhibitor of these
channels or of some other step in the influx mechanism. There is
precedence for this idea, because unsaturated fatty acids (24) and
specifically arachidonic acid (25) have been shown to inhibit
capacitative calcium entry in other systems. In the experiment shown in
Fig. 3, HEK293 cells were treated with
thapsigargin in the absence of extracellular Ca2+, and then
Ca2+ was restored as indicated revealing a robust
capacitative calcium entry. The subsequent addition of 30 µM arachidonic acid caused a transient inhibition of
Ca2+ entry. This inhibition was not always transient,
however, especially when lower concentrations of arachidonic acid were
used. The reversal of the inhibition could reflect only transient
inhibition of capacitative calcium entry or the development of an
arachidonic acid-dependent noncapacitative calcium entry.
Furthermore, this protocol does not provide information on possible
interactions in the reverse direction, i.e. effects of the
capacitative pathway on arachidonic acid-activated Ca2+
signaling. To address this issue, we designed experiments that would
allow us to examine the effect of prior intracellular store depletion
on the arachidonic acid response.
Pre-activation of Capacitative Calcium Entry Inhibits Arachidonic
Acid-induced Ca2+ Entry--
To investigate the effects of
activation of capacitative calcium entry on arachidonic acid-mediated
Ca2+ entry, we carried out the experiment illustrated in
Fig. 4. The membrane permeant
IP3 receptor antagonist, 2-APB (23), has been shown to
inhibit capacitative calcium entry, and there is evidence that it acts
on the channels in a manner that interferes with the signaling
mechanism (26). However, arachidonic acid-induced entry of
Ca2+ is not inhibited by this reagent (see below; Ref. 21).
HEK293 cells in the presence of extracellular Ca2+ were
treated with 100 µM 2-APB as indicated in Fig. 4, and the subsequent addition of thapsigargin resulted in a transient response, indicating inhibition of capacitative calcium entry. When arachidonic acid was subsequently added, a robust [Ca2+]i
rise was observed; because intracellular stores were released
previously by thapsigargin, this indicates that under this experimental
condition, arachidonic acid is capable of the activation of
Ca2+ entry.
In the experiment shown in Fig. 5, we
repeated the above protocol utilizing another reagent that
selectively blocks capacitative calcium entry, 1 µM
Gd3+ (27) (Fig. 1). However, while Gd3+
prevented Ca2+ entry in response to thapsigargin,
arachidonic acid now failed to activate a [Ca2+]i
signal.
This latter result indicates that depletion of Ca2+ stores
by thapsigargin in some manner inhibits the ability of arachidonic acid
to activate the noncapacitative calcium entry pathway. The difference
in the mechanism of action of 2-APB and Gd3+ is therefore
critical to understanding the nature of this interaction. Although the
action of 2-APB on capacitative calcium entry channels may not result
from inhibition of inositol trisphosphate receptors as first proposed
(28), there is nonetheless evidence that its mode of interaction may
involve the interference with the coupling mechanism rather than by
acting simply as a blocker of the channel pore (26). Gd3+,
on the other hand, would be expected to block the channels directly. The known actions of thapsigargin are the inhibition of sarcoplasmic reticulum Ca2+ pumps, depletion of Ca2+ stores,
and entry of Ca2+ through activated capacitative calcium
entry channels (29). Neither inhibition of sarcoplasmic reticulum
Ca2+ pumps nor depletion of Ca2+ stores can be
responsible because 2-APB blocks the interaction (Fig. 5) without
interfering with the ability of thapsigargin to deplete
Ca2+ stores. The entry of Ca2+ cannot be
important because this is blocked by Gd3+, yet this agent
does not prevent the inhibition by thapsigargin of arachidonic
acid-induced entry. We conclude that a 2-APB-sensitive step in the
signaling pathway for capacitative calcium entry acts to inhibit the
activation of noncapacitative calcium entry channels by arachidonic
acid. The nature of this step is unknown. Clearly, we must first
achieve a better understanding of the steps involved in signaling
capacitative calcium entry to determine how this pathway interacts with
and regulates the arachidonic acid pathway or other calcium signaling
pathways. Note that despite the findings indicating that 2-APB does not
block capacitative calcium entry by virtue of its action on inositol
trisphosphate receptors, an inescapable conclusion from the current
findings is that this reagent must be doing something more than simply
acting as a channel blocker.
Although unlikely, an alternative interpretation of the findings in
Figs. 4 and 5 is that somehow treatment with thapsigargin causes
arachidonic acid-activated channels to become
Gd3+-sensitive. To examine this possibility, we repeated
the protocol of Figs. 4 and 5 utilizing both Gd3+ and 2-APB
in combination. If thapsigargin causes arachidonic acid-regulated
channels to become Gd3+-sensitive, the protocol should
result in a block of the arachidonic acid-induced response. If 2-APB
acts to prevent a store-depletion inhibitory effect on the channels,
then arachidonic acid will give a normal response. The results of this
experiment, shown in Fig. 6, show that
the latter is indeed the case.
A somewhat similar mutual antagonism has been described recently for
the interaction between nicotinic- and purinergic-regulated cation
channels (30). In this instance, the authors proposed close proximity
between the interacting receptor/channel types. With respect to the
current findings, there is evidence that arachidonic acid-regulated and
capacitative calcium entry channels are located some undetermined
distance apart in the plasma membrane (19). Also, in the previous study
on ligand-gated channels, the inhibitory interactions resulted in a
lack of additivity, but at least under the experimental conditions in
our study the degree of cross inhibition was even more striking (Figs.
3 and 5).
What determines which of these pathways predominate when multiple
upstream signals occur? The answer to this question may depend on a
number of variables such as the stimulus strength of different
initiating agonists. However, as shown in Fig. 1, when arachidonic acid
is applied, store depletion occurs, but clearly the arachidonic
acid-activated entry predominates. This would suggest that at least
under this experimental protocol, the arachidonic acid pathway takes precedence.
In conclusion, the results of this study demonstrate for the first time
a process of mutual antagonism between two distinct regulated
Ca2+ entry pathways. The function of this mutual antagonism
cannot be known for certain, but it is reasonable to suggest that this represents a mechanism to protect cells from the toxic effects of
excessive [Ca2+]i loads. Thus, these two pathways
exist as alternative pathways for Ca2+ signaling, and the
process of mutual antagonism may ensure that they do not operate simultaneously.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Activation of Gd3+-sensitive
capacitative calcium entry by thapsigargin and
Gd3+-insensitive noncapacitative entry by arachidonic
acid. HEK293 cells were treated with either 1 µM
thapsigargin (A) or 30 µM arachidonic acid
(B) as indicated in the absence of extracellular
Ca2+. A transient rise in [Ca2+]i is
indicative of intracellular Ca2+ release. The readdition of
extracellular Ca2+ as indicated results in Ca2+
entry. The entry of Ca2+ in the thapsigargin-treated cells
is capacitative and is blocked by 1 µM Gd3+
(dotted traces). The entry caused by arachidonic acid is
noncapacitative and is largely insensitive to 1 µM
Gd3+. The results are representative of 4-7 experiments.
F340/F380, the ratio of Fura2 fluorescence due to excitation
at 340 nm to Fura2 fluorescence due to excitation at 380 nm;
AA, arachidonic acid.
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Fig. 2.
Thapsigargin (TG) and
arachidonic acid (AA) release the same intracellular
Ca2+ store. HEK293 cells incubated in the absence of
extracellular Ca2+ were treated with either 30 µM arachidonic acid (solid trace) or 1 µM thapsigargin (dotted trace) (first
arrow). A transient rise in [Ca2+]i
indicates the release of intracellular Ca2+ stores.
Subsequently, the arachidonic acid-treated cells were treated with
thapsigargin, and the thapsigargin-treated cells were treated with
arachidonic acid (second arrow). In both cases, the addition
of the second reagent failed to cause a rise in
[Ca2+]i, indicating that each agent releases the
full Ca2+ store available to the other. Additional stores
of Ca2+ could be released by the Ca2+
ionophore, ionomycin (third arrow). The experiment is
representative of a total of four similar experiments.
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Fig. 3.
Inhibition of capacitative calcium entry by
arachidonic acid. HEK293 cells incubated in the absence of
extracellular Ca2+ were treated with 1 µM
thapsigargin (TG) as indicated. Restoration of extracellular
Ca2+ reveals a robust capacitative calcium entry. The
subsequent addition of arachidonic acid (AA) causes a
transient inhibition of Ca2+ entry. The experiment depicted
is representative of a total of four similar trials.
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Fig. 4.
Arachidonic acid induces Ca2+
entry when capacitative calcium entry is blocked by 2-APB. HEK293
cells incubated in the presence of extracellular Ca2+ were
treated with 100 µM 2-APB as indicated. The subsequent
addition of thapsigargin (TG, dotted trace)
caused a transient rise in [Ca2+]i. The sustained
Ca2+ entry was blocked by 2-APB. The addition of 30 µM arachidonic acid (AA) then activated
sustained Ca2+ entry. This response was similar to that in
control cells that were treated with 2-APB but not stimulated with
thapsigargin (solid trace). This experiment is
representative of a total of six similar experiments.
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Fig. 5.
When capacitative calcium entry is blocked by
Gd3+, arachidonic acid fails to induce Ca2+
entry. The protocol used was similar to Fig. 4 except that instead
of 2-APB, the cells were first treated with the capacitative calcium
entry channel blocker, Gd3+. Again, this resulted in a
block of entry caused by thapsigargin (TG, dotted
trace). Unlike the case for 2-APB, arachidonic acid
(AA) now failed to activate a Ca2+ signal.
However, if the cells were not treated with thapsigargin, arachidonic
acid induced a rise in [Ca2+]i (solid
trace). This experiment is representative of a total of seven
similar experiments.
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Fig. 6.
Arachidonic acid induces Ca2+
entry when capacitative calcium entry is blocked by a combination of
Gd3+ and 2-APB. The protocol used was similar to that
for Figs. 4 and 5. As before, when capacitative calcium entry is
blocked with 100 µM 2-APB, arachidonic acid
(AA) induces a [Ca2+]i signal
(dotted trace); when it is blocked with 1 µM
Gd3+ (solid trace), it does not induce a
[Ca2+]i signal. When a combination of both 2-APB
and Gd3+ is used, the arachidonic acid response is not
blocked. This experiment is representative of a total of four similar
experiments.
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ACKNOWLEDGEMENTS |
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We thank Drs Elizabeth Murphy and Stephen Shears for constructive criticisms.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Pharmacology, Harbin Medical University,
Harbin 150086, Peoples Republic of China.
§ Present address: Eli Lilly and Company, Ltd., Lilly Research Center, Erl Wood Manor, Sunninghill Rd., Windlesham, Surrey GU20 6PH, England.
¶ To whom correspondence should be addressed: Laboratory of Signal Transduction, NIEHS, National Institutes of Health, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1420; Fax: 919-541-7879; E-mail: putney@niehs.nih.gov.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100327200
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ABBREVIATIONS |
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The abbreviations used are: IP3, 1,4,5-trisphosphate; HEK, human embryonic kidney; 2-APB, 2-aminoethyoxydiphenyl borane.
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