From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
Received for publication, August 17, 2000, and in revised form, October 24, 2000
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ABSTRACT |
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We have investigated the signaling pathways
underlying muscarinic receptor-induced calcium oscillations in human
embryonic kidney (HEK293) cells. Activation of muscarinic receptors
with a maximal concentration of carbachol (100 µM)
induced a biphasic rise in cytoplasmic calcium
([Ca2+]i) comprised of release of
Ca2+ from intracellular stores and influx of
Ca2+ from the extracellular space. A lower concentration of
carbachol (5 µM) induced repetitive
[Ca2+]i spikes or oscillations, the continuation
of which was dependent on extracellular Ca2+. The entry of
Ca2+ with 100 µM carbachol and with the
sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor,
thapsigargin, was completely blocked by 1 µM
Gd3+, as well as 30-100 µM concentrations of
the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor,
2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors
is indicative of capacitative calcium entry. Arachidonic acid, a
candidate signal for Ca2+ entry associated with
[Ca2+]i oscillations in HEK293 cells, induced
entry that was inhibited only by much higher concentrations of
Gd3+ and was unaffected by 100 µM 2-APB. Like
arachidonic acid-induced entry, the entry associated with
[Ca2+]i oscillations was insensitive to
inhibition by Gd3+ but was completely blocked by 100 µM 2-APB. These findings indicate that the signaling
pathway responsible for the Ca2+ entry driving
[Ca2+]i oscillations in HEK293 cells is more
complex than originally thought, and may involve neither capacitative
calcium entry nor a role for PLA2 and arachidonic acid.
An increase in the level of intracellular free Ca2+
concentration ([Ca2+]i) plays a central role in
signal transduction for a variety of cellular functions, including
cellular secretion, muscle contraction, cell growth and
differentiation, and apoptosis. Changes in
[Ca2+]i in mammalian cells are mediated by
mobilization of Ca2+ from internal Ca2+ stores
and/or by entry of Ca2+ from the extracellular space. In
many nonexcitable cells Ca2+ signaling by neurotransmitters
or hormones is initiated through cell membrane receptors coupled to
phospholipase C and the production of inositol 1,4,5-trisphosphate
(IP3)1 (1).
IP3 as a second messenger produces a biphasic
Ca2+ signal, comprised of an initial Ca2+
release from endoplasmic reticulum (ER), followed by a sustained Ca2+ plateau due to Ca2+ entry across the
plasma membrane. This Ca2+ entry usually results from the
depletion of intracellular Ca2+ stores and in such
instances is termed "capacitative Ca2+ entry" (2, 3).
This mode of entry presumably involves store-operated Ca2+
channels in the plasma membrane. Although capacitative calcium entry
has been documented in many different cell types, the signal by which
store emptying activates store-operated Ca2+ channels
remains uncertain (4, 5).
In addition to the sustained elevation of [Ca2+]i
seen with high agonist concentrations, a more complex and subtle repetitive cycling of [Ca2+]i, known as
[Ca2+]i spiking or [Ca2+]i
oscillations, often results from lower concentrations of agonists in
some cell types (1, 6, 7). The characteristics of
[Ca2+]i oscillations vary widely among different
cell types, and a single mechanism may be insufficient to account for
the variety of observed responses (1, 7-9). Formation of
IP3 and cyclical release of Ca2+ from
IP3-sensitive stores may underlie the generation of
oscillations induced by agonists (1, 10). However, a
Ca2+-induced Ca2+ release pathway has been
suggested in initiating oscillations by caffeine or other agents
unrelated to IP3 generation (9, 11). Ca2+
influx from the external milieu is currently thought to be activated in
such situations and appears to be needed to sustain
[Ca2+]i oscillations (1, 7). However, the
mechanism whereby Ca2+ entry is triggered during
[Ca2+]i oscillations is not altogether clear.
Some models suggest that capacitative calcium entry provides
Ca2+ entry during oscillations (7, 12). More recently a
novel, noncapacitative mechanism has been proposed that involves
agonist-activated generation of arachidonic acid and arachidonic
acid-induced Ca2+ entry (13-15).
Arachidonic acid is present in cell membranes esterified in
phospholipids and can be released by phospholipase A2
(PLA2) in response to various extracellular stimuli (16,
17). Arachidonic acid can also be generated from diacylglycerol, a
product of phospholipase C or phospholipase D activation, by action of
diglyceride lipase (16). In recent years, an increasing number of
reports have suggested that arachidonic acid directly modulates
cellular responses, including Ca2+ signal transduction. As
for IP3, Ca2+ release from ER and
Ca2+ influx from the extracellular space induced by
arachidonic acid have been demonstrated in a number of cell types
(18-23). However, the mechanisms underlying
[Ca2+]i changes in response to arachidonic acid
are not clear.
In this study, we have used relatively specific pharmacological probes
to analyze and compare capacitative, noncapacitative, and arachidonic
acid-induced Ca2+ entry in HEK293 cells. We confirm earlier
reports of a noncapacitative mechanism associated with
[Ca2+]i oscillations in these cells. However, our
findings indicate a possible role for the IP3 receptor in
this signaling pathway and call into question the role of arachidonic
acid, at least as a direct mediator of Ca2+ entry in this
cell type.
Cell Culture--
Human embryonic kidney 293 (HEK293) cells
obtained from the ATCC were grown at 37 °C in Dulbecco's 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 (24). In brief, coverslips with attached cells
were mounted in a Teflon chamber and incubated in Dulbecco's 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, cells were washed three
times and incubated for 30 min at room temperature (25 °C) in
HEPES-buffered physiological saline solution (HPSS: NaCl, 120 mM; KCl, 5.4 mM;
Mg2SO4, 0.8 mM; HEPES, 20 mM; CaCl2, 1.8 mM; and glucose, 10 mM; with pH 7.4 adjusted by NaOH).
Ca2+-free solutions contained no added CaCl2 in
the HPSS.
In preliminary experiments, we observed that
[Ca2+]i oscillations were not reproducibly
observed in cells loaded with 1 µM Fura2/AM, presumably
due to excessive cytoplasmic Ca2+ buffering. Thus, for
these experiments we used 100 nM Fura2/AM for loading and
1.5 mM extracellular CaCl2 as previously
described (25).
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
(F340/F380).
Mn2+ Quench Measurements--
Mn2+
quench experiments were performed with a group of HEK293 cells in
nominally Ca2+-free medium containing 0.1 or 2 mM MnCl2. Ftot, which is
independent of [Ca2+]i responses (26), was
obtained by a weighted summing of the fluorescence of 340 and 380 nm,
and expressed as the percentage of the initial value in the absence of
extracellular Mn2+.
Materials--
Arachidonic acid and 5,8,11,14-eicosatetraenoic
acid were obtained from BioMol (Plymouth Meeting, PA). Carbachol,
thapsigargin, and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA) were purchased from Calbiochem (La Jolla, CA).
2-Aminoethyoxydiphenyl borane (2-APB) was synthesized as previously
described (27).
Statistics--
For some experiments, average peak responses
(F340/F380) 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).
[Ca2+]i Signaling Responses to Carbachol in
HEK293 Cells--
In Ca2+-containing HPSS, 100 µM carbachol induced a large, somewhat transient increase
in [Ca2+]i
(F340/F380) followed by a
slowly declining but generally sustained elevated level of
[Ca2+]i (Fig.
1A). In nominally
Ca2+-free medium, this same concentration of carbachol
induced a transient increase in [Ca2+]i;
following re-addition of Ca2+ to the medium, a second,
sustained entry of Ca2+ was observed (Fig. 1B),
indicating release of Ca2+ from internal sites and
Ca2+ entry.
[Ca2+]i oscillations have been reported to be
induced by 1 µM carbachol in HEK293 cells transfected
with the M3 muscarinic receptors (28). However, in the
current study in which wild type HEK293 cells were used, we failed to
consistently produce repetitive transient responses of
[Ca2+]i with 1-3 µM carbachol.
When the cells were stimulated with 5 µM carbachol, about
50% of the tested cells (101 of 205) showed oscillatory
[Ca2+]i responses at a frequency of ~0.5-1/min
in the presence of 1.5 mM Ca2+ (Fig.
1C). These robust spikes could last up to 1 h, but the frequency progressively slowed with time. Because all cells did not
oscillate and the frequency varied somewhat among the cells, which did
oscillate, we adopted the protocol shown in Fig. 1C. In this
protocol, a cell was stimulated for about 20 min, the carbachol was
removed by three changes of incubation medium, and then, after an
additional period of about 25 min (data acquisition was halted during
this period), the same cell was again stimulated with 5 µM carbachol for an additional 20 min, generally under an
altered experimental condition. As shown in Fig. 1C, in
normal HPSS, the second stimulation always resulted in an oscillatory response that was similar to, although somewhat slower than the first.
This protocol was utilized for the experiment illustrated in Fig.
1D. In this experiment, 5 min before, and during the second exposure to carbachol, the cell was bathed in a nominally
Ca2+-free medium containing 200 µM BAPTA.
With this protocol, carbachol induced one or two spikes, but sustained
oscillations were not observed. These results confirm that, in wild
type HEK293 cells, as shown previously for cells transfected with the
M3 muscarinic receptor, [Ca2+]i
oscillations produced by a low concentration of carbachol depend on
extracellular Ca2+, presumably indicating a role for
Ca2+ influx.
Effects of Gd3+ on [Ca2+]i
Responses to Thapsigargin and Carbachol in HEK293
Cells--
Gd3+ is a potent inhibitor of agonist-activated
calcium entry (29) and has been shown to discriminate between
capacitative and noncapacitative calcium entry (22). In experiments
utilizing the same protocol as in Fig. 1B, the effects of
Gd3+ on Ca2+ entry due to the SERCA inhibitor,
thapsigargin, were determined. Thapsigargin depletes Ca2+
stores passively by virtue of its ability to inhibit the SERCA pumps on
the endoplasmic reticulum, and the ensuing entry of Ca2+ is
therefore assumed to be the very definition of capacitative calcium
entry (3, 30). As shown in Fig.
2A, Gd3+ inhibited
Ca2+ entry induced by the thapsigargin in a
concentration-dependent manner, and with no significant effect
on the Ca2+ release phase (Fig. 2A and results
not shown). Gd3+ had no significant effect on basal
[Ca2+]i (data not shown). The sensitivity of
thapsigargin-induced capacitative calcium entry to inhibition by
Gd3+ is similar to that reported by Broad et al.
(22).
Similar experiments were carried out utilizing a maximal concentration
of carbachol, and such an experiment is shown in Fig. 2B. Ca2+ entry due
to maximal muscarinic receptor activation appeared similarly sensitive
to inhibition by Gd3+, leading to the conclusion that the
entry is largely or entirely capacitative.
However, significantly different results were obtained when cells were
stimulated to oscillate with the lower, 5 µM
concentration of carbachol. As Fig. 3 illustrates, concentrations of 1, 10, 30, 100, or 500 µM Gd3+ produced little
or no effect on the [Ca2+]i oscillations; only at
the highest concentrations tested, 100 and 500 µM
Gd3+, was there even partial suppression of the oscillatory
frequency. The failure of even these very high concentrations of
Gd3+ to block the oscillations was surprising. However, it
is known that another lanthanide, La3+, can inhibit active
membrane extrusion of Ca2+ at higher concentrations (31).
We determined whether Gd3+ might have a similar action by
examining the time course of the [Ca2+]i response
to thapsigargin in the absence of extracellular Ca2+ and in
the presence of varying concentrations of Gd3+. The decay
of the [Ca2+]i response to thapsigargin under
these conditions is due almost entirely to plasma membrane extrusion
(32). As shown in Fig. 4,
Gd3+ concentrations of 30 µM or greater
caused an augmentation of the thapsigargin-induced
[Ca2+]i signal and a slowing of its decay. Thus,
at these higher concentrations, oscillations may continue due to
"trapping" of intracellular Ca2+, despite an inhibition
of Ca2+ entry. However, at 10 µM
Gd3+, there was no significant augmentation of the
response, indicating that the Ca2+ entry channels
supporting the oscillations are truly less sensitive to
Gd3+ than are capacitative calcium entry channels.
Arachidonic Acid-induced [Ca2+]i Signaling in
HEK293 Cells--
Shuttleworth and his coworkers (13, 21, 28) have
suggested that the noncapacitative calcium entry occurring in HEK293 cells and other cell types in response to low concentrations of muscarinic agonists is mediated by arachidonic acid, released from
membrane lipids by phospholipase A2. Thus, we next examined the effects of Gd3+ on Ca2+ mobilization in
HEK293 cells in response to arachidonic acid. In preliminary
experiments, we found that between 30 and 300 µM arachidonic acid could reproducibly induce both Ca2+
release and Ca2+ entry in a
concentration-dependent manner. However, arachidonic acid
at concentrations > 100 µM occasionally resulted in
[Ca2+]i levels that saturated the indicator
likely due to a nonselective increase in membrane permeability (33).
Concentrations in the range of 5 to 10 µM did not induce
increases in [Ca2+]i in all cells. As shown in
Fig. 5A, 30 µM
arachidonic acid slowly increased the fluorescence ratio, and the
response appeared to occur in two phases. In nominally
Ca2+-free medium, arachidonic acid induced a transient
[Ca2+]i rise followed by a sustained elevation of
[Ca2+]i after restoration of Ca2+ to
the medium (Fig. 5B), indicating that both Ca2+
release and Ca2+ entry are activated by arachidonic acid in
HEK293 cells. To examine the possible involvement of metabolites of
arachidonic acid in the [Ca2+]i responses, we
employed 5,8,11,14-eicosatetraenoic acid, an inhibitor of
cyclooxygenase, lipoxygenases, and cytochrome P450 arachidonic
acid-metabolizing enzymes (34). 20 µM
5,8,11,14-eicosatetraenoic acid had no effect on either
Ca2+ release or Ca2+ entry induced by 30 µM arachidonic acid, indicating that the [Ca2+]i changes induced by 30 µM
arachidonic acid are unlikely to result from an arachidonic acid
metabolite (data not shown). A similar conclusion was reached by
Shuttleworth and Thompson based on a somewhat different strategy
(28).
The pattern of [Ca2+]i signaling induced by
arachidonic acid is reminiscent of that due to thapsigargin; a release of stored Ca2+ followed by activation of Ca2+
entry across the plasma membrane. Thus, we next examined the effects of
Gd3+ on arachidonic acid-induced signaling, because this
lanthanide appears to have relatively selective effects on
store-operated or capacitative calcium entry. At a concentration of 1 µM, which completely blocked Ca2+ entry due
to carbachol and thapsigargin, Gd3+ had no significant
effect on Ca2+ entry in response to 30 µM
arachidonic acid (Fig. 5, B and C). At
concentrations of 3 and 10 µM, Gd3+ inhibited
Ca2+ influx induced by 30 µM arachidonic acid
with complete blockade at 10 µM. Surprisingly, 10 µM Gd3+ also caused a complete abolishment of
arachidonic acid-induced Ca2+ release (Fig. 5, B
and C). After complete inhibition with 10 µM
Gd3+ of Ca2+ release due to 30 µM
arachidonic acid in nominally Ca2+-free medium, a normal
release of [Ca2+]i could be evoked on addition of
1 µM thapsigargin or 100 µM carbachol (not
shown). These results indicate that arachidonic acid induces both
Ca2+ release and Ca2+ entry in HEK293 cells,
and both of these responses are sensitive to inhibition by
Gd3+; however, this pathway is at least 10-fold less
sensitive to Gd3+ than capacitative calcium entry.
Effects of 2-APB on [Ca2+]i Responses Induced
by Carbachol, Thapsigargin, and Arachidonic Acid--
Recent studies
have indicated that capacitative calcium entry involves interactions
between IP3 receptors and the plasma membrane (35). One
piece of evidence for this idea is the sensitivity of capacitative
calcium entry to inhibition by 2-APB (36), a membrane-permeant
inhibitor of the IP3 receptor (27). We next examined the
actions of this reagent as a potential inhibitor of Ca2+
entry responses to agonists, to thapsigargin, and to arachidonic acid.
In unstimulated cells, and in the absence of extracellular
Ca2+, 2-APB at 100 µM slightly augmented the
baseline fluorescence ratio in about 80% of HEK293 cells tested
(n = 48). The increment in the baseline was 8.6 ± 3.2% of that of Ca2+ release by 1 µM
thapsigargin in nominally Ca2+-free medium
(n = 9). A weak inhibitory effect on
Ca2+-ATPase in the ER has been suggested to account for the
rise of [Ca2+]i by high concentrations of 2-APB
(27).
Utilizing a similar protocol as for the Gd3+ experiments,
2-APB produced a concentration-dependent inhibition of
Ca2+ influx induced by 100 µM carbachol (Fig.
6, top) or 1 µM
thapsigargin (Fig. 6, bottom) when Ca2+ was
restored to the bath. Like Gd3+, 2-APB altered the
Ca2+ entry phase with almost the same potency among the two
agonists, with 30 µM 2-APB producing essentially complete
block of Ca2+ entry for both modes of activation. However,
30 µM 2-APB attenuated the Ca2+ release peak
induced by 100 µM carbachol only weakly, and this inhibition was still incomplete with 100 µM 2-APB (Fig.
6). With 100 µM 2-APB, an approximate 20% reduction of
Ca2+ release due to 1 µM thapsigargin could
also be seen (Fig. 6), which may be due to the inhibition of
Ca2+-ATPase in the endoplasmic reticulum and a partial
reduction of the size of the pool sensitive to thapsigargin.
2-APB at 100 µM, a concentration that caused complete
inhibition of capacitative calcium entry, did not alter
Ca2+ entry due to 30 µM arachidonic acid
(Fig. 7). As for thapsigargin, 100 µM 2-APB caused a slight reduction of
[Ca2+]i release in response to 30 µM arachidonic acid (Fig. 7 and results not shown).
These data, including the data obtained with Gd3+, provide
evidence that the mechanisms by which arachidonic acid activates Ca2+ release and Ca2+ influx are different from
those of the store-depleting agents, thapsigargin and carbachol. As
first suggested by Shuttleworth, capacitative calcium entry appears not
to be involved in Ca2+ entry due to arachidonic acid in
HEK293 cells (21).
Effects of 2-APB on [Ca2+]i Oscillations and
Ca2+ Entry in Response to Low Concentrations of
Carbachol--
As shown in Fig. 8, 2-APB
inhibited the repetitive transient [Ca2+]i
responses in a concentration-dependent manner and 100 µM 2-APB completely blocked the sustained oscillatory
response of HEK293 cells to 5 µM carbachol.
The inhibition by 2-APB of the [Ca2+]i response
to 5 µM carbachol was unexpected, because arachidonic
acid-induced Ca2+ signaling was unaffected by this drug.
However, we considered the possibility that this concentration of
carbachol might induce a small influx of Ca2+ that is only
detectable when amplified through calcium-induced calcium release, and
this might depend on functional IP3 receptors. Therefore,
to assess more directly the actions of 2-APB on Ca2+ entry
during [Ca2+]i oscillations, we utilized
Mn2+ quench measurements. Mn2+ enters cells
through divalent cation channels, but quenches Fura2 fluorescence at
all wavelengths (37). Thus, the activity of Ca2+ influx
channels is reported by the rate of Mn2+ quench of Fura2.
In the presence of 0.1 mM Mn2+, in nominally
Ca2+-free medium, a resting rate of Mn2+ quench
was seen in unstimulated cells, and this was blocked when the cells
were pretreated with 100 µM 2-APB (Fig.
9A). 5 µM
carbachol increased Mn2+ quench, and again the rate of
quench in the presence of carbachol was completely blocked by 100 µM 2-APB (Fig. 9B). Because 2-APB essentially
completely blocked even basal Mn2+ quench, we repeated the
experiments with 2 mM Mn2+. Under these
conditions, a basal rate of Mn2+ quench was seen in the
presence of 100 µM 2-APB, but this was not further
increased by 5 µM carbachol (Fig.
10); however, addition of 5 µM arachidonic acid induced a substantial increase in
quench (Fig. 10A). These results demonstrate that 2-APB
attenuated both the resting Mn2+ entry and divalent cation
influx stimulated by 5 µM carbachol, but not that of
exogenous arachidonic acid.
These findings indicate that the signaling mechanism underlying
noncapacitative calcium entry and [Ca2+]i
oscillations may involve signals other than or in addition to
arachidonic acid. There is previous pharmacological evidence for a role
for PLA2; for example, the oscillations are inhibited by
the PLA2 inhibitor, isotetrandrine (28). Data shown in Fig.
11 (A and B)
essentially replicates the previous findings of Shuttleworth and
Thompson (28), showing that isotetrandrine can block
[Ca2+]i oscillations, and the addition of a low
concentration of arachidonic acid can partially restore the response
(in 7 of 14 cells tested). We found that 10 µM
isotetrandrine completely blocked the oscillations in 12 of 21 cells
tested, whereas 20 µM blocked completely in 3 of 5. However, like its close cousin, tetrandrine (38), isotetrandrine can
also function as a calcium channel blocker. We thus tested the effects
of isotetrandrine on the entry of Ca2+ directly activated
by arachidonic acid. As illustrated in Fig. 12A, 10 µM
isotetrandrine consistently inhibited the Ca2+ entry in
response to arachidonic acid (3 of 3 with 10 µM
isotetrandrine; 3 of 4 with 20 µM isotetrandrine). The
inhibition is apparently not due to action as a nonspecific channel
blocker, or to membrane depolarization, because isotetrandrine caused
only slight inhibition of entry in response to thapsigargin (Fig.
12B). These findings call into question the validity of
isotetrandrine as a specific tool to demonstrate PLA2
involvement. In addition, as discussed below, they may indicate that
the effects and pharmacological sensitivity of exogenously added
arachidonic acid do not faithfully reflect the behavior of arachidonic
acid generated endogenously as a component of a physiological signaling
cascade. Furthermore, the relative insensitivity of the
thapsigargin-induced entry to isotetrandrine further supports the view
that the entry driving the [Ca2+]i oscillations
is not capacitative.
In the present study, we initially established that
Gd3+ and 2-APB appear to be relatively specific and potent
inhibitors of capacitative calcium entry. Results shown here indicate
that Ca2+ entry due to two different store-depleting
agents, thapsigargin and carbachol, is sensitive to the inhibitory
effects of Gd3+ and 2-APB in HEK293 cells. Gd3+
from 30 nM to 1 µM and 2-APB from 10 to 100 µM in a concentration-dependent manner
inhibited this Ca2+ entry with similar potency for the two
agonists (Fig. 6 and data not shown), indicating a similar mechanism
for blocking Ca2+ entry, i.e. capacitative
calcium entry activated by store depletion in HEK293 cells. At
concentrations of 1 µM Gd3+ and 100 µM 2-APB, respectively, a complete abolishment of
Ca2+ influx due to carbachol and thapsigargin was observed,
consistent with previous reports in which a complete blockade of
capacitative calcium entry induced by different store-depleting drugs
could be obtained with 1 µM Gd3+ in rat A7r5
cells (22) and with 100 µM 2-APB in DDT1-MF2 cells, A7r5
cells, and HEK293 cells (36). These results clearly demonstrate that
Gd3+ and 2-APB are both potent inhibitors of capacitative
calcium entry. 2-APB has also been shown to modulate IP3
receptors (27), leading Ma et al. (36) to conclude that the
IP3 receptor is somehow involved in the activation of
capacitative calcium entry.
Arachidonic acid, an unsaturated fatty acid produced by the action of
PLA2 or diacylglycerol lipase on membrane lipids, is mobilized in a variety of cell types by the actions of
neurotransmitters and hormones. Arachidonic acid also induces
Ca2+ fluxes (39-41) as well as a variety of
Ca2+-dependent effects in cells, and thus has
been suggested as a second messenger modulating Ca2+ signal
transduction (13, 16, 17, 33). However, the mechanisms underlying
Ca2+ signaling modulation due to arachidonic acid remain
unclear. To better define the mechanisms of calcium signaling in
response to arachidonic acid, we examined the effects of the two
inhibitors of capacitative calcium entry, Gd3+ and 2-APB,
on arachidonic acid-mediated Ca2+ signaling. Arachidonic
acid at concentrations at or above 30 µM released
Ca2+ from intracellular Ca2+ stores and induced
Ca2+ entry (Fig. 5), consistent with findings in other cell
lines (18, 19, 41). Interestingly, we found that 10 µM
Gd3+ completely blocked Ca2+ release in
response to arachidonic acid (Fig. 5, C and D),
but did not affect release due to thapsigargin or carbachol (Fig. 6).
The mechanism for this effect is not known. Direct interaction of
Gd3+ with arachidonic acid seems unlikely, because the
concentration of Gd3+ (10 µM) is less than
that of arachidonic acid (30 µM).
Ca2+ entry induced by arachidonic acid is also attenuated
by Gd3+ but only in concentrations in excess of 1 µM. As for the inhibition of Ca2+ release due
to arachidonic acid, 10 µM Gd3+ was required
to produce complete blockade of Ca2+ entry (Fig. 5,
C and D). A similar result was reported for A7r5 cells (22). The mechanism by which Gd3+ inhibits both
Ca2+ release and influx is not known, nor is it clear as to
whether these two effects are even related. The significant point for the current study, however, is that 1 µM
Gd3+, which is more than sufficient for complete inhibition
of capacitative calcium entry, is without effect when arachidonic is
used as an activator of Ca2+ mobilization.
In addition to Gd3+, 2-APB is emerging as a relatively
specific inhibitor of capacitative calcium entry. Ma et al.
(36) demonstrated that 2-APB blocked capacitative calcium entry in
HEK293 cells, as well as the entry ascribed to the transfected Trp3
channel. In the latter case, previous work had shown that transfected
Trp3 can be activated either through an interaction with
subplasmalemmal IP3 receptors (35) or more directly by
diacylglycerol (42). 2-APB blocked Trp3 channels when activated by
phospholipase C-linked agonists, but not when activated by
diacylglycerol. This finding led Ma et al. (36) to conclude
that 2-APB was not acting as a channel-blocking drug and that its
mechanism of action in the case of Trp3 channels, and probably also in
the case of capacitative calcium entry, involved inhibition of
IP3 receptors. These results have been considered strong
evidence for the conformational coupling model (4, 43) for activation
of capacitative calcium entry, because this model invokes an obligatory
role for the IP3 receptor interacting with plasma membrane
capacitative calcium entry channels (44). Clearly, 2-APB is one of the
more specific inhibitors of capacitative calcium entry. It completely
blocks capacitative calcium entry in concentrations that are without
effect on voltage-operated calcium channels
(27),2 and in the current
study, we have found that it does not block channels activated by
arachidonic acid (Figs. 7 and 10). To our knowledge no other organic
antagonist of capacitative calcium entry channels shows this degree of
specificity (see also Ref. 45). The drug has only one other known site
of action, the IP3 receptor, and it is reasonable for the
present to accept the interpretation of Ma et al. (36) that
this action underlies its actions on calcium entry. However, we note,
as was somewhat evident in the work of Ma et al., that
Ca2+ entry appears to be more sensitive to inhibition by
2-APB than the intracellular, IP3-mediated release of
Ca2+ (Fig. 6). Thus, it is possible that 2-APB may also
have direct, albeit highly specific, actions on capacitative calcium
entry channels, although this distinction is not critical to arguments based on its effects in this study.
Finally, with this background of clear and relatively specific actions
of Gd3+ and 2-APB, we have utilized these reagents to
evaluate the role of the capacitative calcium entry pathway in the
complex Ca2+-signaling response giving rise to
[Ca2+]i oscillations in HEK293 cells (28). In our
study of wild type HEK293 cells, repetitive
[Ca2+]i spikes could be induced by a relatively
low concentration of carbachol, and the generation of this signaling
pattern is dependent on extracellular Ca2+ (Fig.
1D). Although previous models have implicated a role for capacitative calcium entry in the maintenance of
[Ca2+]i oscillations (7, 12), this view has been
recently questioned by Shuttleworth (13). In the current study, the
insensitivity of [Ca2+]i oscillations to
Gd3+ provides clear pharmacological evidence that the entry
pathway activated by low concentrations of carbachol is distinct from the capacitative pathway seen with higher concentrations of carbachol or with store depletion by the SERCA inhibitor, thapsigargin. There is
considerable evidence that arachidonic acid can serve as an activator
of a noncapacitative pathway in HEK293 cells (46). Consistent with this
idea, arachidonic acid-induced entry of Ca2+ was relatively
insensitive to inhibition by Gd3+, curiously, despite the
ability of arachidonic acid to deplete intracellular Ca2+
stores.3 However, the
association between arachidonic acid-induced entry and the entry
associated with oscillations is lost when the effects of 2-APB are
examined. 2-APB was capable of completely inhibiting both the
[Ca2+]i oscillations and increased
Mn2+ quench due to 5 µM carbachol but was
without effect on arachidonic acid-induced entry of either
Ca2+ or Mn2+.
With the evidence presently available, we cannot definitively determine
the mechanisms of Ca2+ signaling that underlie
[Ca2+]i oscillations in HEK293 cells. We suggest
three possible alternatives that may be testable in the future by
continued experimental work:
(i) The simplest explanation for our data is that the entry associated
with [Ca2+]i oscillations is noncapacitative in
nature, but involves some mode of activation other than arachidonic
acid. At least some of the previous evidence for PLA2
involvement (28), based on inhibitory effects of the PLA2
inhibitor, isotetrandrine, is open to question (Fig. 12). Furthermore,
regardless of the precise mechanism of action of 2-APB, its ability to
distinguish clearly between arachidonic acid-induced entry and the
Ca2+ entry associated with [Ca2+]i
oscillations suggests that the arachidonic acid pathway is not involved.
(ii) These arguments notwithstanding, it is still possible, given other
points of evidence that suggest an involvement of PLA2 and
arachidonic acid, that PLA2 is involved in the
oscillations. However, exogenously added arachidonic acid may act
through a different mechanism than arachidonic acid generated by
PLA2 in the cell, perhaps in the vicinity of
Ca2+ channels.
(iii) Finally, we consider that it still is possible that the
Ca2+ entry underlying oscillations is a store-operated
entry, albeit involving different store-operated channels than those
seen with maximal store depletion. Although only
Gd3+-sensitive entry was observed in HEK293 cells following
maximal depletion of Ca2+ stores,
Gd3+-insensitive store-operated channels have been observed
in other experimental systems (29). In the absence of extracellular
Ca2+, at least one [Ca2+]i spike is
always observed with 5 µM carbachol, suggesting that
intracellular release is triggered independently of, and likely prior
to entry. Also, the [Ca2+]i oscillations and
Mn2+ entry due to 5 µM carbachol are both
blocked by 2-APB, a drug with documented specificity for capacitative
calcium channels. Thus, it is possible that, with minimal and
short-lived Ca2+ discharge, a different population of
capacitative calcium entry channels are activated with pharmacological
distinctions and pharmacological similarities to the channels activated
upon maximal store depletion.
The conclusion from this study is that the interplay among various
Ca2+-signaling pathways that result in
[Ca2+]i spikes and oscillations may be much more
complex than originally envisioned. However, some cell types may
utilize a simpler mechanism involving strictly phospholipase C,
intracellular Ca2+ release, and capacitative calcium entry
mechanisms. For example, in mouse lacrimal cells, which produce a
characteristic sinusoidal pattern of [Ca2+]i
oscillations (47), there are no Gd3+-insensitive responses
seen at low agonist concentrations, and the cells appear completely
insensitive to the addition of exogenous arachidonic acid.2
Clearly, additional work will be needed to understand the varied patterns and mechanisms that control the biologically important phenomenon known as [Ca2+]i oscillations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Calcium release, calcium entry, and
[Ca2+]i oscillations due to muscarinic
receptor activation. Fura2-loaded HEK293 cells were activated as
indicated by either 100 µM (A and
B) or 5 µM (C and D)
carbachol. In A, extracellular Ca2+ was present
throughout the experiment. In B, Ca2+ was
initially absent from the medium and was restored to 1.8 mM
were indicated. In C and D, a single HEK293 cell
was activated with 5 µM carbachol, and then the medium
was removed with three consecutive washes, and the cell was allowed to
recover for 25 min. Data collection was interrupted during this
interval of washes and recovery, and is indicated by the
arrow labeled W. Subsequently, the same cell was
activated a second time with 5 µM carbachol. In
D, prior to the second stimulation, the extracellular medium
was changed to one containing 0.2 mM BAPTA and no added
Ca2+. Similar results to the ones depicted were obtained in
a total of five to eight experiments.
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Fig. 2.
Effect of Gd3+ on
thapsigargin-activated Ca2+ entry. In A,
the protocol was identical to that in Fig. 1B, except that 1 µM thapsigargin was utilized as agonist. In one of the
traces (as indicated) 1 µM Gd3+
was added to the medium when Ca2+ was removed and was
present throughout the re-addition of Ca2+. In
B, the agonist was 100 µM carbachol. Results
shown are representative of six (A) and five (B)
independent determinations.
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Fig. 3.
Effect of increasing concentrations of
Gd3+ on [Ca2+]i
oscillations induced by 5 µM
carbachol. The top left panel depicts a control
experiment carried out according to the protocol described for Fig.
1C. In subsequent panels, Gd3+, at the
concentrations shown, was added during the interval indicated. Similar
findings were obtained in a total of four to six experiments.
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Fig. 4.
Effects of Gd3+
on Ca2+ extrusion in
HEK293 cells. To assess inhibitory actions of Gd3+ on
Ca2+ extrusion, cells were activated by 1 µM
thapsigargin (TG) in the absence of external
Ca2+ and in the presence of the indicated concentrations of
Gd3+. Concentrations of Gd3+ of 30 µM or greater delayed the decay of the thapsigargin
transient, indicating a degree of inhibition of plasma membrane
Ca2+ extrusion.
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Fig. 5.
Calcium signaling in HEK293 cells due to
arachidonic acid and the effects of Gd3+. In
A, a Fura2-loaded HEK293 cell was exposed to 30 µM arachidonic acid (AA) as indicated,
resulting in a biphasic rise in [Ca2+]i. The
protocol for B and C was as for Figs. 2 and 3. In
C, summary data are included for both the entry
(filled circles) and release (open circles)
phases of the response to arachidonic acid. The data in C
are means ± S.E. from six to seven experiments.
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Fig. 6.
Effect of 2-APB on Ca2+
release and Ca2+ entry
due to 100 µM carbachol. The
protocol for the two panels is identical to that for Figs.
3B and 3C except that different concentrations of
the membrane-permeant IP3 receptor inhibitor, 2-APB, were
used. In the top panel, the agonist was 100 µM
carbachol, and in the bottom panel the agonist was 1 µM thapsigargin. The results illustrate findings from six
(top) and five (bottom) independent
determinations.
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Fig. 7.
Effect of 2-APB on Ca2+
signaling due to 30 µM arachidonic acid
(AA). 30 µM arachidonic acid was
applied to a HEK293 cell when indicated, in the absence of external
Ca2+, and Ca2+ restored to 1.8 mM
during the indicated interval. The cells were pretreated with either
100 µM 2-APB (dashed line) or the solvent
control, Me2SO (solid line). 100 µM 2-APB failed to inhibit arachidonic acid-induced
Ca2+ entry in a total of six experiments.
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Fig. 8.
Effect of 2-APB on
[Ca2+]i oscillations due to 5 µM carbachol..
The protocol was as for Fig. 4. Sequential stimulations with 5 µM carbachol are shown in A, and the effects
of increasing concentrations of 2-APB on the second stimulation are
depicted in B-D. Similar findings were obtained in a total
of five to six experiments.
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Fig. 9.
Effects of 5 µM carbachol and 100 µM 2-APB on Mn2+
entry into HEK293 cells. The
Ca2+-insensitive fluorescence (Ftot,
see "Materials and Methods") of Fura2-loaded HEK293 cells was
monitored. In control cells (A), addition of 0.1 mM Mn2+ to the medium causes an accelerated
quench of Fura2 (solid line), and this effect is blocked by
2-APB (dotted line). Treatment of the cells with 5 µM carbachol (B) results in an enhanced rate
of Fura2 quench (solid line) that is again completely
blocked by 2-APB (dotted line). A total of seven experiments
produced similar findings.
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Fig. 10.
Effects of 5 µM carbachol, 100 µM 2-APB, and 5 µM arachidonic acid
(AA) on Mn2+ entry into
HEK293 cells. As in Fig. 11, the Ca2+-insensitive
fluorescence (Ftot, see "Materials and
Methods") of Fura2-loaded HEK293 cells was monitored. In
A, in cells treated with 100 µM 2-APB,
addition of 2.0 mM Mn2+ to the medium causes an
accelerated quench of Fura2, but subsequent addition of carbachol does
not further increase the rate of quench (solid line).
However, with this same protocol, arachidonic acid stimulates the rate
of quench (dotted line). In B, addition of 2 mM Mn2+ to control, 2-APB-treated cells
(solid line), or 2-APB-treated cells stimulated with 5 µM carbachol (dotted line) results in a
similar increase in the rate of quench of Fura2. A total of four to
five experiments produced similar findings.
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Fig. 11.
Effects of isotetrandrine on
carbachol-induced [Ca2+]i oscillations. The
protocol for A was as in Fig. 1C. Addition of 10 µM isotetrandrine to the medium blocked the sustained
oscillations due to carbachol (7 of 21 cells). In B, 10 µM isotetrandrine was added during the oscillations, and
the oscillations ceased. Addition of 5 µM arachidonic
acid restored a partial response in 7 of 14 cells tested.
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Fig. 12.
Effects of isotetrandrine on arachidonic
acid- and thapsigargin-induced Ca2+ entry.
In A, Ca2+ entry was activated by 70 µM arachidonic acid. Where indicated, 10 µM
isotetrandrine was added. Similar results were obtained in a total of
three experiments with 10 µM isotetrandrine, and in three
of four experiments with 20 µM isotetrandrine. In
B, 1 µM thapsigargin was used to activate
capacitative calcium entry. The addition of 10 µM
isotetrandrine induced only a slight diminution of Ca2+
entry, and further addition of 20 µM isotetrandrine
inhibited to a slightly greater extent. Similar results were obtained
in a total of five experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Elizabeth Murphy and Jerry Yakel who read the manuscript and provided helpful comments.
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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.
§ 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, November 28, 2000, DOI 10.1074/jbc.M007524200
2 D. Luo, L. M. Broad, G. St. J. Bird, and J. W. Putney, Jr., unpublished observations.
3 The failure of store depletion by arachidonic acid to activate capacitative calcium entry may be due to the previously documented ability of arachidonic acid to inhibit capacitative calcium entry (48).
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ABBREVIATIONS |
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The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; ER, endoplasmic reticulum; PLA2, phospholipase A2; HPSS, HEPES-buffered physiological saline solution; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; 2-APB, 2-aminoethyoxydiphenyl borane; SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPase.
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