During prolonged application of glutamate
(20 min), patterns of increase in intracellular Ca2+
concentration ([Ca2+]i)
were studied in HEK-293 cells expressing metabotropic glutamate
receptor, mGluR1
or mGluR5a. Stimulation of mGluR1
induced an
increase in
[Ca2+]i that consisted of
an initial transient peak with a subsequent steady plateau or an
oscillatory increase in [Ca2+]i The
transient phase was largely attributed to Ca2+
mobilization from the intracellular Ca2+ stores, but the
sustained phase was solely due to Ca2+ influx through the
mGluR1
receptor-operated Ca2+ channel. Prolonged
stimulation of mGluR5a continuously induced [Ca2+]i oscillations through
mobilization of Ca2+ from the
intracellular Ca2+ stores. Studies on mutant receptors of
mGluR1
and mGluR5a revealed that the coupling mechanism in the
sustained phase of Ca2+ response is determined by
oscillatory/non-oscillatory patterns of the initial Ca2+
response but not by the receptor identity. In mGluR1
-expressing cells, activation of protein kinase C selectively desensitized the
pathway for intracellular Ca2+ mobilization, but the
mGluR1
-operated Ca2+ channel remained active. In
mGluR5a-expressing cells, phosphorylation of mGluR5a by protein kinase
C, which accounts for the mechanism of mGluR5a-controlled
[Ca2+]i oscillations, might prevent desensitization and
result in constant oscillatory mobilization of Ca2+ from
intracellular Ca2+ stores. Our results provide a novel
concept in which oscillatory/non-oscillatory mobilizations of
Ca2+ induce different coupling mechanisms during prolonged
stimulation of mGluRs.
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INTRODUCTION |
Ca2+ can transduce many diverse cellular processes.
Such diversity may be achieved by different amplitude and distinct
spatial and temporal patterns of Ca2+ response (1). In B
lymphocytes, the amplitude and duration of Ca2+ signaling
controls differential activation of pro-inflammatory transcriptional
regulators (2). In differentiating neurons, the frequency of
[Ca2+]i oscillations affects
expression of specific neuronal phenotypes such as channel maturation
and neurotransmitter expression (3). Compartmentalization
of Ca2+ signaling is also important
in different cellular processes. For example, cytosolic
Ca2+ signals activate c-fos gene transcription
through the serum response element, but nuclear Ca2+
signals activate it through cyclic AMP response element (4).
Stimulation of two metabotropic glutamate receptor subtypes, mGluR1
and mGluR5a, triggers the release of Ca2+ from the
intracellular stores through inositol 1,4,5-triphosphate (InsP3)1
formation (InsP3/Ca2+ pathway) (5-8). We
recently reported that transient application (1-60 s) of glutamate
induces single-peaked intracellular Ca2+ mobilization in
mGluR1
-transfected cells but elicits
[Ca2+]i oscillations in
mGluR5a-transfected cells (9). The response patterns of the
[Ca2+]i increase depend upon the
identity of a single amino acid, aspartate (at position 854) or
threonine (at position 840), located within the G-protein-interacting
domains of mGluR1
and mGluR5a, respectively. Phosphorylation of
threonine (840) of mGluR5a by PKC interferes with the signal
transduction through mGluR5a. We hypothesized that repetitive
phosphorylation and dephosphorylation of mGluR5a could induce
[Ca2+]i oscillations by signaling on
and off. In mGluR1
, nonphosphorylation at aspartate (854) produces a
non-oscillatory and PKC activator-resistant Ca2+ response
(9). This previous study provides the first evidence that an agonist
can produce oscillatory/non-oscillatory patterns of Ca2+
response by stimulating different receptor subtypes. However, it
remained uncertain whether and how these two mGluRs control different
cellular processes depending on their oscillatory/non-oscillatory Ca2+ responses.
We report here that prolonged stimulation of mGluR1
induced an
increase in [Ca2+]i that consisted of
an initial transient peak and a subsequent steady plateau or an
oscillatory increase in [Ca2+]i. The transient
phase was largely attributed to Ca2+ release from
intracellular Ca2+ stores, but the sustained phase was
solely due to Ca2+ influx through a mGluR1
receptor-operated Ca2+ channel. On the other hand,
prolonged stimulation of mGluR5a continuously induced
[Ca2+]i oscillations
through mobilization of Ca2+ from the intracellular
Ca2+ stores. The coupling mechanism in the sustained phase
of Ca2+ response is determined by
oscillatory/non-oscillatory patterns of the initial Ca2+
response but not by the receptor identity. Thus, during prolonged stimulation of mGluRs, oscillatory/non-oscillatory patterns of Ca2+ response lead to different coupling mechanisms in
Ca2+ signaling.
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MATERIALS AND METHODS |
Fura-2 acetoxymethyl ester (Fura-2/AM) and
phorbol-12-myristate-13-acetate (PMA) were from Wako Pure Chemical
Industries. SK&F96365 and nimodipine were from Funakoshi. The mGluR1
antagonist, 1a-(N-phenyl)carbamoyl-1a,7a-dihydro-7(1H)-hydroxyiminocyclopropa[b]chromen (10) was synthesized in our laboratory.
For construction of the mutant receptors, mGluR1
(T) and mGluR5a(D),
aspartate (854) of mGluR1
and threonine (840) of mGluR5a were
changed into threonine and aspartate, respectively, as described previously (9). The cDNA encoding rat mGluR1
, mGluR5a, or a
mutant receptor was inserted into the eukaryotic expression vector,
pEF-BOS. After transfection of the above plasmids, HEK-293 cells
expressing mGluR1
, mGluR5a, or a mutant receptor were selected with
400 µg/ml geneticin and isolated by a single cloning step (9). These
cells were loaded for 45 min with Fura-2/AM (6 µM) dissolved in balanced salt solution containing 135 mM NaCl,
5.4 mM KCl, 1.8 mM CaCl2, 0.9 mM MgCl2, and 10 mM HEPES (pH 7.4). After incubation, the coverslips were mounted in a laminar flow chamber
with a flow rate of 2 ml/min at 32 °C. The chamber was mounted on a
Nikon inverted stage microscope.
[Ca2+]i measurement was
started at 15 min after superfusion of balanced salt solution. Light
from a Xenon lamp was filtered through either of two different
band-pass filters (340 nm or 380 nm) in the excitation path and
conducted to the specimen on the microscope stage through a diachronic
mirror. The excitation wavelength was constantly switched between 340 nm and 380 nm. The fluorescence emitted from the cells was passed
through a band-pass filter (510 nm). The video images were obtained
using an intensified charge-coupled camera. Output from the camera was
digitized and stored by a computerized imaging system (Hamamatsu, Argus
50). Ratios of sequential 340/380-nm excitation image pairs were
compared with a standard curve for free Ca2+
constructed from shallow solutions of known Ca2+ and Fura-2
concentration. EGTA (1 mM) was included instead of CaCl2 in all experiments using a Ca2+-free
extracellular buffer. The application of reagent or change of external
medium is indicated in each graph by a bar or set of bars.
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RESULTS AND DISCUSSION |
Ca2+ Responses in HEK-293 Cells Expressing
mGluR1
--
In HEK-293 cells expressing mGluR1
, prolonged
application of 30 µM glutamate (20 min) induced an
initial transient peak response followed by an oscillatory or a steady
plateau Ca2+ response (Fig.
1, a and b). 44%
of cells showed [Ca2+]i
oscillations, 39% showed steady plateau.
[Ca2+]i oscillations
observed in the sustained phase of mGluR1
stimulation are
characterized by base-line spikes of relatively constant amplitude. A
minor population of cells showed three types of responses; a single
transient peak response, an initial transient peak with a subsequent
response best described as spike plateau, and an initial peak slowly
descending to the base line followed by a base-line spiking type
of
[Ca2+]i oscillations (data
not shown). The transient peak of
Ca2+ response in mGluR1
-expressing cells was largely
attributed to Ca2+ mobilization from the intracellular
Ca2+ stores, because Ca2+ response was only
slightly reduced in the absence of external Ca2+ (Fig.
5a). In contrast, [Ca2+]i
oscillations during the sustained phase in
mGluR1
-expressing cells were completely abolished in the absence of
external Ca2+ (Fig. 1c).
The oscillatory Ca2+ response in the sustained phase was
blocked by 30 µM SK&F96365, a receptor-operated
Ca2+ channel blocker (11) but not by 10 µM
nimodipine, a voltage-gated Ca2+ channel blocker (12) (Fig.
2, a and b). Steady
plateau Ca2+ response and other types of responses in the
sustained phase of mGluR1
stimulation were also fully dependent on
extracellular Ca2+ and blocked by SK&F96365 but not by
nimodipine (data not shown). Ca2+ influx in the sustained
phase of mGluR1
stimulation is mGluR1
receptor-operated, because
mGluR1
antagonist,
1a-(N-phenyl)carbamoyl-1a,7a-dihydro-7(1H)-hydroxyiminocyclopropa[b]chromen (10), blocked Ca2+ influx in the sustained phase of
Ca2+ response (Fig. 2c).

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Fig. 1.
Ca2+ responses elicited by
prolonged application (20 min) of glutamate in HEK-293 cells expressing
mGluR1 . Two patterns of Ca2+ response observed by
prolonged application (20 min) of 30 µM glutamate (Glu)
consisted of an initial transient peak followed by an oscillatory in
44% of cells (a) or a steady plateau increase in
[Ca2+]i in 39% of cells
(b).
[Ca2+]i oscillations in the sustained
phase of mGluR1 -stimulation were abolished in the absence of
external Ca2+ (Ca2+-free, 1 mM
EGTA) (c). Representative traces are presented.
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Fig. 2.
Effects of Ca2+ channel blockers
and mGluR1 antagonist on the sustained phase of Ca2+
responses evoked by prolonged application (20 min) of glutamate in
mGluR1 -expressed HEK-293 cells. The concentrations of SK&F96365
(receptor-operated Ca2+ channel blocker) (a),
nimodipine (voltage-gated Ca2+ channel blocker)
(b), and
1a-(N-phenyl)carbamoyl-1a,7a-dihydro-7(1H)-hydroxyiminocyclopropa[b]chromen
(mGluR1 antagonist) (c) added to balanced salt solution
were 30, 10, and 50 µM, respectively. An oscillatory
Ca2+ response in the sustained phase was blocked by
SK&F96365 (a) but not blocked by nimodipine (b).
mGluR1 antagonist also blocked an oscillatory Ca2+
response in the sustained phase of mGluR1 stimulation
(c). Representative traces are presented.
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In mGluR1
-expressing cells, in the absence of external
Ca2+, 100 µM carbachol could still mobilize
Ca2+ from intracellular Ca2+ stores during
prolonged application of 30 µM glutamate (Fig. 3), indicating that during prolonged
stimulation of mGluR1
, the InsP3/Ca2+
pathway is turned off before Ca2+ stores are depleted.
These results show that during prolonged stimulation of mGluR1
, the
InsP3/Ca2+-pathway is selectively desensitized,
and Ca2+ entry from SK&F96365-sensitive Ca2+
channel solely contributes to the [Ca2+]i
increase in the sustained phase of mGluR1
stimulation.

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Fig. 3.
Muscarinic receptor stimulation with
prolonged application of glutamate in the absence of external
Ca2+ in HEK-293 cells expressing mGluR1 . In the
absence of external Ca2+ (Ca2+-free, 1 mM EGTA), endogenous muscarinic receptors were stimulated
by 100 µM carbachol (CCh) under prolonged
application of 30 µM glutamate (Glu). Carbachol could
still mobilize Ca2+ from the intracellular Ca2+
stores under these conditions. Representative traces are
presented.
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[Ca2+]i Oscillations in HEK-293
Cells Expressing mGluR1
or mGluR5a--
Prolonged application of
glutamate (20 min) constantly elicited sinusoidal
[Ca2+]i oscillations in
cells expressing mGluR5a (Fig.
4a). In the late phase of
stimulation, these [Ca2+]i
oscillations were not abolished in the absence of external
Ca2+ (Fig. 4b). Thus, prolonged stimulation of
mGluR5a continuously mobilizes Ca2+ from intracellular
stores. In mGluR5a-expressing cells, the frequency of oscillations was
lowered by the removal of external Ca2+. Thus, a source of
extracellular Ca2+ is also involved in the generation of
[Ca2+]i oscillations,
which is in good agreement with the notion that the lack of
Ca2+ influx lengthens the time required for the refilling
of intracellular Ca2+ stores (13-15).

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Fig. 4.
Ca2+ responses elicited by
prolonged application (20 min) of glutamate in HEK-293 cells expressing
mGluR5a. Prolonged application of 30 µM glutamate
(Glu) constantly elicited [Ca2+]i
oscillations in cells expressing mGluR5a (a). In the late
phase of stimulation, these Ca2+ oscillations were not
abolished in the absence of external Ca2+
(Ca2+-free, 1 mM EGTA) (b). The
frequency of oscillations lowered by removal of external
Ca2+ (b). Representative traces are
presented.
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It has been proposed that
[Ca2+]i oscillations can
be classified into two types, base-line spiking and sinusoidal
oscillations, and can be driven by several different mechanisms
(15-17). From this point of view,
[Ca2+]i oscillations seen
in mGluR1
-expressing and mGluR5a-expressing cells are clearly
distinguished between base-line spiking and sinusoidal behavior. The
mechanisms underlying these
two[Ca2+]i oscillations
are also distinct because
[Ca2+]i oscillations in
mGluR1
-expressing cells are fully dependent on external
Ca2+, but those in mGluR5a-expressing cells occur mainly
through mobilization of Ca2+ from the intracellular
stores.
The Effect of PKC Activator on Ca2+
Responses in HEK-293 Cells Expressing mGluR1
--
In
mGluR1
-expressing cells, treatment with 100 nM PMA (18)
reduced but did not abolish the Ca2+ response induced by
transient application (20 s) of glutamate (Fig.
5b). This PMA-resistant
Ca2+ response is fully dependent on the influx of
extracellular Ca2+, because the Ca2+ response
was abolished in the absence of external Ca2+ (Fig.
5c). These results show that activation of PKC is
responsible for desensitization of
InsP3/Ca2+-pathway, but the mGluR1
receptor-operated Ca2+-permeable channel is resistant to
desensitization by PKC in mGluR1
-expressing cells. Dissociation
between InsP3 formation and Ca2+ influx
strongly suggests that neither the formation of inositol phosphate
metabolites nor Ca2+ mobilization is needed for the
activation of the mGluR1
receptor-operated Ca2+
channel.

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Fig. 5.
The effect of PKC activator on
Ca2+ responses elicited by transient application of
glutamate (Glu) in the absence or presence of external Ca2+
in HEK-293 cells expressing mGluR1 . Ca2+ response
elicited by transient application (20 s) of 30 µM
glutamate (Glu) was slightly reduced by removing external
Ca2+ (Ca2+-free, 1 mM EGTA)
(a). 100 nM PMA reduced but not abolished
Ca2+ response elicited by transient application (20 s) of
glutamate (b). This PMA-resistant Ca2+ response
was abolished in the absence of external Ca2+
(c). The number of cells analyzed were 21 (a), 8 (b), and 16 (c). The data are the mean ± S.E. (vertical bars) at each time point.
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Ca2+ Responses in HEK-293 Cells Expressing
Mutant mGluRs--
Different patterns of Ca2+ response in
mGluR1
-expressed and mGluR5a-expressed cells elicited by transient
application (20 s) of glutamate results from a single amino acid
substitution, aspartate of mGluR1
(position 854) or threonine of
mGluR5a (position 840) (9). In cells expressing mGluR1
(T), prolonged
application of 30 µM glutamate (20 min) elicited constant
[Ca2+]i oscillations that
were not abolished in the absence of external Ca2+ (Fig.
6a). On the other hand, in
cells expressing mGluR5a(D), prolonged application of 30 µM glutamate (20 min) induced an initial transient peak
response followed by a steady plateau or an oscillatory Ca2+ response (Fig. 6, b and c), both
of which are identical with those in mGluR1
-expressing cells. 20%
of cells showed [Ca2+]i
oscillations, 63% showed steady plateau. The sustained phase of
Ca2+ response in mGluR5a(D)-expressing cells was abolished
in the absence of external Ca2+ (Fig. 6, b and
c). These results indicate that the coupling mechanism in
the sustained phase of Ca2+ response is determined by
oscillatory/non-oscillatory patterns of the initial Ca2+
response but not by the receptor identity.

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Fig. 6.
Ca2+ responses elicited by
prolonged application (20 min) of glutamate in HEK-293 cells expressing
mutant mGluRs. In cells expressing mGluR1 (T), prolonged
application of 30 µM glutamate (Glu) constantly elicited
[Ca2+]i oscillations, which were not
abolished in the absence of external Ca2+
(Ca2+-free, 1 mM EGTA) (a). In cells
expressing mGluR5a(D), prolonged application of 30 µM
glutamate induced an initial transient peak response followed by a
steady plateau in 20% of cells (b) or an oscillatory
Ca2+ response in 63% of cells (c). The
sustained phase of Ca2+ responses in mGluR5a(D)-expressing
cells were abolished in the absence of external Ca2+
(b and c). Representative traces are
presented.
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In this study, we found that mGluR1
and mGluR5a show distinct
coupling mechanisms during prolonged stimulation by glutamate (20 min).
In the initial phase of stimulation, both mGluR1
and mGluR5a trigger
the release of Ca2+ from the intracellular stores through
InsP3/Ca2+ pathway. In the sustained phase of
stimulation, in mGluR1
-expressing cells,
InsP3/Ca2+-pathway is desensitized, and
Ca2+ entry solely contributes to
[Ca2+]i increase. In
contrast, in mGluR5a-expressing cells, InsP3/Ca2+ pathway is not desensitized, and
Ca2+ is mobilized continuously from the intracellular
Ca2+ stores during the prolonged stimulation. In
mGluR5a-expressing cells, Ca2+ entry is also involved in
the generation of [Ca2+]i
oscillations, because the frequency of oscillations is lowered by the
removal of external Ca2+. At present, it is unclear whether
or not the Ca2+-permeable channels, which are activated
during stimulation of mGluR1
or mGluR5a, are the same. However, the
coupling mechanisms during prolonged stimulation of these two mGluRs
are clearly distinct in that mGluR5a continuously couples to
InsP3/Ca2+ pathway, but mGluR1
does not.
The studies on mutant receptors of mGluR1
and mGluR5a demonstrate
that desensitization of the InsP3/Ca2+ pathway
occurs when the initial Ca2+ response is non-oscillatory.
In contrast, the sinusoidal
[Ca2+]i oscillations seen
in mGluR5a- or mGluR1
(T)-expressing cells prevent the
InsP3/Ca2+ pathway from desensitization during
prolonged stimulation of these receptors. The precise mechanism by
which the sinusoidal [Ca2+]i oscillations avoid
InsP3/Ca2+ pathway desensitization is unclear;
however, our earlier study of mGluR5a-controlled
[Ca2+]i oscillations may
give a hint (9). In that study, we showed that PKC inhibitors
eliminate
[Ca2+]i oscillations and
convert Ca2+ response from an oscillatory to
non-oscillatory pattern in mGluR5a-expressing cells. In contrast, the
PKC activator, PMA, abolishes the Ca2+ response in
mGluR5a-expressing cells (9). We suggested that phosphorylation of
mGluR5a by PKC inactivates mGluR5a, thus resulting in the decrease of
[Ca2+]i, whereas subsequent dephosphorylation of
mGluR5a restores the signal transduction through mGluR5a, thus
regenerating the [Ca2+]i increase. We had
then proposed that repetitive cycles of phosphorylation and
dephosphorylation of mGluR5a generate
[Ca2+]i oscillations (9).
Although not proven by experiments, continuous cycling of
phosphorylation/dephosphorylation of mGluR5a may be provided by
oscillations in the activity of PKC. During mGluR5a stimulation, not
only [Ca2+]i, but also the cellular level of
diacylglycerol, the other bifurcating limb of phosphoinositide pathway
(19), would oscillate. It is conceivable that PKC activity that is
known to be affected by Ca2+ and diacylglycerol would also
oscillate (20). In the present study, we found that PKC is responsible
for desensitization of InsP3/Ca2+ pathway
during mGluR1
stimulation. If PKC activity, which is first
incremented by mGluR5a stimulation, decreases rapidly before the
InsP3/Ca2+ pathway is desensitized, such an
oscillating PKC activity would prevent the
InsP3/Ca2+ pathway from desensitization in
mGluR5a-expressing cells.
In cultured astrocytes, glutamate induces
[Ca2+]i oscillations
through mGluR5 (21, 22). Similar to the observations in
mGluR5a-expressed HEK-293 cells (9), the PKC activator abolishes Ca2+ response in cultured astrocytes (21, 23). Moreover,
both PKC inhibitor and PP1/PP2A phosphatase inhibitor convert
Ca2+ response from an oscillatory to non-oscillatory
pattern, suggesting that the same mechanism underlies the generation of
[Ca2+]i oscillations in
mGluR5-expressed heterologous and native cells (21). Thus, it may also
occur in native cells that oscillatory/non-oscillatory mobilizations of
Ca2+ result in distinct coupling mechanisms during
prolonged stimulation of mGluRs. Although it remains to be elucidated
whether these different coupling mechanisms would indeed establish
diverging cellular processes, our results provide a new insight into
Ca2+ signaling when long lasting stimuli are evoked in
cells.
We thank Dr. Shigetada Nakanishi of Kyoto
University for carefully reading this manuscript.