Diversity of Calcium Signaling by Metabotropic Glutamate Receptors*

Shigeki KawabataDagger , Atsuyuki Kohara§, Rie Tsutsumi§, Hirotsune Itahana, Satoshi Hayashibe, Tokio Yamaguchi§, and Masamichi Okada§parallel

From the Dagger  Molecular Medicine Laboratory, § Neuroscience Research Laboratory, and  Chemistry Laboratory, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., Ibaraki 305, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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, mGluR1alpha or mGluR5a. Stimulation of mGluR1alpha 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 mGluR1alpha 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 mGluR1alpha 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 mGluR1alpha -expressing cells, activation of protein kinase C selectively desensitized the pathway for intracellular Ca2+ mobilization, but the mGluR1alpha -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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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, mGluR1alpha 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 mGluR1alpha -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 mGluR1alpha 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 mGluR1alpha , 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 mGluR1alpha 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 mGluR1alpha 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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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 mGluR1alpha 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, mGluR1alpha (T) and mGluR5a(D), aspartate (854) of mGluR1alpha and threonine (840) of mGluR5a were changed into threonine and aspartate, respectively, as described previously (9). The cDNA encoding rat mGluR1alpha , mGluR5a, or a mutant receptor was inserted into the eukaryotic expression vector, pEF-BOS. After transfection of the above plasmids, HEK-293 cells expressing mGluR1alpha , 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.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Ca2+ Responses in HEK-293 Cells Expressing mGluR1alpha -- In HEK-293 cells expressing mGluR1alpha , 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 mGluR1alpha 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 mGluR1alpha -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 mGluR1alpha -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 mGluR1alpha 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 mGluR1alpha stimulation is mGluR1alpha receptor-operated, because mGluR1alpha 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 mGluR1alpha . 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 mGluR1alpha -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 mGluR1alpha -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 (mGluR1alpha 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). mGluR1alpha antagonist also blocked an oscillatory Ca2+ response in the sustained phase of mGluR1alpha stimulation (c). Representative traces are presented.

In mGluR1alpha -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 mGluR1alpha , the InsP3/Ca2+ pathway is turned off before Ca2+ stores are depleted. These results show that during prolonged stimulation of mGluR1alpha , 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 mGluR1alpha 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 mGluR1alpha . 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.

[Ca2+]i Oscillations in HEK-293 Cells Expressing mGluR1alpha 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.

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 mGluR1alpha -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 mGluR1alpha -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 mGluR1alpha -- In mGluR1alpha -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 mGluR1alpha receptor-operated Ca2+-permeable channel is resistant to desensitization by PKC in mGluR1alpha -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 mGluR1alpha 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 mGluR1alpha . 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.

Ca2+ Responses in HEK-293 Cells Expressing Mutant mGluRs-- Different patterns of Ca2+ response in mGluR1alpha -expressed and mGluR5a-expressed cells elicited by transient application (20 s) of glutamate results from a single amino acid substitution, aspartate of mGluR1alpha (position 854) or threonine of mGluR5a (position 840) (9). In cells expressing mGluR1alpha (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 mGluR1alpha -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 mGluR1alpha (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.

In this study, we found that mGluR1alpha and mGluR5a show distinct coupling mechanisms during prolonged stimulation by glutamate (20 min). In the initial phase of stimulation, both mGluR1alpha and mGluR5a trigger the release of Ca2+ from the intracellular stores through InsP3/Ca2+ pathway. In the sustained phase of stimulation, in mGluR1alpha -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 mGluR1alpha 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 mGluR1alpha does not.

The studies on mutant receptors of mGluR1alpha 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 mGluR1alpha (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 mGluR1alpha 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.

    ACKNOWLEDGEMENT

We thank Dr. Shigetada Nakanishi of Kyoto University for carefully reading this manuscript.

    FOOTNOTES

* 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.

parallel To whom correspondence should be addressed: Neuroscience Research Laboratory, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba-shi Ibaraki 305, Japan. Tel.: 298-52-5111; Fax: 298-56-2515.

1 The abbreviations used are: InsP3, inositol 1,4,5-triphosphate; PKC, protein kinase C; Fura-2/AM, Fura-2 acetoxymethyl ester; PMA, phorbol 12-myristate 13-acetate; mGluR, metabotropic glutamate receptor.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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