Reassessment of the Ca2+ Sensing Property of a Type I Metabotropic Glutamate Receptor by Simultaneous Measurement of Inositol 1,4,5-Trisphosphate and Ca2+ in Single Cells*

Mark S. NashDagger, Ruth Saunders§, Kenneth W. Young, R. A. John Challiss, and Stefan R. Nahorski

From the Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, P. O. Box 138, University Road, Leicester, LE1 9HN, United Kingdom

Received for publication, August 21, 2000, and in revised form, January 25, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta 1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca2+ within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mM Ca2+, increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca2+ concentration. Under nominally Ca2+-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca2+ concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca2+ concentration absent. Depletion of intracellular Ca2+ stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca2+ and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca2+ concentration. However, prolonged activation of phospholipase C requires extracellular Ca2+, while the initial burst of activity is highly dependent on Ca2+ mobilization from intracellular stores.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Group I subfamily of metabotropic glutamate receptors, mGlu11 and mGlu5, couple to phospholipase C (PLC) via Gq/11 proteins to stimulate inositol 1,4,5-trisphosphate (IP3) production and to mobilize intracellular calcium (Ca2+i) stores (1, 2). Recently, the close structural similarity of the amino-terminal domain of mGlu receptors with that of the calcium-sensing receptor led to the suggestion that mGlu receptors may also respond to changes in extracellular calcium concentration ([Ca2+]o) (3-5). Thus, Ca2+o has been reported to both potentiate the stimulation of inositol phosphate (InsP) accumulation by the mGlu1alpha receptor (4) as well as directly activate both Group I mGlu receptors (5). Since Ca2+o also potentiates agonist binding and potency at GABAB receptors (6, 7) Ca2+o sensing may, in fact, be a general property of family 3 G protein-coupled receptors.

The ability to respond to changes in Ca2+o has profound implications for receptor signaling in the central nervous system where local fluctuations in synaptic [Ca2+]o can occur as a result of activation of calcium permeant cation channels (8). Moreover, a novel form of intercellular communication has recently been identified where Ca2+ extruded from one cell, following agonist-driven Ca2+i mobilization, stimulates neighboring cells expressing calcium-sensing receptors (9). Since mGlu receptors are expressed widely in the central nervous system this form of communication may have important consequences for our understanding of neuronal and glial cell interactions. There is, however, indirect evidence against the Ca2+o sensing property of Group I mGlu receptors. Several studies have determined the origin of mGlu receptor-mediated Ca2+i responses; intracellular store release or extracellular Ca2+ entry, by removing Ca2+o. From these, mGlu5 receptor-mediated Ca2+i release in astrocytes, cortical neurons (10-12), and HEK-293 (13), and initial mGlu1alpha receptor responses in HEK-293 (14) and A9 cells (15) were reported to be unaffected by removing Ca2+o. Moreover, the recently published x-ray crystallography structure of the NH2 terminus of the mGlu1alpha receptor identified a high affinity cation-binding site, which suggests that Ca2+ is more likely to be a "scaffold factor" rather than a physiological ligand (16). Given the potential importance of Ca2+o sensing by these receptors such a fundamental question concerning their activation requires an unambiguous answer.

To address this issue we have made real-time concurrent measurements of IP3 and Ca2+i in single cells expressing the mGlu1alpha receptor using a recently developed technique that utilizes an enhanced green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta 1 (eGFP-PHPLCdelta ) to detect IP3 in Fura-2 loaded cells (17-19). PHPLCdelta binds selectively to phosphatidylinositol 4,5-bisphosphate (PIP2) over all other inositol lipids (20) and a fusion construct of this PH domain with eGFP associates with the plasma membrane (18, 19). Hirose et al. (17) recently demonstrated that PHPLCdelta has higher affinity for the soluble head group of PIP2, IP3, and that PLC-induced elevations in IP3 cause translocation of the fusion protein to the cytosol. The extent of membrane association of eGFP-PHPLCdelta can thus be used to evaluate cellular IP3 levels and, by preloading with Fura-2, simultaneous measurements of [Ca2+]i are possible in the same cell. This technique overcomes many of the inherent pitfalls in other assays by allowing simultaneous temporal analysis of two crucial indices of PLC signaling in single cells rather than in cell populations. Our data clearly indicate that the initial response to mGlu1alpha receptor activation is not sensitive to Ca2+o demonstrating that it is not a true Ca2+o-sensing receptor. However, for prolonged IP3 production Ca2+ entry is required. Moreover, Ca2+ mobilization from intracellular stores by IP3 is found to be essential for amplification of the initial response to agonist.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Vector containing the fusion construct between eGFP and the PH domain of PLCdelta 1 was kindly provided by Professor T. Meyer (Stanford University). Detailed information regarding this construct can be found in Stauffer et al. (18). Standard chemicals and biochemicals were from Sigma (Poole, UK) unless otherwise indicated. Radiochemicals were from Amersham Pharmacia Biotech (Amersham, United Kingdom). Quisqualate and 1S,3R-ACPD were from Tocris-Cookson (Bristol, UK). Fura-2 AM was from Molecular Probes (Cambridge, UK) and Dowex anion exchange resin AG1-X8 (200-400 mesh, formate form) from Bio-Rad (Watford, UK). All materials for cell culture were supplied by Life Technologies, Inc. (Paisley, UK).

Cell Culture-- A description of the LacSwitch inducible expression system (Stratagene) used to express human mGlu1alpha in Chinese hamster ovary cells (CHO-lac-mGlu1alpha ) is provided elsewhere (21, 22). For these studies on the CHO-lac-mGlu1alpha cells, maximal levels of receptor expression were used throughout and achieved by preincubation with 100 µM IPTG for greater than 18 h (21). This results in expression levels of ~50,000 receptors per cell (400 fmol/mg protein).2 To minimize the exposure to glutamate, CHO-lac-mGlu1alpha cells were grown in Dulbecco's modified Eagle's medium with GlutamaxTM containing 10% fetal calf serum, proline (44 mg/ml), fungizone (2.5 mg/ml), penicillin (105 units/liter), streptomycin (100 µg/ml), and 300 µg/ml G418. During the induction of receptor expression, cells were maintained in this culture medium except for the substitution of 10% dialyzed serum and the absence of G418. Details for culture of BHK-mGlu1alpha cells can be found elsewhere (4).

Total InsP Assay-- [3H]Inositol phosphate ([3H]InsP) accumulation in CHO cells in response to agonist was determined as described by Hermans et al. (21). Briefly, cells were incubated for 48 h in 24-well multidishes in the presence of 2.5 µCi/ml [3H]inositol. Receptor expression was induced by addition of 100 µM IPTG to the medium ~20 h before experimentation. Growth medium was removed by aspiration and the cells washed with 3 × 1 ml of Krebs-Henseleit buffer (KHB; 10 mM HEPES, 118 mM NaCl, 4.69 mM KCl, 10 mM glucose, 1.18 mM KH2PO4, 4.2 mM NaHCO3, 1.18 mM MgCl2, and 1.3 mM (unless otherwise stated) CaCl2, pH 7.4). A reaction mixture (300 µl total volume) of KHB containing glutamic-pyruvic transaminase (3 units/ml) and pyruvate (5 mM) was added and the cells incubated for 20 min at 37 °C. After incubation with 10 mM LiCl for a further 10 min, cells were challenged with agonists for 15 min and the reaction halted with 500 µl of ice-cold 0.5 M trichloroacetic acid. Where [Ca2+]o was manipulated, the cells were washed and incubated with KHB containing the appropriate [Ca2+]o. Accumulation of [3H]InsP was determined by phase separation of the aqueous cellular constituents and resolution by Dowex anion-exchange chromatography as described previously (22). The levels of membrane [3H]phosphoinositides that remained associated with the cells were determined using the methods described by Batty et al. (23).

Measurement of [Ca2+]i-- Cells were seeded on sterile 22-mm borosilicate coverslips in 35-mm Petri dishes, with a split ratio of ~1:30, and incubated overnight at 37 °C in the presence of 100 µM IPTG. Cells were then washed twice with KHB and incubated with 2 µM Fura-2 AM for 1-2 h at room temperature. After washing, the cells were maintained in KHB until assay. Glutamic-pyruvic transaminase (3 units/ml) and pyruvate (5 mM) was present for all treatments. Agonist-induced changes in [Ca2+]i were determined using a PTI (Photon Technology Deltascan International) system. Fluorescence readings were obtained by excitation at 340 and 380 nm with a frequency of 1 Hz and measurement at 509 nm. Single cells were isolated using shutters in the photomultiplier system. After subtraction of background fluorescence, data were plotted as the 340/380 nm ratio against time. Drugs, in KHB, were perfused (5 ml/min) over the cells at 22 °C.

Dual Measurement of IP3 and [Ca2+]i-- Cells grown on 22-mm diameter borosilicate coverslips were transiently transfected with eGFP-PHPLCdelta by addition of 1 µg of plasmid DNA in a ratio of 1:3 with Fugene 6 (Roche Molecular Biochemicals) used as per manufacturer's instructions. For CHO-lac-mGlu1alpha cells receptor expression was induced the following day by addition of 100 µM IPTG in fresh medium for 20 h. Cells were then perfused with KHB at 37 °C and confocal images of eGFP-PHPLCdelta translocation captured using an UltraVIEW LCI confocal system (PerkinElmer Life Sciences). For dual measurements cells were loaded with Fura-2 AM (as described above) and coverslips mounted on the stage of a Nikon Diaphot inverted epifluorescence microscope. Sequential images were captured at wavelengths above 510 nm after excitation at 340, 380, and 490 nm using an intensified charge-coupled device camera (Photonic Science) connected to a Quanticell 700 (Applied Imaging) system. Four determinations were averaged for each time point, the images were digitized and analyzed post-experiment. To quantify eGFP-PHPLCdelta translocation, an area within the cytoplasm was chosen and the mean fluorescence recorded. The background signal was removed and the data expressed as a ratio of basal fluorescence to that at each time point. The intracellular calcium concentration from the same region was determined as described previously (24) and expressed as [Ca2+]i (nM) above basal.

Data Analysis-- Curve fitting of concentration-dependent data was performed using Prism 3.0 (GraphPad Software, Inc., San Diego, CA) and EC50 values are as provided by non-linear regression analysis using this software. Statistical analysis was performed using the Student's t test and a p value less than 0.05 considered significant.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

InsP Accumulation Is Attenuated by Decreasing [Ca2+]o in Cells Expressing mGlu1alpha Receptors-- Previous studies using BHK cells expressing mGlu1alpha receptors demonstrated sensitivity of these receptors to [Ca2+]o (4). Similar findings were also obtained for CHO cells expressing mGlu1alpha (Fig. 1). Importantly, we demonstrate here that varying the [Ca2+]o, in the absence of quisqualate, from 0 to 4 mM had no effect itself on the accumulation of [3H]InsP (Fig. 1A, diamonds). This differs from the work of Kubo et al. (5), which suggested that calcium was an agonist at Group I mGlu receptors. The data do, however, support the view that Ca2+o potentiates the response of mGlu1alpha receptors to agonist. In nominally [Ca2+]o-free medium minimal stimulation of [3H]InsP accumulation occurred in CHO-lac-mGlu1alpha cells exposed to either the Group I mGlu receptor agonist quisqualate or the partial agonist 1S,3R-ACPD (Fig. 1A). As the [Ca2+]o was raised, [3H]InsP accumulation increased up to a maximum at 1.3 mM [Ca2+]o. This effect was more marked for the partial agonist 1S,3R-ACPD (Fig. 1A, open squares) and for concentrations of agonist that approximate to the EC50 values (Fig. 1A, circles). These were determined in a separate series of concentration dependence experiments in 1.3 mM [Ca2+]o containing KHB and were -6.41 ± 0.08 (log EC50 (M), n = 4, Emax = 19.2-fold) for quisqualate and -4.46 ± 0.11 (log EC50 (M), n = 3, Emax = 16.9-fold) for 1S,3R-ACPD. The effect was not due to a reduction in initial PIP2 levels because comparable levels for the total membrane phosphoinositides were detected for cells in 0 and 1.3 mM [Ca2+]o (data not shown). These cellular levels were unaffected by treatment with 10 µM quisqualate in nominal [Ca2+]o but reduced by 30% in cells in 1.3 mM [Ca2+]o, which represents InsP formation by hydrolysis of PIP2. The combined data thus suggest that maximal receptor activation of PLC requires the presence of both quisqualate and Ca2+o. This supports the view that the mGlu1alpha receptor is an extracellular calcium-sensing receptor capable of detecting changes in [Ca2+]o.


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Fig. 1.   Effect of Ca2+o on stimulation of [3H]InsP accumulation and Ca2+i mobilization by mGlu1alpha receptors. A, CHO-lac-mGlu1alpha cells were preincubated in KHB containing the indicated [Ca2+]o for 30 min before challenging with 30 µM quisqualate (black-square), 1 mM ACPD (), 1 µM quisqualate (), or 30 µM ACPD (open circle ) for an additional 15 min. LiCl (10 mM) was added 10 min before addition of the agonists. Basal stimulation was addition of KHB alone (black-diamond ). Data are expressed as mean ± S.E.M. from four separate experiments. B, representative trace for a single CHO-lac-mGlu1alpha cell challenged for 5 min with 3 µM quisqualate in the presence of the indicated [Ca2+]o. Cells were pretreated with the appropriate concentration for 2 min before and after agonist challenge and perfused with KHB containing 1.3 mM CaCl2 for 15 min between each treatment. The trace has been edited to show only the period of agonist exposure for clarity.

Differential Sensitivity of Peak and Plateau Quisqualate Ca2+i Responses to [Ca2+]o-- To study this phenomenon further, measurements of agonist-induced effects on [Ca2+]i at the mGlu1alpha receptor were made (Fig. 1). A representative trace from a single CHO-lac-mGlu1alpha cell shows the result of a sustained challenge with 3 µM quisqualate (Fig. 1B). The characteristic response, represented by the effect in 1.3 mM Ca2+ containing buffer, is a rapid initial peak due to release of Ca2+ from intracellular stores followed by a sustained phase of Ca2+o entry. Quisqualate dose dependently stimulated this initial increase in peak [Ca2+]i with a log EC50 (M) value of -6.9 ± 0.1. The trace (Fig. 1B) illustrates the typical effect of varying [Ca2+]o on both the peak and sustained phase of the response to maximal agonist concentration. Ca2+o failed to influence the peak response to agonist challenge but increased [Ca2+]i during the sustained phase. In at least six separate experiments, no effect of modifying [Ca2+]o on peak response of mGlu1alpha receptors could be observed. However, the plateau [Ca2+]i was consistently augmented by increasing [Ca2+]o. Similar findings were obtained when submaximal concentrations of quisqualate (0.7 µM) were used (data not shown). Moreover, variations in [Ca2+]o have similar effects on agonist-induced plateau levels of [Ca2+]i in CHO cells expressing m3 muscarinic receptors.3 These results are incompatible with the view that mGlu1alpha receptors are calcium-sensing receptors.

Single Cell Measurement of IP3-- A confocal image through the mid-section of CHO-lac-mGlu1alpha cells 48 h after transfection confirmed the localization of eGFP-PHPLCdelta to the plasma membrane (Fig. 2, A and D). The majority of the fluorescent signal was concentrated over the periphery of the cells, with only a small signal detected in the cytosol. In control cells expressing eGFP alone the signal was located over the cytosol (data not shown). This is consistent with previous work (17-19) demonstrating the association of the PHPLCdelta domain with plasma membrane PIP2. Challenge with 10 µM quisqualate in KHB containing 1.3 mM Ca2+ induced translocation of the fusion protein to the cytosol (Fig. 2B) and this decreased slightly during sustained exposure to the agonist (Fig. 2C). In the nominal absence of Ca2+o the initial peak response to 10 µM quisqualate challenge was similar (Fig. 2E), however, the plateau level of cytosolic fluorescence was markedly lower than in the presence of 1.3 mM Ca2+ (Fig. 2F).


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Fig. 2.   Imaging of agonist-induced changes in [Ca2+]i and eGFP-PHPLCdelta localization in individual CHO-lac-mGlu1alpha cells. Confocal images (A-F) of CHO-lac-mGlu1alpha cells transiently transfected with eGFP-PHPLCdelta and challenged with 10 µM quisqualate in the presence of 1.3 mM (A-C) or nominal (D-F) calcium. Basal (A and D), peak (B and E), and sustained (C and F) responses to agonist are shown. G-L shows dual imaging experiments to detect changes in both [Ca2+]i (G-I) and [IP3]i (J-L) in CHO-lac-mGlu1alpha cells transfected with eGFP-PHPLCdelta and loaded with Fura-2. G-I, psuedocolor images of [Ca2+]i, before (G), 10 s after (H), and ~2 min after (I) prolonged perfusion with 10 µM quisqualate. The eGFP fluorescence associated with this same group of CHO-lac-mGlu1alpha cells at the same times is shown in J-L.

Using an epifluorescence microscope the halo of eGFP fluorescence around the transfected CHO-lac-mGlu1alpha cells was still evident (Fig. 2J). A pseudo-color representation of the [Ca2+]i determined for these resting cells is shown in Fig. 2G. Upon addition of quisqualate (10 µM) a clear increase in the [Ca2+]i occurred (Fig. 2H), which returned to a lower sustained level in the continued presence of the agonist (Fig. 2I). Concurrent with these increases in [Ca2+]i, agonist-induced translocation of the eGFP signal to the cytoplasm was observed (Fig. 2K) and this also decreased during persistent agonist exposure (Fig. 2L).

Initial Response to Agonist Is Not Dependent on Extracellular Calcium-- When the translocation of eGFP-PHPLCdelta to the cytosol in Fig. 2, A-C, is graphically represented as the ratio of fluorescence to the basal against time, IP3 production is found to follow a clear peak and plateau pattern (Fig. 3). In the nominal absence of Ca2+o (determined to be <1 µM free Ca2+) the initial peak is similar, but sustained IP3 production is markedly lower than in the presence of 1.3 mM Ca2+. Analysis of the combined data from 23 cells revealed that while the peak response to 10 µM quisqualate is unchanged the plateau level (determined at 200 s) is decreased by 72 ± 3% (Table I). Similar data were obtained when CHO-lac-mGlu1alpha cells were challenged with the submaximal quisqualate concentration of 1 µM (Table I) with a decrease in sustained production of 81 ± 3% in the absence of Ca2+o. The relatively slight decrease in peak response observed at this concentration can be attributed to small amounts of store depletion during the wash phase.


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Fig. 3.   Effect of Ca2+o on mGlu1alpha receptor-mediated IP3 production in a single CHO-lac-mGlu1alpha cell. Representative traces showing quisqualate (10 µM) (indicated by the dashed line) mediated IP3 production in a single CHO-lac-mGlu1alpha cell in the presence of 1.3 mM (solid line) or nominal (dotted line) calcium containing KHB. After perfusion with KHB containing nominal [Ca2+] the cell was challenged with quisqualate in KHB containing the same [Ca2+] for 3 min and then washed free of agonist. After washing in KHB containing 1.3 mM Ca2+ for 15 min the cell was rechallenged with quisqualate using the same protocol in the presence of 1.3 mM Ca2+. Data are shown as the ratio of cytosolic eGFP fluorescence at each time point relative to the basal.

                              
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Table I
Effect of Ca2+o on agonist-induced eGFP-PHPLCdelta translocation in CHO-lac-mGlu1alpha
Cells transfected with eGFP-PHPLCdelta were exposed to quisqualate in the presence of 1.3 mM or nominal Ca2+, peak and sustained levels of cytosolic eGFP fluorescence were determined and expressed as a ratio relative to the basal. Data are mean ± S.E.M. for 23-28 cells.

For the dual imaging experiments plotting the changes in cytosolic eGFP-PHPLCdelta fluorescence and [Ca2+]i against time revealed the remarkably similar responses to prolonged quisqualate (10 µM) treatment. An initial peak in IP3 production followed agonist addition, which fell to a steady state level, and this was mirrored closely by changes in [Ca2+]i (Fig. 4A). In the nominal absence of Ca2+o the initial peaks in IP3 (expressed as a fluorescent ratio relative to basal; 1.28 ± 0.03 versus 1.33 ± 0.04 in 1.3 mM [Ca2+]o) and [Ca2+]i (501 ± 35 versus 465 ± 37 nM in 1.3 mM [Ca2+]o) were unchanged (Fig. 4B). However, the secondary plateau phase (measured at 200 s) of [Ca2+]i was absent (7 ± 4 nM versus 214 ± 23 nM in 1.3 mM [Ca2+]o) and, although the peak in IP3 production was clearly more prolonged than for [Ca2+]i, by 200 s it had returned to near basal levels (1.04 ± 0.02 versus 1.12 ± 0.03 in 1.3 mM [Ca2+]o) (Fig. 4B). Concentration-dependent effects of quisqualate on IP3 production and [Ca2+]i mobilization were observed in the presence (Fig. 4C) and absence (Fig. 4D) of Ca2+o. Moreover, the peak [Ca2+]i data correlated with the associated change in the eGFP-PHPLCdelta fluorescence ratio (data not shown). The EC50 values for the initial peak height were comparable in 1.3 mM [Ca2+]i (1 µM for IP3 and 0.3 µM for [Ca2+]i) with those in the nominal absence of Ca2+o (0.8 µM for IP3 and 0.4 µM for [Ca2+]i). EC50 values for the plateau levels of IP3 and [Ca2+]i in 1.3 mM Ca2+o were 0.7 and 0.5 µM, respectively. Similarly, glutamate-induced translocation of the fusion protein was not abolished in the nominal absence of Ca2+o (data not shown). The data obtained from analysis of peak heights and plateau levels thus argue very strongly for a failure of extracellular calcium to influence the initial response of PLC to mGlu1alpha receptor activation. However, removing Ca2+o reduces the sustained component of IP3 production.


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Fig. 4.   Comparison of mGlu1alpha receptor-induced IP3 production and Ca2+i mobilization in nominal and 1.3 mM Ca2+o. Representative traces of simultaneous measurements of [Ca2+]i (dotted lines) and cytosolic eGFP-PHPLCdelta fluorescence (solid lines) in single CHO-lac-mGlu1alpha cells exposed to quisqualate (10 µM) (indicated by the dashed lines) in KHB containing 1.3 mM (A) or nominal (B) extracellular calcium. Cells were perfused for 3 min (5 ml/min) with KHB containing no added Ca2+ or 1.3 mM CaCl2 prior to perfusion with quisqualate in the appropriate KHB solution. C and D, dose-response curves for the changes in the [Ca2+]i (dotted lines) and the eGFP-PHPLCdelta ratio (solid lines) in 1.3 mM (C) and nominal (D) [Ca2+]o. Peak (solid symbols) and plateau (open symbols) levels are shown for the increase in both [Ca2+]i above basal (squares) and cytosolic eGFP-PHPLCdelta fluorescence (circles). Data are mean ± S.E.M. of values from greater than 10 individual cells collated from at least three separate experiments. N.B. the values for eGFP-PHPLCdelta translocation are significantly smaller than those obtained confocally because of interference from different focal planes increases the background fluorescence in these non-confocal experiments.

To eliminate the possibility that Ca2+o altered the dynamics of the response to agonist challenge the times at which the signals peaked were determined. No difference was detected in the point at which IP3 or Ca2+ concentration started to rise in the presence or absence of Ca2+o. Moreover, the time at which the response peaked was unaffected by removing Ca2+o, with peak levels of IP3 observed 14.3 ± 0.9 and 13.9 ± 1.1 s after the initial rise in the presence and absence of Ca2+o, respectively. Peak Ca2+i levels were detected 8.5 ± 0.5 and 9.8 ± 0.9 s after the initiation of the response in 1.3 mM and nominal [Ca2+]i, respectively. The values represent mean time (± S.E.M.) from analysis of more than 17 cells exposed to 10 µM quisqualate. Therefore, Ca2+o does not regulate the rate at which the receptor is able to activate PLC after agonist binding or affect the speed of release of Ca2+ from intracellular stores. The rates at which the peak IP3 and Ca2+ responses declined were, however, dependent on Ca2+o with an increased rate evident for both parameters. It is noteworthy that the effect of nominally Ca2+o free conditions was more pronounced on the rate at which [Ca2+]i declines than on that for IP3 such that in the absence of Ca2+o the increase in IP3 is far more prolonged than that of [Ca2+]i (graphically evident in Fig. 4B).

Depletion of Intracellular Stores Attenuates the Initial Response-- Since Ca2+o failed to influence the early receptor-induced events it was interesting to determine whether Ca2+ released from the intracellular stores affected mGlu1alpha receptor signaling. Control cells that were perfused with KHB containing no Ca2+ for 10 min and then challenged with 10 µM quisqualate (Fig. 5A) responded with a transient peak in [Ca2+]i (392 ± 42 nM above basal, n = 22) and a relatively more pronounced peak in [IP3]i (1.40 ± 0.05, n = 20, fluorescent ratio relative to basal). When cells were first challenged with 2 µM thapsigargin (Fig. 5B) there was an increase in [Ca2+]i (249 ± 23 nM above basal, n = 9), which steadily returned to basal as the intracellular Ca2+ stores depleted due to inhibition of the Ca2+-ATPase. Importantly, no effect on IP3 synthesis was detected (1.02 ± 0.01, n = 9) indicating that store Ca2+ does not stimulate PLC in the absence of receptor activation. When quisqualate was then perfused over these cells there was no change in [Ca2+]i (6 ± 3 nM, n = 22) and only a very small increase in IP3 production (1.11 ± 0.03, n = 22).


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Fig. 5.   Effect of thapsigargin pretreatment on the stimulation of IP3 production and Ca2+i mobilization by quisqualate in nominal [Ca2+]o. A, representative trace of a CHO-lac-mGlu1alpha cell perfused with KHB containing no added CaCl2 for 10 min before perfusion with 10 µM quisqualate (indicated by the dot-dash-dot line). Simultaneous measurements of [Ca2+]i (dotted line) and eGFP-PHPLCdelta ratio (solid line) were taken throughout this period. B, example trace of an experiment where cells were perfused for 3 min with KHB (-Ca2+), challenged with 2 µM thapsigargin in Ca2+-free KHB (indicated by the dashed line), reperfused with KHB (-Ca2+) and finally perfused with 10 µM quisqualate (dot-dash-dot line).

Raising [Ca2+]o Differentially Affects IP3 Production and [Ca2+]i Mobilization-- Perfusion of CHO-lac-mGlu1alpha cells with KHB (1.3 mM Ca2+) followed by a 4-min challenge with quisqualate (10 µM) in KHB containing either 1.3 or 2.6 mM Ca2+ revealed an interesting discrepancy between the effect of Ca2+o on IP3 production and [Ca2+]i (Fig. 6). No difference in the peak height or plateau level of translocation of eGFP-PHPLCdelta fluorescence to the cytosol was observed in 1.3 or 2.6 mM [Ca2+]o (Fig. 6A). In contrast, an increase in [Ca2+]i for both parameters after quisqualate challenge was detected (Fig. 6B). Increasing Ca2+o alone had no effect on the [Ca2+]i (data not shown).


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Fig. 6.   Effect of increasing the extracellular Ca2+ concentration on IP3 production and Ca2+i mobilization. CHO-lac-mGlu1alpha cells were preincubated for 3 min with 1.3 mM Ca2+ containing KHB before imaging. After 15 s, 10 µM quisqualate in KHB containing either 1.3 or 2.6 mM Ca2+ was perfused over the cells. Dual measurements of the changes in cytosolic eGFP-PHPLCdelta fluorescence ratio (A) and [Ca2+]i above basal (B) with time were made. The peak (hatched bars) and plateau (open bars) levels of each trace were recorded and expressed as the mean ± S.E.M. of at least 12 individual cells from three separate experiments.

Rat mGlu1alpha Receptor Responds to Ca2+o in a Similar Manner to Human mGlu1alpha Receptor-- To eliminate the possibility that the Ca2+o sensing property of mGlu1alpha receptors reflects species differences since CHO-lac-mGlu1alpha cells express human receptors rather than the rat receptor used previously (4, 5) similar experiments were performed on BHK-mGlu1alpha cells. Transient transfection with eGFP-PHPLCdelta resulted in the enrichment of fluorescence over the plasma membrane (Fig. 7A). Challenge with 1 µM quisqualate induced translocation of the fusion protein to the cytosol and this decreased during sustained agonist exposure (Fig. 7A). In the absence of added Ca2+ the same initial peak response was recorded but sustained increases in cytosolic fluorescence were dramatically decreased (Fig. 7A). This effect is more clearly observed when the translocation data are plotted against time for the response to quisqualate in the presence of 1.3 mM (solid line) or nominal (dashed line) Ca2+o (Fig. 7B). The combined data demonstrate that while peak responses are unchanged plateau levels induced by sustained agonist challenge are significantly decreased (Fig. 7C). Experiments performed using 10 µM quisqualate (maximal agonist concentration) similarly recorded no difference in the peak response in the presence or absence of added Ca2+o, but showed dramatically reduced sustained levels (data not shown).


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Fig. 7.   Effect of Ca2+o on IP3 production in BHK cells expressing the rat mGlu1alpha receptor. BHK-mGlu1alpha cells were transiently transfected with eGFP-PHPLCdelta for 48 h and then imaged with an UltraVIEW confocal system. After perfusion with KHB containing nominal [Ca2+] the cells were challenged with 1 µM quisqualate in buffer with the same [Ca2+] for 3 min and then washed free of agonist. After washing for 10-15 min (1.3 mM Ca2+) cells were re-challenged with quisqualate using the same protocol in the presence of 1.3 mM Ca2+. A, confocal images showing the eGFP fluorescence associated with the basal, peak, and plateau responses induced by 1 µM quisqualate for experiments performed in 1.3 mM or nominal [Ca2+]o. B, representative traces showing the effect of 1 µM quisqualate (horizontal line) on IP3 production in a single BHK-mGlu1alpha cell in the presence of 1.3 mM (solid line) or nominal (dashed line) Ca2+o. The combined data (mean ± S.E.M.) for 10 cells from four separate experiments in the presence of 1.3 mM (open bars) or nominal (solid bars) [Ca2+]o. *, p < 0.01 when compared with response in 1.3 mM [Ca2+]o using Student's paired t test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present work, the establishment of a real-time assay for the measurement of [IP3]i in single cells has revealed that the mGlu1alpha receptor does not show Ca2+o sensing properties. Although previous data from InsP accumulation experiments (Fig. 1; Ref. 4) supported the view that Ca2+o modulates mGlu1alpha receptor activity, no differences in the early phases of IP3 production or Ca2+ mobilization in the presence or absence of Ca2+o were observed. Each of the following parameters appeared unchanged by removal of Ca2+o: the maximal response to quisqualate, the agonist potency, and the dynamics of the peak response for both IP3 production and Ca2+ mobilization. For the purposes of this study nominal [Ca2+]o was used because this maintains the integrity of the intracellular Ca2+ pool for longer compared with chelation of Ca2+ with EGTA, which can rapidly cause store depletion (25). The failure of Ca2+o to affect the initial events in mGlu1alpha receptor signaling suggests that the early processes involved in receptor activation, i.e. agonist binding, coupling to the G proteins, PLCbeta activation and IP3 synthesis and release of Ca2+ from intracellular stores, occur independently of Ca2+o. Clearly this differs from the activation of the calcium-sensing receptor where Ca2+ binding stimulates IP3 production, rapid Ca2+i transients, and coupling to Galpha q/11 (26-28).

In contrast, sustained increases in CHO-lac-mGlu1alpha for both [Ca2+]i and [IP3]i were highly dependent upon Ca2+o. This we believe offers the most probable explanation for the discrepancy between the InsP accumulation assays and the real-time analysis of IP3. Obviously, the contribution of the secondary phase of IP3 production to accumulative measurements made over 15 min is very much greater than the initial transient peak IP3 synthesis, which is lost within 2-3 min. Sustained elevations in Ca2+i during prolonged agonist challenge commonly arise from opening of plasma membrane capacitative Ca2+ channels, possibly synonymous with Trp channels (29), as a result of store depletion. In contrast, activation of mGlu1alpha receptors has been reported to induce a rapid and complete uncoupling from PLC followed by opening of a receptor-operated Ca2+ channel (14). The low level of sustained IP3 synthesis in the absence of Ca2+o observed using the confocal microscope is in disagreement with this suggestion. However, the predominant role of Ca2+-dependent PLCs (e.g. PLCdelta ; 30, 31) during this secondary phase is indicated and is similar to M3 muscarinic receptors where IP3 production was also found to continue in the absence of Ca2+o at a decreased level (32, 33). The data also indicate that the initial peak in [Ca2+]i predominantly represents Ca2+ release from intracellular stores with no involvement of Ca2+o entry. Two distinct phases are evidently involved in the response to activation of mGlu1alpha receptors.

We have previously reported a sensitivity of M3 muscarinic and bradykinin receptor-induced IP3 responses in SH-SY5Y cells to store depletion using thapsigargin (32-34) and an important observation described here is the almost complete dependence of IP3 production by the mGlu1alpha receptor on Ca2+ released from the intracellular stores. This sensitivity of mGlu1alpha receptors to intracellular store Ca2+ may reflect the selective recruitment of PLC isoenzymes responsive to variations in [Ca2+]i. Furthermore, the association of the mGlu1alpha receptor with Homer/Vesl proteins (35, 36) allows for potential cross-linking with IP3 and/or ryanodine receptors (37). Theoretically this could hold mGlu1alpha receptors in close proximity to the site of Ca2+ release from the endoplasmic reticulum such that high local concentrations of Ca2+ may dramatically potentiate the activity of Ca2+-sensitive PLCs. Irrespective of the origins of this sensitivity it is important to note that any inadvertent depletion of Ca2+i stores prior to assaying mGlu1alpha receptor activity will give results that could be erroneously interpreted as demonstrating Ca2+o sensitivity. Numerous authors have reported store depletion during prolonged incubation in Ca2+-free buffers (e.g. Ref. 25).

Kubo et al. (5) identified Ser166 as controlling the Ca2+o sensing properties of rat mGlu1alpha receptors. This apparent inconsistency in Ca2+o sensing property of mGlu1alpha receptors does not reflect species differences, since the rat mGlu1alpha receptor expressed in BHK cells responded in a similar manner to the human receptor. The x-ray crystallography structure of the mGlu1alpha receptor revealed the presence of a high affinity cation-binding site that is likely to be important to the structural integrity of the extracellular domain (16). The high affinity of this site argues against a role in detecting physiological fluctuations in synaptic Ca2+o because the site would always be occupied. A similar argument has been used to propose that GABAB receptors are not physiological Ca2+o sensors (7). However, mutations that interfere with Ca2+ binding might be anticipated to affect signaling through structural changes. This may explain the importance of Ser166, although this residue appears distant to the cation-binding site (16). An alternative explanation for the discrepancy could relate to the expression system used (Xenopus oocytes versus CHO cells) since activation of the Ca2+-activated Cl- current occurs downstream of the events determined in this study. It is likely that these measurements will be more sensitive to Ca2+o influx than the initial IP3 production. In fact we show here that raising Ca2+o increases [Ca2+]i without affecting [IP3] presumably as a consequence of enhanced store-operated Ca2+ entry. Since mGlu1alpha receptors have also been argued to couple to a receptor-operated Ca2+ channel during prolonged agonist exposure (14), it is also conceivable that mutation of Ser166 could influence this process without affecting coupling to PLC.

In conclusion, the presented data are entirely consistent with the failure of Ca2+o to influence the initial activation of PLC by mGlu1alpha receptors. This study also demonstrates the usefulness of imaging of IP3 and [Ca2+]i in single cells by transfection with the eGFP-PHPLCdelta fusion protein (17) and loading with Fura-2. The close similarity of the EC50 value for quisqualate-induced effects on IP3 measured by eGFP-PHPLCdelta translocation with that of Hermans et al. (22) using a radioreceptor assay (22) argues that this is an accurate measure of IP3 production. Moreover, a peak and plateau in IP3 levels is commonly reported (22, 38), while PIP2 levels tend to decrease monophasically to a new steady-state (38) suggesting that PIP2 depletion is not the major signal for translocation as was originally suggested (18, 19). Importantly, our data also confirm for the first time that IP3 follows such a pattern in a single cell since a peak and plateau in IP3 levels obtained for cell populations using radioreceptor assays (22, 38) could equally have arisen through differences in the timing of PLC activation within individual cells. Dual imaging of IP3 and Ca2+i in the same cell has thus allowed investigation of the Ca2+o sensing property of mGlu1alpha receptors without the problems of ambiguity present using more established experimental techniques.

    ACKNOWLEDGEMENTS

We thank Professor T. Meyer for the kind donation of GFP-tagged pleckstrin homology domain and Dr. E. Hermans for preparing the CHO-lac-mGlu1alpha cells.

    FOOTNOTES

* This work was supported by Programme Grants 16895 and 062495/z/00, and an Equipment Grant (061050/z/00) from the Wellcome Trust.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.

Dagger To whom correspondence should be addressed. Tel.: 0116-252-3075; Fax: 0116-252-5045; E-mail: msn2@le.ac.uk.

§ Supported by a Medical Research Council of Great Britain Studentship.

Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M007600200

2 J. V. Selkirk, G. W. Price, S. R. Nahorski, and J. Challiss, unpublished data.

3 R. Saunders, S. R. Nahorski, and J. Challiss, unpublished data.

    ABBREVIATIONS

The abbreviations used are: mGlu1, metabotropic glutamate receptor type 1; mGlu5, metabotropic glutamate receptor type 5; PLC, phospholipase C; G proteins, heterotrimeric GTP-binding regulatory proteins; IP3, inositol 1,4,5-trisphosphate; Ca2+i, intracellular calcium; [Ca2+]o, extracellular calcium concentration; eGFP-PHPLCdelta , enhanced green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta 1; PIP2, phosphatidylinositol 4,5-bisphosphate; CHO-lac-mGlu1alpha , Chinese hamster ovary cells expressing mGlu1alpha receptors; IPTG, isopropyl-beta -D-thiogalactoside; BHK-mGlu1alpha , baby hamster kidney cells expressing mGlu1alpha receptors; [3H]InsP, [3H]inositol phosphates; KHB, Krebs-Henseleit buffer; PH, pleckstrin homology.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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