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
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ABSTRACT |
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Transient transfection of Chinese hamster ovary
or baby hamster kidney cells expressing the Group I metabotropic
glutamate receptor mGlu1 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 mGlu1 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 mGlu1 To address this issue we have made real-time concurrent measurements of
IP3 and Ca2+i in single cells
expressing the mGlu1 Materials--
Vector containing the fusion construct between
eGFP and the PH domain of PLC Cell Culture--
A description of the LacSwitch inducible
expression system (Stratagene) used to express human mGlu1 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-PHPLC 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.
InsP Accumulation Is Attenuated by Decreasing
[Ca2+]o in Cells Expressing mGlu1 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 mGlu1 Single Cell Measurement of IP3--
A confocal image
through the mid-section of CHO-lac-mGlu1
Using an epifluorescence microscope the halo of eGFP fluorescence
around the transfected CHO-lac-mGlu1 Initial Response to Agonist Is Not Dependent on Extracellular
Calcium--
When the translocation of eGFP-PHPLC
For the dual imaging experiments plotting the changes in cytosolic
eGFP-PHPLC
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
mGlu1 Raising [Ca2+]o Differentially Affects
IP3 Production and [Ca2+]i
Mobilization--
Perfusion of CHO-lac-mGlu1 Rat mGlu1 In the present work, the establishment of a real-time assay for
the measurement of [IP3]i in single cells has
revealed that the mGlu1 In contrast, sustained increases in CHO-lac-mGlu1 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 mGlu1 Kubo et al. (5) identified Ser166 as controlling
the Ca2+o sensing properties of rat mGlu1 In conclusion, the presented data are entirely consistent with the
failure of Ca2+o to influence the initial
activation of PLC by mGlu1 with green fluorescent protein-tagged
pleckstrin homology domain of phospholipase C
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 mGlu1
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 mGlu1
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
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.
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
mGlu1
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.
receptor using a recently developed technique
that utilizes an enhanced green fluorescent protein-tagged pleckstrin
homology domain of phospholipase C
1 (eGFP-PHPLC
) to
detect IP3 in Fura-2 loaded cells (17-19). PHPLC
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 PHPLC
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-PHPLC
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
mGlu1
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
in
Chinese hamster ovary cells (CHO-lac-mGlu1
) is provided elsewhere
(21, 22). For these studies on the CHO-lac-mGlu1
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-mGlu1
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-mGlu1
cells can be found elsewhere (4).
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-mGlu1
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-PHPLC
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-PHPLC
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Receptors--
Previous studies using BHK cells expressing mGlu1
receptors demonstrated sensitivity of these receptors to
[Ca2+]o (4). Similar findings were also obtained
for CHO cells expressing mGlu1
(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 mGlu1
receptors to agonist. In nominally
[Ca2+]o-free medium minimal stimulation of
[3H]InsP accumulation occurred in CHO-lac-mGlu1
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 mGlu1
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 mGlu1
receptors. A, CHO-lac-mGlu1
cells were
preincubated in KHB containing the indicated
[Ca2+]o for 30 min before challenging with 30 µM quisqualate (
), 1 mM ACPD (
), 1 µM quisqualate (
), or 30 µM ACPD (
)
for an additional 15 min. LiCl (10 mM) was added 10 min
before addition of the agonists. Basal stimulation was addition of KHB
alone (
). Data are expressed as mean ± S.E.M. from four
separate experiments. B, representative trace for a single
CHO-lac-mGlu1
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.
receptor were made (Fig.
1). A representative trace from a single CHO-lac-mGlu1
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 mGlu1
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 mGlu1
receptors are calcium-sensing receptors.
cells 48 h after
transfection confirmed the localization of eGFP-PHPLC
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 PHPLC
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-PHPLC
localization in individual CHO-lac-mGlu1
cells. Confocal images (A-F) of
CHO-lac-mGlu1
cells transiently transfected with
eGFP-PHPLC
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-mGlu1
cells
transfected with eGFP-PHPLC
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-mGlu1
cells at the same times is shown in
J-L.
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).
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-mGlu1
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
mGlu1 receptor-mediated IP3
production in a single CHO-lac-mGlu1
cell. Representative traces showing quisqualate (10 µM) (indicated by the dashed line) mediated
IP3 production in a single CHO-lac-mGlu1
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.
Effect of Ca2+o on agonist-induced
eGFP-PHPLC translocation in CHO-lac-mGlu1
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.
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-PHPLC
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 mGlu1
receptor activation. However,
removing Ca2+o reduces the sustained component of
IP3 production.
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Fig. 4.
Comparison of mGlu1
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-PHPLC
fluorescence (solid lines) in single CHO-lac-mGlu1
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-PHPLC
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-PHPLC
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-PHPLC
translocation
are significantly smaller than those obtained confocally because of
interference from different focal planes increases the background
fluorescence in these non-confocal experiments.
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-mGlu1 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-PHPLC
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).
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-PHPLC
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).
View larger version (14K):
[in a new window]
Fig. 6.
Effect of increasing the extracellular
Ca2+ concentration on IP3 production and
Ca2+i mobilization. CHO-lac-mGlu1 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-PHPLC
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.
Receptor Responds to Ca2+o in a
Similar Manner to Human mGlu1
Receptor--
To eliminate the
possibility that the Ca2+o sensing property of
mGlu1
receptors reflects species differences since CHO-lac-mGlu1
cells express human receptors rather than the rat receptor used
previously (4, 5) similar experiments were performed on BHK-mGlu1
cells. Transient transfection with eGFP-PHPLC
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).
View larger version (28K):
[in a new window]
Fig. 7.
Effect of Ca2+o on
IP3 production in BHK cells expressing the rat
mGlu1 receptor. BHK-mGlu1
cells were
transiently transfected with eGFP-PHPLC
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-mGlu1
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
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 mGlu1
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 mGlu1
receptor signaling suggests that the early processes involved in
receptor activation, i.e. agonist binding, coupling to the G
proteins, PLC
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 G
q/11 (26-28).
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 mGlu1
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. PLC
;
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 mGlu1
receptors.
receptor on Ca2+
released from the intracellular stores. This sensitivity of mGlu1
receptors to intracellular store Ca2+ may reflect the
selective recruitment of PLC isoenzymes responsive to variations in
[Ca2+]i. Furthermore, the association of the
mGlu1
receptor with Homer/Vesl proteins (35, 36) allows for
potential cross-linking with IP3 and/or ryanodine receptors
(37). Theoretically this could hold mGlu1
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 mGlu1
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).
receptors. This apparent inconsistency in Ca2+o
sensing property of mGlu1
receptors does not reflect species
differences, since the rat mGlu1
receptor expressed in BHK cells
responded in a similar manner to the human receptor. The x-ray
crystallography structure of the mGlu1
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 mGlu1
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.
receptors. This study also demonstrates
the usefulness of imaging of IP3 and
[Ca2+]i in single cells by transfection with the
eGFP-PHPLC
fusion protein (17) and loading with Fura-2.
The close similarity of the EC50 value for
quisqualate-induced effects on IP3 measured by
eGFP-PHPLC
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 mGlu1
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-mGlu1 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.
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-PHPLC, enhanced green fluorescent protein-tagged
pleckstrin homology domain of phospholipase C
1;
PIP2, phosphatidylinositol 4,5-bisphosphate;
CHO-lac-mGlu1
, Chinese
hamster ovary cells expressing mGlu1
receptors;
IPTG, isopropyl-
-D-thiogalactoside;
BHK-mGlu1
, baby hamster
kidney cells expressing mGlu1
receptors;
[3H]InsP, [3H]inositol phosphates;
KHB, Krebs-Henseleit buffer;
PH, pleckstrin homology.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Pin, J. P., and Duvoisin, R. (1995) Neuropharmacology 34, 1-26[CrossRef][Medline] [Order article via Infotrieve] |
2. | Conn, P. J., and Pin, J. P. (1997) Annu. Rev. Pharmacol. Toxicol. 37, 205-237[CrossRef][Medline] [Order article via Infotrieve] |
3. | Kubokawa, K., Miyashita, T., Nagasawa, H., and Kubo, Y. (1996) FEBS Lett. 392, 71-76[CrossRef][Medline] [Order article via Infotrieve] |
4. | Saunders, R., Nahorski, S. R., and Challiss, R. A. (1998) Neuropharmacology 37, 273-276[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Kubo, Y.,
Miyashita, T.,
and Murata, Y.
(1998)
Science
279,
1722-1725 |
6. | Wise, A., Green, A., Main, M. J., Wilson, R., Fraser, N., and Marshall, F. H. (1999) Neuropharmacology 38, 1647-1656[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Galvez, T.,
Urwyler, S.,
Prezeau, L.,
Mosbacher, J.,
Joly, C.,
Malitschek, B.,
Heid, J.,
Brabet, I.,
Froestl, W.,
Bettler, B.,
Kaupmann, K.,
and Pin, J. P.
(2000)
Mol. Pharmacol.
57,
419-426 |
8. | Vassilev, P. M., Mitchel, J., Vassilev, M., Kanazirska, M., and Brown, E. M. (1997) Biophys. J. 72, 2103-2116[Abstract] |
9. | Hofer, A. M., Curci, S., Doble, M. A., Brown, E. M., and Soybel, D. I. (2000) Nat. Cell Biol. 2, 392-398[CrossRef][Medline] [Order article via Infotrieve] |
10. | Nakahara, K., Okada, M., and Nakanishi, S. (1997) J. Neurochem. 69, 1467-1475[Medline] [Order article via Infotrieve] |
11. | Biber, K., Laurie, D. J., Berthele, A., Sommer, B., Tolle, T. R., Gebicke-Harter, P. J., van Calker, D., and Boddeke, H. W. (1999) J. Neurochem. 72, 1671-1680[CrossRef][Medline] [Order article via Infotrieve] |
12. | Prothero, L. S., Richards, C. D., and Mathie, A. (1998) Br. J. Pharmacol. 125, 1551-1561[Abstract] |
13. | Kawabata, S., Tsutsumi, R., Kohara, A., Yamaguchi, T., Nakanishi, S., and Okada, M. (1996) Nature 383, 89-92[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Kawabata, S.,
Kohara, A.,
Tsutsumi, R.,
Itahana, H.,
Hayashibe, S.,
Yamaguchi, T.,
and Okada, M.
(1998)
J. Biol. Chem.
273,
17381-17385 |
15. | Hiltscher, R., Seuwen, K., Boddeke, H. W., Sommer, B., and Laurie, D. J. (1998) Neuropharmacology 37, 827-837[CrossRef][Medline] [Order article via Infotrieve] |
16. | Kunishima, N., Shimada, Y., Tsuji, Y., Sato, T., Yamamoto, M., Kumasaka, T., Nakanishi, S., Jingami, H., and Morikawa, K. (2000) Nature 407, 971-977[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Hirose, K.,
Kadowaki, S.,
Tanabe, M.,
Takeshima, H.,
and Iino, M.
(1999)
Science
284,
1527-1530 |
18. | Stauffer, T. P., Ahn, S., and Meyer, T. (1998) Curr. Biol. 8, 343-346[Medline] [Order article via Infotrieve] |
19. |
Varnai, P.,
and Balla, T.
(1998)
J. Cell Biol.
143,
501-510 |
20. |
Kavran, J. M.,
Klein, D. E.,
Lee, A.,
Falasca, M.,
Isakoff, S. J.,
Skolnik, E. Y.,
and Lemmon, M. A.
(1998)
J. Biol. Chem.
273,
30497-30508 |
21. | Hermans, E., Young, K. W., Challiss, R. A., and Nahorski, S. R. (1998) J. Neurochem. 70, 1772-1775[Medline] [Order article via Infotrieve] |
22. |
Hermans, E.,
Challiss, R. A.,
and Nahorski, S. R.
(1999)
Br. J. Pharmacol.
126,
873-882 |
23. | Batty, I. H., Carter, A. N., Challiss, R. A. J., and Hawthorne, J. N. (1997) Neurochemistry: A Practical Approach , 2nd Ed. , Oxford University Press, Oxford, United Kingdom |
24. | Young, K. W., Pinnock, R. D., and Nahorski, S. R. (1998) Cell Calcium 24, 59-70[Medline] [Order article via Infotrieve] |
25. |
Maloney, J. A.,
Tsygankova, O. M.,
Yang, L.,
Li, Q.,
Szot, A.,
Baysal, K.,
and Williamson, J. R.
(1999)
Am. J. Physiol.
276,
C221-230 |
26. | Brown, E. M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., Hediger, M. A., Lytton, J., and Hebert, S. C. (1993) Nature 366, 575-580[CrossRef][Medline] [Order article via Infotrieve] |
27. | Yamaguchi, T., Chattopadhyay, N., and Brown, E. M. (2000) Adv. Pharmacol. 47, 209-253[Medline] [Order article via Infotrieve] |
28. |
Arthur, J. M.,
Collinsworth, G. P.,
Gettys, T. W.,
Quarles, L. D.,
and Raymond, J. R.
(1997)
Am. J. Physiol.
273,
F129-135 |
29. | Putney, J. W., and McKay, R. R. (1999) Bioessays 21, 38-46[CrossRef][Medline] [Order article via Infotrieve] |
30. | Allen, V., Swigart, P., Cheung, R., Cockcroft, S., and Katan, M. (1997) Biochem. J. 327, 545-552[Medline] [Order article via Infotrieve] |
31. |
Kim, Y. H.,
Park, T. J.,
Lee, Y. H.,
Baek, K. J.,
Suh, P. G.,
Ryu, S. H.,
and Kim, K. T.
(1999)
J. Biol. Chem.
274,
26127-26134 |
32. | Wojcikiewicz, R. J., Tobin, A. B., and Nahorski, S. R. (1994) J. Neurochem. 63, 177-185[Medline] [Order article via Infotrieve] |
33. | Willars, G. B., and Nahorski, S. R. (1995) Mol. Pharmacol. 47, 509-516[Abstract] |
34. | Willars, G. B., Challiss, R. A., Stuart, J. A., and Nahorski, S. R. (1996) Biochem. J. 316, 905-913[Medline] [Order article via Infotrieve] |
35. | Brakeman, P. R., Lanahan, A. A., O'Brien, R., Roche, K., Barnes, C. A., Huganir, R. L., and Worley, P. F. (1997) Nature 386, 284-288[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Kato, A.,
Ozawa, F.,
Saitoh, Y.,
Fukazawa, Y.,
Sugiyama, H.,
and Inokuchi, K.
(1998)
J. Biol. Chem.
273,
23969-23975 |
37. | Tu, J. C., Xiao, B., Yuan, J. P., Lanahan, A. A., Leoffert, K., Li, M., Linden, D. J., and Worley, P. F. Neuron 21, 717-726 |
38. |
Willars, G. B.,
Nahorski, S. R.,
and Challiss, R. A.
(1998)
J. Biol. Chem.
273,
5037-5046 |