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Originally published In Press as doi:10.1074/jbc.M004520200 on August 18, 2000

J. Biol. Chem., Vol. 275, Issue 44, 34252-34259, November 3, 2000
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Expression of Functional Metabotropic Glutamate Receptors in Primary Cultured Rat Osteoblasts

CROSS-TALK WITH N-METHYL-D-ASPARTATE RECEPTORS*

Yuchun GuDagger and Stephen J. Publicover§

From the School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

Received for publication, May 25, 2000, and in revised form, August 14, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Osteoblasts and osteoclasts express functional N-methyl-D-aspartate (NMDA) receptors, which participate in regulation of bone matrix. In rat femoral osteoblasts held in whole cell clamp there is a robust NMDA current but little if any response to L-glutamate. We have investigated expression of metabotropic glutamate receptors (mGluRs) in these cells. By reverse transcription polymerase chain reaction, we have detected expression of mGluR1b (but not mGluR1a, 2, 3, 4, 5, or 6). Blockade of mGluRs with (±)-alpha -methyl-carboxyphenyl-glycine resulted in an enlarged L-glutamate-induced current that resembled the response to NMDA. Conversely, prior stimulation of mGluRs with trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD; mGluR agonist) reduced the NMDA-induced current by 77%. Monitoring of [Ca2+]i showed that NMDA induced a sustained elevation of [Ca2+]i, which was dependent upon [Ca2+]o. Treatment with 1S,3R-ACPD generated an initial transient that was independent of [Ca2+]o, followed by a sustained, [Ca2+]o-dependent phase, a response consistent with phospholipase C-mediated mobilization of stored Ca2+. Investigations of the interaction between the two receptors confirmed inhibitory modulation of the NMDA receptor-induced rise in [Ca2+]i by mGluRs. Parathyroid hormone, which also activates phospholipase C in osteoblasts, had a similar inhibitory effect on the NMDA receptor-induced [Ca2+]i response. Elevation of [Ca2+]i mediated by mGluR activation was reduced by subsequent stimulation of NMDA receptors. This is the first description of mGluRs in bone and shows that complex glutamatergic signaling can occur in this tissue.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is well established that bone cells are regulated in their activity both by circulating hormones (1) and by the interacting effects of a number of locally acting intercellular signals, including prostaglandins, growth factors, cytokines, and nitric oxide (2-9). Recently, it has been reported that functional NMDA1-type glutamate receptors are expressed in a number of bone cell types, including rat and human osteoblasts and osteoclasts, MG-63 osteosarcoma cells, and in bone marrow megakaryocytes (10-15). Osteoblasts, which contain high levels of glutamate (16), express regulatory proteins required for vesicular exocytosis that co-localize with glutamate (17). In vitro, osteoblasts secrete glutamate in a regulated manner.2 Initial histochemical data show that nerve endings able to secrete L-glutamate may also occur within bone (16). Antagonists of NMDA receptors modulate the activities of both osteoblasts and osteoclasts, the bone cells responsible for deposition and resorbtion of bone matrix (10, 14, 18). These findings suggest that glutamate-mediated signaling occurs in bone, in a manner analogous to glutamatergic transmission between neurons, and that it contributes to regulation of bone matrix (19).

The NMDA receptor is part of a complex glutamatergic system in the central nervous system, comprising several receptor types. Glutamate receptors can be divided into iGluRs (including the NMDA type found in bone) and mGluRs. mGluRs are G-protein-linked receptors that stimulate PLC (group 1 mGluRs) or inhibit adenyl cyclase (group 2 and group 3 mGluRs) (20). There is believed to be "cross-talk" between the different glutamate receptor subtypes, primarily by mGluRs acting to regulate activity of iGluRs (20). To date, only iGluRs, primarily the NMDA-type, have been detected in bone cells (10, 11).

The first electrophysiological study of the action of glutamate on bone-derived cells used the human osteoblast-like MG-63 cell line. Bath-applied L-glutamate and NMDA had very similar effects on these cells, with both agonists markedly increasing membrane conductance (21). However, during our studies on NMDA-induced currents in femoral explant-derived osteoblasts of rat (15), we made the surprising observation that the responses to NMDA and L-glutamate differed markedly. Cells that showed a well developed response to bath-applied NMDA gave only a very small current upon application of L-glutamate. This finding suggests the expression in femoral osteoblasts of a second type of glutamate receptor, which negatively modulates the NMDA receptor/channel. We have therefore looked for mGluRs in rat femoral osteoblasts and examined the effects of their activation on osteoblastic NMDA receptor/channels. We report that mGluR-1b receptors are expressed in these cells, that their activation mobilizes stored Ca2+ within the cell, and that they negatively regulate the activity of the osteoblastic NMDA receptor/channel.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Cultures of rat femoral osteoblasts were prepared similarly to the method of Pitsillides et al. (22). Rats (120-130 g) were killed by cervical dislocation. Femora were aseptically removed, stripped of all adhering soft tissues, and passed through four washes of culture medium with 10 times the usual concentration of antibiotics (see below). The epiphyses were removed and the marrow flushed from the cavity with alpha -minimal essential medium. The bone was then sectioned into small fragments that were washed twice with phosphate-buffered saline and once in culture medium (see below) before placing them into 80-mm plastic culture dishes or 20-ml plastic culture flasks (Falcon). Culture medium contained alpha -minimal essential medium supplemented with 10% (v/v) fetal calf serum, pyenicillin (100 µg/ml), streptomycin (50 µg/ml), ascorbate (50 µg/ml), and beta -glycerophosphate (10 mM). Cultures were placed in a humidified atmosphere of 5% CO2 at 37 °C. Medium was changed 24 h later to remove any debris and twice weekly thereafter. The cultures were maintained for 3 weeks, at which point the bone chips were removed, and adherent cells were washed twice with phosphate-buffered saline and detached by treatment with 0·05% (w/v) trypsin and 0.02% (w/v) EDTA. The cells were then seeded, either direct from culture flasks or, after a single passage, onto sterile glass coverslips in 35-mm plastic culture dishes. Seeding density was approximately 3 × 104 cells/ml. After settling, cells flattened and adopted an elongated polygonal outline, usually tapered toward one end.

Cells cultured as described above have been shown to stain positive for alkaline phosphatase and osteocalcin and to produce mineralized matrix (22). We have confirmed osteoblastic phenotype by staining for alkaline phosphatase and using Western blots and radioimmunoassay to demonstrate expression of osteopontin and osteocalcin (23, 24). The cells generated a transient [Ca2+]i elevation in response to treatment with PTH (not shown).

Electrophysiological Recording-- Extracellular saline contained 140 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 10 mM HEPES. pH was adjusted to 7.6 with NaOH. No Mg2+ was included because resting potential of femoral osteoblasts (-60 to -65 mV) is sufficient to cause significant Mg2+ block of NMDA channels (15). Electrodes for voltage clamp measurements were backfilled with saline containing 150 mM CsCl, 5 mM EGTA, 2 mM ATP, 0.5 mM GTP, 10 mM HEPES. pH was corrected to 7.3 with CsOH. Patch electrodes were pulled from filamented 1.5-mm glass capillaries (Clark Electromedical GC150TF) and fire polished. Electrode resistance was 3-7 MOmega .

Cells were used for recordings within 3-24 h after seeding, while cultures were subconfluent. Prior to recording, coverslips were washed thoroughly to remove any traces of culture medium and were transferred to a purpose-built, recording chamber (volume, 300 µl). The chamber was continuously perfused with recording saline at 1.5 ml·min-1. All recordings were carried out at room temperature (20-21 °C) and were made using the whole cell variant of the patch clamp technique. Clumped cells were not used to avoid any effects of gap-junctional contacts between cells. Seals of up to 10 GOmega were achieved prior to breakthrough. After breakthrough, whole cell input resistances were in the order of 1-5 GOmega . Recordings commenced within 1-2 min of breakthrough. Holding potential was -50 mV. Currents were recorded using a Warner PC501A amplifier, with filter cut-off at 2 KHz. The current output (Im) from the amplifier was continuously plotted on a chart recorder for subsequent offline analysis.

Application of drugs was by bath perfusion. Drugs were prepared as concentrated stock solutions in saline immediately prior to use. NMDA was from Sigma, glycine was from Fisons, and (+)-MK-801, 1S,3R-ACPD, and (±)MCPG were from RBI. NMDA and L-glutamate were always co-applied with 10 µM glycine.

The values given in text show the means ± S.E. P values show results of t test (paired or nonpaired, according to the nature of the comparison).

Fluorimetric Monitoring of [Ca2+]i-- Cells were prepared as described above except that, after reseeding, coverslips were placed in 24-well culture plates. The cells were grown with 10% fetal calf serum, 90% alpha -minimal essential medium without phenol red in a humidified atmosphere of 5% CO2 at 37 °C. To maximize the fluorescent signal, cells were grown until nearly confluent before use. 24 h before recording, the medium was changed to 1% bovine serum albumin, 90% Earle's balanced salt solution (EBSS-HEPES; 116.4 mM NaCl, 5.4 mM KCl, 26.2 mM NaHCO3, 1.3 mM NaH2PO4, 2 mM CaCl2, 0.81 mM MgSO4, 4.2 mM HEPES, and 5.5 mM D-glucose, pH 7.4) without phenol red. Immediately before use, the cells were washed three times with EBSS-HEPES and loaded with 1 µM Fura-2-acetoxymethyl-ester (Molecular Probes) in the same buffer at 37 °C for 45 min. The cells were then washed once with EBSS-HEPES without Mg2+. The glass coverslip carrying the cells was inserted into a cuvette containing 2 ml of electrophysiological recording saline (see above) with 10 mM D-glucose. For Ca2+-free experiments, CaCl2 was omitted from the saline, and 1 mM EGTA was included. The cuvette was placed in a thermostatically controlled (37 °C) Perkin-Elmer LS-580 Luminescence Spectrometer. After a period of 5 min for temperature equilibration, the cuvette was alternately excited at 340 and 380 nm, and emission at 505 nm was monitored. Acquisition rate of ratio pairs was 12.5 Hz. Drugs and reagents were added directly to the cuvette, which was stirred continuously. All drugs were made up as concentrated stock solutions in saline immediately prior to use. When drugs were added sequentially there were minor dilution effects. The dilution factors for the various stock solutions used ranged from 1:100 (for NMDA) to 1:1000 (for glutamate and glycine). The greatest dilution occurred in the experiments shown in Fig. 4a, when a total of four additions were made to the cuvette. In these experiments the total increase in volume was 0.85% (such that the final NMDA concentration was 99.2 µM rather than the initial 100 µM). No correction was made for this small effect. At the completion of each experiment, sequential additions of ionomycin (2 µM) and EGTA (10 mM) were made to confirm the validity of the data. Traces were smoothed by using a 50-point moving average. Because calibration of Fura-2 data has inherent problems and is thus prone to inaccuracies (25), data are expressed as the ratio of fluorescence elicited by excitation at 340 and 380 nm. NMDA and L-glutamic acid were from Sigma, glycine was from Fisons, and (+)-MK-801, 1S,3R-ACPD, and (±)MCPG were from RBI. PTH was from Sigma.

Reverse Transcription Polymerase Chain Reaction-- For RNA extraction, confluent osteoblasts were harvested after 3 weeks, using a total RNA isolation kit (Promega) as described by the manufacturer. Whole brain from 120-g Wistar male rats was removed, dropped into liquid nitrogen, and homogenized at 4 °C according to the protocol provided with the RNA isolation kit.

Synthesis of the First Strand cDNA from Total RNA-- The first strand cDNA was synthesized from total RNA (2 µg) in 20 µl of reaction mixture (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 40 units of RNasin (RNase inhibitor, Promega), 500 µM each dATP, dCTP, dGTP, and dTTP, 10,000 units/ml super II reverse transcriptase (Life Technologies, Inc.)) on a 2.5 µM random primer. The reverse transcriptase reaction was carried out at 42 °C for 55 min, and the reaction was stopped by raising the temperature to 70 °C for 15 min. Preparation of cDNA from femoral osteoblasts was carried out on four separate occasions, each from a separate culture. One to three PCR reactions, for each set of primers, were carried out on each of these samples to confirm validity of the findings.

PCR Primers-- Specific primers against mGluR1, mGluR1 mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, and NMDAR-2C were synthesized by MWG (Ebersberg, Germany; Table I). Primers for mGluR2 and NMDAR-2C were designed on the basis of published sequences. All other primers for glutamate receptors were previously published pairs (Table I). Primers for rat hypoxanthine phosphoribosyltransferase were a gift from Prof. Alicia El Haj (University of Keele, Keele, UK).

                              
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Table I
PCR primers and protocols
Column 3 (Size) shows predicted size of product in bp; column 4 (Source) shows source of primer sequence. Where cell is blank, primers were designed. Last five columns show temperatures, durations, and cycle numbers for PCR reactions.

PCR-- PCR was performed with Taq DNA polymerase (Promega, Madison) in 25 µl of reaction mixture containing each primer at a final concentration of 0.6 µM.

PCR protocols were as given in Table I. For mGluR2, mGluR3, and mGluR5, the reaction was carried out using a touchdown program. The PCR amplification products were electrophoresed on 1.5% agarose gels and visualized using ethidium bromide under ultraviolet light. The bands of interest were excised, and DNA was extracted from the Gel using an extraction kit (Promega). Sequencing was carried out by Alta Biocience (Birmingham, UK).

To assess the relative expression of mRNA for mGluR1b in rat osteoblasts and rat brain, we used serial dilution (10-fold steps) of the cDNA templates. The reaction was then carried out as described above, but products were assessed after 26 cycles of reaction. The minimum dilution necessary to prevent formation of a detectable product was used to estimate the relative levels of mGluR1b cDNA in the rat brain and rat femoral osteoblast templates. Primers for a 300-bp region of rat hypoxanthine phosphoribosyltransferase were used to check that there was similar representation of this housekeeping gene in the brain and osteoblast cDNA samples.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reverse Transcription PCR

To investigate the possible expression of mGluRs in rat femoral explant-derived osteoblasts, reverse transcription PCR was carried out using primers directed against rat mGluR1a, mGluR1b, mGluR2, mGluR3, mGluR4, mGluR5, and mGluR6. Products of the appropriate size were detected with all six primer pairs in rat brain (Fig. 1, a-d). mGluR1 and mGluR5 occurred as both a and b subtypes. Using rat femoral osteoblast cDNA, we detected the presence of message for mGluR1b of appropriate size (Fig. 1a; 378 bp) but no other mGluR types. Control reactions carried out with total osteoblast RNA gave no product. Sequencing showed that osteoblast mGluR1b product was identical to the rat brain mGluR1b sequence.


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  		Fig. 1.   Detection of mGluR and NMDAR-2C receptor subunits by PCR. a, expression of mGluR1. Lane 1 shows products from rat whole brain cDNA; lane 2 is rat liver cDNA; and lane 3 is rat femoral osteoblast cDNA. Predicted products are 294 bp (mGluR1a) and 378 bp (mGluR1b). Both receptors are expressed in brain, but only the mGluR1b product is detected in cDNA from osteoblasts. b, expression of mGluR2. Lane 1 shows product from rat whole brain cDNA; lane 2 shows femoral osteoblast cDNA. Predicted product size is 456 bp. mGluR2 is expressed in brain, but no product is detected with osteoblast cDNA. c, expression of mGluR3 (lanes 1-4), mGluR4 (lanes 9-12), and mGluR5 (lanes 5-8). Lanes 1, 5, and 9 show products from rat whole brain cDNA; lanes 2, 6, and 10 two are rat femoral osteoblast cDNA; lanes 3, 7, and 11 are rat liver cDNA; and lanes 4, 8, and 12 are rat osteoblast total RNA. Predicted product sizes are 261 bp for mGluR3, 301 bp for mGluR4, 336 bp for mGluR5a, and 431 bp for mGluR5b. All four subunits were detected in rat whole brain cDNA, but there were no products from liver cDNA, osteoblast cDNA, or osteoblast total RNA. d, expression of mGluR6. Lane 1 shows products from rat whole brain cDNA; lane 2 is rat femoral osteoblast cDNA. Predicted product size is 460 bp. mGluR6 is expressed in brain, but no product is detected with osteoblasts. e, estimation of relative expression of mGluR1b in rat brain cDNA and osteoblast cDNA by serial dilution of the cDNA template. Upper panel shows rat brain cDNA (in which both mGluR1a and mGluR1b products are detected). mGluR1b can be detected using undiluted template and at 10-1, 10-2, and 10-3 dilutions but not at 10-4. Lower panel shows osteoblast cDNA. mGluR1b can be detected at 10-1 and 10-2 dilutions but not at 10-3 or 10-4. Arrows show expected position of mGluR1b product (378 bp). f, expression of NMDAR-2C. Lane 1 shows product from rat whole brain; lane 2 shows product from rat osteoblast cDNA; and lane 3 shows that there is no product from osteoblast total RNA. Predicted product size is 359.

The intensity of the band for mGluR1b product generated from osteoblast cDNA appeared weaker than that for the brain (Fig. 1a; n = 10). To confirm this difference, the cDNA templates from osteoblasts and from brain were serially diluted in 10-fold steps (down to 10-4) before carrying out the PCR reaction. The level of dilution necessary to prevent detectable formation of a product from osteoblast cDNA was 10-2-10-3, but a dilution of 10-3-10-4 of the brain cDNA was required (Fig. 1e). Similar results were obtained in each of three separate experiments.

Recent findings suggest that the NMDAR-2C subunit may be required for inhibitory modulation of the NMDA receptor by mGluRs (Ref. 26 and see "Discussion"). PCR using femoral osteoblast cDNA and NMDAR-2C primers gave a product of the appropriate size (359 bp), the sequence of which was identical to the relevant portion of the rat brain NMDAR-2C sequence.

Electrophysiology

Currents Induced by NMDA and L-Glutamate-- Functional NMDA receptors are present in approximately 25% of femoral osteoblasts in primary culture (15). Addition of 100 µM NMDA plus 10 µM glycine to the inflow of the recording chamber induced prolonged, slowly desensitizing inward currents with a mean peak amplitude of 77.8 ± 7.4 pA (mean ± S.E.; n = 9). When the same cells were superfused, with 100 µM L-glutamate plus 10 µM glycine, currents were very small, often appearing as little more than an increase in noise. Mean glutamate current amplitude was 17.4 ± 0.9 pA (n = 9; p < 0.00002 compared with effect of NMDA; Fig. 2a). The effect of L-glutamate plus 10 µM glycine was investigated in a further 20 cells in which the action of NMDA was not measured. Currents greater than 20 pA were never seen.


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  		Fig. 2.   Modulation of NMDA receptor currents by mGluR activation. a, blockade of mGluRs restores NMDA receptor current. Left and center panels shows whole cell currents elicited by 100 µM NMDA and 100 µM L-glutamate (shaded bars; both co-applied with 10 µM glycine) respectively, in the same cell. The response to NMDA was completely inhibited by 100 nM (+)-MK-801 (filled bar). When L-glutamate was reapplied, in the presence of 500 µM (±)MCPG (right panel, open bar), the response was greatly enhanced, reaching a peak similar to that seen with NMDA. The (±)MCPG-enhanced response to L-glutamate was blocked by 100 nM (+)-MK-801. Scale bar shows 60 pA and 80 s. Similar data were obtained from three other cells. b, stimulation of mGluRs inhibits the NMDA receptor current. Left panel shows response to 100 µM NMDA with 10 µM glycine. Right panel shows response to NMDA with 10 µM glycine after stimulation of mGluRs with 300 µM 1S,3R-ACPD (open bar). The response to NMDA was greatly reduced and resembled that seen during superfusion with L-glutamate. Scale bar shows 20 pA and 40 s. Similar data were obtained from three other cells.

Effect of an mGluR Antagonist on the Currents Induced by L-Glutamate-- To investigate the possible involvement of mGluR receptors in the differing effects of NMDA and L-glutamate, we used the mGluR antagonist (±)MCPG. After superfusion with NMDA and L-glutamate (both with 10 µM glycine), we reapplied L-glutamate plus 10 µM glycine in the presence of 500 µM (±)MCPG. Inclusion of (±)MCPG converted the small currents induced by 100 µM L-glutamate (18.5 ± 2.7 pA; n = 4) to larger responses (87.5 ± 18.7 pA n = 4; p < 0.025 compared with L-glutamate alone) that were similar in amplitude to those evoked by NMDA (89.0 +18.0 pA; n = 4; p > 0.2). This glutamate-induced current was blocked by 100 nM (+)-MK-801 (Fig. 2a).

Effect of an mGluR Agonist on the Currents Induced by NMDA-- Because blockade of mGluR receptors enabled glutamate to induce inward currents similar to those evoked by NMDA, we investigated the effect of the mGluR agonist 1S,3R-ACPD on the NMDA response. 100 µM NMDA and 10 µM glycine were applied briefly to confirm the presence of an NMDA current and to determine peak current amplitude (65.5 ± 12.0 pA; n = 4). When NMDA and glycine were reapplied to the cells in combination with 300 µM 1S,3R-ACPD, the peak current amplitude was reduced to 15.0 +2.2 pA. (n = 4; p < 0.02 compared with amplitude in the absence of 1S,3R-ACPD; Fig. 2b).

[Ca2+]i Changes in Stimulated Cells

Effect of NMDA-- 100 µM NMDA plus 10 µM glycine induced a sustained rise in [Ca2+]i (n = 10). This response was reversed, in a dose-dependent manner, by (+)-MK-801 (Fig. 3a). When Ca2+-free medium was used (0 Ca2+, 1 mM EGTA), no response to NMDA was seen (Fig. 3a; n = 5).


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  		Fig. 3.   Elevation of [Ca2+]i upon stimulation of NMDARs and mGluRs. a, left panel, addition of 100 µM NMDA (with 10 µM glycine; shaded bar) to the cuvette caused a sustained rise in [Ca2+]i. This effect was reversed by (+)-MK-801 (filled bars) in a dose-dependent manner. Similar data were obtained in four other experiments. Right panel, application of 100 µM NMDA (with 10 µM glycine) to cells bathed in Ca2+-free medium caused no increase in [Ca2+]i. Similar data were obtained in four other experiments. Scale bar shows Delta ratio 340/380 and time in seconds for both traces. b, left panel, application of 300 µM 1S,3R-ACPD (shaded bar) caused a [Ca2+]i peak followed by a sustained increase in [Ca2+]i (see also Fig. 4b). Similar results were obtained in two other experiments. Right panel, application of 300 µM 1S,3R-ACPD to cells bathed in Ca2+-free medium caused a transient increase in [Ca2+]i, which then fell back to control levels within 100 s. Similar results were obtained in two other experiments. Scale bar shows Delta ratio 340/380 and time in seconds for both traces.

Elevation of [Ca2+]i Induced by 1S,3R-ACPD-- In Ca2+-containing medium, 300 µM 1S,3R-ACPD induced a transient rise in [Ca2+]i, lasting approximately 90 s, followed by a sustained plateau (Fig. 3b; n = 7). When 1S,3R-ACPD was applied in Ca2+-free saline, a similar transient was seen, but [Ca2+]i then fell to levels similar to those before 1S,3R-ACPD addition (Fig. 3b; n = 3). Addition of 75 nM ionomycin, to deplete internal stores of Ca2+, caused a small, sustained rise in [Ca2+]i. Subsequent addition of 1S,3R-ACPD did not cause an elevation of [Ca2+]i (data not shown).

Interactions between NMDA Receptors, mGluR1b Receptors, and PTH Receptors in Modulating [Ca2+]i

To observe interactions between the two types of glutamate receptors in their modulation of [Ca2+]i, we investigated the effects of sequential additions of agonists. Upon addition of 100 µM NMDA and 10 µM glycine to the cuvette, a sustained increase in [Ca2+]i was observed, as described above (n = 5). When 200 µM 1S,3R-ACPD was added to the cuvette, the elevated [Ca2+]i fell within 1-2 min to a value approximately midway between the peak response and that before addition of NMDA, and thereafter continued to fall more slowly (Fig. 4a). When (±)MCPG and (+)-MK-801 were then added together, [Ca2+]i fell, within 5 min, to levels close to the starting, basal level. However, it was noticeable that, when these antagonists were added, there was an initial increase in [Ca2+]i, lasting approximately 90 s, before the level fell (Fig. 4a). Similar results were obtained in four other experiments.


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  		Fig. 4.   Interaction between mGluRs and NMDARs. a, left panel, 1S,3R-ACPD inhibits NMDA-induced elevation of [Ca2+]i. Addition of 100 µM NMDA (with 10 µM glycine; open bar) caused a sustained elevation of [Ca2+]i. Subsequent addition of 1S,3R-ACPD (200 µM; shaded bar) caused a marked reduction in the NMDA-induced elevation. Addition of (±)MCPG (500 µM) and (+)-MK-801 (200 nM) to the cuvette (filled bar) caused [Ca2+]i to fall to control levels, although there was a transient (90 s) increase in [Ca2+]i on addition of the drugs. Similar results were obtained in four other experiments. Right panel, PTH inhibits NMDA-induced elevation of [Ca2+]i. Addition of 100 µM NMDA (with 10 µM glycine; open bar) caused a sustained elevation of [Ca2+]i. Subsequent addition of PTH (100 nM; shaded bar) caused a marked reduction in the NMDA-induced elevation. Similar results were obtained in two other experiments. Scale bar shows Delta ratio 340/380 and time in seconds for both traces. b, NMDAR activation inhibits elevation of [Ca2+]i by 1S,3R-ACPD. Addition of 300 µM 1S,3R-ACPD to the cuvette (open bar) caused a [Ca2+]i transient followed by a sustained elevation. Subsequent addition of 100 µM L-glutamate and 10 µM glycine (shaded bar) caused a reduction in the plateau phase of the [Ca2+]i signal. Addition of the mGluR antagonist (±)MCPG (500 µM; filled bar) then caused a rise in [Ca2+]i. Similar results were obtained in three other experiments. Scale bar shows Delta ratio 340/380 and time in seconds.

Because the effect of activation of mGluR1b is believed to be exerted through stimulation of Gq and PLC (20), we examined the effect of PTH, which is also able to activate this pathway (27, 28). After application of NMDA the [Ca2+]i response was allowed to develop, and 100 nM PTH was then added to the cuvette. The effect of PTH was similar to that of 1S,3R-ACPD, causing [Ca2+]i to fall rapidly to a point approximately midway between the basal and NMDA-enhanced levels (Fig. 4a). Similar results were obtained in two other experiments.

We also investigated whether prior addition of 1S,3R-ACPD would blunt the response to subsequent activation of NMDA receptors. Fig. 4b shows the results obtained when glutamate plus 10 µM glycine was applied after stimulation of mGluRs. 300 µM 1S,3R-ACPD caused an initial [Ca2+]i transient followed by a sustained plateau. Subsequent addition of 100 µM L-glutamate and 10 µM glycine (stimulating both NMDA receptors and mGluRs) reduced [Ca2+]i (Fig. 4b; n = 5). When (±)MCPG was added to block mGluRs, [Ca2+]i rose again to a sustained plateau (Fig. 4b). Similar results were obtained in three other experiments.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The data described here are, to our knowledge, the first evidence that bone cells express mGluRs. PCR analysis of cDNA from cultured femoral osteoblasts indicates that these cells express exclusively the type 1b mGluR. Stimulation of group 1 mGluRs (mGluR1 and mGluR5) activates PLC and consequent generation of IP3 and diacylglycerol, although an increase in [cAMP] has been reported (20). In accordance with the predicted linkage of mGluR1b to PLC, we observed an elevation of [Ca2+]i upon exposure of the cells to 1S,3R-ACPD. The initial phase of this response was not sensitive to [Ca2+]o and thus reflects mobilization of stored Ca2+. A subsequent, sustained phase of the [Ca2+]i response to 1S,3R-ACPD required extracellular Ca2+ and probably reflects capacitative Ca2+ influx. The occurrence of capacitative influx in osteoblasts has been proposed on the basis of the effects of thapsigargin and A23187 (29, 30).

mGluRs Inhibits NMDA Receptors-- Our interest in expression of mGluRs in bone cells was initiated because currents induced by activation of the NMDA (15) were not seen during superfusion with L-glutamate. We report here that blockade of mGluRs with (±)MCPG restored the NMDAR current, and the effect of NMDA was strongly inhibited by 1S,3R-ACPD, confirming that the difference between the effects of NMDA and L-glutamate was the result of inhibitory cross-talk between mGluRs and NMDARs. Fluorimetric measurements showed that the action of NMDA to elevate [Ca2+]i was also subject to mGluR modulation, being partially suppressed by 1S,3R-ACPD.

The majority of previous studies have demonstrated enhancement of NMDA responses upon activation of mGluRs (31-34), but inhibitory effects mediated through group 1 (PLC-linked) mGluRs have also been reported (35-38). The inhibitory pathway varies, being via generation of diacylglycerol and protein kinase C activation (mimicked by 4beta -phorbol 12,13-dibutyrate; Ref. 35) or by a staurosporine-resistant mechanism (38). The nature of mGluR-mediated modulation of NMDARs may be determined by expression of NMDAR-2 subtypes. Pizzi et al. (37) observed a neuroprotective action of mGluR 1/5 activation in cerebellar granule cells. This effect was exerted through a protein kinase C-mediated, inhibitory effect of group 1 mGluR stimulation on glutamate-induced elevation of [Ca2+]i (37). Pizzi et al. reported that this effect correlated specifically with expression of NMDAR-2C. In NMDAR-2C-depleted neurons, both group 1 mGluR-activation and protein kinase C stimulation enhanced NMDA receptor-mediated Ca2+ influx (26). Our finding that NMDAR-2C is expressed in rat osteoblasts, where the influence of mGluR activation is inhibitory, is in accord with this idea. NMDAR-2D is also known to be expressed in bone cells (11).

It has been reported that compounds that interact with mGluRs may also act at NMDARs, primarily as antagonists in the presence of subsaturating doses of agonist (39). The modulation of NMDA currents and [Ca2+]i responses, by mGluR ligands, reported here might therefore reflect a direct action on the NMDAR. However, we consider this most unlikely for the following reasons: (i) We have measured currents induced in osteoblasts by both 100 µM and 1 mM NMDA (in the presence of saturating doses of glycine as used in this study) and observed no significant difference in current amplitude.3 We are therefore confident that 100 mM NMDA is a saturating or nearly saturating dose. (ii) At the doses used in our experiments (100 µM NMDA, 10 µM glycine), 1S,3R-ACPD may have a minor inhibitory effect (up to15%) on the amplitude of NMDA currents (39). We observed 75-80% inhibition of the response to NMDA. (iii) At the NMDA receptor agonist dosages that we employed, (±)MCPG, like 1S,3R-ACPD, may act as a weak antagonist of the NMDA receptor (39). However, (±)MCPG enhanced the response of the osteoblastic NMDA receptor, consistent with a reduction of mGluR-mediated inhibition of NMDARs. (iv) PTH, which stimulates Gq similarly to activation of mGluR1b (20, 28), has an effect on [Ca2+]i that is strikingly similar to that of 1S,3R-ACPD. This observation is consistent with actions of both 1S,3R-ACPD and PTH to modulate [Ca2+]i through their activation of PLC, rather than a direct effect of 1S,3R-ACPD on the NMDA receptor. We conclude that our data reflect actions of these compounds on the mGluR1b receptor not direct effects on the NMDAR.

NMDARs Inhibit the Response to mGluR Activation-- As well as inhibition of NMDA currents by activation of mGluRs, our measurements of [Ca2+]i also suggest that NMDAR activation may inhibit mGluR receptor-induced Ca2+ signaling. When L-glutamate was applied to cells 250 s after 1S,3R-ACPD (during the [Ca2+]o-dependent phase of the response), [Ca2+]i fell to a lower level than in the presence of 1S,3R-ACPD alone. An inhibitory effect of NMDAR activation on PLC has been described previously in CA1 neurons (40). A similar effect in cerebral cortex was irreversible, possibly reflecting NMDA-induced cellular damage (41). When (±)MCPG and (+)-MK-801 were added to cells in which both NMDARs and mGluRs were stimulated (with NMDA and 1S,3R-ACPD), there was an initial increase in [Ca2+]i, lasting approximately 90 s before [Ca2+]i returned to control levels. This brief elevation of [Ca2+]i probably reflects a difference in the rate at which blockade of the two receptor-mediated pathways becomes effective, such that one becomes temporarily released from inhibition. The mGluR-mediated signal may persist because of a delay between blockade of mGluRs by (±)MCPG and the return of IP3 and diacylglycerol to resting levels. Alternatively, because the action of (+)-MK-801 on NMDARs is by open channel block (42), blockade of NMDARs may proceed relatively slowly after application of the drug. However, in the present study the effect of (+)-MK-801 was normally complete within 10-20 s (Figs. 2 and 3).

Functional Significance-- The cells that we have used in this study are a primary culture derived from a load-bearing bone. These cells maintain osteoblastic characteristics in culture, including expression of alkaline phosphatase, osteopontin, and osteocalcin (22, 24); the ability to produce mineralized matrix (22); mechanosensitivity (production of nitric oxide, Ca2+ signaling, and up-regulation of osteopontin and osteocalcin; Refs. 22 and 24); and responsiveness to PTH, vitamin D and prostaglandin E2 (23). The co-expression of mGluRs and NMDARs described here is therefore likely to be characteristic of osteoblasts in vivo. Data reported by others and ourselves are consistent with a role for NMDARs in the control of bone matrix turnover (10-15, 21, 43). The discovery of functional, PLC-linked mGluRs in osteoblasts indicates that glutamate signaling may exert complex effects on the regulation of bone cells. Firstly, the receptor cross-talk that we describe here will be of significance in regulating the effect of glutamate signaling and may itself be developmentally regulated by selective expression of different NMDAR-2 s, as has been described in the brain (26, 37, 44, 45). Interestingly, it appears that negative modulation of NMDA receptors by mGluRs does not occur in cultured osteoclasts, because the NMDA receptor currents evoked in these cells by L-glutamate and NMDA are similar (12, 14). Activation of glutamatergic receptors in osteoblasts and osteoclasts can therefore elicit different [Ca2+]i signals. Secondly, activation of G-protein-linked PLC is believed to be of significance in osteoblastic responses to many stimuli including prostaglandins and PTH (27, 46-48), bradykinin (49), and purinoceptors (50, 51). Responses to stretch may also involve generation of IP3 (52, 53). Stimulation of IP3 signaling by glutamate receptors is therefore likely to interact with the effects of other stimuli, which activate this pathway. Furthermore, NMDAR activation by glutamate may inhibit IP3-mediated Ca2+ signaling activated by other signals, and activation of phospholipase C by other stimuli may regulate Ca2+-influx through osteoblastic NMDA receptors. Our data suggest that such interaction occurs between PTH-mediated activation of PLC and NMDARs (Fig. 4a). The complex glutamatergic system in bone cells may provide a target for pharmacological manipulation of bone mass.

    ACKNOWLEDGEMENTS

Our thanks to Min Fang and Dr. Long Lu for carrying out high pressure liquid chromatography to confirm drug purity, to Jackson Kirkman-Brown and Emma Punt for assistance with the fluorimetric measurements, to Dr. Justin St. John for advice on PCR and to Prof. R. H. Michell for critical reading of the 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.

Dagger Recipient of an Overseas Research Student award.

§ To whom correspondence should be addressed. Tel.: 44-121-414-5455; Fax: 44-121-414-5925; E-mail: s.j.publicover@bham.ac.uk.

Published, JBC Papers in Press, August 18, 2000, DOI 10.1074/jbc.M004520200

2 Genever, P. G., and Skerry, T. M. (2000) J. Bone Miner. Res. 15, Suppl. 1, 378

3 Y. Gu and S. J. Publicover, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; iGluRs, ionotropic glutamate receptors; mGluRs, metabotropic glutamate receptors; PCR, polymerase chain reaction; (±)MCPG, (±)-alpha -methyl-carboxyphenyl-glycine; 1S, 3R-ACPD, trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid; IP3, inositol(1,4,5)trisphosphate, PTH, parathyroid hormone; PLC, phospholipase C; bp, base pair(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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