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RECEPTORS AND SIGNAL TRANSDUCTION

Role of calcium channels in carboxyl-terminal parathyroid hormone receptor signaling

Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 8 November 2005 ; accepted in final form 3 February 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major systemic regulator of calcium homeostasis that activates PTH/PTHrP receptors (PTH1Rs) on target cells. Carboxyl fragments of PTH (CPTH), secreted by the parathyroids or generated by PTH proteolysis in the liver, circulate in blood at concentrations much higher than intact PTH-(1–84) but cannot activate PTH1Rs. Receptors specific for CPTH fragments (CPTHRs), distinct from PTH1Rs, are expressed by bone cells, especially osteocytes. Activation of CPTHRs was previously reported to modify intracellular calcium within chondrocytes. To further investigate the mechanism of action of CPTHRs in osteocytes, cytosolic free calcium concentration ([Ca²⁺]_i) was measured in the PTH1R-null osteocytic cell line OC59, which expresses abundant CPTHRs but no PTH1Rs. [Ca²⁺]_i was assessed by single-cell ratiometric microfluorimetry in fura-2-loaded OC59 cells. A rapid and transient increase in [Ca²⁺]_i was observed in OC59 cells in response to the CPTH fragment hPTH-(53–84) (250 nM). No [Ca²⁺]_i signal was observed in COS-7 cells, in which CPTHR binding also cannot be detected. Neither hPTH-(1–34) nor a mutant CPTH analog, [Ala^55–57]hPTH-(53–84), that does not to bind to CPTHRs, increased [Ca²⁺]_i in OC59 cells. The [Ca²⁺]_i response to hPTH-(53–84) required the presence of extracellular calcium and was blocked by inhibitors of voltage-dependent calcium channels (VDCCs), including nifedipine (100 nM), -agatoxin IVA (10 nM), and -conotoxin GVIA (100 nM). We conclude that activation of CPTHRs in OC59 osteocytic cells leads to a rapid increase in influx of extracellular calcium, most likely through the opening of VDCCs.

calcium influx; osteocytes

We previously reported the isolation and characterization of clonal osteocytic cells expressing high levels of CPTHRs that had been derived from fetuses in which most exons encoding the PTH1R had been ablated by gene targeting to eliminate potentially confounding effects of coexpressed PTH1Rs (4). These clonal osteocytic (OC) cell lines expressed 1,900,000 to 3,400,000 CPTHR-binding sites per cell, a level 6- to 10-fold higher than that observed on osteoblastic cells obtained from the same fetal calvarial bones and at least 5-fold higher than on ROS 17/2.8 cells (5). Abundant CPTHR expression in the osteocytic cells was associated with biological responses to CPTH peptides, including regulation of apoptosis and connexin-43 expression (5). Thus these OC cells provide a useful model of CPTHR action in bone cells. These and other observations, including evidence of effects of extended CPTH fragments such as PTH-(7–84) on bone resorption in vitro and in vivo, suggest that CPTHRs may play a significant physiological role in modulating the functions of normal bone cells (21).

We recently described the use of synthetic truncated and/or alanine-substituted CPTH peptides to define critical residues within the intact PTH-(1–84) hormone required for effective interaction with CPTHRs on osteocytes (7). These included the tripeptide sequence Arg²⁵-Lys²⁶-Lys²⁷, the dibasic sequence Lys⁵³-Lys⁵⁴, and three additional residues within the hPTH-(55–84) sequence: Asn⁵⁷, Lys⁶⁵, and Lys⁷² (7). Functional analysis of these mutant PTH peptides demonstrated a strong correlation between binding affinity and biological effect (cell survival) (7).

The signaling properties of CPTHRs are incompletely defined. Erdmann et al. (8) previously reported that CPTH peptides, such as hPTH-(52–84) and others that included the sequence hPTH-(73–76), could induce [Ca²⁺]_i signaling in human fetal chondrocytes. The increase in cytoplasmic Ca²⁺ concentrations in the chondrocytes was blocked by depletion of extracellular calcium but not by various inhibitors of intracellular calcium release, suggesting a mechanism involving calcium influx (8).

Osteocytes play an important role in sensing extracellular mechanical stress, and the mechanical signals mediated by osteocytes may regulate the overall metabolism of cells in bone tissue (14). Mechanotransduction in bone is complex in nature, being influenced by many modulators such as PTH, prostanoids, and extracellular [Ca²⁺]. Influx of extracellular calcium through voltage-dependent calcium channels (VDCCs) is implicated in osteocytic mechano- and hormonal transduction (14).

Here, we report that COOH-terminal fragments of PTH elicit increases of [Ca²⁺]_i in osteocytes via influx through VDCCs. These data suggest that the CPTHRs might regulate osteocyte biology via an increase in intracellular calcium.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cell cultures. The clonal, conditionally immortalized OC59 osteocytic cell line was isolated by enzymatic digestion from calvarial bones of 18.5-day-old tsA58(+)/PTH1R(–/–) fetuses, as previously described (5). Briefly, single colonies were isolated from the primary mixed calvarial cells by limiting dilution, expanded, and then screened and selected for specific binding of ¹²⁵I-[Tyr³⁴]-hPTH-(19–84), as previously reported (5). Cells were cultured at 33°C in a humidified atmosphere (95% air-5% CO₂) using growth medium [-MEM containing 10% FBS (lot no. 1011961; Life Technologies, Gaithersburg, MD) and 1% penicillin-streptomycin]. ROS 17/2.8 rat osteosarcoma cells were cultured in DMEM-F12 medium supplemented with 10% FBS, 2 mM glutamine, 1% nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin. RAW 264.7 cells were obtained from ATCC and were maintained in DMEM (Sigma, St. Louis, MO) supplemented with 10% FBS, glutamine, penicillin, and streptomycin, as described above. COS-7 cells were maintained in DMEM containing 2 mM L-glutamine and 10% FBS. PC12 cells were maintained in Ham’s F-12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate; 15% horse serum and 2.5% fetal bovine serum, as well as 2.5% PC12, ROS 17/2.8, RAW 264.7, and COS-7 cells, were maintained at 37°C in a humidified atmosphere of 95% air-5% CO₂. For measurement of [Ca²⁺]_i, cells were pretreated with 0.3 mM 8-Br-cAMP for 16 h, starting on day 2 of culture, because previous studies (13) on ROS 17/2.8 rat osteosarcoma cells had demonstrated that such treatment increased CPTHR expression on the surface of these cells. We had also observed in preliminary studies that this maneuver augmented both CPTHR expression and the [Ca²⁺]_i response to CPTH peptides in OC59 cells.

Human PTH peptides. Recombinant hPTH-(1–84), [Tyr³⁴]-hPTH-(19–84), and [Tyr34]-hPTH-(24–84) were gifts of Chugai Pharmaceutical (Shizuoka, Japan); hPTH-(7–84) and hPTH-(53–84) were purchased from Bachem (Torrance, CA). All other PTH fragments were synthesized at the Massachusetts General Hospital Peptide and Oligonucleotide Core Laboratory (Boston, MA).

Determination of [Ca²⁺]_i. [Ca²⁺]_i was monitored by ratio microfluorimetry using the fura-2 method. In brief, cells were plated into Lab-Tek II chambered cover glasses (Nalge Nunc International, Naperville, IL) at a density of 10,000 cells per well and cultured as described above. For measurement of [Ca²⁺]_i, cells were transferred to room temperature and washed with a balanced salt solution (BSS; 127 mM NaCl, 3.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM CaCl₂, 0.8 mM MgCl₂, 5 mM glucose, and 10 mM HEPES, pH 7.4) before being loaded with 5 µM fura-2 acetoxymethyl ester (with 0.05% Pluronic F-127; Molecular Probes, Eugene, OR) for 45 min at room temperature, washed in BSS, and further incubated at room temperature for 30 min to allow discharge of uncleaved fura-2 AM. In cells prepared in this manner, fura-2 fluorescence was diffuse without a punctate distribution pattern. Fluorescence was measured in single cells excited alternatively at 340 and 380 nm using a Deltascan dual-wavelength fluorimeter (Photon Technologies, Lawrenceville, NJ) directed through the stage of an inverted Nikon Diaphot 200 microscope (Melville, NY) with a cutoff filter at 400 nm. Emissions were monitored in real time with a Sensys charge-coupled device camera (Photometrics, Tucson, AZ) and analyzed using the Poenie-Tsien ratio with Imagemaster 2 software (Photon Technologies). At the conclusion of each experiment, fura-2 fluorescence was calibrated to [Ca²⁺]_i by treating the cells with 5 µM ionomycin to achieve the maximum Ca²⁺-bound dye ratio at 340/380 nm and fluorescence at 380 nm excitation (R_max and S_b2), followed by treatment with 5 mM EGTA to determine Ca²⁺-free dye ratio at 340/380 and fluorescence at 380 nm of excitation (R_min and S_f2). These parameters were used to estimate [Ca²⁺]_i using the equation of Grynkiewicz et al. (10). Calcium transients monitored in the absence of extracellular calcium are reported as a ratio of fura-2 fluorescence at 340 nm excitation over that at 380 nm. (S)-(–) BAY K8644 (B133) and thapsigargin were obtained from Sigma.

Western blot analysis. OC59, COS-7, and PC12 cells were cultured as previously described (see Cell cultures). For protein isolation, cell cultures were rinsed three times with ice-cold PBS, and cell layers were harvested into protein extraction buffer (RIPA buffer) consisting of 50 mM Tris·HCl (pH 7.5), 135 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 2 mM EDTA, 50 mM NaF, 2 mM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF. Cell pellets were homogenized in RIPA buffer, incubated for 60 min at 4°C, and centrifuged at 1,000 rpm for 5 min. The resulting supernatants were collected and stored at –80°C. Total protein concentration was measured using a bicinchoninic acid protein assay (Pierce, Rockford, IL). Equal amounts of protein from OC59, COS-7, and PC12 cell layers were used in the assay. Samples were mixed with equal volumes of 2x SDS sample buffer, boiled for 5 min, centrifuged for 5 min, and subjected to SDS-PAGE using a 10% polyacrylamide gel. Membranes were blocked with 5% milk, 1% BSA in Tris-buffered saline, and 0.1% Tween 20 (TBS-T) for 2 h and incubated overnight at 4°C with a rabbit polyclonal anti-VDCC -subunit reactive with multiple VDCC types (catalog no. ab6298-50; Novus Biologicals, Littleton, CO). Blots were washed three times in TBS-T, each time for 15 min, and then incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:600) (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. Signals were detected by enhanced chemiluminescence (Pierce).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
[Ca²⁺]_i response to CPTH peptides in PTH1R-null osteocytes. CPTHR signaling was examined in the established PTH1R-null OC cell line (OC59) because these cells have two important advantages: they do not express the PTH1R and they do express high levels of the CPTHR (5). On the basis of previous analysis of CPTHR binding in these cells, several hPTH peptides were utilized to examine CPTHR signaling, including hPTH-(1–84), hPTH-(7–84), [Tyr³⁴]-hPTH-(13–84), [Tyr³⁴]-hPTH-(19–84), [Tyr³⁴]-hPTH-(24–84), and hPTH-(53–84). We previously demonstrated that these peptides could efficiently bind to the CPTH receptor in OC59 osteocytic cells (5), ROS 17/2.8 rat osteoblastic cells (8), and RAW 264.7 murine monocytic cells (6).

To investigate whether CPTH receptor activation could elicit [Ca²⁺]_i signals in OC59 cells, we first stimulated the cells with the CPTHR ligand, hPTH-(53–84) (250 nM). As shown in Fig. 1B, the hPTH-(53–84) peptide induced a transient elevation in [Ca²⁺]_i that reached a maximum within 20 s after peptide addition and returned to baseline within 10–15 s. Similar signals were observed using the same concentration of longer human PTH peptides, including PTH-(1–84), PTH-(7–84), [Tyr³⁴]PTH-(13–84), [Tyr³⁴]PTH-(19–84), and [Tyr³⁴]PTH-(24–84) (data not shown). The addition of vehicle alone (balanced salt solution) induced no increase in [Ca²⁺]_i (Fig. 1A), indicating that the PTH effect was specific for the peptide and not a consequence of mechanical stimulation of the cells. Interestingly, cells were heterogeneous with respect to the increase in [Ca²⁺]_i induced by CPTHR. As shown in Fig. 1E, in any given field an average of 55–65% of fura-loaded cells demonstrated the CPTH-induced increase in [Ca²⁺]_i. However, synchronization of the cells by serum starvation did not increase the percentage of responsive cells, suggesting that distribution of unsynchronized cells across the cell cycle does not account for this phenomenon (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Regulation of cystolic free calcium concentrations ([Ca2+]i) by carboxyl fragments of parathyroid hormone (CPTH) peptide in cells expressing carboxyl-terminal PTH receptors (CPTHRs). Cai2+ was assessed by single-cell ratiometric microfluorimetry, as described in MATERIALS AND METHODS. Cells were plated in 4-well chamber slides at a density of 10,000 cells/well, pretreated on day 2 of culture with 0.3 mM 8 Br-cAMP for 16 h, washed twice with balanced salt solution (BSS), and, before loading with fura 2-AM at a final concentration of 5 µM for 45 min at room temperature, washed several times with BSS and then incubated further with BSS for another 30 min at room temperature in the dark to allow discharge of uncleaved fura-2AM. Human PTH-(53–84) (250 nM) (or BSS buffer alone) was added at the time indicated by the arrow. Subsequent calibration was performed by addition of ionomycin (5 mM) followed by EGTA (5 mM). A: OC59 cells treated with BSS alone. B: OC59 cells treated with hPTH-(53–84). Inset, 360/380 ratio in response to PTH peptide (P), ionomycin (I), and EGTA (E). C: ROS 17/2.8 cells treated with hPTH-(53–84). D: RAW 264.7 cells treated with hPTH-(53–84). Similar results were obtained using OC14 osteocytic cells (not shown). E: percentage of cells (means ± SD) responding to hPTH-(53–84) treatment was calculated after the number of cells showing PTH-dependent calcium influx at 10 s compared with the total number of ionomycin-responsive cells in each microscopic field were counted (6 fields examined). Experiments shown were repeated a minimum of 3 times with similar results.

Specificity of [Ca²⁺]_i response to CPTH receptor peptides. To examine the specificity of CPTHR [Ca²⁺]_i signaling, OC59 cells were treated with PTH peptides that do not bind effectively to the CPTHR, including the amino-terminal fragment hPTH-(1–34) (Fig. 2A), mutant [Ala^55–57]hPTH-(53–84) (Fig. 2B), and truncated hPTH-(57–84) (Fig. 2C). None of these peptides induced a [Ca²⁺]_i response, indicating a strong correlation between binding affinity and signaling.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Specificity of calcium signal induced by CPTH/CPTHR interaction. Cells were prepared, and microfluorimetric measurements were performed and calibrated as described in the legend to Fig. 1. A–C: [Ca2+]i following addition to OC59 cells of hPTH-(1–34) (A), [Ala55–57]hPTH-(53–84) (B), or hPTH-(57–84) (C), each at 250 nM. D: [Ca2+]i in COS-7 cells treated with hPTH-(53–84) (250 nM). E: percentage of OC59 cells responding to hPTH-(1–84), hPTH-(7–84), or hPTH-(53–84), added at the indicated concentrations, is shown as means ± SD (n = 4). Experiments were each repeated a minimum of 3 times with similar results.

It is known that hPTH-(53–84) binds to CPTHRs on OC59 cells with 10- to 100-fold lower apparent affinity than either hPTH-(1–84) or hPTH-(7–84), both of which exhibit IC₅₀s of 30 nM (7). As shown in Fig. 2E, comparable responses to hPTH-(1–84) and hPTH-(7–84) were observed at both 10^–10 M and 10^–8 M, as expected, whereas 10^–10 M hPTH-(53–84) was inactive and 10^–8 M hPTH-(53–84) elicited only a partial response, consistent with its substantially lower affinity for the receptor.

Extracellular calcium is the source of increased [Ca²⁺]_i induced by CPTH/CPTHR interaction. To investigate the source of the [Ca²⁺]_i response to CPTH peptides, OC59 cells were washed for several seconds with calcium-free BSS containing 0.1 mM EGTA immediately before PTH peptide treatment. Depletion of extracellular calcium with EGTA in this way completely blocked the increase in [Ca²⁺]_i otherwise induced by hPTH-(53–84), consistent with a mechanism involving influx of extracellular calcium (Fig. 3A).

View larger version (11K): [in this window] [in a new window]   Fig. 3. Source of calcium signal induced by CPTH/CPTHR interaction in OC59 osteocytes. Cells were prepared, and microfluorimetric measurements were performed and calibrated as described in legend to Fig. 1. A: OC59 cells were prewashed with calcium-free BSS containing 0.1 mM EGTA for several seconds, BSS containing EGTA was replaced with calcium-free BSS, and hPTH (53–84) (250 nM) was then immediately added. B: OC59 cells were pretreated for 6 min with thapsigargin (10–8 M) before addition of hPTH (53–84) (250 nM). C: OC59 cells were exposed to 10 µM Gd3+ and then immediately treated with hPTH-(53–84) (250 nM). Experiments shown were repeated 3 times with similar results.

We further tested the source of the [Ca²⁺]_i using gadolinium chloride (Gd³⁺), which competitively blocks a variety of VDCCs in cells from many tissues and organs, including stretch-sensitive ionic channels in the sarcolemma of skeletal muscle fibers (3). In OC59 cells pretreated with Gd³⁺ (10 µM), the addition of hPTH-(53–84) failed to induce an increase in [Ca²⁺]_i (Fig. 3C). Note that in all of these experiments involving inhibitors, subsequent addition of the ionophore ionomycin during the calibration procedure induced the expected substantial increase in [Ca²⁺]_i (not shown in Fig. 3, see Fig. 4, insets).

View larger version (34K): [in this window] [in a new window]   Fig. 4. Effect of voltage-dependent calcium channel (VDCC) inhibitors on cytosolic calcium response to hPTH-(53–84) in OC59 cells. A: Western blot analysis of VDCC -subunits, using a polyclonal antibody capable of detecting multiple VDCC -subunits, expressed in OC-59, PC12, or COS-7 cells (equal amounts of protein were loaded per lane; see MATERIALS AND METHODs). B–F: OC59 cells were prepared, and microfluorimetric measurements of [Ca2+]i were performed and calibrated as described in legend to Fig. 1. B: OC-59 cells treated with KCl (70 mM). C: OC-59 cells treated with (S)-(–) BAY K8644 (1 µM). Cells were pretreated with VDCC inhibitors nifedipine (100 nM) (D), conotoxin (100 nM; E), or agatoxin (10 mM; F) for 2 min before addition of hPTH-(53–84) (250 nM). Ionomycin was added subsequent to hPTH-(53–84) in the continued presence of inhibitor and, in each case, induced the expected increase in [Ca2+]i (see F, inset).

In addition, several VDCC inhibitors, including nifedipine (100 nM; Fig. 4D), conotoxin (10 nM; Fig. 4E), and agatoxin (100 nM; Fig. 4F), blocked calcium influx mediated by hPTH-(53–84).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Herein, we report that CPTHR activation in PTH1R-null osteocytes induces a rapid and transient increase in intracellular cytosolic free calcium. This calcium signal is due to an influx of extracellular calcium, possibly through VDCCs or a VDCC-like channel(s).

To study CPTHR signaling, we used osteocytic cells (OC59) derived from mice that genetically lack functional PTH1Rs. This approach eliminated potential confounding effects of coexpressed PTH1Rs and allowed us to focus directly upon CPTHR-dependent actions of intact PTH and other extended CPTH fragments that may interact with the PTH1R. We (5) previously reported that these clonal osteocytic cells express a high density of CPTHRs that is 6- to 10-fold greater than that observed on the osteoblastic cells obtained from the same bones. Thus the very high abundance of expressed CPTHRs on osteocytic cells, in addition to their lack of PTH1Rs, provides a suitable model to study CPTH receptor signaling.

We found that hPTH-(53–84) elicited a rapid and marked increase (100 nM) of [Ca²⁺]_i in OC59 osteocytes as well as in other cells expressing CPTHRs, including ROS 17/2.8 osteoblast cells and RAW 264.7 monocytic osteoclast precursor cells. These data suggest that increased [Ca²⁺]_i may be involved generically in CPTHR signaling in different bone cells. In 1996, Erdmann et al. (8) described induction of cytoplasmic Ca²⁺ transients by hPTH-(52–84) in isolated human fetal chondrocytes, an event that occurred several minutes after addition of the peptide. The relatively delayed nature of the [Ca²⁺]_i signal in the cells studied by Erdmann et al. (8) could be explained by a mechanism whereby one or more serial events triggered by activated CPTHRs are required before augmented calcium influx can occur. The tempo of the [Ca²⁺]_i response observed in our studies was much more accelerated, suggesting a more direct coupling of CPTHR activation to relevant calcium channels.

Another difference between our results and those of Erdmann et al. (8) relates to the structural features of the CPTH ligand required for the [Ca²⁺]_i response. Thus we found that hPTH-(57–84), which cannot be shown to displace the CPTHR radioligand ¹²⁵I-[Tyr³⁴]-hPTH-(19–84) from CPTHRs on OC59 cells (5), was similarly incapable of triggering rapid [Ca²⁺]_i transients in these cells. Erdmann et al. (8), in contrast, observed (delayed) [Ca²⁺]_i responses in chondrocytes following application of hPTH-(57–76), hPTH-(61–80), or hPTH-(64–84) [none of which would be predicted to bind with measurable affinity to osteocyte CPTHRs (5, 7)], although they did not study hPTH-(57–84) per se (8). The explanation for this apparent disparity is not yet clear but could relate to differences in cell types studied, differences in technique, or possibly the existence of distinct subtypes of CPTHRs on chondrocytes vs. osteoblastic cells.

The [Ca²⁺]_i in response to CPTHR signaling was heterogeneous in that not all cells in a given microscopic field responded. Previous studies of chondrocytes also reported a heterogeneous response (8). Synchronization of our cells by serum starvation did not increase the percentage of responsive cells, suggesting that distribution across the cell cycle does not account for the heterogeneous CPTH response. We also considered phenotypic drift within this established cell line as a possible explanation, but heterogenity of the calcium response was still observed following subcloning of OC59 cells (data not shown). These findings suggest that other variable(s) must affect receptor expression or the postreceptor/effector coupling process.

In the present study, the specificity of the signal induced by CPTH/CPTHR interaction was investigated using ligands that do not bind effectively to the CPTH receptor, including hPTH-(1–34) and binding-defective mutant and short CPTH peptides. We also tested COS-7 cells, which do not express CPTH receptors. In none of these circumstances were the calcium signals otherwise elicited by human hPTH-(53–84) in OC59 cells observed. We also examined calcium signals induced by low concentrations (10^–10 M) of hPTH peptides that have different binding affinities to CPTHRs (5, 7). PTH peptides with high binding affinity, such as hPTH-(1–84) and hPTH-(7–84), induced vigorous calcium signals at 10^–10 M, whereas hPTH-(53–84), which binds with roughly 100-fold lower affinity, was inactive at 10^–10 M and only partially active at 10^–8 M. Collectively, these data strongly suggest that calcium influx is a specific response to CPTHR activation.

To investigate whether the increase in intracellular calcium induced by CPTHR activation was due to calcium inflow from extracelluar or intracellular sources, we eliminated extracellular calcium by washing the cells briefly with EGTA. Calcium chelation in the extracelluar fluid completely abolished the [Ca²⁺]_i induced by CPTHR activation, indicating that extracellular calcium is the source of the [Ca²⁺]_i. Also, Gd³⁺, which is known to interfere with calcium influx through calcium channels (16), inhibited calcium signaling in response to CPTHR activation. On the contrary, depletion of intracellular calcium stores with thapsigargin did not inhibit the CPTH-induced calcium signal. In fact, thapsigargin prolonged the recovery phase of the hPTH-(53–84)-induced [Ca²⁺]_i transient, which suggests that smooth endoplasmic reticular calcium-ATPases ordinarily may be involved in terminating the CPTHR [Ca²⁺]_i signal. Collectively, these observations suggest that extracellular calcium is the major source of cytosolic calcium influx in response to CPTHR signaling. This result is consistent with previous obervations of Fukayama et al. (9), who showed increased uptake of extracellular ⁴⁵Ca by SaOS-2 human osteosarcoma cells in response to CPTH peptides, and of Erdmann et al. (8), who observed that the increase in cytoplasmic Ca²⁺ concentrations in the chondrocytes treated with hPTH-(52–84) was blocked by depletion of extracellular calcium but not by various inhibitors of intracellular calcium release.

Previous in vivo studies in parathyroidectomized rats have shown that hPTH-(7–84) can rapidly lower serum calcium without increasing urinary calcium (23). Although this may reflect inhibition of calcium release from bone (27), our findings are consistent with the possibility that some portion of the acute hypocalcemic response could result from CPTH-triggered increased net calcium entry and sequestration within cells expressing CPTHRs.

To assess whether the calcium influx involved membrane calcium channels, and more specifically VDCCs, we pretreated the cells with different VDCC blockers, including nifedipine, agatoxin, and conotoxin, each of which completely abolished the calcium influx. These data suggested that calcium influx through VDCCs is involved in CPTHR signaling. In 2002, Throckmorton et al. (28) demonstrated that treatment of a human umbilical vein endothelial cell line with PTH-(1–84) induced the appearance of VDCCs with subsequent calcium influx and PKC translocation through a calcium-phospholipid pathway (28). Similar induction of VDCC expression by PTH was reported in MLO-Y4 osteocytic cells by Gu et al. (11). The ability of different VDCC blockers to interfere with the cytosolic free calcium response to CPTH treatment suggests that either multiple VDCCs are involved in mediating the signal or that the specific VDCC involved in mediating the signal could be blocked with nifedipine, agatoxin, and conotoxin. Pharmacological modifications of cation channels by inorganic and organic blockers are so far extremely limited; various blockers have been described but unfortunately lack high specificity for these channels (12).

The mechanism of VDCC opening in response to CPTH treatment remains to be elucidated. One possibility is that CPTH peptides directly interact with VDCCs, leading to their opening. In this regard, it is known that VDCC -subunits can directly interact with different extracellular agonists and antagonists with subsequent opening or closing, respectively. For example, BAY K8644 acts as an L-type Ca²⁺ channel agonist (29), whereas nifedipine, agatoxin, verapamil, and conotoxin act as antagonists (17). Alternatively, messenger molecule(s), produced in response to CPTHR stimulation, may interact with VDCC proteins to elicit subsequent opening of the VDCC channels. One example of this indirect mechanism is the G protein-dependent activation of VDCCs that occurs via interaction of liberated G protein -subunits with VDCCs (19). There is no evidence to date implicating G protein activation in signal transduction by CPTHRs, however.

Although the mechanism of CPTH receptor/VDCC interaction is unclear, there is strong evidence that CPTH receptors and VDCCs are jointly involved in mediating certain cellular processes in bone cells, such as intercellular communication (5, 15). For example, stimulation of osteocytes and osteoblasts with CPTH promotes cell-to-cell communication (25). Similarly, in human osteoblastic cells, gap junction-dependent intercellular communication also required activation of VDCC L-type calcium channels and subsequent influx of extracellular calcium (15). The propagation of gap junction-dependent intercellular communication was abolished by chelation of extracellular calcium and by VDCC blockers (15). Collectively, these observations suggest that CPTHR and VDCCs might be components of the same regulatory pathway.

In conclusion, calcium influx through VDCCs is involved in CPTHR signaling, and this signaling process is potentially involved in regulation of osteocyte function.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institutes of Health Grants AR-47062 (to F. R. Bringhurst), DK-02889, and DK-65032 (to P. Divieti) and an educational grant from NPS Pharmaceuticals.

	ACKNOWLEDGMENTS

 
We thank J. T. Potts, E. Nemeth, and K. Krapcho for discussion, advice, and critical reading of this manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: P. Divieti, Endocrine Unit, W501, Massachusetts General Hospital, Boston, MA 02114 (e-mail: divieti{at}helix.mgh.harvard.edu)

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.

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