Activation of L-type Calcium Channels Is Required for Gap Junction-mediated Intercellular Calcium Signaling in Osteoblastic Cells*

Niklas Rye JørgensenDagger §, Stefan Cuoni TeilmannDagger , Zanne HenriksenDagger , Roberto Civitelli, Ole Helmer SørensenDagger , and Thomas H. Steinberg

From the Dagger  Osteoporosis and Metabolic Bone Unit, Department of Endocrinology, Copenhagen University Hospitals, Copenhagen Hospital Corporation DK-2650 Hvidovre, Denmark and the  Department of Internal Medicine, Washington University School of Medicine, Washington University, St. Louis, Missouri 63110

Received for publication, June 13, 2002, and in revised form, November 5, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Connexin43 (Cx43)1-mediated gap junctional communication regulates bone matrix production in vitro (1) and is required for normal bone formation in vivo (2). Modulation of gap junctional communication in vitro alters osteocalcin production by transcriptional regulation. In vivo, detailed analysis of Cx43-deficient mice has revealed distinctive skeletal abnormalities. However, the mechanisms by which Cx43-mediated gap junctional communication alter bone formation are poorly understood. One such mechanism may be by the coordination of intercellular calcium signaling among osteoblastic cells. A well described in vitro model, and one which may mimic the effects of mechanical forces on bone cells, is that of mechanically induced intercellular calcium signaling or intercellular calcium waves (ICW).

Two mechanisms for the propagation of mechanically induced ICW in osteoblastic cells have been described. In the UMR 106-01 rat osteoblastic cell line, mechanical stimulation of a single osteoblast leads to propagation of fast (~15 µm/s) ICW that require activation of P2Y (purinergic) receptors on neighboring cells, presumably by released nucleotides, and subsequent generation of inositol trisphosphate and release of intracellular calcium stores. In the ROS 17/2.5 rat osteoblastic cell line, slow ICW require gap junctional communication. Unlike gap junction-mediated ICW that have been identified in many other cell models, gap junction-mediated ICW in ROS cells do not require release of intracellular calcium stores and do not require diffusion of IP3 through gap junction pores.

In human osteoblastic cells, both of these mechanisms for the propagation of ICW have been identified and have different kinetics. Mechanical stimulation of human osteoblast-like cells results in fast calcium waves that require activation of P2Y receptors. If these waves are blocked by desensitization of P2Y receptors, slow gap junction-dependent ICW are unmasked.

In this study we have investigated the mechanism of slow gap junction-dependent ICW in osteoblastic cells and found that these ICW involve plasma membrane depolarization, activation of L-type voltage-operated calcium channels, and influx of extracellular calcium.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Chemicals-- The rat osteoblastic cell lines, ROS 17/2.8 (ROS) and UMR 106-01 (UMR) and UMR cells transfected with the gene coding for the gap junction protein, connexin43 (UMR/Cx43) (3), were grown in Minimum Essential Medium supplemented with 10% heat-inactivated calf serum, non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and penicillin/streptomycin (Invitrogen). Geneticin (200 µg/ml) was added to the medium of UMR/Cx43 cells. The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C with medium change every 2-3 days.

Human osteoblastic cells were isolated from human bone marrow obtained from healthy volunteers, age 20-36, by puncture of the posterior iliac spine. All participants had read and signed informed consent, and the study was approved by the local ethics committee. The marrow material was collected in phosphate-buffered saline with Ca and Mg (Invitrogen) containing 100 units/ml Heparin (Sigma). The mononuclear fraction of the marrow was isolated on a Lymphoprep (Nycomed) gradient and plated. The next day the supernatant was aspirated to remove the non-adherent cells. Adherent cells were then grown in Minimal Essential Medium without phenol red (Invitrogen), supplemented with 10% heat-inactivated fetal calf serum and penicillin/streptomycin. The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C, with medium change every 7 days. After 4-5 weeks of culture when cells reached ~80% confluence, the medium was supplemented with 100 nM dexamethasone (Sigma) for 7-10 days to enhance osteoblastic differentiation. For calcium imaging experiments, cells were plated on 25-mm No. 1 glass coverslips for 2-4 days and used at 70-90% confluence.

The fluorescent calcium indicator, fura-2, was used for calcium imaging and measurements and purchased from Molecular Probes (Eugene, OR). Nucleotides were from Roche Molecular Biochemicals. The endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was purchased from Calbiochem and used in a final concentration of 50 nM. All other chemicals were from Sigma. The gap junction inhibitor, heptanol, was made fresh on the day of experiment as a 1:4 heptanol:ethanol solution and used in a final concentration of 3.5 mM. alpha -glycyrrhetinic acid (AGA), also a gap junction inhibitor, was used in a final concentration of 2 or 5 µM. Nifedipine, an inhibitor of L-type voltage-operated calcium channels (VOCC) was used in a concentration of 1, 5, or 10 µM.

Calcium Imaging-- Measurement of intracellular calcium concentration was performed using the fluorescent calcium indicator, fura-2. Cells in monolayers adherent to non-coated glass coverslips were incubated at 37 °C in Minimum Essential Medium containing 5 µM fura-2/AM for 30 min and then incubated in fresh medium without dye for an additional 20 min. Coverslips were affixed to a teflon chamber and mounted in a PDMI-2 open perfusion microincubator (Medical Systems Corp., Greenvale, NY), maintained at 37 °C with superfused CO2, on a Zeiss Axiovert 135 microscope. Imaging was performed with a Metamorph/Metafluor imaging system (Universal Imaging, West Chester, PA), with excitation wavelengths of 340 and 380 nm for acquiring ratio images of fura-2. Probenecid (Sigma) at a concentration of 1 mM was added throughout the experiments to prevent dye leakage. Intercellular calcium waves were initiated by mechanical stimulation of a single cell using a borosilicate glass micropipette affixed to an Eppendorf 5171 micromanipulator.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ROS Cells, UMR/Cx43 Cells, and Human Osteoblastic Cells Propagate Intercellular Calcium Waves with Different Kinetics-- Intercellular calcium waves were initiated by mechanically stimulating single cells in monolayers. As previously shown, stimulation of a single ROS cell caused a rise in the intracellular calcium concentration in the stimulated cell. Subsequently, an intercellular calcium wave was transmitted to neighboring cells with a time lag of 15-30 s (Fig. 1). This intercellular calcium wave propagated to 2.4 cells and with slow kinetics (wave propagation of 2-4 µm/s) as previously defined (1). In UMR/Cx43 cells, mechanical stimulation of a single cell initiated intercellular calcium waves as well, but these waves were faster (15 µm/s) and propagated to an average of 23 cells with only a short time lag between neighboring cells (Fig. 1). These kinetics are similar to those previously defined in the parental UMR cells and have previously been shown to require activation of P2Y receptors but not gap junctional communication. In human osteoblastic cells, the mechanically induced intercellular calcium waves propagated with a velocity of 10-12 µm/s and extended to 7.6 cells with an intermediate time lag between cells.


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Fig. 1.   Intercellular calcium waves in ROS 17/2.8, UMR/Cx43, and human osteoblastic cells (HOB). A single cell was stimulated mechanically, causing an increase in the intracellular calcium concentration, which subsequently propagates to adjacent cells. Numbers indicate seconds after mechanical stimulation. Scale bar indicates relation between pseudocolors and intracellular calcium concentrations. Note the differences in kinetics for the propagation of the waves in the three cell types.

ROS Calcium Waves Require Intact Gap Junctional Communication-- The gap junction inhibitor AGA was used to confirm that inhibition of gap junctional communication blocked the spread of intercellular calcium signals in ROS cells. Monolayers of ROS cells were loaded with fura-2, and ICW were induced before and after the addition of 2 or 5 µM AGA. Mechanically induced ICW extended to 2.8 cells before addition of AGA and to 0.3 cells after AGA was added (Fig. 2). No difference was seen between 2 or 5 µM AGA. After washing the AGA away with phosphate-buffered saline containing 0.2% bovine serum albumin, mechanically induced ICW were partially restored, propagating to 1.4 cells. AGA did not inhibit mechanically induced ICW in UMR cells; waves propagated to 17 cells before adding AGA and to 24 cells after adding AGA (2 µM) to the medium.


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Fig. 2.   The maximum calcium wave extension in ROS 17/2.8 cells and in a primary culture of human osteoblastic cells with and without ATP (100 µM) desensitization of the P2Y receptors, in columns named HOB and HOB (after ATP), respectively. The effectS of different inhibitory conditions are shown. ATP, desensitization of purinergic P2Y receptors by adenosine-triphosphate. AGA, the gap junctional inhibitor alpha -glycyrrhetinic acid (2 µM). low [Ca2+]o, extracellular calcium is removed. Nifedipine, a blocker of L-type VOCC (1 µM). n.d., not done.

The gap junction inhibitor heptanol was also used to confirm the involvement of gap junctions in the propagation of ICW in osteoblastic cells. The initial untreated wave in the ROS cells extended to an average of 2.8 cells. Freshly mixed heptanol was added to the bathing solution in a final concentration of 3.5 mM. After 5 min, ICW were almost totally blocked, with a calcium signal propagated only to 0.3 cells. As with AGA, wave propagation could be partially restored by removing heptanol (1.3 cells/wave). Thus, the propagation of intercellular calcium waves in ROS cells, but not in UMR cells, requires intact gap junctional communication, in agreement with previous data (4).

ICW in ROS Cells, but Not in UMR Cells, Require Influx of Extracellular Calcium through L-type Voltage-gated Calcium Channels-- We have previously shown that intracellular calcium stores are not required for the propagation of ICW in ROS cells (4). To confirm that gap junction-dependent ICW required influx of extracellular calcium, ICW were assessed after the bathing medium was exchanged for calcium-free medium (Fig. 2). ICW propagation was assessed within 2 min of medium exchange; at this time the cells retained ionomycin-releasable intracellular calcium stores (not shown). In contrast, P2-dependent ICW in UMR cells extended farther in calcium-free medium than in calcium-containing medium (48 cells/wave versus 30 cells/wave).

The above results implied that a plasma membrane calcium channel was involved in gap junction-dependent ICW in ROS cells. To assess the involvement of L-type calcium channels in the wave mechanism, monolayers of ROS cells were treated with nifedipine, an antagonist of L-type voltage-gated calcium channels. Nifedipine was used in a final concentration of 1, 5, or 10 µM. At all concentrations, nifedipine inhibited ICW propagation (Fig. 2). Replacing the bathing solution with nifedipine-free medium restored the ability of the cells to propagate ICW. In contrast, UMR waves were not inhibited by nifedipine treatment, and in fact propagated farther: 28 cells/wave before addition of nifedipine and 50 cells/wave in the presence of nifedipine.

ROS Calcium Waves Require Membrane Depolarization-- Because L-type calcium channels are activated by membrane depolarization, prior membrane depolarization would inhibit ICW by preventing membrane depolarization at the time of ICW generation. For these experiments, ROS cells were depolarized using medium containing 70 mM KCl. Before depolarization, ICW propagated to 2.6 cells/wave. After the cells were switched to the high potassium medium, there was a calcium response in all cells because of depolarization-induced activation of VOCC. After the cells had returned to resting [Ca2+]i, another mechanical stimulus was applied, and the wave now propagated only to 0.6 cells. Changing back to the original medium with physiological potassium concentration restored ICW propagation (1.8 cells/wave). In UMR cells, this protocol again had the reverse effect on ICW propagation; the number of cells in the wave increased from 21 to 33, similar to the result seen after nifedipine treatment or removal of extracellular calcium.

Nifedipine-sensitive ICW Require Gap Junctional Communication-- Because ICW in ROS cells require gap junctional communication, the above data suggest that the L-type calcium channel activation responsible for ICW is dependent on gap junctional communication in ROS cells. We tested this hypothesis by examining ICW in UMR/Cx43 cells, which express both their native P2Y2 receptors and transfected Cx43 gap junction protein (4). We first confirmed that the fast calcium wave in these cells, like UMR ICW, did not require extracellular calcium as follows. Single cells in fura-2-loaded monolayers of UMR/Cx43 cells were stimulated mechanically. A fast wave was seen, similar to the wave in untransfected UMR cells. The wave spread to an average of 24 cells. Next, the medium was exchanged for calcium-free medium, and ICW were assessed within 2 min. In calcium-free medium the wave spread to an average of 34 cells/stimulated cell.

Next, we confirmed that the slow ICW unmasked by desensitization of ATP receptors in UMR/Cx43 required gap junctional communication. The fast P2 receptor-dependent ICW were blocked by desensitizing the receptors with 100 µM ATP, which inhibits further response to ATP without depleting intracellular calcium stores (4). Mechanical stimulation of single UMR/Cx43 cells now revealed a slow ROS-like wave that extended to 3.0 cells. This slow ICW was absent in untransfected UMR cells similarly treated. Finally, AGA (2 µM) was added to the bathing solution of UMR/Cx43 cells, and upon mechanical stimulation ICW were almost completely blocked (0.5 cells in wave). Again, the AGA effect was reversed by washing the cells in solution not containing AGA (2.3 cells in wave).

Finally we asked whether these slow ICW, which were present in UMR/Cx43 but not in UMR, required L-type calcium channels and plasma membrane depolarization. When ICW were elicited in UMR/Cx43 cells in medium containing 1 µM nifedipine before desensitization of P2 receptors, ICW propagated to 30 cells compared with 25 cells without nifedipine. In high potassium experiments performed as described above, adding potassium increased the wave from 31 cells before to 36 cells after. In contrast, when these experiments were performed in UMR/Cx43 after desensitization of P2 receptors, both nifedipine- and potassium-induced membrane depolarization blocked the slow gap junction-dependent ICW.

Slow ICW in Human Osteoblasts Require Activation of L-type Calcium Channels-- Human osteoblastic cells propagate both fast P2 receptor-dependent ICW and slow gap junction-dependent ICW (5). As was seen in UMR/Cx43 cells, 5 µM AGA did not block fast ICW propagation in the human cells; fast ICW propagated to 7.6 cells before addition of AGA and 6.9 cells after addition of AGA. Addition of ATP to human osteoblastic cells resulted in a widespread calcium response and desensitization so that subsequent addition of ATP did not cause calcium transients. After ATP desensitization, mechanical stimulation elicited a slow ICW that propagated to an average of 4.7 cells. The slow ICW were inhibited by 5 µM AGA, confirming that the slow ICW unmasked after desensitization of P2 receptors require gap junctional communication.

We then asked whether gap junction-dependent ICW seen in ATP-desensitized human osteoblastic cells were inhibited by removal of extracellular calcium, plasma membrane depolarization by high external potassium, or VOCC inhibitors as summarized in Table I. Removal of extracellular calcium resulted in inhibition of the slow wave (Fig. 2). Slow ICW were also blocked by the L-type calcium channel inhibitors nifedipine (Fig. 2), verapamil, or nitrendipine or membrane depolarization in high extracellular potassium (Fig. 3). To confirm these treatments did not inhibit the P2Y-dependent wave, experiments were performed in cells that were not exposed to ATP. No inhibition of fast ICW was seen after adding nifedipine in a final concentration of 1 µM to the bathing solution, with an average extent of the wave to 4.9 cells in the wave before nifedipine and 5.1 cells in the wave after nifedipine (Fig. 2).

                              
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Table I
Maximal extent of calcium transients expressed as number of cells in wave
Maximal number of cells in a wave after adding different agents. ND, not done; n, number of experiments; S.E., standard error of the mean.


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Fig. 3.   The average number of cells in calcium waves in human osteoblastic cells after ATP desensitization and subsequent blocking of either L-type VOCC by nifedipine (1 µM) or depolarizing the plasma membrane by increasing extracellular potassium concentration to 70 mM.


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Fig. 4.   A schematic presentation of the two mechanisms for mechanically induced calcium wave propagation in human cells. Top panel, a mechanical stimulus increases intracellular calcium concentration in the stimulated cell. A diffusible messenger traverses the gap junctions to the neighboring cell with subsequent membrane depolarization. Activation of VOCC mediates extracellular calcium entry into the cytosol, thereby increasing the intracellular calcium concentration. Bottom panel, mechanical stimulation of a single cell with subsequent increase in intracellular calcium concentration. Nucleotides (probably ATP) are released to the extracellular space, binding to P2 receptors on the neighboring cell. Inositol trisphosphate (IP3) is generated in these cells, binding to receptors of the endoplasmic reticulum, releasing calcium from the intracellular stores. Cx43 indicates high permeability gap junctions containing connexin 43.

Gap Junction Hemichannels Are Not Required for ICW Propagation-- In low extracellular calcium, some cells form gap junction hemichannels at the plasma membrane that may mediate release of ATP and propagation of ICW that appear to be gap junction-dependent (6). We asked whether osteoblastic cells express functional gap junction hemichannels by monitoring uptake of the fluorescent dye Lucifer Yellow added to the extracellular medium. In ROS cells and UMR/Cx43 cells no uptake of dye was seen in medium containing either normal calcium or no extracellular calcium (data not shown). In human osteoblastic cells, no Lucifer Yellow uptake was seen in medium containing extracellular calcium. However, in calcium-free medium, Lucifer Yellow uptake was seen in ~10% of cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These studies show that slow gap junction-mediated intercellular calcium waves in osteoblastic cells require the influx of extracellular calcium through L-type VOCC, as summarized in Table I. The propagation of gap junction-dependent ICW was abolished in the absence of extracellular calcium. Depolarization of the plasma membrane with high potassium inhibited the waves, presumably by preventing the activation of L-type calcium channels by rapid changes in the plasma membrane potential. Finally, L-type calcium channel antagonists blocked wave propagation. Nifedipine in concentrations as low as 1 µM inhibited ICW; electrophysiological studies have shown that nifedipine concentrations of 10 µM selectively blocked the L-type VOCC with no effects on T-type VOCC in osteoblastic cells (7).

Although it is unclear what is diffusing through gap junctions to mediate these ICW, the requirement for plasma membrane depolarization and activation of VOCC suggests that ionic traffic through gap junctions may be responsible (Fig. 4). This mechanism would be reminiscent of the transmission of calcium transients in excitable tissues such as cardiac myocytes. However, the ICW in osteoblastic cells are much slower than in excitable tissues, suggesting some fundamental difference in mechanism or in receptor density.

This model for the propagation of gap-junction-dependent ICW differs from the model for ICW propagation originally proposed for respiratory epithelia and invoked for other cell types, including hepatocytes and glial cells. In this previously described model, ICW are propagated by diffusion of inositol trisphosphate through gap junction channels and subsequent release of intracellular calcium stores (8, 9). Although ROS cells are well coupled by gap junctions comprised of Cx43, they do not propagate mechanically stimulated ICW via IP3 diffusion, demonstrated by the observation that depletion of intracellular calcium stores has no effect on mechanically induced ICW propagation in these cells (4). It is unclear why these cells do not propagate ICW by diffusion of IP3 through gap junctions. One possible explanation is that osteoblastic cells may not express sufficient levels of Cx43 to permit the amount of IP3 diffusion that is required for these ICW to occur. Another possibility is that different connexins may be required to allow ICW mediated by IP3 diffusion.

In primary osteoblastic cells, ICW that require IP3-mediated release of intracellular calcium stores can occur. However, these ICW do not require gap junctional communication; they involve activation of P2 receptors by extracellular ATP. This mechanism for ICW propagation occurs in many cells because G protein-coupled P2Y receptors are widely expressed. These P2-mediated ICW may complicate analysis of ICW propagation and must be excluded before ICW can be attributed to gap junctional communication. We have previously shown that monolayers of hamster tracheal epithelia (4) and mouse nasal epithelia (10) propagate IP3-dependent ICW that do not require gap junctional communication but do require activation of P2 receptors.

In glioma cells, connexins participate in P2 receptor-mediated ICW by forming plasma membrane hemichannels that release ATP (6). We could not detect gap junction hemichannel formation in the rat osteoblastic cell lines, and in human osteoblastic cells we demonstrated hemichannel formation in only 10% of cells and only under low calcium conditions. It is therefore unlikely that hemichannels play a role in the gap junction-mediated ICW seen here. If gap junction hemichannels did participate in osteoblast ICW, they would have to do so by mediating calcium influx rather than by mediating ATP release.

These studies demonstrate that gap junction-mediated ICW in ROS cells are a good model for the gap junction-dependent coordination of ICW that occurs in human osteoblastic cells. Intercellular calcium signaling in human osteoblastic cells is complex because it involves both gap junction-dependent and P2Y-receptor-dependent mechanisms, but the two mechanisms can be studied independently, as demonstrated in this study and our previous work on ICW in human osteoblastic cells and can be modeled independently using the ROS and UMR rat osteoblastic cell lines.

In contrast to gap junction-dependent ICW, P2Y-dependent ICW in UMR cells and primary osteoblastic cells were actually increased after removal of extracellular calcium, VOCC inhibition, or addition of potassium to depolarize the cells. These fast P2Y-mediated waves therefore seem to be inhibited by calcium influx via VOCC activation for reasons that are not readily apparent.

Gap Junction-mediated intercellular communication is important for production of bone matrix proteins in vitro (1) and for normal bone development in vivo (2). The current studies suggest that activation of L-type calcium channels might be an important component of the signaling pathway by which gap junctional communication influences bone development or turnover. The current work highlights the importance of L-type VOCC in osteoblast calcium signaling and suggests the possibility that regulation of the expression and activity of VOCC may be important in modulating the coordination of calcium signaling that occurs among osteoblasts. Osteoblasts have been demonstrated to express plasma membrane VOCC that can mediate calcium entry (11, 12). VOCC have been implicated in regulation of protein expression in bone cells. Applying strain to osteoblasts in vitro in the presence of Bay K 8644, an agonist of L-type VOCC, has been shown to increase the expression of certain proteins specific for bone formation, such as osteopontin and osteocalcin (13). In vitro fluid shear stress applied to the human osteosarcoma cell line SaOS-2 increased TGF-beta mRNA expression (14), a growth factor involved in bone formation. Verapamil, an antagonist of L-type VOCC, inhibited this increase. Another L-type VOCC antagonist, nifedipine, prevented loading-related increases in prostaglandin E2, nitric oxide, and glucose-6-phosphate dehydrogenase in rat osteoblasts, but not in osteocytes (15). It is possible that the ICW characterized here are involved in these processes.

In conclusion, we have shown that the gap junction-dependent propagation of intercellular calcium signals in ROS rat osteoblastic cells requires influx of extracellular calcium through L-type VOCC and that the gap junction-dependent component of intercellular calcium signaling in human osteoblastic cells occurs by the same mechanism. These findings suggest that regulation of the expression or activity of L-type calcium channels may be a means by which calcium signaling is modulated in bone cells and that modulation of these channels may influence bone turnover.

    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.

§ To whom correspondence should be addressed: Osteoporosis Research Clinic, Dept. 545, Copenhagen University Hospital Hvidovre, Kettegaard Allé 30, DK-2650 Hvidovre, Denmark. Tel.: 45-36-32-32-32; Fax: 45-36-32-36-40; E-mail: niklas@dadlnet.dk.

Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M205880200

    ABBREVIATIONS

The abbreviations used are: Cx43, connexin43; ICW, intercellular calcium waves; AGA, alpha -glycyrrhetinic acid; VOCC, voltage-operated calcium channels.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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