0099-2399/93/1909-0462/$03.00/0 JOURNAL OF ENDOOONTICS Copyright © 1993 by The American Association of Endodontists

Printed in U.S.A. VOL. 19, NO. 9, SEPTEMBER 1993

Responses of Osteoblastic Cells (UMR 106) Exposed to Elevated Extracellular Calcium

Craig S. Haga, DDS, MS, and Paula H. Stern, PhD

Bone cells can be exposed to high calcium in the course of endodontic treatment. To investigate the effects of high calcium on bone cell function, re- sponses of a rat osteoblast cell line (UMR 106) were examined. Responsiveness of the cells to parathy- roid hormone, prostaglandin FI=, and ionomycin was assessed by measuring calcium transients elicited by these stimuli. Raising the medium calcium from 1.8 to 50 mM did not alter cell responsiveness. Pre- treatment of the cells with the calcium pump inhibitor sodium vanadate prevented parathyroid hormone effects and slightly decreased prostaglandin FI, el- fects in both normal and high calcium. The effect of ionomycin was prolonged in high calcium when van- adate was present. The results suggest that cells of the osteoblast phenotype can maintain calcium sig- naling in the presence of high extracellular calcium. These processes could play a role in the therapeutic effectiveness of high calcium in endodontic treat- ment.

Calcium hydroxide is commonly used in endodontic treat- ment to prevent root resorption (l). In addition, it is applied to promote restoration and prevent loss of mineralized tissue when iatrogenic perforation has occurred (2). These proce- dures could potentially expose bone cells to high concentra- tions of calcium. To determine whether bone cells are able to respond normally in the presence of high calcium, we exposed clonal osteosarcoma cells to markedly elevated (50 mM) cal- cium concentrations and examined their ability to generate rapid signals to physiological stimuli. The physiological agents whose effects were investigated were parathyroid hormone (PTH), which increases bone resorption (3) and can inhibit or increase bone formation (4, 5) and prostaglandin F~, (PGF), which is a poor stimulator of resorption (6), but can increase bone formation in vivo (7).

MATERIALS AND METHODS

Cell Culture

UMR 106 osteoblast-like osteosarcoma cells were grown in 75-cm 3 plastic flasks at 37"C in a humidified 5% CO2 atmos-

462

phere in Dulbecco's modified Eagles medium supplemented with 2.8 mM L-glutamine, 15% heat-inactivated horse serum, and 100 units/ml sodium-penicillin G. The medium was changed every 3 days. The cells reached confluence within 5 to 7 days and were then used in the experiment.

Intracellular Calcium Measurements

Cells were harvested from culture dishes by rapid (3-min) treatment with 0.01% trypsin and 0.02% EDTA in a phos- phate-buffered saline. The cells were then washed and loaded with 2 ~m fluo-3-acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min at room temperature in a buffer containing 145 mM NaCI, 5 mM HC1, 1.8 mM, or 50 mM CaCI2, 1 mM MgC12, 10 mM glucose, 10 mM HEPES, 1% bovine serum albumin, and 2.5 mM probenecid. The buffer was adjusted to pH 7.4. The cells were washed twice after the dye was loaded and then resuspended in a loading buffer. Approximately 0.5 to 1.5 million cells/ml were used for each experiment. Two milliliters of the cell suspension were added to a continuously stirred cuvette. A Perkin-Elmer LS-5B luminescence spectrophotometer was used to measure the fluorescence. Excitation was at 505 nm; emission was meas- ured at 530 nm (8).

The parathyroid hormone was bovine 1-34 peptide (Bachem, Torrance, CA). The stock solution concentration was l0 -5 M dissolved in l mM HCI. Prostaglandin F~o (Sigma, St. Louis, MO) stock solution concentration was 1 mM in 100% ethanol. The ionomycin (Calbiochem, La Jolla, CA) stock solution was l mM dissolved in 100% ethanol Vanadate (Sigma) stock solution was 1 mM in deionized water.

Statistics

Univariate statistics were used to analyze the responses to multiple exposures to PTH, PGF, or ionomycin within a single experiment. The mean, standard error, and the coeffient of variation were recorded. To compare the responses to PTH, PGF, or ionomycin in low and high calcium between experi- ments, the unpaired t test was used.

RESULTS

PTH, PGF, and ionomycin all elicited rapid calcium tran- sients in UMR 106 cells (Fig. 1). Similar responses were

Vol. 19, No. 9, September 1993

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FIG 1. Responses of UMR 106 cells in 1.8 mM calcium (above) and 50 mu calcium (below). Cells were incubated as described in Materials and Methods. Cell concentration was 0.75 million cells/ml. Additions were as follows: PTH, 15 #1 of 10 -s M; PGF~,, 10 pJ of 10 -3 M; ionomycin, 3 #1 of I M. Fluorescence was monitored at 530 rim, with excitation at 505 nm.

TABLE 1. Reproducibility of responses from a cell suspension in a single experiment (50 mm calcium concentration)*

Run PTH PGF1. Ionomycin

1 6.2 16.5 13.6 2 5.1 17.6 14.0 3 4.3 19.4 17.2 4 3.9 22.2 18.1 5 2.8 18.8 14.0

Mean _+ SE 4.46 _+ 0.57 14.9 .+_+ 3.21 15.4 _+ 0.9

Coefficient of variation 0.28 0.48 0.94

* Values represent the change in fluorescence in A arbitrary units.

obtained in both normal (1.8 mM) and high (50 mM) calcium. Responses to each treatment were highly reproducible. Table 1 shows the effects of PTH, PGF, and ionomycin added to five separate cuvettes of cells within a single experiment. All of the measurements from this experiment are shown. Table 2 shows the reproducibility of the response between experi- ments. No statistically significant differences were observed between responses in high and low calcium.

The similarity of the responses in high and low calcium suggest that the UMR cells have highly responsive pumping mechanisms which maintain intracellular calcium concentra- tions. To further examine this, NaVO3, an inhibitor of the

Calcium Effects on Cells 463

TABLE 2. Reproducibility of responses between experiments*

Calcium Coefficient Treatment Mean SE

(mM) of Variation

PTH 1.8 5.10 0.84 0.405 PTH 50 5.17 1.52 0.778

PGFI= 1.8 14.61 2.61 0.506 PGFI= 50 14.07 3.31 0.623

Ionomycin 1.8 19.51 3.41 0.463 Ionomycin 50 19.25 4.36 10.561

"Data are means of responses from three experiments. Values repreee~t the changes in fluoresc~xm in A arbitrary units.

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FIG 2. Effects of NaVO3 on the responses of UMR cells in 1.8 mM calcium (above) and 50 mM calcium (below). Cell concentration was 0.79 million cells/ml. NaVO3, 2/d of 1 M, was added where indicated. Other treatments were as described for Fig. 1.

ATP-dependent calcium uptake into intracellular stores (9) was used. NaVO3 inhibited the effect of PTH and slightly decreased the response to PGF (Fig. 2). In addition, in the high calcium the fluorescent signal after treatment with io- nomycin failed to recover normally; a higher baseline level of calcium was obtained (Fig. 2).

No transients were elicited by addition of the ethanol solvent (data not shown).

DISCUSSION

With the use of high concentrations of calcium hydroxide in endodontic procedures, various cells can potentially be

464 Haga and Stern

exposed to high calcium. In the current study we examined the effects of high calcium on bone cells of the osteoblast phenotype, since the beneficial effects of calcium hydroxide to induce formation of mineralized tissue could be due to the actions of the high calcium on this cell type.

Cell function was determined by measuring intracellular calcium signaling in response to hormonal stimuli. Calcium is an important cellular second messenger (10). Signaling is initiated when a hormone or a local factor binds to a mem- brane receptor. This can open a channel to allow influx of extraceilular calcium. Alternatively, receptor activation can cause the hydrolysis of membrane phospholipids, causing release of a molecule, inositol-l,4,5-PO4, which in turn re- leases calcium from intracellular stores.

The UMR 106 cell line was used because it is a well- established clonal cell line having the osteoblast phenotype. The dye, fluo-3, fluoresces when bound to calcium. The dye is introduced into the cell as the acetoxymethyl ester which is cleaved by intracellular esterases (I 1). In this way, intracellular calcium can be measured by the fluorimeter. Fluo-3 has a high fluorescence yield at wavelengths in the visible range when bound to calcium (11). This is advantageous because it avoids interference from other cell constituents that fluoresce in the ultraviolet range. Disadvantages with the use of fluo-3 are that there cannot be a calibration of absolute concentra- tion with the dye because there is no way to saturate the dye. Also, there is no shift in wavelength when calcium is bound to the dye, and thus ratios cannot be used for calibration (11 ). Therefore, we have used the standard procedure for this dye (11) of measuring the change in arbitrary units to determine the responses of the cells to the treatments.

A medium calcium concentration of 1.8 mM in the presence of 1 mg/ml bovine serum albumin was used to simulate physiological extracellular calcium. In normal bone metabo- lism the osteoclasts and osteoblasts are constantly resorbing and forming bone. When the osteoclasts resorb bone they release the calcium from the bone and thus can raise the extracellular calcium. There is evidence that the concentration of the calcium at the site of remodeling is around 50 mM under acidic conditions (12). The 50 mr, l extracellular calcium concentration was used to simulate this high calcium environ- ment.

Parathyroid hormone and prostaglandin FI~ both increase intracellular calcium by allowing entry of extracellular cal- cium and releasing calcium from intracellular stores (13-15). Ionophores increase the passive flux of calcium by combining it into a membrane-permeable complex (16). When intracel- lular calcium is released, various active transport mechanisms rapidly pump calcium from the cell to maintain a normal intracellular concentration (I 7).

Responses to PTH, PGF~, and ionomycin in UMR cells were quite similar at 1.8 mr, t or 50 mM calcium. These effects were highly reproducible within and between experiments. In the presence of NaVO3, which inhibits ATP-dependent cal- cium transport from the cytoplasm, the cells did not fully recover from ionomycin stimulation in high calcium. Under these conditions, cells were able to pump some of the calcium out of the cell, but the calcium failed to return to the baseline. In low calcium, the cell were able to return the response to the normal baseline probably because the gradient was not as great. The observation that some responses were normal in the presence of NaVO3 probably indicates that transport

Journal of Endodontics

systems other than those sensitive to NaVO3 are involved in the removal of calcium from the cytoplasm. The mechanism of the impaired response to PTH in presence of NaVO3 is not known. NaVO3 has been shown to affect osteoblasts, promot- ing mitogenesis and inhibiting osteoblastic acid phosphatase- like phosphotyrosyl protein phosphatase activity (18). In these properties, NaVO3 acted similarly to sodium fluoride which inhibits parathyroid hormone effects on bone (19). The greater sensitivity of PTH than of PGF to NaVO3 suggests that the mechanisms of the two stimulators may differ.

The results demonstrate that cells of the osteoblast pheno- type exhibit a remarkable ability to withstand elevated cal- cium. Studies to determine whether the effects seen in the UMR 106 ceils can be reproduced in normal bone cells will be of interest. Although the findings do not reveal how elevated calcium can lead to mineralization they do indicate that normal cellular signaling, which may be involved in the mineralization process, can be elicited under these conditions. The findings are of potential relevance for the efficacy of calcium hydroxide as used in endodontic procedures.

This research was supported by a great from the Research and Education Foundation of the American Association of Endodontics (to C. S. H.), a graduate student award from Northwestern University Dental school (to C. S. H.), and NIH Grant AR 11262 (to P. H. S.). The technical assistance of Agnes Tatrai is gratefully acknowledged. We thank Dr. Edward Osetek for his support and interest.

Dr. Haga is a member of the Department of Graduate Endodontics and Dr. Stem is a member of the Department of Pharmacology, Northwestern Univer- sity, Chicago, IL. Address requests for reprints to Dr. Paula Stem, Department of Pharmacology, Northwestem University, 303 E. Chicago Avenue, Chicago, IL 60611.

References

1. Andrea.sen JO. Treatment of fractured and avulsed teeth. J Dent Child 1971 ;38:29-49.

2. Heitharsay GS. Stimulation of root formation in incompletely developed pulpless teeth. Oral Surg 1972;29:620-30.

3. Ralsz LG. Bone resorption in tissue culture. Factors influencing the response to parathyroid hormone. J Clin Invest 1965;44:103-16.

4. Kream BE, Rowe DW, Gworek SC, Raisz LG. Parathyroid hormone alters collagen synthesis and proootlagen mRNA levels in fetal rat calvada. Proc Natl Acad Sci USA 1980;77:5654-8.

5. Hock JN, Fonseca J. Anabolic effect of human synthetic parathyroid hormone-(1-24) depends on growth hormone. Endocrinology 1990; 127:1804- 10.

6. Klein DC, Raisz LG. Prostaglandin: stimulation of bone resorption in tissue culture. Endocrinology 1970;85:657-765.

7. Norrdin RW, Jee WSS, High WB. The role of prostaglandin in bone in vivo. Prostaglandins Leukot Essent Fatty Acid 1990;41:139-49.

8. Tatrai A, Lakatos P, Thompson S, Stem PH. Effects of endothelin-1 on signal transduction in UMR-106 osteoblastic cells. J Bone Miner Res 1992;7:1201-9.

9. Fohr K J, Wahl Y, Engling R, Kemmer JP, Granzel M. Decavanadate displaces inositol 1,4,5-tdphosphate (IP3) from its receptor and inhibits IP3 induced calcium release in permealized pancreatic acinar call. Cell Calcium 1991 ;12:735-42.

10. Exton JH. Mechanisms involved in calcium-mobilizing angonist re- sponses. Adv Cyclic Nucieotide Protein Phosphorylation Res 1986;20:211-62.

11. Minta A, Kao JPY, Tsien RY. Fluorescent indicators of cyctosolic calcium based on rhodamine and fluorescain chromophores. J Biol Chem 1989;264:8171-8.

12. Eeckhout Y, Delaisse J-M, Ledent P, Vaes G. The proteinases of bone resorption. In: Glanert AM, ed. The control of tissue damage. Amsterdam: Elsevier, 1988:297-313.

13. Reid IA, Civitelli R, Halstead LR, Avioli LV, Hruska KA. Parathyroid hormone acutely elevates intracellular calcium in osteoblastic cells. Am J Physiol 1987;252:E45-E51.

14. Yamaguchi DT, Hahn T J, tida-Klein A, Kleeman CR, Muellem SJ. Parathyroid hormone-activated calcium channels in an osteoblast-like clonal

Vol. 19, No. 9, September 1993

call line: cAMP-dependant and cAMP-independent calcium channels. Biol Chem 1987;262:7711-8.

15. Yamaguchi DT, Hahn TJ, Beeker PG, Kleeman CR, Muallem S. Rela- tionship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells. J Biochem 1990;263:10745-53.

16, Stem PH. Ionophores: chemistry, physiology and potential applications to bone biology. Clin Orthop Ret Res 1977;122:273-98.

Calcium Effects on Cells 465

17. Carafoli E. The calcium pumping ATPase of the plasma membrane. Annu Rev Physio11991 ;53:531-7.

18. Lau KHW, Tanimoto H, Baylink DJ. Vanadate stimulates bone pro- liferation and bone collagen synthesis in vitro. Endocrinology 1988;123: 2858-67.

19. RaJsz LG, Taves DR. The effect of fluoride on parathyroid function and responsiveness in the rat. Calcif Tissue Res 1967;1:219-28.

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