This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mailland, M. Articles by Seuwen, K. Search for Related Content PubMed PubMed Citation Articles by Mailland, M. Articles by Seuwen, K. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL

Stimulation of Cell Proliferation by Calcium and a Calcimimetic Compound

Research, NOVARTIS Pharma AG, CH-4002 Basel, Switzerland; and Laboratoire de Neurobiologie Cellulaire et Moléculaire du Centre National de la Recherche Scientifique (M.R.), F-91198 Gif-sur-Yvette, France

Address all correspondence and requests for reprints to: Dr. Klaus Seuwen, Research, NOVARTIS Pharma AG, Building S-360/401, CH-4002 Basel, Switzerland. E-mail: Klaus.Seuwen{at}Pharma.Novartis.Com

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Some mesenchymal cells respond to stimulation by specific cations with increased cell proliferation. In the present study we have investigated whether the parathyroid/kidney/brain calcium-sensing receptor (PCaR) can mediate such mitogenic responses. We have expressed the recombinant rat PCaR in CCL39 hamster fibroblasts, which do not express a detectable endogenous cation sensor. The transfected cells responded to increased extracellular calcium concentrations ([Ca²⁺]_e) with strong inositol phosphate (IP) formation, which was insensitive to pertussis toxin treatment of cells. We could not detect negative coupling of the receptor to adenylyl cyclase. The calcimimetic NPS R-568 left-shifted the concentration-response curve for [Ca²⁺]_e-induced IP formation and increased the maximal response. In [³H]thymidine incorporation experiments, increasing [Ca²⁺]_e from 1 to 4 mM was found to stimulate DNA synthesis weakly, but significantly. A strong potentiation of this response was observed in the presence of NPS R-568. [Ca²⁺]_e and NPS R-568 also synergized to increase cell numbers in cultures maintained in defined medium. In contrast to our expectations, no significant stimulation of IP formation or cell proliferation could be observed after stimulation of cells with the reported PCaR agonist gadolinium (Gd³⁺) or with aluminum (Al³⁺), which stimulates osteoblast proliferation. Gd³⁺ actually inhibited IP formation stimulated by increased [Ca²⁺]_e as well as by thrombin and AlF₄^-, indicating toxicity. However, submaximal receptor stimulation by Gd³⁺ was evident when intracellular calcium transients were measured in fluo-3-loaded cells. Our data show that PCaR can stimulate cell proliferation when expressed in an appropriate cellular context. However, it is unlikely that PCaR mediates the strong mitogenic effects elicited by the cations Gd³⁺ and Al³⁺ observed in osteoblasts.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
OCCASIONAL reports showing increased cell proliferation in response to cations appeared many years ago (1, 2). The possible physiological role of such responses remained obscure. However, the observation that bone-forming osteoblasts respond to calcium and other cations (3, 4) provided a rational link to physiology; thus, this cation-sensing mechanism might participate in regulation of the skeletal apposition of calcium circulating in plasma.

The circulating levels of calcium are mainly controlled by parathyroid cells that secrete the calcium-mobilizing PTH in response to a lowering of plasma calcium (5). Brown and co-workers recently demonstrated (6) that parathyroid calcium sensing is accomplished by a G protein-coupled receptor, PCaR. This receptor is activated by increased extracellular calcium concentrations ([Ca²⁺]_e), but also responds to strontium, magnesium, and polycations such as neomycin B. In addition, transition metal ions such as Gd³⁺ have been reported to act as very potent agonists at this receptor (5, 6, 7, 8, 9). In fact, Gd³⁺ was used to obtain the bovine PCaR complementary DNA (cDNA) through expression cloning (6).

The present study was undertaken to investigate whether PCaR is able to trigger cell proliferation in mesenchymal cells. As a model system we chose CCL39 Chinese hamster lung fibroblasts, which are a well characterized system with respect to mitogenic signaling (10, 11). It should be noted that these cells, like many other fibroblasts, are relatively refractory to mitogenic stimulation by G protein-coupled receptors, activating exclusively phosphoinositide turnover (10, 11, 12, 13).

Experiments with bovine and human parathyroid cells have indicated that PCaR mediates the stimulation of phosphoinositide turnover and the inhibition of adenylyl cyclase, i.e. signals through both G_q and G_i proteins (5, 14). In CCL39 cells transfected with the rat PCaR cDNA (15, 16), we could not detect negative modulation of adenylyl cyclase, but we measured a strong coupling to phosphoinositide turnover that was further potentiated by the calcimimetic drug NPS R-568, a compound acting like a positive allosteric modulator of PCaR function (17). Increased [Ca²⁺]_e plus NPS R-568 strongly stimulated cell proliferation in a pertussis toxin (PTX)-insensitive manner.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
[2-³H]Adenine and [methyl-³H]thymidine were obtained from Amersham (Zurich, Switzerland). Myo-[³H]inositol from American Radiolabeled Chemicals was obtained through Anawa Trading (Wangen, Switzerland). Cell culture media were purchased from Bioconcept (Basel, Switzerland). Recombinant basic fibroblast growth factor (FGF) was obtained from Bachem (Bubendorf, Switzerland), and human -thrombin was purchased from Roche (Buchs, Switzerland). PTX, forskolin, fluo-3, and bovine insulin were purchased from Sigma (Buchs, Switzerland). Antibiotic G418 was obtained from Life Technologies (Basel, Switzerland). NPS R-568 was synthesized at Sandoz (Basel, Switzerland).

Cells and cell culture
CCL39 cells are an established line of Chinese hamster lung fibroblasts (American Type Culture Collection, Rockville, MD). The cells were routinely grown in a 1:1 mixture of DMEM and Ham’s F-12 (DMEM/F12) supplemented with 10% FCS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). This medium mixture contains 1.05 mM Ca²⁺. To obtain cells expressing the rat PCaR, CCl39 cells were transfected with the rat brain PCaR cDNA cloned into the pRK5 expression vector (15) using electroporation. Transfected cells were selected using antibiotic G418 (500 µg/ml).

Inositol phosphate (IP) and cAMP formation
Cells were seeded into 24-well plates and grown to confluence. For determinations of cAMP formation, cells were labeled with [³H]adenine (74 MBq/ml) in serum-free medium for 2–3 h. Thereafter, cells were washed twice in a phosphate-free HEPES-buffered salt solution that we call modified HBS or mHBS (130 mM NaCl, 5.4 mM KCl, 0.5 mM CaCl₂, 0.9 mM MgSO₄, 10 mM glucose, and 20 mM HEPES, pH 7.4). Phosphate was omitted to allow high concentrations of Ca²⁺ to be used in the assays. The relatively low concentration of basal calcium was chosen to maintain the PCaR safely in an inactive state before increasing [Ca²⁺]_e or adding other metal ions. We have verified that both adenylyl cyclase as well as phosphoinositide turnover activity can be measured normally under these assay conditions. In some experiments with Gd³⁺ (Figs. 2 and 3), MgCl₂ was substituted for MgSO₄, as sulfate ions may form insoluble complexes with Gd³⁺. To assess inhibition of adenylyl cyclase by cations or other agents, cells were incubated at 37 C in mHBS containing 5 µM forskolin, 1 mM isobutylmethylxanthine, and test compounds for 15 min. Thereafter, medium was aspirated, and cells were extracted by adding ice-cold 5% trichloroacetic acid. The extracts were analyzed for [³H]cAMP using batch column chromatography (18).

View larger version (24K): [in this window] [in a new window]   Figure 2. Effect of Gd3+ on IP formation. CaRc6 cells were challenged with different [Ca2+]e in the absence or presence of 10 or 100 µM Gd3+. The inset shows the effect of 100 µM Gd3+ on the response to the AlF4- complex (ALF; 10 µM AlCl3 plus 0.5 mM NaF) and thrombin (THR; 10 nM). Error bars indicate the SEM of triplicate determinations.

View larger version (13K): [in this window] [in a new window]   Figure 3. Stimulation of intracellular calcium transients by increased [Ca2+]e (A), NPS R-568 (B), and Gd3+ (C). In A and C, the basal calcium concentration of the buffer was 0.2 mM; in B, 2 mM was used. Average changes (mean ± SEM) in intracellular calcium derived from several identical experiments are given next to the corresponding representative traces.

When PTX was used in experiments, cells were pretreated with the toxin (100 ng/ml) for at least 3 h during labeling with [³H]adenine or [³H]inositol.

Intracellular free calcium
Cells grown on round glass coverslips were loaded with the intracellular calcium indicator fluo-3 (5 µM; 60 min, 37 C) in a buffer containing 120 mM NaCl, 22 mM NaHCO₃, 6 mM KCl, 0.2 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, and 5 mM HEPES, pH 7.4. After loading, coverslips were mounted on an inverted Zeiss IM35 microscope (Zeiss, Zurich, Switzerland) in a thermostated perfusion chamber (37 C). Experiments were carried out using mHBS containing MgCl₂ instead of MgSO₄ as incubation buffer. The fluorescence excitation wavelength was set at 480 nm; emission was measured at 510 nm. Signals were recorded with a Deltaron HR 1700 MOS sensor camera (Fuji Photochemicals, Tokyo, Japan). Single cell calcium signals were calibrated as described by Kao et al. (20) using ionomycin and EGTA.

DNA synthesis
Cells were seeded into 96-well plates and grown to confluence. To synchronize cells in the G0/G1 phase of the cell cycle, cultures were serum deprived for 24 h. Thereafter, cells were incubated in serum-free medium containing [³H]thymidine (74 MBq/ml), insulin (1 µg/ml), and the indicated concentrations of cations or growth factors for an additional 24 h. The plates were then washed, and radioactivity incorporated into trichloroacetic acid-insoluble material was counted. When PTX was used in experiments, cells were pretreated with the toxin for at least 3 h during the serum deprivation phase. We have verified that the antibiotics present in our medium do not activate PCaR at the concentrations employed.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CCL39 cells expressing rat PCaR show calcium-induced IP formation
Untransfected CCL39 fibroblasts did not show stimulation of phosphoinositide turnover, cell proliferation, or a modulation of adenylyl cyclase activity in response to variations in [Ca²⁺]_e between 1–10 mM (data not shown). After transfection of these cells with the rat PCaR cDNA, we isolated cell populations responding to increased [Ca²⁺]_e with a strong increase in IP formation. None of the cell populations showed a significant inhibition of adenylyl cyclase. Individual cell clones were isolated, and several clones expressing rat PCaR stably were characterized. The experiments described below were carried out using clone 39CaRc6, which showed strong responses to [Ca²⁺]_e in phosphoinositide turnover assays that were stable for more than 20 passages in culture. Figure 1 shows IP formation as a function of [Ca²⁺]_e and illustrates the effect of a first generation calcimimetic drug, NPS R-568 (17). The EC₅₀ for [Ca²⁺]_e was 4.15 ± 0.15 mM (mean ± SEM; n = 7), which is in good agreement with previously reported values (5, 6, 7, 8, 18). The concentration-response curve for [Ca²⁺]_e was significantly left-shifted, and the maximal response was increased by NPS R-568. The calcimimetic acted with an apparent EC₅₀ of 0.3 µM when IP formation was measured at a [Ca²⁺]_e of 2.5 mM as a function of drug concentration (not shown). Stimulation of IP formation by Ca²⁺ and NPS R-568 was insensitive to pretreatment of cells with PTX (data not shown). We also found activation of the rat PCaR by strontium (EC₅₀ = 8 mM), magnesium (EC₅₀ = 15 mM), and polycations such as neomycin B (EC₅₀ = 0.3 mM), but not with Al³⁺, which acts as a strong mitogen for osteoblasts (3, 4). Unexpectedly, we were unable to detect receptor activation by the transition metal Gd³⁺ in this type of assay. This is illustrated in Fig. 2. Gd³⁺ alone did not stimulate IP formation if applied between 1–100 µM. In contrast, the cation applied at 100 µM almost fully suppressed the signal stimulated by increased [Ca²⁺]_e, which seemed to indicate antagonism. However, this effect was clearly not specific for Ca²⁺-induced signals. The same inhibition was observed when phosphoinositide turnover was stimulated with the AlF₄^- complex or with thrombin, a strong activator of this signaling pathway in CCL39 cells (11). From these data we conclude that Gd³⁺ cannot act as a full functional agonist on PCaR because of nonspecific inhibition of the phosphoinositide signaling system. No inhibitory effects on IP production were observed with Al³⁺ applied between 1–100 µM (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 1. Stimulation of IP formation by increased extracellular calcium and potentiation of the response by NPS R-568. Error bars indicate the SEM of triplicate determinations.

Calcium and NPS R-568 stimulate DNA synthesis and cell proliferation
DNA synthesis reinitiation experiments were carried out in serum-free DMEM/F12 medium in the presence of 1 µg/ml insulin. As the medium contained 0.8 mM phosphate, it was not possible to raise [Ca²⁺]_e above 5 mM without inducing calcium phosphate precipitates immediately. Under our assay conditions, calcium alone applied at a final concentration of 2–4 mM was found to stimulate DNA synthesis significantly (Fig. 4A). However, a striking potentiation of this response was observed in the presence of 1 µM NPS R-568. Thus, the mitogenic response measured with 2 mM Ca²⁺ and 1 µM NPS R-568 was comparable to the response to thrombin or 3% FCS. These data reflect the synergistic action of the cation and the calcimimetic at the level of phosphoinositide breakdown, as shown above. At a [Ca²⁺]_e of 1 mM, the calcimimetic also increased the serum effects, as expected for growth factors acting in conjunction. Neither calcium nor NPS R-568 exerted similar effects in untransfected CCL39 cells (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 4. Stimulation of [3H]thymidine incorporation by increased [Ca2+]e and potentiation by NPS R-568. In A, CaRc6 cells were stimulated with the indicated calcium concentrations or with FCS in the absence (filled symbols/bars) or presence (open symbols/bars) of NPS R-568 (1 µM). In B, the effect of PTX (open bars) is shown. Growth factors used for comparison in B are thrombin (THR; 10 nM), FGF (50 ng/ml), and FCS (3%), tested at a [Ca2+]e of 1 mM. Error bars indicate the SEM (n = 4).

As expected, PTX did not affect the mitogenic action of Ca²⁺, Ca²⁺ plus NPS R-568, or FGF, whereas the responses to thrombin and FCS were clearly inhibited (Fig. 4B).

To confirm and extend the DNA synthesis reinitiation data, we measured increases in cell numbers in cultures of 39CaRc6 cells maintained in serum-free medium supplemented with Ca²⁺ and NPS R-568 as indicated in Fig. 5. After 2 days in defined medium, significant cell proliferation was detected in cultures supplemented with Ca²⁺ and the calcimimetic. However, saturating concentrations of NPS R-568 (1 µM and higher) were not tolerated in long term cell culture, indicating toxicity. Optimal results were obtained with 0.3 µM of the compound. It is probable that more selective and potent calcimimetics would give rise to still stronger increases in cell number.

View larger version (33K): [in this window] [in a new window]   Figure 5. Stimulation of cell proliferation by calcium, calcium plus NPS R-568, and growth factors. CaRc6 cells were grown to confluence in 24-well plates, serum deprived for 24 h, and restimulated as indicated. After 2 days, cell numbers were determined using a Coulter counter. Cells were stimulated with thrombin (THR; 10 nM), FGF (50 ng/ml), or FCS (10%) at a [Ca2+]e of 1 mM. As in the [3H]thymidine incorporation experiments, 1 µg/ml insulin was included in the DMEM/F12 medium. Error bars indicate the SEM (n = 4). Asterisks indicate a significant difference from control ([Ca2+]e = 1 mM) at P < 0.05 (*) and P < 0.01 (**), respectively (by two-tailed t test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The experiments reported here show that the rat PCaR expressed in fibroblasts is functional. It is coupled to the phosphoinositide signaling system and has the potential to trigger cell proliferation. Our experiments did not reveal a significant coupling of the receptor to PTX-sensitive G_i proteins as experiments with bovine and human parathyroid cells had suggested (5, 14). However, our results seem to confirm data reported in abstract form, showing that the rat PCaR is exclusively coupled to phosphoinositide turnover (21).

In contrast to published data (5, 6, 7, 8), we could not detect a significant activation of the rat PCaR by Gd³⁺ when we measured IP formation. In fact, we found that this transition metal strongly inhibited IP formation stimulated by a variety of agonists in several cell systems at concentrations above 10 µM (Fig. 2 and results not shown). However, a productive interaction with the calcium receptor could be demonstrated in measurements of intracellular calcium transients with fluo-3, where Gd³⁺ gave rise to clearly measurable signals, albeit of lower amplitude than the signals elicited by increased [Ca²⁺]_e or NPS R-568. Similar differences in the efficacy between Ca²⁺ and Gd³⁺ were observed recently by Pearce et al. (22) working with 293 cells expressing the human PCaR. At first glance these rather negative results with Gd³⁺ seem surprising, as Gd³⁺ was the agonist used to isolate the bovine PCaR cDNA through expression cloning (6). However, it is possible that the phosphoinositide signaling system in oocytes and various mammalian cells shows a different sensitivity to inhibition by Gd³⁺, thus leading to a different apparent pharmacology of the PCaR.

A second surprising result of the present study was the strong mitogenic response triggered by the rat PCaR in CCL39 cells. In this cell system, as in most other mesenchymal cells, receptors coupled exclusively to phosphoinositide turnover do not stimulate cell proliferation particularly well (10, 12, 13). A probable explanation for our results supported by preliminary data could be a relatively high activity of phosphoinositide turnover activity maintained over time in the presence of NPS R-568, which may create a situation similar to that observed after expression in fibroblasts of a desensitization-defective mutant of the neurokinin NK-2 receptor (13).

It is interesting to compare the mitogenic effects elicited by PCaR with those stimulated by the to date uncloned osteoblast calcium receptor, which may be a close relative of PCaR (4, 23). Both receptors stimulate [³H]thymidine incorporation within the same concentration range of [Ca²⁺]_e (i.e. between 1–4 mM), and both receptors are stimulated by Gd³⁺. However, whereas 10 µM of Gd³⁺ strongly stimulates cell proliferation in osteoblasts, this concentration was not effective in our fibroblasts expressing rat PCaR. Toxicity precludes the use of higher concentrations of the transition metal in [³H]thymidine incorporation experiments. Also, whereas bone cells respond very well to Al³⁺ (3, 4), we found that this cation does not interact with the rat PCaR. Furthermore, no stimulation of phosphoinositide turnover or intracellular calcium transients is detected in MC3T3-E1 cells stimulated by increased [Ca²⁺]_e or Gd³⁺ (4) (Mailland, M., et al., manuscript in preparation). These data suggest that a different molecular entity is involved in mediating the mitogenic effects of cations in bone cells. This contention is further supported by the fact that we and others were unable to detect PCaR by PCR or Northern analysis in osteoblasts and that NPS R-568 does not modulate the mitogenic effects of cations on bone cells (Mailland, M., et al., manuscript in preparation).

	Acknowledgments

 
We thank Rainer Gamse for support and helpful discussions, Anna Teti for discussion and critical reading of the manuscript, and Barbara Wilmering and Viviane Schiff for expert technical assistance.

Received December 27, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Smith JB 1984 Aluminum ions stimulate DNA synthesis in quiescent cultures of swiss 3T3 and 3T6 cells. J Cell Physiol 118:298–304[CrossRef][Medline]
2. Jones TR, Antonetti DL, Reid TW 1986 Aluminum ions stimulate mitosis in murine cells in tissue culture. J Cell Biochem 30:31–39[CrossRef][Medline]
3. Quarles LD, Wenstrup RJ, Castillo SA, Drezner MK 1991 Aluminum-induced mitogenesis in MC3T3–E1 osteoblasts: potential mechanism underlying neoosteogenesis. Endocrinology 128:3144–3151[Abstract/Free Full Text]
4. Quarles LD, Hartle JE, Middleton JP, Zhang J, Arthur JM, Raymond JR 1994 Aluminum-induced DNA synthesis in osteoblasts: mediation by a G-protein coupled cation sensing mechanism. J Cell Biochem 56:106–117[CrossRef][Medline]
5. Brown EM 1991 Extracellular Ca²⁺ sensing, regulation of parathyroid cell function, and role of Ca²⁺ and other ions as extracellular (first) messengers. Physiol Rev 71:371–411[Free Full Text]
6. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC 1993 Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature 366:575–580[CrossRef][Medline]
7. Brown EM, Fuleihan GE, Chen CJ, Kifor O 1990 A comparison of the effects of divalent and trivalent cations on parathyroid hormone release, 3',5'-cyclic-adenosine monophosphate accumulation, and the levels of inositol phosphates in bovine parathyroid cells. Endocrinology 127:1064–1071[Abstract/Free Full Text]
8. Garrett JE, Capuano IV, Hammerland LG, Hung BC, Brown EM, Hebert SC, Nemeth EF, Fuller F 1995 Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. J Biol Chem 270:12919–12925[Abstract/Free Full Text]
9. Ruat M, Snowman AM, Hester LD, Snyder SH 1996 Cloned and expressed rat Ca²⁺-sensing receptor. J Biol Chem 271:5972–5975[Abstract/Free Full Text]
10. Pouyssegur J, Seuwen K 1992 Transmembrane receptors and intracellular pathways that control cell proliferation. Annu Rev Physiol 54:195–210[Medline]
11. Van Obberghen Schilling E, Vouret Craviari V, Chen YH, Grall D, Chambard JC, Pouyssegur J 1995 Thrombin and its receptor in growth control. Ann NY Acad Sci 766:431–441[Medline]
12. Seuwen K, Kahan C, Hartmann T, Pouyssegur J 1990 Strong and persistent activation of inositol lipid breakdown induces early mitogenic events but not Go to S phase progression in hamster fibroblasts. Comparison of thrombin and carbachol action in cells expressing M1 muscarinic acetylcholine receptors. J Biol Chem 265:22292–22299[Abstract/Free Full Text]
13. Alblas J, van Etten I, Moolenaar WH 1996 Truncated, desensitization-defective neurokinin receptors mediate sustained MAP kinase activation, cell growth and transformation by a Ras-independent mechanism. EMBO J 15:3351–3360[Medline]
14. Chen CJ, Barnett JV, Congo DA, Brown EM 1989 Divalent cations suppress 3',5'-adenosine monophosphate accumulation by stimulating a pertussis toxin-sensitive guanine nucleotide-binding protein in cultured bovine parathyroid cells. Endocrinology 124:233–239[Abstract/Free Full Text]
15. Ruat M, Molliver ME, Snowman AM, Snyder SH 1995 Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals. Proc Natl Acad Sci USA 92:3161–3165[Abstract/Free Full Text]
16. Riccardi D, Park J, Lee WS, Gamba G, Brown EM, Hebert SC 1995 Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci USA 92:131–135[Abstract/Free Full Text]
17. Nemeth EF, Steffey ME, Fox J 1996 The parathyroid calcium receptor: a novel therapeutic target for treating hyperparathyroidism. Pediatr Nephrol 10:275–279[Medline]
18. Salomon Y 1979 Adenylate cyclase assay. Adv Cyclic Nucleotide Res 10:35–55[Medline]
19. Seuwen K, Lagarde A, Pouyssegur J 1988 Deregulation of hamster fibroblast proliferation by mutated ras oncogenes is not mediated by constitutive activation of phosphoinositide-specific phospholipase C. EMBO J 7:161–168[Medline]
20. Kao JPY, Harootunian AT, Tsien RY 1989 Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J Biol Chem 264:8179–8185[Abstract/Free Full Text]
21. Rogers KV, Dunn CK, Hebert SC, Brown EM, Nemeth EF 1995 Pharmacological comparison of bovine parathyroid, human parathyroid, and rat kidney calcium receptors expressed in Hek 293 cells. J Bone Miner Res [Suppl 1] 10:S483 (Abstract)
22. Pearce SHS, Bai M, Quinn SJ, Kifor O, Brown EM, Thakker RV 1996 Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells. J Clin Invest 98:1860–1866[Medline]
23. Quarles LD, Hinson TK, Seldin MF, Siddhanti SR 1996 Identification of a novel calcium-sensing receptor related gene localized to mouse chromosome 7. J Bone Miner Res [Suppl 1] 11:S141 (Abstract)

This article has been cited by other articles:

				Z. Saidak, M. Brazier, S. Kamel, and R. Mentaverri Agonists and Allosteric Modulators of the Calcium-Sensing Receptor and Their Therapeutic Applications Mol. Pharmacol., December 1, 2009; 76(6): 1131 - 1144. [Abstract] [Full Text] [PDF]

				T. De Santis, V. Casavola, S. J. Reshkin, L. Guerra, B. Ambruosi, N. Fiandanese, R. Dalbies-Tran, G. Goudet, and M. E. Dell'Aquila The extracellular calcium-sensing receptor is expressed in the cumulus-oocyte complex in mammals and modulates oocyte meiotic maturation Reproduction, September 1, 2009; 138(3): 439 - 452. [Abstract] [Full Text] [PDF]

				T. Kawata, Y. Imanishi, K. Kobayashi, T. Miki, A. Arnold, M. Inaba, and Y. Nishizawa Parathyroid Hormone Regulates Fibroblast Growth Factor-23 in a Mouse Model of Primary Hyperparathyroidism J. Am. Soc. Nephrol., October 1, 2007; 18(10): 2683 - 2688. [Abstract] [Full Text] [PDF]

				T. Bouschet, S. Martin, and J. M. Henley Receptor-activity-modifying proteins are required for forward trafficking of the calcium-sensing receptor to the plasma membrane J. Cell Sci., October 15, 2005; 118(20): 4709 - 4720. [Abstract] [Full Text] [PDF]

				M. Yamauchi, T. Yamaguchi, H. Kaji, T. Sugimoto, and K. Chihara Involvement of calcium-sensing receptor in osteoblastic differentiation of mouse MC3T3-E1 cells Am J Physiol Endocrinol Metab, March 1, 2005; 288(3): E608 - E616. [Abstract] [Full Text] [PDF]

				N. Chattopadhyay, S. Yano, J. Tfelt-Hansen, P. Rooney, D. Kanuparthi, S. Bandyopadhyay, X. Ren, E. Terwilliger, and E. M. Brown Mitogenic Action of Calcium-Sensing Receptor on Rat Calvarial Osteoblasts Endocrinology, July 1, 2004; 145(7): 3451 - 3462. [Abstract] [Full Text] [PDF]

				C. Petrel, A. Kessler, P. Dauban, R. H. Dodd, D. Rognan, and M. Ruat Positive and Negative Allosteric Modulators of the Ca2+-sensing Receptor Interact within Overlapping but Not Identical Binding Sites in the Transmembrane Domain J. Biol. Chem., April 30, 2004; 279(18): 18990 - 18997. [Abstract] [Full Text] [PDF]

				J. Tfelt-Hansen, N. Chattopadhyay, S. Yano, D. Kanuparthi, P. Rooney, P. Schwarz, and E. M. Brown Calcium-Sensing Receptor Induces Proliferation through p38 Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase But Not Extracellularly Regulated Kinase in a Model of Humoral Hypercalcemia of Malignancy Endocrinology, March 1, 2004; 145(3): 1211 - 1217. [Abstract] [Full Text] [PDF]

				S. U. Miedlich, L. Gama, K. Seuwen, R. M. Wolf, and G. E. Breitwieser Homology Modeling of the Transmembrane Domain of the Human Calcium Sensing Receptor and Localization of an Allosteric Binding Site J. Biol. Chem., February 20, 2004; 279(8): 7254 - 7263. [Abstract] [Full Text] [PDF]

				C. Petrel, A. Kessler, F. Maslah, P. Dauban, R. H. Dodd, D. Rognan, and M. Ruat Modeling and Mutagenesis of the Binding Site of Calhex 231, a Novel Negative Allosteric Modulator of the Extracellular Ca2+-sensing Receptor J. Biol. Chem., December 5, 2003; 278(49): 49487 - 49494. [Abstract] [Full Text] [PDF]

				J. F. Staub, E. Foos, B. Courtin, R. Jochemsen, and A. M. Perault-Staub A nonlinear compartmental model of Sr metabolism. II. Its physiological relevance for Ca metabolism Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2003; 284(3): R835 - R852. [Abstract] [Full Text] [PDF]

				O. Kifor, R. J. MacLeod, R. Diaz, M. Bai, T. Yamaguchi, T. Yao, I. Kifor, and E. M. Brown Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells Am J Physiol Renal Physiol, February 1, 2001; 280(2): F291 - F302. [Abstract] [Full Text] [PDF]

				E. M. Brown and R. J. MacLeod Extracellular Calcium Sensing and Extracellular Calcium Signaling Physiol Rev, January 1, 2001; 81(1): 239 - 297. [Abstract] [Full Text] [PDF]

				S. U. Goebel, P. L. Peghini, P. K. Goldsmith, A. M. Spiegel, F. Gibril, M. Raffeld, R. T. Jensen, and J. Serrano Expression of the Calcium-Sensing Receptor in Gastrinomas J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4131 - 4137. [Abstract] [Full Text]

				T. B. DRÜEKE Cell Biology of Parathyroid Gland Hyperplasia in Chronic Renal Failure J. Am. Soc. Nephrol., June 1, 2000; 11(6): 1141 - 1152. [Full Text]

				M. Pi, S. C. Garner, P. Flannery, R. F. Spurney, and L. D. Quarles Sensing of Extracellular Cations in CasR-deficient Osteoblasts. EVIDENCE FOR A NOVEL CATION-SENSING MECHANISM J. Biol. Chem., February 4, 2000; 275(5): 3256 - 3263. [Abstract] [Full Text] [PDF]

				S. Johnson and A. Knox Autocrine production of matrix metalloproteinase-2 is required for human airway smooth muscle proliferation Am J Physiol Lung Cell Mol Physiol, December 1, 1999; 277(6): L1109 - L1117. [Abstract] [Full Text] [PDF]

				M. J. Rutten, K. D. Bacon, K. L. Marlink, M. Stoney, C. L. Meichsner, F. P. Lee, S. A. Hobson, K. D. Rodland, B. C. Sheppard, D. D. Trunkey, et al. Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells Am J Physiol Gastrointest Liver Physiol, September 1, 1999; 277(3): G662 - G670. [Abstract] [Full Text] [PDF]

				C.-L. Tu, W. Chang, and D. D. Bikle The Extracellular Calcium-sensing Receptor Is Required for Calcium-induced Differentiation in Human Keratinocytes J. Biol. Chem., October 26, 2001; 276(44): 41079 - 41085. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mailland, M. Articles by Seuwen, K. Search for Related Content PubMed PubMed Citation Articles by Mailland, M. Articles by Seuwen, K. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL