©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcitonin Increases Cytosolic Free Calcium Concentration via Capacitative Calcium Influx (*)

Anna Teti (1)(§), Rossella Paniccia (2) (3), Steven R. Goldring (4)

From the (1)Department of Experimental Medicine, School of Medicine, University of L'Aquila, 67100 L'Aquila, Italy, (2)Istituto Dermopatico dell'Immacolata, 00167 Rome, Italy, (3)Institute of Histology and General Embryology, University ``La Sapienza,'' 00161 Rome, Italy, and the (4)Arthritis Unit, Massachusetts General Hospital, and New England Deaconess and Baptist Hospitals, Harvard Medical School, Boston, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The calcitonin receptor has been proposed to function as an extracellular Ca concentration ([Ca]) sensor (Stroop, S. D., Thompson, D. L., Kuestner, R. E., and Moore, E. E.(1993) J. Biol. Chem. 268, 19927-19930). To test this hypothesis we studied the LLC-PK renal tubular cells and the PC cells, a cell line stably transfected with the cloned porcine calcitonin receptor. [Ca] was measured by fura-2 single cell microfluorometry. Addition to the cells equilibrated in 1.25 mM Ca-containing media of 1-10 mM extracellular Ca did not result in a significant increase of [Ca]. Treatment with 10M salmon calcitonin (sCT) elicited a rapid, persistent elevation of [Ca]. Addition of 1-10 mM extracellular Ca in the presence of sCT induced a significant [Ca]elevation, about 10-fold that observed in the absence of the hormone. Ca influx was inhibited by lanthanum. The rise of [Ca] at elevated [Ca]was not due to a Ca sensing mechanism with release of Ca from intracellular stores, since it was prolonged, and was not abolished by prior depletion of Ca stores with 10M thapsigargin. On the contrary, this agent potentiated Ca influx after addition of 1-10 mM Ca by 13-fold versus control. Prior stimulation of [Ca] with 10M arginine-vasopressin had similar effects, enhancing the subsequent Ca influx. Enhancement of Ca influx by sCT was confirmed by increased Mn quenching of fura-2 fluorescence. In conclusion, arginine-vasopressin or calcitonin enhance Ca influx in LLC-PK cells via a Ca release-activated conductance, probably dependent upon capacitative Ca entry. Thus, these effects are not unique to the calcitonin receptor and argue against the receptor functioning as a [Ca] sensor.


INTRODUCTION

Characterization of a calcitonin receptor (CTR)()cloned from a porcine renal epithelial cell line, LLC-PK, predicts a 482-amino acid binding protein with high affinity for salmon calcitonin (sCT) (dissociation constant K 6 nM)(1) . This CTR belongs to a subfamily of G-protein-linked receptors which, based on similarity in amino acid sequence, includes, for example, the parathyroid hormone/parathyroid hormone related peptide receptor (32% amino acid identity and 56% similarity) (2) and the secretin receptor (30% identity, 58% similarity)(3) . These receptors are likely to represent a new family of G-protein-coupled receptors, associated with, among other activities, the regulation of Ca homeostasis.

The ligand for the CTR, calcitonin (CT), is a 32-amino acid peptide hormone secreted by the thyroid C cells in response to elevated serum Ca levels. CT administration results in a reduction of the Ca concentration in the extracellular fluid ([Ca])(4) . This is accomplished via inhibition of the bone resorbing activity of the osteoclast (5, 6) and enhanced renal calcium excretion(7, 8) . Most cellular effects of CT are associated with activation of the adenylyl cyclase pathway(9, 10) . However, recently it has become apparent that the CTR is also associated with the phospholipase C enzyme pathway(11, 12) , which induces breakdown of membrane phosphoinositol lipids to yield inositol 1,4,5-triphosphate (InsP) and diacylglycerol. The two second messenger molecules, in turn, stimulate Ca release into the cytoplasm from intracellular pools, and activate the serine/threonine protein kinase C, respectively(13, 14) .

A recent report by Stroop et al.(15) has suggested that the CTR functions as a [Ca] sensor. A [Ca] sensor has been proposed as a mechanism that allows the cell to sense increases of [Ca] in the millimolar range, and to respond with significant elevations of the cell signal cytosolic free Ca concentration ([Ca])(16, 17) . Should the CTR operating as a [Ca] sensor be confirmed, the observation would be of great interest, in view of the fact that only selected cell types, including the CTR-expressing osteoclasts(16, 17, 18) , retain this unique Ca sensing activity. The secretion of CT in hypercalcemic conditions (4) would thus be predicted to activate the calcium sensor activity of the CTR resulting in modulation of osteoclast function. A synergistic role of the two factors, CT and elevated [Ca], would reciprocally potentiate the cellular responses.

The [Ca] sensing receptor, recently cloned and characterized from bovine parathyroid glands(18) , predicts a 120-kDa seven-trans membrane domain receptor, which shares limited similarity to the metabotropic glutamate receptor(19) . Its extracellular domain contains clusters of acidic amino acid residues, which are likely to represent a Ca binding sequence, whereas the intracellular domain shows sequences similar to those of other G-protein-coupled receptors. The predicted structure of the CTR is unrelated to that of the [Ca] sensing receptor, since it lacks similarity in amino acid sequence and does not possess extracellular putative Ca-binding consensus sequences(1) . This observation argues against the CTR functioning as a [Ca] sensor.

The present studies were undertaken to more rigorously analyze the CTR for Ca sensing function. We have performed single cell [Ca] studies, by fura-2 microfluorometry, in CTR-positive cells in vitro employing the LLC-PK cell line(1) , and the PC cells, a line derived from MC-3T3-E cells stably transfected with the CTR cloned from LLC-PK cells(15) . In nonstimulated conditions, both cell types were insensitive to an elevated [Ca], whereas upon stimulation with sCT they acquired the capability of responding to an increase of [Ca] with a [Ca]elevation. Characterization of the mechanism inducing the [Ca]-dependent [Ca] elevation, however, demonstrated that this was exclusively due to Ca influx across the plasma membrane, induced by a nonspecific post-receptor event that was not unique for the CTR but was shared by arginine-vasopressin (AVP), another Ca-mobilizing hormone.


EXPERIMENTAL PROCEDURES

Materials

Dulbecco's modified and modified minimum essential media, fetal bovine serum, reagents, and sterile plasticware for cell culture were from Flow Laboratory (Irvine, CA). Fura-2 acetoxymethyl ester (fura-2/AM) was from Molecular Probes (Eugene, OR). Ionomycin and fatty acid-free bovine serum albumin were from Calbiochem (La Jolla, CA). Salmon calcitonin was kindly donated by Drs. Francesco Bartucci and Vera Calcagno, Sandoz Prodotti Farmaceutici S.p.A. (Milan, Italy). All other reagents were from Sigma.

Cell Cultures

LLC-PK cells are a porcine kidney epithelial cell line previously characterized in our laboratory (9). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics and fed once a week. At confluence, cells were split 1:20 by standard trypsin procedures.

PC cells were obtained by stable transfection of MC-3T3-E cell line as described previously(11) . Cells were grown in modified minimum essential medium supplemented with 2.5% fetal bovine serum, fed once a week, and split 1:3 every 7-14 days. MC-3T3-E cells were grown in the same condition as PC cells.

Buffer

Bathing medium through all experiments was a modified Krebs-Henseleit solution (KHH) with or without 1.25 mM Ca, buffered with 20 mM HEPES, and containing 0.2% bovine serum albumin.

Measurement of [Ca]

[Ca]of nonconfluent monolayers of LLC-PK, PC, and MC-3T3-E cell lines was measured by dual wavelength fluorescence of cells loaded with the Ca-sensitive intracellular probe fura-2(16, 20) . Cells, seeded on glass coverslips at a density of 100,000/3.5-cm diameter dish, were loaded with 3 µM fura-2/AM in serum-free medium, at 37 °C for 60 min. Measurements were performed in single cells, at 340 and 380 nm excitation wavelengths, with an AR-CM fluorometer (Spex Industries, Inc., Edison, NJ) connected with a Diaphot TMD inverted microscope (Nikon Corp., Tokyo, Japan) equipped with a Nikon CF X40 objective. Emissions were collected by a photomultiplier carrying a 510-nm cut-off filter and recorded by an ASEM Desk 2010 computer (ASEM S.p.A, Buia, Italy), which automatically calculated real-time 340/380 ratios. Calibration of the signal was obtained at the end of each observation by adding 5 µM ionomycin to saturate the dye to maximal fluorescence, followed by 7.5 mM EGTA plus 60 mM Tris-HCl, pH 10.5, to release Ca from fura-2 and obtain minimal fluorescence. [Ca] was calculated according to previously described formulas(20) .

Statistics

Data are presented as average ± S.E. Statistical analysis was performed by analysis of variance (ANOVA). p < 0.05 was conventionally considered to indicate statistical significance. For the dose-response data, when ANOVA revealed a statistical significance, Student's t test was used.


RESULTS

Basal [Ca] in LLC-PK, PC and MC-3T3-E cells is indicated in . In all cell types [Ca]remained stable over a period of at least 30 min. No spontaneous fluctuations were observed.

Fig. 1A and show events elicited in LLC-PK, PC, and MC-3T3-E cells by treatment with 1 and 10 mM Ca. Unless otherwise specified, Ca was added to the 1.25 mM Ca-containing KHH buffer; therefore, the final [Ca] was 2.25 and 11.25, respectively. This elevation of the [Ca] did not show ability to elicit a [Ca] response in the cells. No significant differences were observed in high Ca-treated cells versus control [Ca] levels.


Figure 1: Effect of 1 and 10 mM Ca on [Ca] of single LLC-PK cells. A, traces representing real-time fura-2 fluorescence ratio (excitation wavelength 340 and 380 nm, emission 510 nm) in a single LLC-PK cell challenged with 1 and 10 mM Ca. Initial [Ca] in the medium = 1.25 mM. Calibration of fluorescence as [Ca] has been obtained upon addition of 5 µM ionomycin, followed by 7.5 mM EGTA plus 60 mM Tris (pH 10.5). B, a single LLC-PK cell was treated with 10M sCT, which elicited a [Ca] increase. When [Ca] stabilized, the cell was sequentially treated with 1 and 10 mM Ca, which further increased [Ca]. Initial [Ca] in the medium = 1.25 mM.



LLC-PK and PC cells were similarly sensitive to sCT, whereas MC-3T3-E cells failed to show sensitivity to the hormone. illustrates the effect of 10M sCT on [Ca]. The hormone induced a severalfold increase of [Ca]over basal levels in the two CT-sensitive cell types. Responses were transient, with a [Ca] peak followed by a decrease to lower levels.

Fig. 1B and show events elicited by addition of 1 and 10 mM Ca in LLC-PK, PC and MC-3T3-E cells pretreated with 10M sCT. Ca was added to the cells during the sustained [Ca] elevation induced by sCT. Addition of Ca resulted in a significant [Ca] elevation in LLC-PK and PC, but not in MC-3T3-E cells ().

Mechanism of Ca-induced [Ca] increase in sCT treated LLC-PKcells

LLC-PK cells were utilized to investigate the mechanisms responsible for the Ca-dependent [Ca]transients induced by sCT. Fig. 2shows concentration-dependent curves obtained in LLC-PK cells treated with the doses of [Ca] as indicated on the abscissa. These curves were constructed computing peak [Ca] in cells equilibrated in Ca-free KHH, to which Ca was added to the final desired concentration from 1 M CaCl stock solution. In the absence of sCT, cells showed low sensitivity to elevated [Ca], even though responses were approximately 2-fold higher compared to those observed when the same doses of CaCl were added to cells equilibrated in the 1.25 mM Ca-containing KHH. Treatment with 10M sCT significantly enhanced the [Ca]-induced [Ca]increase. Maximal response was observed at 10 mM Ca, with EC = 6 mM Ca.


Figure 2: Concentration-dependent curves of [Ca]-induced [Ca] increases in single LLC-PK cells. Cells were challenged with the doses of Ca indicated on the abscissa, in the presence or in the absence of 10M sCT. Each point shows mean ± S.E. of at least six independent experiments. Curves were significantly different (p < 0.01), as assessed by ANOVA followed by Student's t test.



In Fig. 3A the effect of chelation of extracellular Ca by EGTA is shown. Addition of 1.25 mM EGTA slightly reduced [Ca]. In this circumstance, cells responded to sCT with a rapid transient increase of [Ca], which peaked at 133 ± 27 nM (n = 6), a level approximately 4-fold lower than that observed in the presence of extracellular Ca. Then, [Ca]rapidly returned toward base line. No sustained phase was observed. Subsequent addition of extracellular Ca resulted in a rapid, sustained [Ca] elevation. This indicates that sCT treatment has two effects. First, it is capable of stimulating Ca release from intracellular storing organelles, even in Ca-free conditions. Second, sCT stimulates a large inward Ca flux from the extracellular environment, abolished by removal of extracellular Ca by EGTA. This is further confirmed by the experiment shown in Fig. 3B. A single LLC-PK cell has been stimulated by sCT to obtain the [Ca] increase. During the sustained phase, the cell was treated with LaCl, an agent known to block all Ca influx mechanisms across the plasma membrane(21) . In the presence of La, [Ca] rapidly dropped to near basal levels, indicating once again the sustained phase to be dependent upon Ca influx. Addition of 1-10 mM Ca in the presence of La failed to induce a significant [Ca] rise compared to untreated cells (Fig. 3B, I). This indicates that Ca-dependent [Ca] increase stimulated by sCT largely depends on gating of Ca channels of the plasma membrane.


Figure 3: Effect of EGTA and La on the Ca influx in single LLC-PK cells. A, [Ca] in the KHH was reduced by addition of the Ca-chelating agent EGTA. The cell was then treated with 10M sCT, followed by 1 mM Ca. Initial [Ca] in the medium = 1.25 mM. Predicted [Ca] upon addition of EGTA = 0 mM. B, a single LLC-PK cell was treated with 10M sCT. During the sustained [Ca] increase phase, 10M La was added to abolish Ca influx. Further addition of 1 and 10 mM Ca in the presence of La failed to stimulate a significant [Ca] rise. Initial [Ca] in the medium = 1.25 mM.



To rule out the potential confounding effects of modifications in Ca efflux, and to confirm that actual Ca influx was responsible for the changes in [Ca], we performed experiments with Mn quenching of the fura-2 dye to monitor influx of another extracellular divalent cation. Mn permeates the cell through the same channels employed by Ca, rapidly quenching fura-2 fluorescence by irreversible binding(22) . In Fig. 4A, addition of sCT in the presence of Mn resulted in a rapid increase of fura-2 fluorescence, followed by a slow progressive reduction. Similar fura-2 quenching was observed in cells pretreated with sCT and then with 1 and 10 mM Ca prior to addition of Mn (Fig. 4B). This further indicates that both the sustained phase observed during stimulation with sCT and the [Ca] increase observed in cells further challenged with high Ca are due to massive influx of divalent cations across the plasma membrane.


Figure 4: Mn quenching of fura-2 fluorescence. In A, a single LLC-PK cell was treated with 10M Mn, then with 10M sCT. In B, a single LLC-PK cell was treated with 10M sCT, then with 1 and 10 mM Ca prior to addition of Mn. Initial [Ca] in the medium = 1.25 mM. The drop of fura-2 fluorescence, measured at 340 nm excitation wavelength, indicates quenching of the dye by the Mn that permeates the cell.



The [Ca]-dependent elevation of [Ca] could not be explained by Ca release from intracellular stores, as it was persistent (Fig. 1B). However, to further examine the role of Ca stores in the sCT-induced Ca influx, we performed experiments involving Ca depletion by thapsigargin, a specific inhibitor of the CaATPase of the endoplasmic reticulum(23) . Fig. 5A shows the effect of thapsigargin on the signal induced by sCT. Thapsigargin transiently increased [Ca] to 558 ± 39 (n = 4) and completely abolished the response of the cell to sCT, indicating that Ca release from the stores is involved not only in the sCT-induced peak increase of [Ca], but also in the secondary Ca influx across the plasma membrane. This is further demonstrated in experiments (not shown) in which Ca depletion was obtained by treatment with 1 µM of the Ca ionophore ionomycin, in which we observed similar complete inhibition of the response to sCT. We next examined whether depletion of intracellular Ca stores modulated the response of the cells to elevated extracellular Ca. To accomplish this, we treated the LLC-PK cells with thapsigargin and then added 1-10 mM Ca (Fig. 5B). In this circumstance, thapsigargin not only failed to prevent, but, similar to sCT, significantly stimulated Ca entry into the cells (I). Finally, we depleted Ca stores with a physiologic stimulus. LLC-PK cells are known to express Ca mobilizing receptors for AVP. Therefore, we pretreated the cells with AVP and observed a rapid [Ca]transient with a peak and a sustained phase (Fig. 6). Similar to sCT, addition of 1-10 mM Ca in the presence of AVP greatly stimulated [Ca] increases with a similar pattern (Fig. 6, I). This indicates that the effect of sCT on Ca influx in LLC-PK cells is not unique for the CTR but is shared by other Ca mobilizing receptors, such as the V-type AVP receptor.


Figure 5: Effect of thapsigargin on the [Ca] in single LLC-PK cells. A, trace representing [Ca] in a single LLC-PK cell treated with the endoplasmic reticulum CaATPase inhibitor thapsigargin (TPS). Thapsigargin depleted the intracellular Ca storing organelles, inducing a transient [Ca] increase and preventing the response of the cell to 10M sCT. B, a single cell was pretreated with 10M thapsigargin (TPS) prior to addition of 1 and 10 mM Ca. Note that Ca store depletion by thapsigargin stimulated [Ca]-induced [Ca] increases.




Figure 6: Effect of AVP on [Ca] of a single LLC-PK cell. A single cell was first challenged with 10M AVP, which elicited a [Ca] transient, then with 1 and 10 mM Ca. This maneuver stimulated Ca influx similar to that observed in sCT-treated cells.




DISCUSSION

Cultured LLC-PK and PC cells express abundant CTRs (1, 11) and therefore represent an excellent model for studying the signal transduction pathways activated by sCT. The two cell lines respond to sCT with typical, biphasic [Ca] transients, a feature of the signaling pathway involving phospholipase C(13) . The initial rise has been demonstrated to result from a direct effect of InsP on InsP-activated Ca channels in intracellular Ca storing organelles(11) . The sustained elevation that follows the initial peak is due to persistent Ca entry across the plasma membrane(11) . The amplitude of the sustained phase, which is responsible for the maintenance of [Ca] higher than basal for several minutes, suggests that the predicted Ca influx mechanism shows a large conductance, determined by an extensive Ca movement across the plasma membrane.

This study provides evidence that the sCT-induced Ca conductance is significantly stimulated when the Ca gradient on the two sides of the plasma membrane is increased. This causes a [Ca] increase, which is prolonged in nature and lasts several minutes. Such a feature argues against the activation of a [Ca] sensing, since this should result in a transient [Ca] rise that would be abolished by agents that deplete intracellular Ca stores(16, 17, 24, 25) .

Our results indicate that activation of the CTR in LLC-PK cells induces a nonspecific Ca release-activated Ca influx. This is demonstrated by several observations. First, the release of Ca is produced not only by sCT, but also by activation of another Ca mobilizing receptor, such as the V-type AVP receptor. Second, agents that nonspecifically stimulate Ca release from intracellular stores, such as thapsigargin, promote Ca internalization similar to that induced by sCT and AVP. As a result of these observations, we can expect that activation of the Ca conductance does not require receptor occupancy. This rules out the involvement of InsP as a direct second messenger necessary for determining the sCT-induced Ca-dependent [Ca] rise. The relevance of the activity of membrane Ca channels is further demonstrated by experiments involving blockade of Ca influx from the extracellular environment. La, a nonspecific inhibitor of all Ca entry mechanisms, is significantly active in blocking both the sustained phase induced by sCT and the additional [Ca] increase due to addition of Ca to the bathing buffer. Experiments performed with Mn further confirm that influx of divalent cations is already occurring upon stimulation with sCT, and that it proceeds when an elevated [Ca] is created.

According to these results, we hypothesize that the CTR does not function as a [Ca] sensor. This argues against the data reported by Stroop et al.(15) , who studied a recombinant human CTR transfected in baby hamster kidney (BHK) cells. In our hands, the mechanism stimulated by sCT resembles that described first by Casteels and Droogmans (26) and then by Takemura and Putney (27), which has been termed ``capacitative Ca entry,'' or ``store-operated Ca entry pathway''(28) . According to Putney and Bird(29) , a capacitative Ca entry is stimulated by Ca mobilizing signals, through activation of PLC-coupled receptors. It is largely accepted that InsP, produced by phospholipase C-dependent membrane phosphoinositol lipid breakdown, is responsible for such Ca influx via a secondary, indirect mechanism(30) . The most compelling evidence for this hypothesis comes from the observation that inhibitors of the microsomal CaATPase, such as thapsigargin, which cause depletion of intracellular stores without InsP production, mimic the effect of surface membrane InsP-linked agonists to activate Ca entry (29). Regardless of the primary process that produces intracellular Ca pool depletion, capacitative Ca entry allows the emptied stores to be rapidly refilled, in order to restore the resting conditions(26, 27, 28, 29, 30) . The mechanism producing the capacitative Ca influx has not yet been fully elucidated. It implies a system for communication between the Ca pool and the plasma membrane, such that the permeability of the plasma membrane would be increased when the intracellular pool is empty(27) . To date two fundamental mechanisms for the retrograde signal for capacitative Ca entry have been considered; (i) a diffusible messenger is produced or released when the intracellular stores are depleted, diffuses to the plasma membrane and activates Ca entry(31, 32, 33) , or (ii) the emptying of the intracellular Ca stores causes a conformational change in the organelle and/or its surface proteins, and this information is delivered to the plasma membrane either by direct coupling (34) or via the cytoskeleton(35) .

In conclusion, our study provides evidence indicating that a Ca release Ca influx mechanism is operating in the CTR-expressing LLC-PK and in the PC cells stably transfected with the CTR. This mechanism resembles the recently described capacitative Ca entry observed in cells challenged with Ca mobilizing agents. Such Ca influx is not due to Ca release from intracellular stores, but rather is stimulated by Ca store depletion. These results seem to rule out the CTR functioning as a [Ca] sensor.

  
Table: Effect of sCT on [Ca] of single LLC-PK, PC, and MC-3T3-E cells


  
Table: Effect of 1 and 10 mM Ca on [Ca] of LLC-PK, PC, and MC-3T3-E cells


  
Table: Effect of La, thapsigargin, and AVP on Ca influx in LLC-PK cells



FOOTNOTES

*
This work was supported by Italian Ministry of University Grant quota 40% 92/6079 and National Council of Research Grant 93/4693CT04 (to A. T.), grants from the Italian Ministry of University (to the Institute of Histology and General Embryology, University ``La Sapienza,'' Rome), and from United States Public Health Service Grants AR-03564 and DK-46773 (to S. R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dipartimento di Medicina Sperimentale, Universit de L'Aquila, Via Vetoio, Coppito 2, 67100 L'Aquila, Italy. Tel.: 39-6-4976-6575; Fax: 39-6-446-2854.

The abbreviations used are: CTR, calcitonin receptor; CT, calcitonin; sCT, salmon calcitonin; InsP, inositol 1,4,5-triphosphate; AVP, arginine-vasopressin; ANOVA, analysis of variance; KHH, Krebs-Henseleit solution.


ACKNOWLEDGEMENTS

We thank Drs. Elio Ziparo, Mario Molinaro, and Mario Stefanini who kindly made available to us the equipment of the Institute of Histology and General Embryology, University ``La Sapienza'' of Rome. We also acknowledge the excellent technical support of Giancarlo Sciortino.


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