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Calcitonin (CT) Rapidly Increases Na⁺/H⁺ Exchange and Metabolic Acid Production: Effects Mediated Selectively by the C1a CT Receptor Isoform

CIHR Group in Skeletal Remodeling, Department of Physiology, Division of Oral Biology (A.S., S.J.D.), and Department of Pharmacology & Toxicology (P.C.), Faculty of Medicine & Dentistry, The University of Western Ontario, London, Canada N6A 5C1; and Departments of Cell Biology and Orthopedics and the Yale Cancer Center (W.C.H., R.B.), Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Dr. S. J. Dixon, Department of Physiology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1. E-mail: jeff.dixon{at}fmd.uwo.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two isoforms of the calcitonin receptor are expressed in rabbit: the common C1a isoform and the calcitonin receptor e13 isoform, which has a deletion in the seventh transmembrane domain. Using microphysiometry, we investigated the effects of calcitonin on proton efflux from HEK293 cells stably transfected with C1a, calcitonin receptor e13, or empty vector. In C1a-expressing cells only, calcitonin rapidly induced a biphasic elevation in proton efflux consisting of an initial transient and a sustained plateau, accompanied by an increase in lactate efflux. Inhibitors of Na⁺/H⁺ exchange abolished only the initial transient, whereas removal of extracellular glucose abolished only the sustained plateau. These data suggest that activation of Na⁺/H⁺ exchange mediates the initial transient, whereas increased glucose metabolism underlies the sustained plateau. Because both receptor isoforms activate adenylyl cyclase, the lack of effect of calcitonin on proton efflux from calcitonin receptor e13-expressing cells argued against involvement of cAMP in activating proton efflux. Similarly, studies involving elevation or buffering of cytosolic free Ca²⁺ concentration argued against involvement of Ca²⁺. Activation of PKC mimicked the plateau phase of calcitonin-induced proton efflux from C1a cells, whereas inhibition or depletion of PKC suppressed it. Activation of proton transport and production are novel cellular responses to calcitonin, mediated selectively by the C1a receptor isoform via a mechanism involving PKC.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
CALCITONIN (CT) IS a 32-amino acid hormone synthesized and secreted by the parafollicular cells of the thyroid gland. CT acts primarily on bone and kidney to reduce plasma Ca²⁺ levels by inhibiting bone resorption and promoting Ca²⁺ excretion. Therefore, CT is administered therapeutically in the treatment of diseases characterized by high bone turnover, including Paget’s disease of bone, malignancy-associated hypercalcemia, and osteoporosis (1). Mice homozygous for knockout of CALC I (the gene encoding CT and CT gene-related peptide) develop osteopenia, indicating a role for CT in skeletal homeostasis in vivo (2).

The CT receptor (CTR), a G protein-coupled receptor, has been identified in a number of tissues, and the existence of splice variants has been reported (3). The most common isoform of the CTR, C1a, was originally cloned from porcine kidney (4) and is also expressed in human, mouse, rat, and rabbit cells (5, 6, 7, 8, 9, 10). A novel isoform in rabbit, CTRe13, is generated by deletion of exon 13 during mRNA processing, resulting in the absence of 14 amino acid residues in the putative 7th transmembrane spanning domain. This deletion reduces the affinity of the CTRe13 isoform for CT. The dissociation constant (K_d) for binding of salmon CT to the CTRe13 isoform is 9 nM, whereas the K_d for the C1a isoform is 0.2 nM (10). Both isoforms activate adenylyl cyclase, leading to production of cAMP and activation of PKA. In contrast, only the C1a isoform couples to G_q, activating PLC, which in turn leads to Ca²⁺ mobilization and activation of PKC. It has been suggested that CT-induced elevations in cAMP and cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) have two separate effects on osteoclasts: abolition of cell motility, and induction of cellular retraction, respectively (11). Studies have shown that in addition to G_s and G_q, the C1a isoform couples to G_i, and that the inhibition of adenylyl cyclase by G_i is negatively regulated by PKC (12). The C1a isoform induces Shc phosphorylation and Erk1/2 activation by mechanisms involving G_i, PKC, and cytosolic Ca²⁺ (13).

Expression of C1a and CTRe13 isoforms vary in a tissue-specific manner, with CTRe13 accounting for less that 15% of the total CTR mRNA in rabbit osteoclasts, kidney, and brain, but comprising at least 50% of the transcripts in skeletal muscle and lung (10). Characterization of the signaling pathways activated by these two receptor isoforms is crucial for understanding the function of these receptors in osteoclasts and other target cells.

G protein-coupled receptors regulate critical intracellular processes such as ion transport and cellular metabolism. Ion transport plays important physiological roles in both bone and kidney, which are key targets for CT. However, little is known about regulation of ion transport and metabolism by the C1a and CTRe13 CTR isoforms. The purpose of this study was to determine the effects of CT on proton efflux from human embryonic kidney cells transfected with either the C1a or CTRe13 isoform. We examined the ability of CT to modulate proton efflux and investigated underlying mechanisms and signaling pathways. Here, we report novel cellular responses to CT mediated specifically by the C1a receptor isoform.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials and solutions
-MEM (catalog no. 12571) buffered with HCO₃^- (26 mM), PBS (catalog no. 14040), FBS (catalog no. 26140), antibiotic solution (penicillin, 10,000 U/ml; streptomycin, 10,000 µg/ml; Amphotericin, 25 µg/ml, catalog no. 15240), trypsin solution (Ca²⁺ and Mg²⁺ free, 0.05% trypsin and 0.53 mM EDTA, catalog no. 25300), G418 (catalog no. 11811), HCO₃^-- and glucose-free DMEM (catalog no. 23800), and HCO₃^--free MEM (catalog no. 41500 and catalog no. 41200) were obtained from Gibco Laboratories (Burlington, Ontario, Canada). Bovine albumin (fraction V, fatty acid free) was from Roche Molecular Biochemicals (Laval, Quebec, Canada; catalog no. 775835). DMEM (catalog no. D-7777), amiloride (catalog no. A-7410), bafilomycin A₁ (catalog no. B-1793), 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) (catalog no. C-3912), isobutylmethylxanthine (IBMX, catalog no. I-5879), and lysophosphatidic acid (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) (LPA) (catalog no. L-7260) were obtained from Sigma (St. Louis, MO). Amiloride and CPT-cAMP were dissolved in standard superfusion medium just before use, whereas IBMX was dissolved in dimethylsulfoxide. Indo-1 penta(acetoxymethyl)ester (indo-1 AM, catalog no. I-1223), 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM, catalog no. B-6769), and thapsigargin (catalog no. T-7459) were obtained from Molecular Probes, Inc. (Eugene, OR). BAPTA-AM, indo-1 AM, and thapsigargin were dissolved in dimethyl sulfoxide and stored in aliquots at -20 C. Salmon CT was obtained from Bachem (Torrance, CA; catalog no. H-2260) or Rorer Pharmaceutical (Fort Washington, PA). Stock solutions of CT were prepared in 0.2% sodium acetate and 0.05% sodium chloride with 1 mg/ml bovine albumin and stored in aliquots at -80 C. 1-[6-((17ß-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122, catalog no. 662035), 1-[6-((17ß-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione (U-73343 catalog no. 662041), bisindolylmaleimide I (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide, catalog no. 203291), staurosporine (catalog no. 569397), and ionomycin (catalog no. 407952) were obtained from Calbiochem (San Diego, CA); and stock solutions of all these agents were prepared in dimethyl sulfoxide and stored in aliquots at -80 C. Cariporide (HOE 642) was generously provided by Aventis Pharma Deutschland GmbH (Frankfurt am Main, Germany). Cariporide was dissolved in water and stored in aliquots at -20 C. Stock solution of TPA (13-O-tetradecanoyl phorbol-13-acetate) was prepared in dimethyl sulfoxide and stored at -20 C. 2-Deoxy-D-[1,2-N-³H]glucose (26 Ci/mmol) was purchased from NEN Live Science Products, Inc. (Boston, MA).

Standard superfusion medium, used in experiments monitoring proton efflux, was HCO₃^--free MEM (catalog no. 41500) supplemented with HEPES (1 mM) and bovine albumin (1 mg/ml) and adjusted to 290 ± 5 milliosmoles/liter with NaCl, and pH 7.30 ± 0.02 with NaOH. Glucose-free superfusion medium was DMEM (catalog no. 23800) without glucose or HCO₃^-, supplemented with HEPES (1 mM), L-glutamine (2 mM), bovine albumin (1 mg/ml), and adjusted to 290 ± 5 milliosmoles/liter and to pH 7.30 ± 0.02. The buffering power of the superfusion solutions was determined over a pH range of 7.0–7.3 at 37 C and found to be 1.4 ± 0.1 mM/pH unit for standard medium and 1.3 ± 0.1 mM/pH unit for glucose-free medium.

For experiments measuring changes in [Ca²⁺]_i, harvested cells were resuspended in HCO₃^--free MEM (catalog no. 41200) supplemented with HEPES (20 mM) adjusted to 290 ± 5 milliosmoles/liter and pH 7.30 ± 0.02. During [Ca²⁺]_i measurements, cells were placed in continuously stirred Na⁺-HEPES buffer (135 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose, and 20 mM HEPES, 290 ± 5 milliosmoles/liter, pH 7.30 ± 0.02).

Cells and culture
We used HEK293 cells, which stably express the rabbit CTR isoform C1a (C1a cells), the CTRe13 isoform (CTRe13 cells), or empty vector (pBK-CMV). CT activates adenylyl cyclase in both C1a cells and CTRe13 cells, and the concentrations of CT required to produce half-maximal (EC₅₀) cAMP responses are approximately 0.06 nM and 0.6 nM, respectively. Maximum cAMP responses are similar in both cell types (10). Cells were maintained in DMEM (catalog no. D-7777) supplemented with HCO₃^- (24 mM), heat-inactivated FBS (10% vol/vol), G418 (500 µg/ml), and antibiotic solution (1% vol/vol), pH 7.30 ± 0.02, in 5% CO₂ at 37 C. For experiments monitoring proton efflux, cells were harvested by exposure to trypsin solution and seeded at a density of 7–10 x 10⁴ cells/cm² on porous polycarbonate membranes (Transwell, 12-mm diameter, 3-µm pore size; Corning, Inc. Costar, Corning, NY) in -MEM (catalog no. 12571) supplemented with HCO₃^- (26 mM), FBS (10% vol/vol), and antibiotics (1% vol/vol), pH 7.30 ± 0.02. Cultures were grown in 5% CO₂ at 37 C for 48 h.

For studies of [Ca²⁺]_i, lactate efflux or glucose uptake, cells were seeded (5 x 10⁴ cells/cm²) on 60-mm culture dishes (Becton Dickinson and Co., Lincoln Park, NJ) in supplemented -MEM for 48–72 h. For experiments examining lactate efflux, growth medium was replaced with -MEM supplemented with bovine albumin (1 mg/ml) and antibiotic solution (1% vol/vol), on the day of the experiment.

Measurement of proton efflux
Cells adhering to the polycarbonate membranes were placed in microflow chambers and positioned above silicon-based potentiometric sensors, which can detect changes in extracellular pH of as little as 10^-3 units (Cytosensor microphysiometer, Molecular Devices, Sunnyvale, CA) (14). Cells were continuously superfused at a rate of 100 µl/min with medium at 37 C. Superfusion media with low buffering power were used to enhance the small alterations in pH_o arising because of efflux of protons from cells. Each chamber was supplied with medium from one of two reservoirs by a computer-controlled valve. Test substances were directly introduced in superfusion medium, and changes in proton efflux were monitored. The lag time between a valve switch and the arrival of test solutions at the microflow chambers was 4–5 sec.

Surface potential of each silicon sensor, corresponding to the extracellular pH (pH_o), was plotted as a voltage-time trace. At 37 C, 61 mV corresponds to 1 pH unit. To measure the rate of acidification (net cellular efflux of proton equivalents), fluid flow to cells was stopped periodically for 30 sec. During this time, acid accumulated in the microflow chamber (vol, 2.8 µl) causing pH_o to decrease. Measurement of acidification rate was obtained by linear least-squares fit to the slope of the pH_o-time trace during the time when fluid flow to the cells was stopped. Net cellular efflux of proton equivalents (proton efflux) was calculated from the acidification rate, based on the buffering power of the superfusion solution and volume of the microflow chamber. In experiments examining the role of cytosolic Ca²⁺ in CT-induced proton efflux, cells were loaded with BAPTA-AM (50 µM in conditioned medium) for 30 min at 37 C before placement in microflow chambers. To test for possible nonspecific interactions with silicon sensors, cultures were lysed using detergent solution and then superfused with CT solution or test media.

Measurement of [Ca²⁺]_i
Cells expressing C1a, CTRe13, or empty vector, were loaded with indo-1 by incubation in conditioned medium with indo-1 AM (2 µM) for 30 min at 37 C. Cells were then washed and harvested by 1-min exposure to trypsin solution. Conditioned medium was added to inactivate trypsin; and subsequently, cells were sedimented and resuspended in HEPES-buffered MEM (catalog no. 41200). Aliquots of cell suspension were sedimented and resuspended in 2 ml of continuously stirred Na⁺-HEPES buffer in a fluorometric cuvette maintained at 37 C. Test substances were added directly to the cuvette.

[Ca²⁺]_i was monitored using a dual-wavelength fluorimeter (Model RF-M2004, from Photon Technology International, South Brunswick, NJ) at 355 nm excitation and emission wavelengths of 405 and 485 nm. The system software was used to subtract background fluorescence and calculate the ratio, R, which is the fluorescence intensity at 405 nm divided by the intensity at 485 nm. [Ca²⁺]_i was determined from the relationship [Ca²⁺] = K_d [(R-R_min)/(R_max-R)]ß, where K_d (for the indo-1-Ca²⁺ complex) was taken as 250 nM, R_min and R_max were the values of R at low and saturating concentrations of Ca²⁺, respectively, and ß was the ratio of the fluorescence at 485 nm measured at low and saturating Ca²⁺ concentration (15).

Measurement of lactate efflux and glucose uptake
Extracellular lactate was measured using a spectrophotometric assay based on generation of NADH via catalytic action of lactate dehydrogenase (Sigma lactate assay kit, catalog no. 826). Cultures were incubated with CT or vehicle in serum-free -MEM (catalog no. 12571) containing bovine albumin (1 mg/ml) in 5% CO₂ at 37 C. Samples of media (100 µl) were collected for lactate determination; and, at the end of each experiment, cells were harvested for protein measurement.

Initial rates of glucose uptake by C1a cells were measured using radiolabeled 2-deoxyglucose, as described previously (16). Briefly, cultures were incubated for 1 min at 23 C with 2-deoxy-D-[³H]glucose (60 µM, specific activity adjusted with unlabeled 2-deoxyglucose to 3.3 mCi/mmol) in glucose-free transport buffer. Incubations were terminated by washing with ice-cold isosmotic Tris-sucrose solution and cells were harvested for protein determination and scintillation counting.

Data analysis and statistics
Proton efflux was normalized as a percentage of basal efflux in standard superfusion medium before addition of test substance or change of superfusion solution. This normalization compensated for differences in cell numbers among the chambers. Basal levels of [Ca²⁺]_i were determined 50 sec before application of test substance. Peak Ca²⁺ responses were quantified as the maximum elevation of [Ca²⁺]_i above basal levels induced by the test substance. Results are presented as representative traces or as means ± SE of the number of samples indicated. Sigmoid curves were fit by nonlinear regression using Prism (GraphPad Software, Inc., San Diego, CA). Comparisons among means were performed by ANOVA followed by a Tukey-Kramer test for multiple comparisons. Differences were accepted as statistically significant at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of CT on proton efflux
Proton efflux from HEK293 cells, stably transfected with the C1a receptor isoform, CTRe13 isoform, or empty vector, was monitored using microphysiometry. Basal proton efflux in standard superfusion medium remained steady for periods of at least 6 h. In C1a cells, superfusion with CT (10 nM) caused a rapid biphasic increase in proton efflux characterized by an initial transient (which reached maximum in 1.5–3 min and lasted approximately 8 min) and a sustained plateau (which lasted for periods of at least 4 h) (Fig. 1A). Treatment with vehicle did not cause any appreciable change in proton efflux. Because the CTRe13 isoform binds CT with lower affinity than does the C1a isoform, higher concentrations of CT were used to test CTRe13 cells. In contrast to its effect on C1a cells, CT (0.1–1 µM) did not elicit detectable changes in proton efflux from either CTRe13 cells (Fig. 1B) or control cells transfected with empty vector (Fig. 1C).

View larger version (28K): [in this window] [in a new window]   Figure 1. CT causes a biphasic increase in proton efflux from C1a cells. HEK293 cells transfected with the CTR isoform C1a (A), CTRe13 (B), or empty vector (C) were cultured on porous polycarbonate membranes, and proton efflux was monitored by microphysiometry. Cells were superfused with standard medium and, at 1.5-min intervals, superfusion was interrupted for 30 sec to measure acidification rate. Proton efflux (net efflux of proton equivalents) was calculated from the acidification rate and expressed as a percentage of basal proton efflux. Where indicated by the shaded areas, C1a cells were superfused with CT (10 nM) or vehicle (Veh), and CTRe13 cells and control cells were superfused with CT (100 nM). Results are representative responses from at least three separate cell preparations. Mean values of basal proton efflux per sample were (in nmol proton equivalents/min): C1a cells, 0.19 ± 0.02 (mean ± SE, n = 128); CTRe13 cells, 0.10 ± 0.02 (n = 21); Vector, 0.11 ± 0.02 (n = 8).

View larger version (31K): [in this window] [in a new window]   Figure 2. Increase in proton efflux from C1a cells is dependent on CT concentration. C1a cells were superfused with standard medium, and proton efflux was monitored. A, Representative time courses of changes in proton efflux induced by the indicated concentrations of CT. B and C, Cell samples were superfused with the indicated concentrations of CT in standard medium for 15–21 min. B, Maximum amplitude of the initial phase (above basal) was measured within 4.5 min of application of CT. C, Data are amplitude of the sustained plateau phase, quantified as the average increase in proton efflux above basal, 21–25.5 min after application of CT. Data in B and C are means ± SE from at least four separate cell preparations. EC50s were 30 and 10 pM CT in B and C, respectively. Mean value of basal proton efflux per sample was 0.196 ± 0.002 nmol proton equivalents/min (n = 107).

View larger version (20K): [in this window] [in a new window]   Figure 3. Effect on proton efflux of sequential treatments with CT. C1a cells were superfused with standard medium, and proton efflux was monitored by microphysiometry. Where indicated by the shaded areas, cells were superfused with CT (10 nM) or vehicle (Veh) in standard medium. Results are from parallel samples and are representative of responses from four separate cell preparations. Values of basal proton efflux per sample were (in nmol proton equivalents/min): CT, 0.07; Veh, 0.12.

C1a cells were treated with amiloride (500 µM) or vehicle in standard superfusion medium. Treatment with amiloride caused a slight decrease in proton efflux (to 95% of basal levels). Subsequently, cells were treated with CT (10 nM) in the continued presence of amiloride or vehicle. Amiloride abolished the transient component of the initial phase of the CT response (Fig. 4A). Cells were then superfused with either amiloride or vehicle, before returning to standard superfusion medium. A rapid overshoot in proton efflux was observed upon washout of amiloride. Subtracting the response to CT in the presence of amiloride from the control response revealed the amiloride-sensitive component (Fig. 4Aii). This component accounts for the transient portion of the initial phase of the CT response. Consistent with the pattern observed in Fig. 4Aii, an increase in the set point of the Na⁺/H⁺ exchanger, to a more alkaline value of cytosolic pH, causes a transient increase in proton efflux by the exchanger, lasting only until a new alkaline steady-state value is reached.

View larger version (33K): [in this window] [in a new window]   Figure 4. The initial transient component of the CT-induced increase in proton efflux is inhibited by NHE blockers. C1a cells were superfused with standard medium, and proton efflux was monitored. A (i), Parallel samples were exposed to vehicle (Veh) or amiloride (Amil, 500 µM) for the period indicated by the horizontal bar beneath the graph. After equilibration for 7.5 min, cultures were superfused with CT (10 nM) where indicated by the shaded area, in the continued presence of Veh or amiloride. A (ii), The amiloride-sensitive component of the CT response, obtained by subtracting the proton efflux in the presence of amiloride from the proton efflux in the presence of Veh. A (iii), Bars represent amplitudes of initial phase and sustained plateau induced by CT in the presence of Veh or amiloride. Data are changes in proton efflux above basal expressed as percentage of basal efflux (means ± SE of four samples from three separate cell preparations; *, P < 0.05 for the effect of amiloride). Mean values of basal proton efflux per sample were (in nmol proton equivalents/min): 0.12 ± 0.02 for Veh and 0.20 ± 0.05 for amiloride. B, The same protocol described in A was used to investigate the effects of cariporide (Carip, 5 µM, a selective blocker of NHE-1) on the increase in proton efflux induced by CT (10 nM). Data in B (iii) are means ± SE of three samples from two separate cell preparations, * P < 0.05 for the effect of Carip. Mean values of basal proton efflux per sample were (in nmol proton equivalents/min): 0.07 ± 0.02 for Veh and 0.09 ± 0.03 for Carip.

It has been previously shown that CT stimulates H⁺ secretion from rat kidney intercalated cells via a bafilomycin A₁-sensitive mechanism (20), likely the vacuolar H⁺-ATPase. We used the specific inhibitor, bafilomycin A₁, to examine the possible involvement of the vacuolar H⁺-ATPase in CT-induced proton efflux. C1a cells were treated with bafilomycin (100–500 nM) or vehicle in standard superfusion medium. Treatment with bafilomycin did not affect basal proton efflux. Proton efflux induced by 10 nM CT in the presence of bafilomycin A₁ (initial phase, 79 ± 11%; sustained plateau, 38 ± 3%; n = 7) was not significantly different from responses in the presence of vehicle (initial phase, 65 ± 5%; sustained plateau, 31 ± 4%; n = 8), arguing against a role for the vacuolar H⁺-ATPase.

Dependence of CT-induced proton efflux on extracellular glucose
We next considered the possibility that the sustained plateau phase of the CT-induced increase in proton efflux is caused by enhanced metabolism of glucose. Parallel samples of C1a cells were superfused with standard medium (containing 5 mM glucose) or glucose-free medium. Superfusion with glucose-free medium rapidly decreased basal proton efflux to 43 ± 5% of control levels (Fig. 5Ai, mean ± SE, n = 8), indicating that basal efflux is highly sensitive to changes in the availability of glucose. Cells were then exposed to CT (10 nM) in the presence or absence of glucose. We observed that the removal of glucose virtually abolished the sustained plateau phase of the CT response, leaving the initial transient component intact. Upon reintroduction of glucose, proton efflux recovered to levels comparable with the sustained plateau in control cells exposed to CT in the presence of glucose (Fig. 5Aii).

View larger version (29K): [in this window] [in a new window]   Figure 5. Dependence of the CT-induced increase in proton efflux on extracellular glucose. Proton efflux from C1a cells was monitored using microphysiometry. A (i), Parallel samples were initially superfused with standard medium. Subsequently, samples were superfused with either glucose-free medium (0 glucose, open circles) or standard medium (5 mM glucose, closed circles) for the period indicated by the horizontal bar beneath the graph. After equilibration for 7.5 min, cultures were superfused with CT (10 nM) where indicated by the shaded area, in the continued presence of appropriate media. A (ii), Amplitude of the plateau calculated at the times indicated by I and II in A (i). The filled bar represents the response of cells in the continuous presence of 5 mM glucose. The unfilled bar represents the response of cells exposed to glucose-free medium. The amplitude of the plateau at I was calculated as the change in proton efflux from the level immediately before superfusion with CT. The amplitude of the plateau after return to standard (glucose-containing) superfusion medium (II) was calculated as the change in proton efflux from the level immediately before the initial change in superfusion medium. Amplitudes are expressed as percentage of basal proton efflux and are means ± SE of 8–10 samples from at least 6 separate cell preparations; *, P < 0.05 for the effect of glucose removal. Mean values of basal proton efflux per sample were (in nmol proton equivalents/min): 0.15 ± 0.03 for 5 mM glucose and 0.17 ± 0.04 for glucose-free. B (i), Parallel samples of C1a cells were superfused with standard medium. Cells were then treated with CT (10 nM) or vehicle (Veh) where indicated by the shaded area. Subsequently, both samples were superfused with glucose-free medium where indicated by the horizontal bar. B (ii), Values of proton efflux calculated at the times indicated by III and IV in B (i). Data are expressed as percentage of basal proton efflux (calculated immediately before superfusion with CT or Veh) and are means ± SE of 6–7 samples from 5 separate cell preparations; *, P < 0.05 for the effect of CT. Mean values of basal proton efflux per sample were (in nmol proton equivalents/min): 0.40 ± 0.07 for CT and 0.30 ± 0.10 for Veh.

Based on its dependence on extracellular glucose, it is possible that lactic acid production plays a role in the sustained plateau phase of proton efflux. To investigate the effect of CT on lactic acid efflux, C1a cells were incubated with either CT (10 nM) or vehicle, and CTRe13 cells were incubated with CT (1 µM) or vehicle for 2 h. CT caused a significant increase in lactate efflux from C1a cells but had no significant effect on lactate efflux from CTRe13 cells (Fig. 6). The amplitude of the CT-induced increase in lactate efflux (50%) in C1a cells was comparable with that of the sustained increase in proton efflux (44%) induced by CT. Thus, the plateau phase of the CT-induced increase in proton efflux seems to arise by enhanced glycolysis. We next examined whether this effect involved enhanced glucose transport. CT had no significant effect on the initial rate of 2-deoxyglucose uptake (380 ± 63 nmol [³H]deoxyglucose/g cell protein·min in control cells; 358 ± 69 nmol [³H]deoxyglucose/g cell protein·min in cells pretreated with 10 nM CT for 15 min, mean ± SE, n = 6 separate experiments). These findings indicate that CT-induced proton efflux does not arise simply from an increase in the rate of glucose uptake.

View larger version (13K): [in this window] [in a new window]   Figure 6. Effect of CT on lactate production. C1a and CTRe13 cells were treated with CT (10 nM or 1 µM, respectively) or vehicle (Veh) in -MEM containing bovine albumin (1 mg/ml) for 2 h. Samples of extracellular medium were collected for lactate determination, and cells were harvested for protein determination. Lactate efflux is expressed as nmol lactate/(µg cell protein·h). Values are means ± SE of at least three samples from two separate experiments; *, P < 0.05 for the effect of CT.

To further investigate the involvement of cAMP in regulating proton efflux, we examined the effects of CPT-cAMP (a membrane-permeable, phosphodiesterase-resistant analog of cAMP). Maximally effective concentrations of CPT-cAMP (0.1–1 mM) induced only a small increase in proton efflux from C1a cells, to 7 ± 1% above basal (n = 21). Moreover, CPT-cAMP had no significant effect on the subsequent response of C1a cells to CT (10 nM) (Table 1). Next, we examined the effects of the phosphodiesterase inhibitor, IBMX. IBMX (40 µM) induced a small increase in proton efflux but did not potentiate the subsequent response of C1a cells to CT (1–10 nM) (Table 1). Even at higher concentrations, IBMX did not potentiate the response to CT. In C1a cells treated with IBMX (0.1–1 mM), the increase in proton efflux induced by 1 nM CT (initial phase, 84 ± 9%; sustained plateau, 58 ± 6%; n = 7) was not significantly different from the response to CT in vehicle-treated control cells (initial phase, 81 ± 13%; sustained plateau, 56 ± 7%; n = 6). Taken together, these data suggest that elevation of cAMP does not underlie the CT-induced increase in proton efflux from C1a cells.

View larger version (17K): [in this window] [in a new window]   Figure 7. Effect of CT and LPA on [Ca2+]i. C1a and CTRe13 were loaded with Ca2+-sensitive dye indo-1 and suspended in Ca2+-containing Na+-HEPES buffer in a fluorometric cuvette at 37 C with continuous stirring. [Ca2+]i was monitored by fluorescence spectrophotometry. A, Where indicated by the arrows, CT (1 nM for C1a cells or 100 nM for CTRe13 cells) or LPA (10 µM) was added directly to the cuvette. Traces are representative of the responses of at least 3 separate cell preparations. B, Single applications of CT were made to individual samples of C1a or CTRe13 cells at the concentrations indicated. Data are amplitude of maximal elevations of [Ca2+]i above resting levels and are means ± SE of 3–14 samples for each point, from 2 separate cell preparations. Error bars for CTRe13 data were smaller than the symbols.

Next, we examined the dependence of the amplitude of the Ca²⁺ response on CT concentration (Fig. 7B). The EC₅₀ for the effects of CT on elevation of [Ca²⁺]_i in C1a cells was 1–3 nM, similar to that reported for CT-induced production of inositol phosphates in C1a cells (10) but 100-fold greater than the EC₅₀ obtained for effects of CT on proton efflux (compare Figs. 2, B and C, with 7B).

We then investigated whether activation of phospholipase C played a role in the CT-induced elevation of [Ca²⁺]_i in C1a cells. Cells were pretreated with U-73122, a PLC inhibitor, or U-73343, an inactive analog (3 µM, 10.5 min), and were resuspended in Na⁺-HEPES buffer containing inhibitor or inactive analog. U-73122 virtually abolished the Ca²⁺ response to CT, implicating PLC in this response. In contrast, after pretreatment with U-73343, CT (1 nM) induced an elevation in [Ca²⁺]_i similar to responses observed in untreated C1a cells (Table 2).

View larger version (16K): [in this window] [in a new window]   Figure 8. Elevation of [Ca2+]i does not increase proton efflux. A, Samples of indo-1-loaded C1a cells were suspended in Na+-HEPES buffer, and [Ca2+]i was monitored by fluorimetry. Where indicated by arrows, CT (10 nM), the calcium ionophore, ionomycin (100 nM), or the SERCA inhibitor, thapsigargin (1 µM), were directly added to the cuvette. Data are representative of responses from at least three separate cell preparations. B and C, Proton efflux from C1a cells was monitored using microphysiometry. Where indicated by the shaded area, cells were treated with CT (10 nM), ionomycin (Iono, 1 µM) or thapsigargin (Thaps, 1 µM) in standard medium. Traces are representative of responses of three samples from at least two separate cell preparations. Mean values of basal proton efflux per sample in standard medium were (in nmol proton equivalents/min): 0.52 ± 0.09 (n = 3) for ionomycin-treated cells and 0.5 ± 0.3 for thapsigargin-treated cells.

To further assess the role of CT-induced elevation of [Ca²⁺]_i in proton efflux, we loaded cells with the intracellular Ca²⁺ chelator, BAPTA. We have previously reported that loading C1a cells using BAPTA-AM (50 µM, 30 min) reduced the amplitude of the elevation of [Ca²⁺]_i induced by CT (1 nM), from 319 ± 37 nM to 53 ± 4 nM above basal (13). In the present study, C1a cells were loaded with BAPTA under the same conditions, and proton efflux in response to CT was monitored. CT-induced increases in proton efflux were still observed in BAPTA-loaded cells, indicating that a large elevation of [Ca²⁺]_i is not necessary for this response (data not shown).

Effect of PKC activation on proton and lactate efflux
In a number of tissues, PKC plays a role in agonist-mediated activation of Na⁺/H⁺ exchange and glycolysis (22, 23, 24). Therefore, we investigated the possible role of PKC in mediating the CT-induced increase in proton and lactate efflux.

TPA is a potent activator of the conventional and novel PKC isoforms. In parallel samples of C1a cells, TPA (100 nM) caused a sustained increase in proton efflux, mimicking the plateau phase of the response to CT (Fig. 9A). When vehicle-treated control cells were subsequently treated with CT (10 nM), they responded with a biphasic elevation in proton efflux. However, in cells treated acutely with TPA, CT elicited the initial transient increase in proton efflux but no further increase in the sustained plateau phase. When both samples were treated again with TPA (100 nM), no additional increases in proton efflux were observed.

View larger version (28K): [in this window] [in a new window]   Figure 9. Effect of a PKC activator on proton efflux from C1a cells. C1a cells were superfused with standard medium, and proton efflux was monitored using microphysiometry. A (i), Parallel samples were treated acutely with the PKC activator, TPA (100 nM), or vehicle (Veh) where indicated by the first shaded area. Subsequently, both samples were treated with CT (10 nM) where indicated by the second shaded area. Both samples were then treated with TPA (100 nM) where indicated by the third shaded area. A (ii), Changes in proton efflux above basal were quantified at the times indicated by Roman numerals in A (i) and are expressed as percentage of basal values calculated immediately before superfusion with TPA or Veh. Data are means ± SE of three to five samples from three separate cell preparations; *, P < 0.05 for the effect of TPA. Mean values of basal proton efflux per sample were (in nmol proton equivalents/min) 0.08 ± 0.03 for Veh (n = 3) and 0.10 ± 0.02 for TPA (n = 5). B (i), Parallel samples of C1a cells were superfused with standard medium. Cells were then treated with TPA (100 nM) or Veh where indicated by the shaded area. Subsequently, both samples were superfused with glucose-free medium where indicated by the horizontal bar. B (ii), Values of proton efflux calculated at the times indicated by IV and V in B (i). Data are expressed as percentage of basal proton efflux (calculated immediately before superfusion with TPA or Veh) and are means ± SE of three to four samples from two separate cell preparations; *, P < 0.05 for the effect of TPA. Mean values of basal proton efflux per sample were (in nmol proton equivalents/min): 0.28 ± 0.09 for TPA and 0.30 ± 0.10 for Veh.

Because CT failed to elicit an increase in proton or lactate efflux from CTRe13 cells, we investigated the responsiveness of these cells to TPA. First, parallel samples of CTRe13 cells were treated with CT (1 µM) or vehicle. In both cases, subsequent application of TPA (100 nM) caused a sustained increase in proton efflux similar to that seen in C1a cells (after CT, 41 ± 2% above basal, n = 10; following vehicle, 61 ± 11% above basal, n = 2; Fig. 10A). These data rule out the possibility that CT activates a signaling mechanism that inhibits PKC-induced proton efflux. We next examined the effects of TPA on lactate efflux from C1a and CTRe13 cells. TPA (100 nM) stimulated lactate efflux from both C1a and CTRe13 cells (Fig. 10B). As shown in Fig. 6, basal lactate efflux from CTRe13 cells was considerably lower than that from C1a cells; however, the absolute effect of TPA on lactate efflux was almost as much in CTRe13 cells as in C1a cells, indicating that the CTRe13 cells are capable of increasing glycolysis in response to activation of PKC.

View larger version (20K): [in this window] [in a new window]   Figure 10. Effect of a PKC activator on proton and lactate efflux. A, CTRe13 cells were superfused with standard medium, and proton efflux was monitored using microphysiometry. Parallel samples were treated with CT [1 µM, A (i)]; or vehicle (Veh) A (ii) followed by the PKC activator, TPA (100 nM) where indicated by the shaded areas. Values of basal proton efflux per sample were 0.02 nmol/min for CT-treated and 0.05 for Veh-treated cells. Traces are representative of responses of 10 samples from 3 separate preparations for A (i) and 2 samples from 1 preparation for A (ii). B, C1a and CTRe13 cells were incubated with or without TPA (100 nM) in -MEM containing bovine albumin (1 mg/ml) for 2 h. Samples of extracellular medium were collected for lactate determination, and cells were harvested for protein determination. Lactate efflux is expressed as nmol lactate/(µg cell protein·h). Values are means ± SE of at least 6 samples from 2–3 separate experiments; *, P < 0.05 for the effect of TPA.

Effect of PKC inhibition or depletion on CT-induced increase in proton efflux
To further assess the role of PKC in the CT-induced increase in proton efflux, we first investigated the effect of the kinase inhibitor staurosporine. On its own, staurosporine (100 nM) did not affect basal proton efflux. However, staurosporine partially inhibited both the initial transient and the sustained plateau phase of the increase in proton efflux induced by CT (10 nM, Fig. 11A). To confirm the effectiveness of staurosporine, we examined its effect on responses to TPA. In vehicle-treated C1a cells, TPA increased proton efflux to plateaus of 27 ± 5% above basal (mean ± SE, n = 4). On the other hand, pretreatment with staurosporine (100 nM, 15 min) completely abolished the response to TPA, confirming its effectiveness (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 11. Effect of protein kinase inhibitors on CT-induced increase in proton efflux. C1a cells were superfused with standard medium, and proton efflux was monitored. A (i), Parallel samples were exposed to Veh or staurosporine (100 nM) for the period indicated by the horizontal bar beneath the graph. After equilibration, cultures were superfused with CT (10 nM) where indicated by the shaded area, in the continued presence of vehicle (Veh) or staurosporine. A (ii), Bars represent amplitudes of initial transients and sustained plateaus induced by CT in the presence of Veh or staurosporine (Stauro). Data are expressed as percentage of basal values and are means ± SE of 12 samples from 5 separate cell preparations; *, P < 0.05 for the effect of staurosporine. Mean values of basal proton efflux per sample were (in nmol proton equivalents/min): 0.21 ± 0.03 for Veh and 0.23 ± 0.03 for staurosporine (n = 12). B, The same protocol as described in A above was used to investigate the effects of bisindolylmaleimide I (Bisindo, 100 nM) on the increase in proton efflux induced by CT (10 nM). B (ii), Means ± SE of 7 samples from 4 separate cell preparations; *, P < 0.05 for the effect of Bisindo. Mean values of basal proton efflux per sample were (in nmol proton equivalents/min): 0.10 ± 0.02 for Veh and 0.09 ± 0.02 for Bisindo.

Last, PKC was depleted by chronic treatment with TPA (100 nM for at least 16 h). Depletion of PKC reduced the sustained plateau of the response to CT (10 nM) by approximately 75%, though having little or no effect on the initial transient (Fig. 12). As expected, depletion of PKC virtually abolished subsequent responses to TPA. Taken together, these findings indicate a role for PKC in mediating the sustained plateau phase of CT response in C1a cells.

View larger version (29K): [in this window] [in a new window]   Figure 12. Effect of chronic pretreatment with TPA on proton efflux. C1a cells were incubated with TPA (100 nM, 16 h) in serum-free -MEM to deplete protein kinase C. TPA-pretreated and untreated cells were then superfused with standard medium, and proton efflux was monitored using microphysiometry. Where indicated by the shaded areas, parallel samples were superfused with CT [10 nM, A (i)] or TPA [100 nM, A (ii)]. B, Bars represent the amplitudes of the sustained plateau induced by CT or TPA. Data are changes in proton efflux above basal expressed as percentage of basal efflux (means ± SE of three to six samples from two to three separate cell preparations); *, P < 0.05 for the effect of TPA pretreatment. Mean values of basal proton efflux per sample were (in nmol proton equivalents/min): 0.17 ± 0.04 for untreated (n = 9) and 0.27 ± 0.05 for TPA-pretreated (n = 9).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The plateau phase of the response remained sustained in the continued presence of CT and after wash out. After treatment with a maximal concentration of CT, no additional increase in proton efflux was observed in response to a subsequent treatment. A similar sustained response and refractoriness to subsequent challenge has been described for the activation of adenylyl cyclase by the CTR in T47D cells (25). The sustained response in both cases is probably attributable to the nearly irreversible binding of CT to its receptor at physiological pH (3). Prolonged activation of signaling pathways downstream from the C1a isoform may also contribute to the sustained elevation of proton efflux. Internalization of the CTR (26) prevents responses to subsequent treatments with CT, and it is also possible that the internalized receptor continues to transduce signals, in turn maintaining the sustained increase in proton efflux.

Mechanisms underlying the effects of CT on proton efflux
Blockers of Na⁺/H⁺ exchange (amiloride and cariporide) had negligible effects on basal proton efflux, indicating little activity of the exchanger under resting conditions. In contrast, both the inhibitors abolished the initial transient component of the CT-induced proton efflux. CT seems to rapidly stimulate the exchanger, giving rise to a transient increase in proton efflux that lasts only until a more alkaline steady-state cytosolic pH is established. Use of the Cytosensor microphysiometer allowed us to resolve stimulation of Na⁺/H⁺ exchange more rapidly than could be accomplished using conventional approaches. On washout of amiloride or cariporide from CT-treated samples, a transient overshoot in proton efflux occurred, consistent with the recovery of Na⁺/H⁺ exchange activity. At the concentrations used in our studies, cariporide selectively blocks the NHE-1 isoform (19), which is endogenously expressed in HEK293 cells. Taken together, these findings are consistent with NHE-1 mediating the initial transient component of the CT response. In the rabbit renal distal convoluted tubule, CT activates a latent HCO₃^--dependent mechanism, to induce recovery of cytosolic pH after an acid load (27). Under the conditions used in our studies, it is unlikely that HCO₃^- influx contributed to efflux of proton equivalents, because all the superfusion solutions used in the microphysiometer must be nominally HCO₃^- -free to avoid the production of gas bubbles.

In HEK293 cells, basal proton efflux drops to approximately 50% on removal of extracellular glucose. In C1a cells, the sustained plateau, but not the initial transient, is dependent on extracellular glucose. On reintroduction of glucose, proton efflux recovers rapidly to levels comparable with those in control cells treated with CT in the presence of glucose. This indicates that signaling pathways downstream from the CTR have been activated in the absence of glucose, but the sustained plateau is manifested only upon reintroduction of extracellular glucose. Interestingly, CT increased both lactate efflux and proton efflux (during the sustained plateau) by approximately 50% above basal rates but did not alter the rate of glucose uptake. Thus, our data clearly demonstrate that the sustained plateau component of the CT response arises from an increased rate of glycolysis, leading to efflux of lactic acid, which may be mediated by a member of the family of monocarboxylate transporters. A recent study demonstrated that activation of heterologously expressed human CTRs leads to gradual acidification of the culture medium over a period of 1–3 d (28). Additionally, it has been shown that CT transiently increases proton efflux from neonatal mouse calvariae (29). However, the mechanisms underlying these effects were not characterized in either study.

Signal transduction pathways mediating CT-induced proton efflux from C1a cells
The C1a receptor couples through multiple G proteins to modulate the adenylyl cyclase/PKA pathway and the PLC/Ca²⁺/PKC pathway. In contrast, the CTRe13 isoform couples to the adenylyl cyclase/PKA pathway but not to the PLC/Ca²⁺/PKC pathway. It is possible that CT-induced activation of Na⁺/H⁺ exchange in C1a cells is mediated by the -subunit of a heterotrimeric G protein activated by C1a, but not CTRe13, as shown for G_q and G13 in other systems (17). Second-messenger-dependent signaling pathways may mediate the effects of CT on glycolysis and Na⁺/H⁺ exchange. The lack of effect of CT on proton efflux from CTRe13 cells indicates that cAMP-dependent pathways do not mediate an increase in proton efflux. This was further confirmed by the small response to the cAMP analog CPT-cAMP and the inability of the phosphodiesterase inhibitor IBMX to potentiate CT responses in C1a cells.

Elevation of [Ca²⁺]_i activates NHE-1 in certain systems via a Ca²⁺/calmodulin site on the exchanger (17). In our study, the EC₅₀ for the effect of CT on proton efflux was markedly less than its EC₅₀ for elevation of [Ca²⁺]_i, indicating that if Ca²⁺ were to induce proton efflux, only small elevations of [Ca²⁺]_i would be required to elicit the response. However, the Ca²⁺ ionophore, ionomycin, and the SERCA inhibitor thapsigargin suppressed proton efflux, in spite of causing elevations in [Ca²⁺]_i similar to those induced by CT. Moreover, the response to CT was still observed in cells loaded with the intracellular Ca²⁺ chelator BAPTA. Thus, CT-induced elevation of [Ca²⁺]_i does not stimulate proton efflux from C1a cells.

PKC-mediated activation of proton production and efflux has been reported in other systems (22, 23, 24, 30, 31, 32). Several observations indicate that PKC underlies at least part of the sustained plateau phase of the CT response in C1a cells. First, TPA [a potent activator of the conventional and novel PKC isoforms (, ßI, ßII, , , , , and )] causes a sustained glucose-dependent increase in proton and lactate efflux, mimicking the plateau phase of the CT-response. Second, in cells treated chronically with TPA to deplete PKC, the sustained plateau phase of the CT response is markedly suppressed. Third, in cells treated acutely with TPA, CT evokes only a transient increase in proton efflux, without further increase in the plateau phase. Fourth, in C1a cells previously treated with CT, TPA fails to elicit any further increase in proton efflux. Thus, activation of PKC seems to underlie the sustained plateau phase of the response to CT. An alternative explanation is that TPA leads to desensitization of the CTR via PKC-mediated phosphorylation. However, this is unlikely because cells treated with TPA (acutely or chronically) still responded to CT with a transient increase in proton efflux (Figs. 9A and 12Ai). Furthermore, CT induces full elevation of [Ca²⁺]_i in cells acutely treated with TPA (data not shown). Our findings are in keeping with previous reports showing that activation of PKC stimulates 6-phosphofructo-2-kinase activity, glycolysis, and lactate production in hepatocytes and fibroblasts (23, 24). It is possible that the sustained plateau phase of the CT response is mediated by either Ca²⁺-sensitive or -insensitive PKC isoforms, because chelating cytosolic Ca²⁺ with BAPTA does not reduce basal [Ca²⁺]_i in C1a cells (134 ± 19 nM in control cells vs. 186 ± 12 nM in BAPTA-loaded cells, n = 7). In this regard, both CT and TPA activate PKC (a Ca²⁺-sensitive conventional PKC isoform) in a porcine renal tubule cell line that possesses native CTRs (33).

The kinase inhibitor staurosporine and the more specific PKC inhibitor bisindolylmaleimide I partially inhibit the effects of CT on proton efflux. At the concentration used in our studies, staurosporine inhibits PKC (isoforms , ßI, ßII, , , and ), PKA, protein kinase G, CaM kinase, and myosin light chain kinase. Staurosporine partially inhibited both the initial transient and the sustained plateau of the CT response. On the other hand, bisindolylmaleimide I, a more potent and selective inhibitor of PKC (isoforms , ßI, ßII, , , and ), primarily inhibited the plateau phase. These findings establish that a PKC-dependent pathway underlies, at least in part, the sustained plateau phase of the CT response. Though bisindolylmaleimide completely blocks the effects of TPA on proton efflux from C1a cells, it only partially inhibits the CT response, suggesting that, in addition to PKC, an as-yet-unidentified mechanism is also involved.

Possible physiological significance of the effects of CT on proton efflux
CT regulates Ca²⁺ homeostasis by inhibiting osteoclastic bone resorption and enhancing renal Ca²⁺ excretion. Expression of the C1a and CTRe13 CTR isoforms varies in a tissue-specific manner, with CTRe13 accounting for 10–15% in osteoclasts, kidney, and brain and at least 50% in skeletal muscle and lung (10). The differences in expression pattern and signaling between the two isoforms may be involved in regulation of CT actions. In bone and kidney, ion transport processes play major roles in skeletal remodeling, maintenance of cellular volume, transepithelial transport, and calcium and acid-base homeostasis. Secretion of proton equivalents into the renal tubular lumen is mediated by two transporters, the Na⁺/H⁺ exchanger and H⁺-ATPase (34). Activation of Na⁺/H⁺ exchange may play a role in the regulation by CT of both Na⁺ reabsorption and proton secretion in the kidney. In osteoclasts, activation of the basolateral Na⁺/H⁺ exchanger may decrease the availability of protons for the ruffled border (apical) vacuolar H⁺-ATPase, thus rapidly diminishing resorptive activity.

In conclusion, we report, for the first time, that CT acts selectively through the C1a receptor isoform to activate a biphasic increase in proton efflux. Na⁺/H⁺ exchange mediates the initial transient, whereas glucose-dependent metabolic acid production is responsible for the sustained plateau phase. Activation of PKC underlies the sustained plateau phase of the response to CT.

	Acknowledgments

 
We thank Ms. Elizabeth Pruski for expert technical assistance with cell culture and lactate assays, Dr. Svetlana V. Komarova and Dr. Stephen M. Sims (Department of Physiology, University of Western Ontario) for helpful comments and advice, and Dr. Udo Albus (Aventis Pharma, Germany) for providing cariporide.

	Footnotes

 
This work was supported by the Canadian Institutes of Health Research [Grant MOP-10854 (to S.J.D.) and Studentship Award (to A.S.)] and by National Institutes of Health [Grant DE-04724 (to R.B.)]. P.C. is a Research Scholar of the Heart and Stroke Foundation of Canada. A preliminary report of this work has appeared in the abstracts of the American Society for Bone and Mineral Research, 2000.

Abbreviations: BAPTA-AM, 1,2-Bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester; [Ca²⁺]_i, cytosolic free Ca²⁺ concentration; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; CT, calcitonin; CTR, CT receptor; EC₅₀, concentration required to produce 50% of maximum effect; IBMX, isobutylmethylxanthine; indo-1 AM, indo-1 penta(acetoxymethyl)ester; K_d, dissociation constant; LPA, lysophosphatidic acid; NHE, Na⁺/H⁺ exchanger; SERCA, sarco-endoplasmic reticulum Ca²⁺-ATPase; TPA, 13-O-tetradecanoyl phorbol-13-acetate.

Received February 23, 2001.

Accepted for publication June 11, 2001.

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