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Endocrinology Vol. 138, No. 12 5282-5292
Copyright © 1997 by The Endocrine Society

ARTICLES

Parathyroid Hormone-Induced Calcium Release from Intracellular Stores in a Human Kidney Cell Line in the Absence of Stimulation of Cyclic Adenosine 3',5'-Monophosphate Production1

Anne-Sixtine Jobert, Christine Leroy, Daniel Butlen and Caroline Silve

INSERM U 426, Faculté de Médecine Xavier Bichat, and Institut Fédératif de Recherche "Cellules Epithéliales," Université Paris VII, Paris Cedex 18, France 75870

Address all correspondence and requests for reprints to: Caroline Silve, M.D., Ph.D., INSERM U 426, Faculté de Médecine Xavier, 16 rue Henri Huchard, BP 416, 75870 Paris cedex 18, France. E-mail: silve{at}bichat.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH-induced mobilization of cytosolic Ca2+ in a human kidney cell line (HEK/W) occurring in the absence of cAMP stimulation was characterized and compared with that obtained in the same cells stably transfected by the PTH/PTH-related peptide (PTHrp) receptor (HEK/T). In both cell lines, N-terminal fragments of PTH and PTHrp induced a concentration-dependent biphasic stimulation in [Ca2+]i: a transient peak followed by a slow linear increase. These increases in [Ca2+]i were inhibited by the PTH antagonist [Nle8,18,Tyr34]bPTH(3–34). The transient peaks were due to calcium release from intracellular stores, as they resisted quenching of calcium in the extracellular buffer and were abolished by prior emptying of intracellular stores. These peaks differed, however, both in latency period and in magnitude, in the two cell lines. The phospholipase C inhibitor U73122 inhibited the PTH-induced increase in [Ca2+]i in HEK/T cells, but not in HEK/W. Similarly, PTH-induced inositol phosphate (InsPs) production was detected in HEK/T but not in HEK/W cells. PTH-induced calcium release in HEK/W cells was inhibited by the simultaneous presence of ryanodine and U73122. Low level PTH/PTHrp receptor messenger RNA expression was demonstrated by ribonuclease protection in HEK/W cells, although no specific binding of [125I]PTHrP(1–34) could be detected. Amplification products for the PTH/PTHrp receptor 1, but no other isoforms, were detected by RT-PCR in HEK/W cells. As expected, HEK/T cells responded to PTH by a 500-fold stimulation in cAMP production and expressed large numbers of PTH/PTHrp receptors, as shown by [125I]PTHrp binding. These results demonstrate that the signal transduction pathways activated by PTH in HEK/W and HEK/T cells are different. Because the major difference in these cell lines is the number of PTH/PTHrp receptors expressed, these results suggest that the transduction of signals by the PTH/PTHrp receptor is controlled by receptor number in such a way that PTH stimulates an increase in intracellular calcium in the absence of stimulation of InsPs and cAMP production in cells expressing low levels of PTH/PTHrp receptor, but stimulates calcium release through an InsPs pathway and induces cAMP production in cells expressing large numbers of PTH/PTHrp receptors. The control of receptor number may be one of the mechanisms through which PTH effects are regulated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH STIMULATES cAMP formation and increases the intracellular calcium concentration [Ca2+]i in various cell types (1). In certain cells, however, PTH stimulates only one of the two transduction pathways (2, 3), demonstrating that the coupling of PTH receptor to signal transduction pathways in these cells is different from that in cells responding to PTH by both a stimulation of cAMP production and [Ca2+]i. This observation raises the possibility that the pathways involved in intracellular calcium increases in one or the other cases might be different. Indeed, different mechanisms have been implicated in the stimulation of [Ca2+]i by PTH, including an increase of intracellular calcium release in the absence (4, 5, 6) or after stimulation of inositol phosphate (InsPs) production (7, 8), and the activation of calcium channels (9, 10). The molecular basis for these differing responses has not been fully characterized, and various hypotheses are still being considered. Such differences in responses could be due to the presence of multiple PTH/PTH-related peptide (PTHrp) receptor isoforms (1). However, a PTH/PTHrp receptor has been cloned (1, 12), and it was subsequently demonstrated that this receptor, when expressed in transfected cells, could increase both cAMP production and [Ca2+]i (7, 12). In studies performed using the cloned receptor, increases in [Ca2+]i have been shown to occur in the presence (7, 8) and the absence (5, 6) of phospholipase C activation. Thus, for at least these responses, it is not necessary to invoke different receptor isoforms to explain the activation of different signal transduction pathways by PTH. Other possible mechanisms include differences in 1) the number of receptors expressed (13, 14, 15); 2) cellular signal transduction machinery (13); and 3) the agonist used [e.g. the utilization of N-terminal, midregion or C-terminal fragments of PTH or PTHrp (16, 17, 18)].

We have observed a stimulation of intracellular calcium by PTH in the absence of cAMP production in a human kidney cell line otherwise able to produce both second messengers in response to other agonists. Such stimulation of intracellular calcium by PTH in the absence of stimulation of cAMP production has been previously demonstrated by Orloff et al. (3) in keratinocytes. In their studies, however, Orloff et al. did not explore the mechanisms involved in this selective PTH-induced calcium increase. The purpose of the present study was 2-fold: 1) to characterize the PTH-induced intracellular calcium increase in the human kidney cell line occurring in the absence of cAMP stimulation; 2) to investigate the molecular basis for such a response. To do so, PTH responses were compared in wild-type cells and in the same cells stably transfected by the PTH/PTHrp receptor, cells that responded to PTH with both an increase in intracellular calcium and in cAMP production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides, reagents, and materials
Human (h) PTH(1–34), hPTHrp(1–34), hPTH(1–84), and [Nle8,18, Tyr34] bovine (b) PTH(3–34)amide, Fura2-AM, bradykinin, ryanodine, BSA, bacitracin, cytidine, and carbachol were purchased from Sigma (St. Louis, MO). U73122 was obtained from ICN Biomedicals (Costa Mesa, CA), and (D-Trp12, Tyr34)bPTH(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)amide was purchased from Bachem (Torrence, CA). Dowex resin AG1-X8 (200–400 mesh, formate form) was obtained from Bio-Rad Laboratories (Richmond, CA). Culture solutions, media, G418, and lipofectamine were purchased from GIBCO-BRL (Gaithersburg, MD). [125I]Na (2300 Ci/mmol), and [3H] myo-inositol (117 Ci/mmol) were purchased from Amersham (Les Ulis, France). Plasticware was obtained from Costar (Cambridge, MA).

Cell culture
HEK 293 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Cells were maintained in a humidified atmosphere of 95% air, 5% CO2, at 37 C in DMEM/HAM F12 medium supplemented with heat-inactivated newborn calf serum and FCS (5% each) and 1 mM glutamine. Certain experiments were repeated using HEK 293 cells obtained from INSERM U 344 (Paris, France) (referred to as HEK/W/U344) and cultured under the same conditions described above.

Transfection
To obtain cells stably expressing the hPTH/PTHrp receptor, wild-type HEK 293 cells from the ATCC (referred to HEK/W) were cotransfected with a pSV2 neo plasmid and 3- to 20-fold excess of a full-length hPTH/PTHrp receptor complementary DNA (cDNA) inserted in the pCDNA 1 plasmid, kindly provided by H. Jüppner, using lipofectamine and following the manufacturer’s instructions. Selection by G418 (750 µg/ml) for neomycin resistance was started 24 h after transfection. PTH/PTHrp receptor-containing clones were identified by measuring the stimulation of cAMP production by a submaximal dose of hPTH(1–34) (7.2 x 10-8 M). The clone selected for this study responded to PTH by a 500-fold stimulation of cAMP production over basal levels (see Results).

Stimulation of cAMP production
Confluent cells in 24-well culture plates were stimulated by adding 5 µl of 10 mM acetic acid containing 0.1% BSA in the absence (control) or with various amounts of PTH peptide to wells containing 250 µl DMEM/HAM F12 supplemented with 1 mM isobutylmethylxanthine and 0.1% BSA for 10 min at 37 C as described (19). Stock solutions of PTH peptides were prepared in 10 mM acetic acid. cAMP produced was measured using a previously described RIA after acetylation of the samples (20). Proteins were measured with the Coomassie protein assay reagent (Pierce Chemical Co, Rockford, IL). Results are expressed as nanomoles of cAMP or picomoles of cAMP/mg protein/10 min.

Measurement of cytosolic calcium concentrations
Measurement of the cytosolic calcium concentration [Ca2+]i was performed on cells in suspension as described (19). Confluent cells in 56- cm2 dishes were detached by versene and resuspended at 0.5–1 x 106 cells/ml in DMEM/HAM F12 containing 1% BSA. Cells were loaded with 3 µM Fura2-AM for 45 min at 37 C, washed twice with PBS buffered with 20 mM HEPES (pH 7.4–7.6) containing 1 mM CaCl2 and 0.1% BSA (PBS/Ca/BSA), and resuspended in PBS/Ca/BSA at 0.5–1 x 106 cells/ml. Two milliliters of loaded cells were transferred into a plastic cuvette at 30 C under constant stirring, and fluorescence was monitored continuously in a F2000 Hitachi spectrofluorimeter (Hitachi Scientific Instruments, Mountain View, CA) at excitation and emission wavelengths of 340/380 and 510 nm, respectively. Maximal and minimal cytosolic [Ca2+]i were determined by adding 50 µM digitonin and 10 mM EGTA, respectively. Cytosolic [Ca2+]i was calculated as described (21) using a dissociation constant (Kd) of 224 nM. Stock solutions of test substances or vehicles alone were added directly to the cell suspension through an injection port. The different agonists used were: 1) PTH peptides in 20 µl 10 mM acetic acid (final concentrations: 10-9 to 10-6 M), 2) ryanodine in 20 µl H2O (final concentration: 100 µM); 3) carbachol in 2 µl H2O (final concentration: 1 mM); 4) U73122 in 2 µl dimethylsulfoxide (final concentration: 10 µM); and 5) bradykinin (final concentration: 10-6 M) in 2 µl H2O. Vehicles had no significant effect on [Ca2+]i.

Stimulation and measurement of InsPs production
InsPs production was determined as described (22, 23, 24). Confluent cells in 56-cm2 culture plates were loaded overnight with [3H]myo-inositol (2 µCi/ml) in culture medium containing 1 mM cytidine. The cells were detached and washed five times in medium containing 25 mM HEPES-NaOH, pH 7.4, 137 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 0.44 mM NaH2PO4, 0.33 mM Na2HPO4, 1 mM MgCl2, 1 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, 3 mM lactic acid, 10 mM sodium acetate, and 0.1% BSA (medium A). Cells were then resuspended in medium A supplemented with 10 mM LiCl and 0.1% bacitracin (3–5 x 105 cells/ml), and incubations were performed at 30 C in the absence (basal) or presence of agonists (0.1 µM hPTH(1–34), 5 min or 1 mM carbachol, 20 min). In preliminary experiments, it was determined that maximum stimulation of InsPs production was obtained 5 min after stimulation by 0.1 µM hPTH(1–34) and 20 min after stimulation by 1 mM carbachol. Stimulation was stopped by adding 200 µl ice-cold 5 mM EDTA-NaOH, pH 7.0, and 1 ml ice-cold chloroform-methanol (1:2 vol/vol), and placing the samples at 4 C. Chloroform (300 µl) and water (300 µl) were then added, and the samples were stirred and then centrifuged for 10 min at 3,000 x g at 4 C to separate the phases. One milliliter of the hydrophilic phase from each sample was diluted with 3 ml of 3 mM HEPES-NaOH, pH 7.0, before chromatography. The remaining hydrophobic phase was mixed with 500 µl methanol and 500 µl H2O at 4 C and centrifuged for 5 min at 3,000 x g. Radioactivity incorporated in total phosphoinositides was determined by counting the resulting lower phases after evaporating to dryness. The hydrophilic neutralized phase from each sample was applied to a chromatography column containing 0.25 g Dowex resin AG1-X8. Free inositol, glycerophosphoinositol (GroPIns), and total InsPs were successively and respectively eluted by 8 ml of the following buffers: 3 mM HEPES-NaOH pH 7.0 (inositol), 30 mM ammonium formate (GroPIns), 1 M ammonium formate, and 0.1 M formic acid (InsPs). The radioactivity contained in each eluate and in phosphoinositide extracts was determined by scintillation spectroscopy, and the results were corrected for variable quenching. Results are presented as the ratio of the total 3H-labeled InsPs over total incorporated radioactivity (inositol+GroPIns+InsPs+PI). The percentage of total radioactivity incorporated in inositol and GroPIns, respectively, represented 50–75% and 0.4–0.7% of total radioactivity and were not significantly changed by PTH stimulation.

Iodination of PTHrp(1–34)
Five micrograms of PTHrp(1–34) (Sigma) were labeled with 1 mCi [125I] Na in 50 mM Na phosphate buffer (pH 7.5) with 3 x 0.6 µg chloramine T for 1 min at room temperature (19, 25). The reaction was stopped with 20 µl of 1 mM ß-mercaptoethanol, 20 µl of 49 mM N-acetyl-tyrosine, and 200 µl of 60 mM KI. After removal of an aliquot for trichloroacetic acid precipitation, the product of iodination was purified using a SEP-PAK C18 cartridge (Waters, Milford, MA). The column was washed with 6 ml 0.1% trifluoroacetic acid (TFA) and 30–40 ml 10% acetonitrile in 0.1% TFA, and the iodinated peptide was eluted with 40% acetonitrile in 0.1% TFA.

[125I]PTHrp binding
Binding reactions were performed as described (19, 26) for 4 h at 16 C on confluent cells grown in 24-well plates in 250 µl binding buffer [50 mM Tris-HCl (pH 7.7), 100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 0.5% FCS, and 5% horse serum] with 1 x 105 cpm [125I]PTHrp, in the absence or presence of unlabeled hPTH(1–34) at concentrations ranging from 7.2 x 10-11 to 7.2 x 10-7 M. At the end of the incubation, an aliquot of incubation medium was removed to determine the radioactivity, cells were washed and solubilized in 0.5 M NaOH, and cellular radioactivity was measured in a {gamma}-counter. Results are expressed as specific [125I]PTHrp bound/100 µg protein. Nonspecific binding was that which occurred in the presence of 7.2 x 10-7 M hPTH(1–34). Protein content per well was measured as described above.

RNA extraction
Total RNA was extracted from cells grown in 56-cm2 dishes according to the method of Chomczynski and Sacchi (27) and stored at -80 C. Messenger RNA (mRNA) was extracted from HEK/W using the Dynabeads mRNA direct kit (Dynal, Compiegne, France) following the manufacturer’s instructions.

RNAse protection analysis
A 312-bp fragment of the hPTH/PTHrp receptor was amplified from the full-length PTH/PTHrp receptor cDNA inserted in the pcDNA1 plasmid using the primers C and D (Table 1Go) and cloned into the pCRII plasmid using the TA cloning system (Invitrogen, San Diego, CA). The plasmid containing the hPTH/PTHrp fragment was linearized by digestion with Spe1, and the riboprobe synthesized using T7 RNA polymerase. RNAse protection analyses were performed as described (28, 29). The unprotected PTH/PTHrp receptor riboprope migrated at 442 bp.


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  		Table 1. Sequence and position of primers used to amplify the PTH/PTHrp receptor

 
Amplification of PTH/PTHrp receptor sequences by RT-PCR
cDNA was synthesized by reverse transcription of mRNA (HEK/W) and total RNA (HEK/T). Five to 10 µl of cDNA were amplified in a final volume of 50 µl using 1 U of Taq DNA Polymerase (GIBCO-BRL) in the presence of 1.5 mM MgCl2, 0.25 mM of each deoxynucleotide, and 0.5 pmol of each primer. The sequences of the oligonucleotide primers used for amplification of the hPTH/PTHrp receptor were based on the human cDNA sequence reported by Schipani et al. (30) and are described in Table 1Go.

Statistical analysis
Unless otherwise stated, results are expressed as mean ± SEM. Statistical analyses were performed using Student’s t test or analyzed by one-way ANOVA. Comparisons between individual groups were made using the Fisher least significant difference t test (31). P values of less than 0.05 were considered as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTH-induced calcium responses in HEK cells
Basal intracellular [Ca2+]i was slightly higher in HEK/W cells compared with HEK/T cells, but the difference did not reach significance (106.6 ± 15.1 and 81.5 ± 3.2 nM, respectively, in HEK/W and HEK/T, n = 9–11, P > 0.05). In HEK/W and HEK/T cells, hPTH(1–34) (10-7 M) induced a biphasic calcium response that consisted of a first transient increase with a return to baseline within 2 to 3 min followed by a second increase of lower amplitude at 4 min (Fig. 1Go). The initial transient peak differed between the two cell lines both in the length of the latency period and by a modest difference in the amplitude of the response. In HEK/W cells, hPTH(1–34) produced a 1.5 ± 0.2-fold increase over basal in [Ca2+]i (peak minus basal: 57.6 ± 6.0 nM, n = 11) with 20–30 sec latency period after PTH addition. In contrast, in HEK/T cells, 10-7 M hPTH(1–34) induced an immediate transient increase with a 2.1 ± 0.1-fold increase in [Ca2+]i over basal levels (peak minus basal: 91.3 ± 11.5 nM, n = 9; P < 0.05 comparing HEK/W and HEK/T cells). The second rise in [Ca2+]i was similar in both cell lines (Fig. 1Go). Identical results were observed when cells were stimulated with 10-7 M hPTHrp(1–34) and 10-7 M hPTH(1–84) (data not shown). Bradykinin (10-6 M), used as a control, elicited a similar increase in [Ca2+]i in both cell lines: stimulation fold over basal: 1.67 ± 0.14 (peak minus basal: 67.7 ± 6.6 nM) and 1.8 ± 0.8 (peak minus basal: 65.9 ± 4.8 nM), respectively, in HEK/W and HEK/T cells (Fig. 1Go, n = 9–11). Identical results were obtained in a similar series of experiments carried out using HEK/W/U344 cells, i.e. wild-type HEK293 cells derived from a different clone (data not shown).



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  		Figure 1. Intracellular calcium rises induced by PTH and bradykinin in HEK/W ({circ}) and HEK/T ({triangleup}). [Ca2+]i was monitored by fluorimetry following the addition of 10-7 M hPTH(1–34) at 40 s ({uparrow}) and 10-6 M bradykinin at 380 s ({uparrow}). The tracings are the mean of nine to 11 separate experiments. SEM values were less than 15% of the means for all points and are not presented.

 
Increases in [Ca2+]i were induced in a concentration-dependent manner by PTH in both HEK/W and HEK/T cells. The stimulation-fold increases over basal were similar in both cell lines for hPTH(1–34) at concentrations ranging from 10-9 M to 5 x 10-8 M. At 5 x 10-8 M hPTH(1–34), the increase in [Ca2+]i reached a plateau in HEK/W cells, whereas 10-7 M hPTH(1–34) was required to induce maximal [Ca2+]i in HEK/T cells (Fig. 2Go). Analysis of the data indicated that the EC50 for the calcium effect of PTH was approximately 10-8 M in HEK/W cells, and 7 x 10-8 M in HEK/T cells. No significant stimulation was observed for PTH concentrations below 10-9 M in either cell line.



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  		Figure 2. Stimulation of [Ca2+]i by increasing concentrations of hPTH(1–34) ({uparrow}) and by 10-6 M bradykinin ({uparrow}) in HEK/W (A-E) and in HEK/T (F-K). Intracellular calcium was monitored with fura-2 on cells in suspension as described in Materials and Methods. In panel L, results are expressed as fold stimulation over basal (HEK/W: •; HEK/T: {blacktriangleup}). Results are representative of those obtained in two separate experiments. The concentrations of PTH used are indicated in the figure.

 
Addition of the PTH antagonist [Nle8,18,Tyr34] bPTH(3–34) amide 2 min before that of 10-7 M hPTH(1–34) inhibited the PTH(1–34)-induced calcium response cells in a concentration-dependent fashion in HEK/W (Fig. 3Go, A-E) and HEK/T cells (Fig. 3Go, F-J). The antagonist alone did not stimulate [Ca2+]i for concentrations up to 10-7 M and did not inhibit the calcium responses induced by 10-6 M bradykinin (Fig. 3Go). Similar results were obtained with the antagonist [D-Trp12,Tyr34]bPTH(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)amide (not shown).



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  		Figure 3. Dose-dependent inhibition of PTH(1–34)-induced [Ca2+i] increases by [Nle8,18,Tyr34] bPTH(3–34) amide in HEK/W (A-E) and HEK/T (F-J). [Nle8,18,Tyr34] bPTH(3–34) amide ({Uparrow}) was added 2 min before hPTH(1–34) (10-7 M) ({uparrow}). [Ca2+]i was monitored with fura-2 as described in Materials and Methods. The concentrations of [Nle8,18,Tyr34] bPTH(3–34) amide used were (in molar concentration) A and F: 0; B and G: 10-10; C and H: 10-9; D and I: 5 x 10-8; E and J: 10-7. For each concentration of antagonist, a different aliquot of cells from the same cell preparation was used. Intracellular calcium rises induced by bradykinin ({uparrow}) were also monitored and were not affected by pretreatment with bPTH(3–34). Similar results were obtained in two separate experiments.

 
Role of extracellular calcium influx and calcium release from intracellular stores in PTH-induced calcium responses in HEK/W and HEK/T cells
To determine whether PTH-induced calcium responses were caused by an influx of extracellular calcium and/or by the release of calcium from intracellular stores, cells were stimulated by 10-7 M hPTH(1–34) in the absence of extracellular calcium (Ca2+EC). To do so, the calcium response was tested after the addition of an excess of the calcium chelator EGTA (5 mM). The initial transient rise in [Ca2+]i induced by PTH still occurred in both HEK/W and HEK/T cells in the absence of Ca2+EC, whereas the second increase in [Ca2+]i was abolished in both cell types (Fig. 4Go, A and B).



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  		Figure 4. PTH-induced [Ca2+]i rise in the absence of extracellular calcium in HEK/W (A) and in HEK/T (B). hPTH(1–34) (10-7 M) ({uparrow}) was added to the medium 2–3 min after the addition of 5 mM EGTA. [Ca2+]i was monitored with fura-2 on cells in suspension as described in Materials and Methods. Tracings are representative of five experiments.

 
The involvement of intracellular pools of calcium in the transient calcium increases in response to PTH was further confirmed by studying PTH stimulation after intracellular calcium pools were depleted by pretreatment with carbachol. As expected from results obtained in other systems (32, 33), carbachol alone (1 mM) induced a large transient increase in [Ca2+]i in both cell lines (Fig. 5Go; stimulation fold: 5.03 ± 0.85 and 5.79 ± 0.33 in HEK/W and HEK/T, respectively; n = 5). When added after carbachol, PTH no longer induced calcium transients in either HEK/W or HEK/T cells (Fig. 5Go, B-D). These results demonstrated that the transient PTH-induced calcium peaks in both HEK/W and HEK/T cells were dependent on release from intracellular calcium pools.



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  		Figure 5. Effect of pretreatment with carbachol on PTH-induced [Ca2+]i increases in HEK/W (A and B) and HEK/T (C and D) cells. Carbachol (1 mM) (B and D) ({Uparrow}) was added 3.5 to 4.5 min before 10-7 M hPTH (1–34) ({uparrow}). Intracellular calcium was monitored with fura-2 on cells in suspension as described in Materials and Methods. Results in each panel are representative of three separate experiments.

 
Pathways involved in PTH-induced calcium responses in HEK/T and HEK/W cells
Two major pools of calcium can participate in the increase of cytoplasmic calcium in response to various agonists. From which of these pools calcium is released depends on the mechanisms that are activated following stimulation (34). One mechanism involves the activation of phospholipase C, resulting in the production of inositol trisphosphate (IP3) as second messenger (34). A second system is mediated by ryanodine receptors (RyR) (35). To evaluate the pathway(s) involved in PTH-induced calcium responses in HEK/T and HEK/W cells, PTH stimulation was performed in cells following pretreatment with U73122 (an inhibitor of phospholipase C), ryanodine (an inhibitor of RyR), or the two agents together. U73122 (10 µM) did not modify the calcium transient elicited by PTH in HEK/W cells (Fig. 6Go, E and F). In contrast, U73122 totally inhibited the PTH-induced [Ca2+]i increase in HEK/T cells (Fig. 6Go, A and B). U73122 alone had no effect on basal [Ca2+]i in either HEK/W or HEK/T cells (Fig. 6Go, B and F). Pretreatment of HEK/W or HEK/T cells with 100 µM ryanodine, a concentration known to inhibit RyR (35), had no effect on PTH-induced increase in [Ca2+]i (Fig. 6Go, C and G). Surprisingly, in HEK/W cells pretreated with both 100 µM ryanodine and 10 µM U73122, the PTH-induced increase in [Ca2+]i was dramatically reduced (Fig. 6Go, E and H, stimulation fold over basal: 1.54 compared with 1.10, respectively, in untreated and treated cells). Ryanodine alone (100 µM) (Fig. 6Go, C and G) or in the presence of U73122 (10 µM) (Fig. 6Go, D and H) had no effect on basal [Ca2+]i in HEK/W and HEK/T cells. Pretreatment of HEK/W cells with 15 µM dantrolene, another RyR inhibitor (36), inhibited the PTH-induced increase in [Ca2+]i by 20.2 ± 2.3% (n = 6, P < 0.05), but had no significant effect on the PTH-induced increase in [Ca2+]i in HEK/T.



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  		Figure 6. Effect of pretreatment with U73122 and/or ryanodine on PTH-induced [Ca2+]i increases in HEK/T (A-D) and HEK/W (E-H) cells. A and E, 10-7 M hPTH(1–34) alone; B and F, 10 µM U73122 ({Uparrow}) were added 5 min before 10-7 M hPTH(1–34) ({uparrow}). C and G, 100 µM ryanodine ({uparrow}) were added 5 min before 10-7 M hPTH(1–34) ({uparrow}). D and H, 10 µM U73122 ({Uparrow}) and 100 µM ryanodine ({uparrow}) were added 5 min before 10-7 M hPTH(1–34) ({uparrow}). Results in each panel are representative of three separate experiments.

 
To further document the difference in PTH-induced calcium responses observed in HEK/T and HEK/W cells, the susceptibility of both lines to inhibition of calcium signaling in response to bradykinin was studied. In the majority of tissues, bradykinin has been reported to induce activation of phosphoinositide metabolism (37). In contrast to the PTH-induced calcium response, the response to bradykinin was similar in both HEK/W and HEK/T cells (Fig. 1Go). In both lines, bradykinin increased [Ca2+]i in the absence of extracellular calcium, and U73122 completely inhibited the bradykinin-induced increase in [Ca2+]i, thus confirming that bradykinin stimulates the release of calcium from IP3-sensitive intracellular pools. As observed for PTH-induced responses, pretreatment of both HEK/W and HEK/T cells by carbachol abolished the stimulation of intracellular calcium by bradykinin (data not shown).

PTH-induced stimulation of InsPs production
To confirm the activation of phospholipase C by PTH in HEK/T, InsPs production was measured after stimulation by PTH in both cell lines. No stimulation of InsPs production by PTH (10-7 M, 5 min) could be detected in HEK/W cells ([3H]-InsPs: 0.69 ± 0.02 and 0.67 ± 0.01% of total radioactivity incorporated, respectively, in PTH-untreated and treated cells, n = 3, NS). In contrast, PTH increased InsPs production in HEK/T cells ([3H]-InsPs: 0.52 ± 0.01 and 0.65 ± 0.04% of total radioactivity incorporated, respectively, in PTH-untreated and -treated cells; stimulation-fold over basal: 1.24 ± 0.08; n = 3, P < 0.05). These results are in agreement with the observation that U73122 inhibited PTH-induced calcium response in HEK/T cells but not in HEK/W cells. Carbachol, used as a control, produced a significant increase in [3H]InsPs production in both cell lines (stimulation fold over basal: 2.02 ± 0.06 and 4.52 ± 0.30, respectively, in HEK/W and HEK/T cells, n = 3).

PTH-induced cAMP production in HEK/W and HEK/T cells
Basal cAMP values were significantly lower in HEK/W compared with HEK/T cells (17.97 ± 1.25 and 55.71 ± 5.42 pmol/mg protein, respectively, n = 15, P < 0.05). No significant stimulation of cAMP production by hPTH(1–34) could be detected in HEK/W for concentrations up to 2.4 x 10-7 M. A small increase was observed when cells were stimulated with 7.2 x 10-7 M hPTH (1–34) (Fig. 7Go, stimulation-fold over basal: 2.27 ± 0.18, n = 15, P < 0.05). In contrast, hPTH(1–34) elicited a strong concentration-dependant increase in cAMP production in HEK/T cells. Maximal stimulation was obtained with 7.2 x 10-7 M hPTH(1–34) (571.7 ± 56.2-fold stimulation over basal, n = 9–15). Analysis of the data indicated that the EC50 for the stimulation of cAMP production by PTH was approximately 3 x 10-9 M. Similar results were obtained when cAMP production was stimulated with hPTHrp(1–34) or hPTH(1–84) (data not shown). Carbachol pretreatment did not significantly modify PTH-induced stimulation of cAMP production (Table 2Go). Forskolin and PGE2 stimulated cAMP production to the same extent in both cell lines (data not shown). The antagonist [Nle8,18,Tyr34] bPTH(3–34) amide did not stimulate cAMP production in either cell line for concentrations up to 10-7 M (data not shown).



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  		Figure 7. Stimulation of cAMP production by increasing concentrations of hPTH(1–34) in HEK/W (•) and HEK/T ({blacktriangleup}) cells. cAMP production in the presence of 1 mM isobutylmethylxanthine was performed as described in Materials and Methods. Data are expressed as the mean ± SEM; n = 9–15. Error bars for HEK/W are smaller than the symbols.

 

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  		Table 2. Effect of carbachol pretreatment on PTH-induced cAMP production in HEK/W and HEK/T cells

 
Characterization of [125I]PTHrP(1–34) binding to HEK/W and HEK/T cells
No specific binding of [125I]PTHrp(1–34) could be detected in HEK/W cells (Fig. 8Go). As expected, specific binding of [125I]PTHrp(1–34) was detected in HEK/T. Specific binding represented 11.8 ± 0.51% of total radioactivity added (n = 3), and it was competitively inhibited by increasing concentrations of unlabeled hPTH(1–34). Half-maximal displacement of bound PTHrp was obtained with approximately 7 x 10-9 M hPTH (1–34). PTH/PTHrp receptor number per cell (calculated by Scatchard analysis of radioligand binding data) was approximately 3.5 x 105 receptors per cell.



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  		Figure 8. Competitive inhibition of [125I]hPTHrp(1–34) binding by increasing concentrations of unlabeled hPTH(1–34) in HEK/W ({blacksquare}) and HEK/T ({blacktriangleup}) cells. Results were corrected for nonspecific binding measured in the presence of 7.2 x 10-7 M hPTH(1–34) and are expressed as the mean ± SEM (n = 3). The insert presents the Scatchard plot of the binding data for HEK/T cells (r = 0.91). No Scatchard plot could be generated from the binding data in HEK/W cells (r = 0.57).

 
Expression of PTH/PTHrp receptor mRNA in HEK/W and HEK/T cells
To study PTH/PTHrp receptor mRNA expression, RNAse protection analysis was performed on total RNA from HEK/W and HEK/T cells using a human PTH/PTHrp receptor probe specific for a 312-bp fragment coding for a portion of the N-terminal extracellular domain of the receptor. Results clearly demonstrated the presence of a specific signal for the PTH/PTHrp receptor mRNA in HEK/W cells (Fig. 9Go). The intensity of the signal was proportional to the quantity of total RNA used in the assay. No signal was detected in HEK/W using 5 µg RNA, while 50 and 100 µg RNA gave signals of increasing intensity. Similar results were obtained with the HEK/W/U344. As expected, an intense signal was obtained when as little as 10 µg RNA from HEK/T cells was used (Fig. 9Go).



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  		Figure 9. Expression of PTH/PTHrp receptor mRNA measured by RNAse protection assay in HEK/W and HEK/T cells. Different concentrations of total RNA from HEK/W cells (lanes 1 and 4, 50 µg; lanes 2 and 5, 100 µg; lane 3, 5 µg), from HEK/W/U344 cells (lane 6, 5 µg; lane 7, 50 µg; lane 8, 100 µg), and from HEK/T cells (lane 9, 10 µg) were hybridized with a 125I-labeled antisense hPTH/PTHrp receptor riboprobe, as described in Materials and Methods. mw, Molecular weight standards; lane rb, hPTH/PTHrp receptor riboprobe alone.

 
To further characterize the PTH/PTHrp receptor mRNA expressed in HEK/W cells, cDNA was prepared from HEK/W and HEK/T cells and used as a substrate to amplify the entire PTH/PTHrp receptor sequence in three overlapping fragments, using three pairs of primers (C/B, E/F, I/J) (Table 1Go). The pair C/B amplifies exons S, E1, E2, and the beginning of exon G [according to the nomenclature of Kong et al. (38)], the pair E/F extends from exon E1 to the junction of exon M3/M4, and the pair I/J extends from exon M3 to the end of exon T (Table 1Go). When the pairs E/F and I/J were used, amplification products obtained for cDNA from HEK/W and HEK/T cells migrated as single bands of expected size (871 and 1010 bp, respectively) (Fig. 10Go). When the pair C/B was used, a single band of expected size (451 bp) was obtained in the HEK/T, while in the HEK/W, a smaller amplification product migrating between 300 and 400 bp was detected in addition to the 451-bp product. This smaller amplification product corresponds to the amplification of a previously described alternatively spliced mRNA of the PTH/PTHrp receptor (19).



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  		Figure 10. Amplification of PTH/PTHrp receptor cDNA prepared from HEK/W mRNA (lanes 2, 4, 6) and HEK/T total RNA (lanes 1, 3, 5) using the oligonucleotide pairs C/B (lanes 1 and 2), E/F (lanes 3 and 4), and I/J (lanes 5 and 6). Oligonucleotide pairs C/B, E/F, and I/J are described in Table 1Go, and amplify the entire receptor cDNA in three overlapping fragments. Amplified products were separated on a 1% agarose gel and stained with ethidium bromide. The mol wt markers are a 100-bp ladder (lanes mw).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results demonstrated that 1) in the untransfected human embryonic kidney cell line HEK293 the agonists PTH(1–34), PTHrp(1–34), and PTH(1–84) increased [Ca2+]i, but did not stimulate cAMP and InsPs production; 2) these cells expressed PTH/PTHrp receptor mRNA, although no binding of iodinated PTHrp(1–34) could be detected; 3) in the same cell line transfected by the PTH/PTHrp receptor and expressing a high number of receptors, PTH agonists increased [Ca2+]i via a phospholipase C sensitive pathway and stimulated cAMP and InsPs production.

We found a readily observable, reproducible, and sensitive stimulation of intracellular [Ca2+]i in wild-type HEK293 cells obtained from two different sources. In addition, the presence of PTH/PTHrp receptor mRNA was clearly identified in the cells by RT-PCR and RNAse protection analysis. Our results contrast with previously published work reporting the absence of a PTH-induced increase in intracellular [Ca2+]i in wild-type HEK293 cells (8, 39), and the absence of PTH/PTHrp receptor mRNA expression evaluated by Northern blotting (40). The reasons for the discrepancy between these and our results remain unclear. Nevertheless, the demonstration in this human renal cell line of a robust, reproducible, and sensitive PTH-induced increase in [Ca2+]i permitted the further characterization of PTH-induced calcium release occurring in the absence of stimulation of cAMP production.

In HEK/W cells, PTH-induced increases in [Ca2+]i were observed for PTH concentrations as low as 10-9 M. In contrast, PTH in concentrations as high as 2.4 x 10-7 M did not stimulate cAMP production. The absence of stimulation of cAMP production could not be explained by a deficiency in the Gs/adenylate-cyclase system, as PGE2 and forskolin stimulated cAMP production under the same assay conditions. In addition, PTH-stimulated cAMP production was easily detected in the same HEK293 cells following transfection with a plasmid that induced the expression of large numbers of PTH/PTHrp receptors (see below). These results are similar to those obtained by Orloff et al. (3) for cultured human keratinocytes, in which a failure of PTH or PTHrp to stimulate cAMP production in otherwise adenylate-cyclase-competent cells with functional Gs and catalytic subunits was demonstrated.

The PTH-induced increase in [Ca2+]i in HEK/W cells was further characterized and compared with that obtained in the same HEK cells stably transfected with PTH/PTHrp receptors (HEK/T). The HEK/T cells expressed 1) high levels of PTH/PTHrp receptor mRNA, as determined by RNAse protection analysis; 2) high PTH/PTHrp receptor number, as demonstrated by the binding of [125I]PTHrp; and 3) responded to PTH by a 500-fold stimulation of cAMP and by an increase in [Ca2+]i. Comparisons of the PTH-induced [Ca2+]i increases showed that in both HEK/W and/T cell lines, PTH agonists induced a biphasic stimulation: a transient peak with a return to baseline followed by a slow linear increase over time. The initial increase was due to calcium release from intracellular stores, as it resisted quenching of calcium in the extracellular buffer and was abolished by prior emptying of the intracellular calcium stores. Although the transient peak in [Ca2+]i was due to a release from intracellular stores in both HEK/W and HEK/T cells, the response differed in both time course and magnitude, suggesting that the pathways involved in the PTH-induced calcium response were different in the two cell lines. In support of this hypothesis, the phospholipase C inhibitor U73122 inhibited the PTH-induced calcium increase in the HEK/T cells, but not in the HEK/W cells. Furthermore, PTH-stimulated InsPs production was detected in the HEK/T but not in HEK/W cells. In both cell lines, significant formation of InsPs in response to carbachol could be detected, demonstrating that this signaling pathway was potentially operational in both cell lines. Interestingly, ryanodine, when added with the phospholipase C inhibitor U73122, inhibited PTH-induced calcium increase in HEK/W cells, indicating that activation of the RyR plays a role in the PTH-induced calcium release in HEK/W cells. Taken together, these data clearly demonstrate that the pathways involved in PTH-induced calcium release in HEK/W and HEK/T cells are different and suggest that PLC activation is the dominant pathway in HEK/T cells, but not in HEK/W cells. Although clearly not the dominant pathway responsible for the release of [Ca2+]i from HEK/W cells, it is possible that low levels of IP3 production occurred in these cells but were below the sensitivity of the assays used in our study. In particular, the requirement for both ryanodine and U73122 to inhibit calcium release by HEK/W cells is compatible with this possibility. Measurement of the IP3 fraction of InsPs, a more sensitive approach for the detection of IP3 production, might be useful in resolving this issue.

Until recently, the production of IP3 appeared to account for calcium release from intracellular stores in non-muscle cells in most cases. It is now clear, however, that several independent pathways exist, and that agonist-induced calcium release from intracellular stores may occur without a rise in intracellular InsPs. To our knowledge, the present study is the first to demonstrate ryanodine-sensitive calcium release triggered by PTH.

Differences in the expression of activities involved in the transduction of signals through the PTH/PTHrp receptor and presence of PTH/PTHrp receptor isoforms have both been proposed to explain the selective activation of a single transducing pathway following PTH stimulation. In this study, two cell lines, differing only by the level of PTH/PTHrp receptor expression, were compared. It is difficult to exclude the possibility that transfection of cells with the PTH/PTHrp receptor did not modify activities necessary for signal transduction, and such effects could contribute to the differences observed in our study. Nevertheless, both HEK/T and HEK/W cells had an equivalent capacity to generate cAMP and increase [Ca2+]i in response to other agonists, suggesting that changes in postreceptor signal transduction are unlikely to entirely explain the differences observed in these two cell lines in response to PTH. In regard to PTH/PTHrp receptor isoform expression, several findings in our study argue against the expression of a PTH/PTHrp receptor isoform that differs from the classic type I PTH/PTHrp receptor. First, a similar increase in [Ca2+]i was obtained by PTH and PTHrp, demonstrating that the PTH receptor involved recognized both PTH and PTHrp, as demonstrated for the classic PTH/PTHrp receptor (1). The receptor involved thus was not the type II PTH receptor (41). Second, a PTH-stimulated increase in [Ca2+]i was produced by a N-terminal fragment of PTH (and PTHrp), not by C-terminal or midregion fragments, indicating that a PTH receptor isoform specifically recognizing C- or midregion PTH fragments was not involved (16, 17, 18). Third, PTH-stimulated calcium release in HEK/W was inhibited in a concentration-dependent fashion by two specific PTH/PTHrp receptor antagonists ([Nle8,18,Tyr34]bPTH(3–34)-amide and [D-Trp12,Tyr34]bPTH(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)amide). This confirmed that the PTH effect was mediated by a classic PTH receptor that bound and was activated by the N-terminal portion of the molecule. Fourth, PTH/PTHrp receptor mRNA was clearly demonstrated using a RNAse protection assay and RT-PCR. Furthermore, no other amplification products suggesting the presence of other functional receptor isoforms were detected. In this context, it is noteworthy that we (19) and others (42) have not been able to identify the presence of functional isoforms resulting from alternate splicing of the PTH/PTHrp receptor, other than the isoform identified by Jüppner et al. (12). Of course, none of these findings conclusively identifies the PTH/PTHrp receptor as the initiator of the response observed. Nevertheless, taken together, these results strongly suggest that the PTH-induced increase in [Ca2+]i demonstrated for HEK/W cells was mediated by the classic PTH/PTHrp receptor. The absence of detectable [125I]PTHrp binding suggests that the number of receptors expressed was too low to be detected by this technique, as proposed by Orloff et al. (3) for keratinocytes.

Our results provide a third possible explanation for the seemingly discrepant results obtained in prior studies; that is, a regulation by receptor number of the transduction signals activated. Such a regulation by receptor number has been demonstrated for the PTH/PTHrp receptor (1, 15) and for other receptors (14). It is usually accepted that PTH stimulation of an IP3 response requires a higher level of receptor expression than PTH stimulation of cAMP production. Our results are in agreement with these results, as we detected InsPs production in response to PTH only in cells expressing a high level of receptors. The results presented here extend that observation, however, and suggest that the differing PTH responses demonstrated in HEK/W and HEK/T cells are determined by the level of PTH/PTHrp receptor expression in such a way that PTH induces an increase in [Ca2+]i in the absence of either InsPs or cAMP production in cells expressing low numbers of PTH/PTHrp receptors through a pathway sensitive to ryanodine, while in cells expressing a high number of receptors, PTH can stimulate cAMP production and induces calcium release through an increase in the production of InsPs. Consistent with this hypothesis, PTH-induced calcium release was previously demonstrated to be associated with phospholipase C activation in HEK293 cells transfected with the PTH/PTHrp receptor and expressing 4 x 106 receptors per cell (7), but to occur in the absence of phospholipase C activation in cells expressing 1.5 x 105 receptors per cell (5). Interestingly, the ryanodine-sensitive pathway for calcium release is no longer detected in cells expressing large numbers of PTH/PTHrp receptors. The extinction of this pathway is not likely to be a direct effect of an increase in receptor number, but may result from other changes in PTH signaling induced by the increased number of receptors.

In conclusion, our data suggest that changes in PTH/PTHrp receptor number can modify both the extent of calcium signaling via phospholipase C and ryanodine receptor pathways, as well as the stimulation of cAMP production by PTH. As PTH/PTHrp receptor number and PTH effects vary as a function of cell differentiation, this may be one of the molecular mechanisms through which PTH effects are regulated.


   Footnotes
 
1 This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université Paris 7, Faculté Xavier-Bichat, Association pour l’Utilisation du Rein Artificiel, and Laboratoire de Recherches Physiologiques. Back

Received April 9, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Segre GV 1996 Receptors for parathyroid hormone and parathyroid hormone related peptide. In: Bilezikian JP, Raisz LG (eds) Principles in Bone Biology. Academic Press, New York, pp 377–403
  2. Whitfield JF, Chakravarthy BR, Durkin JP, Isaacs RJ, Jouishomme H, Sikorska M, Williams RE, Rixon R H 1992 Parathyroid hormone stimulates protein kinase C but not adenylate cyclase in mouse epidermal keratinocytes. J Cell Physiol 150:299–303[CrossRef][Medline]
  3. Orloff JJ, Ganz MB, Ribaudo AE, Burtis WJ, Reiss M, Milstone LM, Stewart AF 1992 Analysis of PTHrP binding and signal transduction mechanisms in benign and malignant squamous cells. Am J Physiol 262:E599–E607
  4. Babich M, Choi H, Johnson RM, King KL, Alford GE, Nissenson RA 1991 Thrombin and parathyroid hormone mobilize intracellular calcium in rat osteosarcoma cells by distinct pathways. Endocrinology 129:1463–1470[Abstract]
  5. Seuwen K, Boddeke HGWM 1995 Heparin-insensitive calcium release from intracellular stores triggered by the recombinant human parathyroid hormone receptor. Br J Pharmacol 114:1613–1620[Medline]
  6. Tong Y, Zull J 1995 Functional expression and signaling properties of cloned human parathyroid hormone receptor in Xenopus oocytes. J Biol Chem 271:8183–8191[Abstract/Free Full Text]
  7. Abou-Samra AB, Jüppner H, Force T, Freeman MW, Kong XF, Schipani E, Urena P, Richards J, Bonventre JV, Potts JT, Kronenberg HM, Segre GV 1992 Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium. Proc Natl Acad Sci USA 89:2732–27367[Abstract/Free Full Text]
  8. Pines M, Fukayama S, Costas K, Meurer E, Goldsmith PK, Wu X, Muallem S, Behar V, Chorev M, Rosenblatt M, Tashjian AH, Suva LJ 1996 Inositol 1-,4-,5-trisphosphate-dependent Ca2+ signaling by the recombinant human PTH-PTHrP receptor stably expressed in a human kidney cell line. Bone 18:381–389[Medline]
  9. Yamaguchi DT, Hahn TJ, Iida-Klein A, Kleeman CR, Muallem S 1987 Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. J Biol Chem 262:7711–7718[Abstract/Free Full Text]
  10. Gesek FA, Friedman PA 1992 On the mechanism of parathyroid hormone stimulation of calcium uptake by mouse distal convoluted tubule cells. J Clin Invest 90:749–758
  11. Tanaka H, SmogorzewskiI M, Koss M, Massry SG 1995 Pathways involved in PTH-induced rise in cytosolic Ca2+ concentations of rat renal proximal tubule. Am J Physiol 268:F330–F337
  12. Jüppner H, Abou-Samra AB, Freeman M, Kong XF, Schipani E, Richards J, Kolakowski LF, Hock J, Potts JT, Kronenberg HM, Segre GV 1991 A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 254:1024–1026[Abstract/Free Full Text]
  13. Raymond JR 1995 Multiple mechanisms of receptor-G protein signaling specificity. Am J Physiol 269:F141–F158
  14. Zhu X, Gilbert S, Birnbaumer M, Birnbaumer I 1994 Dual signaling potential is common among Gs-coupled receptors and dependent on receptor density. Mol Pharmacol 46:460–469[Abstract]
  15. Guo J, Iida-Klein A, Huang X, Abou-Samra AB, Segre GV, Bringhurst FR 1995 Parathyroid hormone (PTH)/PTH-related peptide receptor density modulates activation of phospholipase C and phosphate transport by PTH in LLC-PK1 cells. Endocrinology 138:3884–3891
  16. Whitfield JF, Isaacs RJ, Chakravarthy BR, Durkin JP, Morley P, Neugebauer W, Williams RE, Willick G, Rixon RH 1994 C-terminal fragments of parathyroid hormone-related protein, PTHrP-(107–111) and (107–139), and the N-terminal PTHrP-(1–40) fragment stimulate membrane-associated protein kinase C activity in rat spleen lymphocytes. J Cell Physiol 158:518–522[CrossRef][Medline]
  17. Inomata N, Akiyama M, Kubota N, Jüppner H 1995 Characterization of a novel parathyroid hormone (PTH) receptor with specificity for the carboxyl-terminal region of PTH-(1–84). Endocrinology 136:4732–4740[Abstract]
  18. Wu S, Pirola CJ, Green J, Yamaguchi DT, Okano K, Jüppner H, Forrester JS, Fagin JA, Clemens TL 1993 Effects of N-terminal, midregion, and C-terminal parathyroid hormone-related peptides on adenosine 3',5'-monophosphate and cytoplasmic free calcium in rat aortic smooth muscle cells and UMR-106 ostoblast-like cells. Endocrinology 133:2437–2444[Abstract]
  19. Jobert AS, Fernandes I, Turner G, Coureau C, Prie D, Nissenson A, Friedlander G, Silve C 1996 Expression of alternatively spliced isoforms of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger RNA in human kidney and bone cells. Mol Endocrinol 10:1066–1076[Abstract]
  20. Le Goas F, Amiel C, Friedlander G 1991 Protein kinase C modulates cAMP content in proximal tubular cells: role of phosphodiesterase inhibition. Am J Physiol 261:F582–F587
  21. Grynkiewiciz G, Poenie M, Tsien RY 1985 A new generation of Ca++ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
  22. Guillon G, Kirk CJ, Balestre MN 1986 Characterization of specific V1a vasopressin-binding sites on a rat mammary tumor cell line. Biochem J 240:189–196[Medline]
  23. Berrridge MJ, Downes CP, Hanley MR 1982 Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem J 206:587–595[Medline]
  24. Ammar A, Rajerison R.M, Roseau S, Bloch-Faure M, Butlen D 1994 Frog glomerular vasotocin receptors resemble mammalian V1b receptors. Am J Physiol 267:R1198–R1208
  25. Nissenson RA, Diep D, Strewler GJ 1988 Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies. Binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney. J Biol Chem 263:12866–12871[Abstract/Free Full Text]
  26. Jüppner H, Abou Samra AB, Uneno S, Gu WX, Potts Jr JT, Segre GV 1988 The parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells. J Biol Chem 263:8557–8560[Abstract/Free Full Text]
  27. Chomczynski P, Sacchi N 1987 Single step-method of RNA isolation by acid-guanidium-thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  28. Turner G, Coureau C, Riszk Rabin M, Escoubet B, Hruby M, Walrant O, Silve C 1995 Parathyroid hormone (PTH)/PTH-related protein receptor messenger ribonucleic acid expression and PTH response in a rat model of secondary hyperparathyroidism associated with vitamin D deficiency. Endocrinology 136:3751–3758[Abstract]
  29. Ausubel AU, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K 1993 Current Protocols in Molecular Biology. Wiley and Sons, New York
  30. Schipani E, Karga H, Karaplis AC, Potts JT, Kronenberg HM, Segre GV, Abou-Samra AB, Jüppner H 1993 Identical complementary deoxyribonucleic acids encode a human renal and bone parathyroid hormone (PTH)/PTH-related peptide receptor. Endocrinology 132:2157–2165[Abstract]
  31. Snedecor GW, Cochran WG 1972 Statistical Methods. Iowa State University Press, Ames, IA, p 272
  32. Cox DA, Cohen ML 1995 5-Hydroxytryptamine2B receptor signaling in rat stomach fundus: role of voltage-dependent calcium channels, intracellular calcium release and protein kinase C. J Pharmacol Exp Ther 272:143–50[Abstract/Free Full Text]
  33. Abu-Shakra SR, Cole AJ, Adams RN, Drachman DB 1994 Cholinergic stimulation of skeletal muscle cells induces rapid immediate early gene expression: role of intracellular calcium. Brain Res 26:55–60[CrossRef]
  34. Petersen OH 1996 New aspects of cytosolic calcium signaling. News Physiol Sci 11:13–17[Abstract/Free Full Text]
  35. Sutko JL, Walian PJ 1996 Ryanodine receptor Ca2+ release channels: does diversity in form equal diversity in function? Physiol Rev 76:1027–1072[Abstract/Free Full Text]
  36. Reid IR, Civitelli R, Halstead LR, Avioli LV, Hruska KA 1987 Parathyroid hormone acutely elevates intracellular calcium in osteoblastlike cells. Am J Physiol 253:E45–E51
  37. Watson S, Arkinstall S 1994 The G-Protein Linked Receptor FactsBook. Academic Press. London, pp 67–70
  38. Kong XF, Schipani E, Lanske B, Joun H, Karperien M, Defize LHK, Jüppner H, Potts JT, Segre GV, Kronenberg HM, Abou-Samra AB 1994 The rat, mouse and human genes encoding the receptor for parathyroid hormone and parathyroid hormone-related peptide are highly homologous. Biochem Biophys Res Commun 200:1290–1299[CrossRef][Medline]
  39. Pines M, Adams A.E, Stueckle S, Bessale R, Rashti-Behar V, Chorev M, Rosenblatt M, Suva LJ 1994 Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: lack of interaction with a C-terminal human PTH peptide. Endocrinology 135:1713–1716[Abstract]
  40. Behar V, Pines M, Nakamoto C, Greenberg Z, Bisello A, Stueckle SM, Bessalle R, Usdin TB, Chorev M, Rosenblatt M, Suva LJ 1996 The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor. Endocrinology 137:2748–2757[Abstract]
  41. Usdin TB, Gruber C, Bonner TI 1995 Identification and functional expression of a receptor selectively recognizing parathyroid hormone, the PTH2 receptor. J Biol Chem 270:15455–15458[Abstract/Free Full Text]
  42. Usa T, Segre GV 1996 Two alternatively spliced PTH/PTHrP receptor transcripts are expressed in rat kidney. J Bone Miner Res 11[Suppl 1]: (Abstract M492)



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