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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
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 
-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 1
) 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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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.
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Results |
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). 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. 1). 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. 1, 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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). 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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, A-E) and HEK/T cells (Fig. 3, 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. 3). 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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, A and B).
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; 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. 5, 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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, E and F). In contrast, U73122 totally
inhibited the PTH-induced [Ca2+]i increase in
HEK/T cells (Fig. 6, A and B). U73122 alone had no effect on basal
[Ca2+]i in either HEK/W or HEK/T cells (Fig. 6, 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. 6, 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. 6, E
and H, stimulation fold over basal: 1.54 compared with 1.10,
respectively, in untreated and treated cells). Ryanodine alone (100
µM) (Fig. 6, C and G) or in the presence of U73122 (10
µM) (Fig. 6, 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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). 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. 7, 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 2).
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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). 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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). 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. 9).
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). 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 1). 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. 10). 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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Discussion |
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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.
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Footnotes |
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Received April 9, 1997.
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  A. Couvineau, V. Wouters, G. Bertrand, C. Rouyer, B. Gerard, L. M. Boon, B. Grandchamp, M. Vikkula, and C. Silve PTHR1 mutations associated with Ollier disease result in receptor loss of function Hum. Mol. Genet., September 15, 2008; 17(18): 2766 - 2775. [Abstract] [Full Text] [PDF]
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 T. M. Murray, L. G. Rao, P. Divieti, and F. R. Bringhurst Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands Endocr. Rev., February 1, 2005; 26(1): 78 - 113. [Abstract] [Full Text] [PDF]
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 A. ValIn, C. Guillen, and P. Esbrit C-Terminal Parathyroid Hormone-Related Protein (PTHrP) (107-139) Stimulates Intracellular Ca2+ through a Receptor Different from the Type 1 PTH/PTHrP Receptor in Osteoblastic Osteosarcoma UMR 106 Cells Endocrinology, July 1, 2001; 142(7): 2752 - 2759. [Abstract] [Full Text] [PDF]
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 M. Teixeira, C. Bernard, E. Ferrary, and D. Butlen Purine and pyrimidine nucleotide-sensitive phospholipase A2 in ampulla from frog semicircular canal Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2001; 280(2): R519 - R526. [Abstract] [Full Text] [PDF]
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 A. D. Short and C. W. Taylor Parathyroid Hormone Controls the Size of the Intracellular Ca2+ Stores Available to Receptors Linked to Inositol Trisphosphate Formation J. Biol. Chem., January 21, 2000; 275(3): 1807 - 1813. [Abstract] [Full Text] [PDF]
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G. Boguslawski, L. V. Hale, X.-P. Yu, R. R. Miles, J. E. Onyia, R. F. Santerre, and S. Chandrasekhar Activation of Osteocalcin Transcription Involves Interaction of Protein Kinase A- and Protein Kinase C-dependent Pathways J. Biol. Chem., January 14, 2000; 275(2): 999 - 1006. [Abstract] [Full Text] [PDF]
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 I. Fernandes, R. Beliveau, G. Friedlander, and C. Silve NaPO4 cotransport type III (PiT1) expression in human embryonic kidney cells and regulation by PTH Am J Physiol Renal Physiol, October 1, 1999; 277(4): F543 - F551. [Abstract] [Full Text] [PDF]
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) and HEK/T (
).
[Ca2+]i was monitored by fluorimetry
following the addition of 10-7 M hPTH(1–34)
at 40 s (
) and 10-6 M bradykinin at
380 s (
). Results are
representative of those obtained in two separate experiments. The
concentrations of PTH used are indicated in the figure.
) was added 2 min before hPTH(1–34)
(10-7 M) (
) and HEK/T (
