The Ca2+-sensing receptor: a
target for polyamines
Stephen J.
Quinn,
Chian-Ping
Ye,
Rubin
Diaz,
Olga
Kifor,
Mei
Bai,
Peter
Vassilev, and
Edward
Brown
Endocrine-Hypertension Division, Department of Medicine, Brigham
and Women's Hospital, Boston, Massachusetts 02115
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ABSTRACT |
The Ca2+-sensing receptor
(CaR) is activated at physiological levels of external
Ca2+
(Cao) but is expressed in a
number of tissues that do not have well-established roles in the
control of Cao, including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown by
our current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic free
Ca2+
(Cai), inositol phosphate
production, and the activity of a nonselective cation channel. Spermine
stimulated Cai transients in
CaR-transfected HEK cells, with a concentration producing a
half-maximal response (EC50) of ~500
µM in the presence of 0.5 mM
Ca2+, whereas sustained increases
in Cai had an
EC50 of ~200 µM. The order of
potency was spermine > spermidine >> putrescine. Elevation of
Cao shifted the
EC50 for spermine sharply to the
left, with substantial stimulation below 100 µM. Addition of
subthreshold concentrations of spermine increased the sensitivity of
CaR-expressing HEK cells to Cao.
Parathyroid hormone secretion from bovine parathyroid cells was
inhibited by 50% in the presence of 200 µM spermine, a response
similar to that elicited by 2.0 mM
Cao. These data suggest that
polyamines could be effective agonists for the CaR, and several
tissues, including the brain, may use the CaR as a target for the
actions of spermine and other endogenous polycationic agonists.
spermine; polycationic agonists; calcium
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INTRODUCTION |
A CALCIUM-SENSING RECEPTOR (CaR) has been cloned that
allows CaR-expressing cells to sense external
Ca2+
(Cao) within a physiological
range close to 1.5 mM (3, 5). Initially cloned from bovine parathyroid
cells, this receptor is highly expressed in tissues involved in
regulating Cao, including the
parathyroid, calcitonin-secreting cells of the thyroid (C-cells), and
several regions of the kidney (3, 19). Interestingly, the CaR is also
distributed in a number of other tissues that do not have
well-established roles in the control of
Cao. These include several regions
of the brain such as the hippocampus, as well as the pituitary, the
collecting duct of the kidney, the lung, and the intestines (2, 3, 7,
8, 10, 21). In many of these tissues, the physiological role of the CaR
is not understood. One possibility is that it senses endogenous ligands other than Cao, thus allowing the
CaR to function in a number of specialized capacities in different
CaR-expressing tissues.
One class of putative CaR agonists is endogenous polyamines,
polycationic molecules that include spermine and spermidine. Indeed,
spermine was the first organic compound believed to act on the CaR
receptor (16), and we now provide evidence that spermine can act
directly on the CaR to inhibit parathyroid hormone (PTH) secretion.
Endogenous polyamines are known to influence a number of cell
functions, including proliferation and differentiation. In addition,
there is growing support for the concept of polyamines acting as
neurotransmitters or neuromodulators (9, 12, 25). For example,
spermine, through modulation of the
N-methyl-D-aspartate (NMDA) receptor, has been implicated in a variety of processes in the
central nervous system (CNS), such as long-term potentiation and
neurotoxicity (6). Some of these effects on the CNS require external
polyamines but are NMDA independent, suggesting alternative sites of
action, one of which could be the CaR. Our studies indicate that
polyamines are effective agonists for the CaR. Furthermore, several
tissues, including the brain, could potentially use the CaR as a target
for the actions of spermine and other endogenous polyamines.
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MATERIALS AND METHODS |
Cell Preparations
Preparation of dispersed bovine parathyroid cells.
Dispersed parathyroid cells were prepared from parathyroid glands of 1- to 3-wk-old calves, using digestion with collagenase and
deoxyribonuclease, and PTH secretion was assessed by radioimmunoassay as previously described (13).
Culturing and maintenance of CaR-transfected and untransfected
HEK-293 cells.
These cell lines were the generous gift of Dr. Kimberly Rogers (NPS
Pharmaceuticals, Salt Lake City, UT). The CaR-expressing HEK-293 cell
line (5001) was stably transfected with the human parathyroid CaR (10)
and selected by hygromycin resistance. The transfected HEK-293 cells
express the CaR on the membrane surface and are responsive to addition
of CaR agonists to the external medium (K. Rogers, personal
communication). Cells were grown in Dulbecco's modified Eagle's
medium with 10% fetal bovine serum and 200 µg/ml hygromycin.
Western analysis of plasma membrane proteins.
Crude plasma membrane was isolated from HEK-293 cells as described
previously (1). After determination of the protein
concentration in the crude plasma membrane preparation, an appropriate
amount of membrane protein (4 µg) was subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis using a linear gradient of
polyacrylamide. The proteins on the gel were electrotransferred to a
nitrocellulose membrane. After blocking with 5% milk, the blot was
incubated with primary antibody (no. 4641, kindly provided by Drs.
Forrest Fuller and Rachel Simin, NPS Pharmaceuticals), with and without the synthetic peptide of the bovine parathyroid CaR (amino acids 215-237) against which it was raised (as a control for nonspecific staining) and with a secondary goat anti-rabbit antibody conjugated to
horseradish peroxidase (Sigma; diluted 1:500). The CaR protein was
detected with an enhanced chemiluminescence system (Amersham).
Measurement of cytosolic free
Ca2+ using the
cell population system.
Coverslips with near-confluent HEK cells were loaded with fura
2-acetoxymethyl ester and placed diagonally into thermostated cuvettes
equipped with a magnetic stirrer. The bath solution was stirred at
37°C, and CaR agonists were added to the desired final concentration. Excitation monochrometers were centered at 340 and 380 nm, with emission light collected at 90° using a long-pass emission
filter. The ratio of emitted light at 340-nm excitation to that at
380-nm excitation and in vitro calibrations were used to estimate
cytosolic free Ca2+
(Cai), as previously described
(1). The Hill coefficient was determined using a curve-fitting
algorithm from an Enzfitter software package.
Measurement of [3H]inositol
phosphates.
The transfected cells were labeled with
[3H]inositol (~10
µCi/106 cells) overnight in
medium 199 (with 1% penicillin-streptomycin, 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, pH 7.5, and 15% bovine serum), washed with media (10 mM LiCl,
0.5 mM MgSO4, 0.5 mM
CaCl2, and 2 mg/ml bovine serum
albumin in Eagle's solution), and then incubated with polyvalent
cations for 30 min. The reactions were terminated with a final
concentration of 10% trichloroacetic acid (TCA). After sedimentation
of precipitated debris and removal of TCA by ether extraction, inositol
phosphates (IP) in the aqueous phase were separated on Dowex
anion-exchange columns. The radioactive inositol monophosphate,
bisphosphate, trisphosphate, and tetrakisphosphate were eluted stepwise
with 0.2, 0.4, 0.8, and 1.2 M formate containing 0.1 M formic acid, respectively, and quantitated using a liquid scintillation counter, as
previously described (4).
Electrophysiological Technique
Cell-attached patch.
Micropipettes were coated with Sylgard. The tip resistances for
single-channel experiments were 8-15 M
. Junction potential was
compensated using the electronic circuit of the amplifier. Seal
resistances of >10 G
were used in single-channel experiments to
guarantee a good resolution of small currents. The single-channel currents were filtered at 2-3 kHz through an eight-pole Bessel filter, digitized (50-200 µs/point) and analyzed with an IBM
computer, a Labmaster board (Scientific Solutions, Solon, OH), and
programs based on the Fastlab (Indec Systems, Sunnyvale, CA) and pCLAMP (Axon Instruments) systems. Voltage stimuli were applied with the same
setup.
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RESULTS |
A clonal HEK-293 cell line (5001) was stably transfected with the human
CaR and selected and maintained by hygromycin resistance. This
transfected HEK cell line expressed the human CaR at high levels (Fig.
1). On Western analysis of
plasma membrane proteins isolated from HEK-293 (5001) cells stably
transfected with the human CaR, there were immunoreactive bands between
140 and 205 kDa as well as additional bands of higher molecular masses
(Fig. 1, lane
2), and all bands were ablated
following preabsorption of the antibody with specific peptide
(Fig. 1, lane
1). The pattern of immunoreactive
bands was similar to that found for crude plasma membrane preparations
prepared from bovine parathyroid glands, whereas crude plasma membrane
preparations from wild-type HEK-293 cells showed no immunoreactive
bands (1).

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Fig. 1.
Western analysis of Ca2+-sensing
receptor (CaR) in crude plasma membrane preparations isolated from
HEK-293 cells stably transfected with human CaR. Crude plasma membrane
proteins were isolated from HEK-293 (5001) cells that had been stably
transfected with human CaR. Each protein sample (4 µg) was subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis as
described in MATERIALS AND METHODS.
CaR was stained with an anti-receptor antibody (4641) with
(lane 1) or without
(lane 2) preincubation with specific peptide against which it was raised, as a control for nonspecific staining. Similar Western analysis of wild-type HEK-293 cells showed no
immunoreactive bands. Molecular mass in kDa is shown at
left.
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Stably transfected HEK-293 (5001) cells exhibited CaR-mediated cell
activation following incubation with established CaR agonists, including Cao, neomycin, and
Gd3+ (Fig.
2). The dose-response relationships for
Cao-elicited increases in
Cai are shown in Fig.
3 for peak and sustained
Cai responses. The peak
Cai transient reached a
near-maximal response at Cao concentrations of
5 mM, with a half-maximal response at ~3.5 mM.
The sustained phase of the Cai
response attained maximal levels at
Cao concentrations of
3.5 mM,
with a half-maximal response at ~2 mM
Cao. Untransfected HEK cells
showed no change in Cai following
application of neomycin and Gd3+
or with elevation of Cao
(n = 5).

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Fig. 2.
Cytosolic free Ca2+
(Cai) responses to agonist
activation of HEK-293 cells expressing CaR. A clonal HEK-293 cell line
(5001) stably transfected with human CaR was used to test different
agonists for CaR. Polycationic CaR ligands included 5 mM external
Ca2+
(Cao;
A), 200 µM neomycin
(B), and 100 µM
Gd3+
(C). Agonist was added immediately
prior to Cai transient and remained for duration of recording.
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Fig. 3.
Dose-response relationship for Cao
stimulation of Cai responses in
HEK cells expressing CaR. Cai
recordings from CaR-expressing HEK (5001) cells stimulated by
Cao were analyzed for peak and sustained Cai responses. Peak
response is maximal level attained during a 5-min stimulation with a
given Cao concentration. Sustained response is Cai value at end of
5-min stimulation. Concentration producing a half-maximal response
(EC50) was ~3.5 mM for
transient rise and ~3 mM for sustained elevation in
Cai.
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Polyamines produced a similar Cai
response in this CaR-transfected HEK (5001) cell line (Fig.
4). The HEK cells exhibited transient
Cai increases followed by plateau
Cai responses at high spermine
concentrations, >300 µM, and showed sustained responses without
clear Cai peak responses at lower
spermine levels. The half-maximal response was observed at ~500 µM
for the transient rise and at ~200 µM for the sustained elevation
(Fig. 5). Spermidine elicited similar
Cai responses but at higher
concentrations than spermine, showing a concentration producing a
half-maximal response (EC50) of
~4 mM (Fig. 6), whereas concentrations of
putrescine as high as 10 mM gave little or no
Cai response. These polyamines did
not elicit Cai responses in the
untransfected HEK cells (n = 5). These
data show that spermine and spermidine are both effective agonists of
the CaR, resulting in Cai changes
similar to those found with activation of the CaR by
Cao.

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Fig. 4.
Cai responses to
spermine activation of HEK-293 cells expressing CaR. Spermine elicited
both initial transients and sustained increases in
Cai. Spermine concentrations were
700 (A), 300 (B), 200 (C), and 100 (D) µM. Arrow, initiation of
spermine application. Untransfected HEK-293 cells showed no change in
Cai following application of
spermine or other polyamines. Experiments were performed in media
containing 0.5 mM Ca2+ and 0.5 mM
Mg2+.
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Fig. 5.
Dose-response relationship for spermine stimulation of
Cai responses in HEK cells
expressing CaR. Cai recordings
from CaR-expressing HEK cells stimulated by spermine were analyzed for
peak and sustained Cai responses.
Peak response is maximal level attained during a 5-min stimulation with
a given Cao concentration.
Sustained response is Cai value at
end of 5-min stimulation. EC50 was
~500 µM for transient rise and ~200 µM for sustained elevation
in Cai. Experiments were performed
in media containing 0.5 mM Ca2+
and 0.5 mM Mg2+.
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Fig. 6.
Dose-response relationship for spermine and spermidine stimulation of
transient Cai response in HEK
cells expressing CaR. Cai
recordings from CaR-expressing HEK (5001) cells stimulated by spermine
or spermidine were analyzed for peak and sustained Cai responses. Peak response is
maximal Cai level attained during a 5-min stimulation with a given
Cao concentration.
EC50 was ~500 µM for spermine
and ~4 mM for spermidine. Experiments were performed in media
containing 0.5 mM Ca2+ and 0.5 mM
Mg2+. Note log scale for polyamine
concentration.
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The activation of the CaR appeared to be cooperative, with steep
dose-response relationships for stimulation by
Cao and spermine. The Hill
coefficient for Cao stimulation
was 4.73 ± 0.08 (mean ± SE; n = 12), whereas the Hill coefficient for spermine stimulation was 3.39 ± 0.13 (n = 10). A heterologous
positive cooperativity can also be seen between CaR agonists. For
example, Cao was known to
potentiate the secretory response of parathyroid cells to extracellular Mg2+ and vice versa. Such
interacting behavior between Cao
and the polyamines could have important physiological consequences when the levels of both Cao and
polyamine may change. In the presence of
Cao concentrations >0.5 mM, the
CaR was activated by lower concentrations of spermine (Fig.
7), with the threshold shifting from 200 to
10 µM as Cao was elevated to 2.5 mM. The EC50 demonstrated a
similar change, from 500 to <200 µM. The maximal
Cai responses to spermine addition
were similar, regardless of the
Cao levels between 0.5 and 2.5 mM,
suggesting that the agonist effects were not additive. Spermine could
also elicit transient Cai
responses in the absence of Cao;
however, these Cai changes were
smaller, and no sustained Cai
elevations were observed (n = 6).

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Fig. 7.
Effect of Cao on
spermine stimulation of Cai
transients in HEK cells expressing CaR. Dose-response curves were
determined for spermine stimulation in presence of various
concentrations of Cao. With
addition of Cao, CaR shows greater
sensitivity to spermine. In presence of
Cao between 0.5 and 2.5 mM,
threshold shifts from 200 to 10 µM and 50% effective concentration
(EC50) demonstrates a similar
change, from 500 to <200 µM spermine.
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A similar effect was found in the
Cao-evoked
Cai response as the spermine
concentration was elevated (Fig. 8),
leading to a CaR more sensitive to
Cao. In the presence of spermine
concentrations between 20 and 200 µM, the threshold for activation of
the CaR shifted from 1.5 mM to <1.0 mM
Cao. The
EC50 also showed a leftward shift
from 3.5 to <3.0 mM Cao. The
maximal Cai response elicited by
5.5 mM Cao was similar in the
presence of 0-200 µM spermine.

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Fig. 8.
Effect of spermine on Cao
stimulation of Cai transients in
HEK cells expressing CaR. Dose-response curves were determined for
Cao stimulation in presence of
different concentrations of spermine. With addition of spermine, CaR
becomes more sensitive to Cao. In
presence of spermine concentrations between 20 and 200 µM, threshold
of CaR shifts from 1.5 to <1 mM
Cao.
EC50 also shows a leftward shift
from 3.5 to 2.5 mM Cao.
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Increased production of IP was observed during stimulation of
CaR-transfected HEK cells with Cao
and spermine (Fig. 9), consistent with
activation of phospholipase C (PLC) leading to the formation of
inositol trisphosphate and the release of
Ca2+ stores, resulting in a
transient rise in Cai. The levels
of IP at 5 mM Cao were 15-fold
higher than those present at 0.5 mM
Cao. Spermine (1,000 µM)
stimulated IP production eightfold at 0.5 mM
Cao, with a threshold effect
observed at 300 µM. This action of spermine was potentiated by
increasing Cao from 0.5 to 1.5 mM,
with a threshold spermine concentration of 100 µM and an
EC50 of ~500 µM
spermine. IP production stimulated by spermine at 2.5 mM
Cao appeared more blunted, likely
due to the strong activation of the PLC-IP system by this level of
Cao.

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Fig. 9.
Spermine stimulation of inositol phosphate (IP) accumulation in HEK-293
cells expressing CaR. Increased production of IP is observed during
stimulation of CaR-transfected HEK (5001) cells with spermine and
Cao. Spermine (1,000 µM)
stimulated IP production 8-fold, with a threshold effect at 300 µM,
in media containing 0.5 mM Ca2+
and 0.5 mM Mg2+. This spermine
effect was potentiated by increasing
Cao from 0.5 to 1.5 mM. At a
Cao of 5 mM, IP production rose
15-fold higher than production at 0.5 mM
Cao. cpm, Counts/min.
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The CaR-transfected HEK cells were also studied using the on-cell
patch-clamp technique with untransfected HEK-293 cells employed as
controls. A nonselective cation channel was observed in both CaR-transfected and untransfected HEK cells. This channel had a mean
conductance of ~50 pS, exhibited similar permeabilities for
Na+,
K+, and
Ca2+, and showed little voltage
dependency of its open-state probability (Po) (29, 30).
Under basal conditions,
Po was ~0.1,
and the CaR agonists Cao and
neomycin increased
Po by three- to
fivefold when added to the external bath. Spermine also activated this nonselective cation channel and raised
Po to a similar
extent (Fig. 10). No glutamate receptor
agonists were present in the pipette or bath solutions. This channel is
observed in both untransfected and CaR-transfected HEK cells;
however, only the transfected cells show activation by spermine (Fig.
10) or the other CaR agonists (30). The activation of this
nonselective channel did not appear to be voltage dependent.

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Fig. 10.
Spermine activation of a nonselective cation channel in HEK-293 cells
expressing CaR. A nonselective cation channel was observed in both
untransfected (A) and
CaR-transfected (B) HEK-293 cells. This channel had a mean conductance of 45-50 pS, exhibited similar permeabilities for Na+,
K+, and
Ca2+, and showed little voltage
dependency of its open-state probability (Po). Arrow,
spermine addition. C: spermine (300 µM) increased Po by at least
4-fold in CaR-expressing HEK-293 cells but had no effect on
untransfected HEK-293 cells. CaR agonists
(Cao and neomycin) also raised
Po to a similar
extent in transfected HEK (5001) cell line.
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To better understand the potential physiological impact of polyamines
on tissues expressing endogenous CaR, the effect of spermine on PTH
secretion was assessed in bovine parathyroid cells. These PTH secretion
experiments showed dose-dependent inhibition of hormone secretion as
Cao was increased between 0.5 and
2 mM (Fig. 11). In the presence of 0.5 mM
Cao, spermine produced a similar 50% decline in PTH secretion between 100 and 200 µM spermine, with
little additional change in hormone secretion at higher spermine concentrations.

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Fig. 11.
Spermine inhibition of parathyroid hormone (PTH) secretion from bovine
parathyroid cells. Paired experiments with bovine parathyroid cells
showed that both Cao
(A) and spermine
(B) were effective inhibitors of PTH
secretion. There was a steep inhibition of hormone release between 0.75 and 1.25 mM Cao and between 100 and 200 µM spermine, with 60 and 40% decreases in basal PTH
secretion, respectively. Spermine experiments were performed in media
containing 0.5 mM Ca2+ and 0.5 mM
Mg2+. Parathyroid cells appeared
to be more sensitive than CaR-expressing HEK-293 (5001) cells to
changes in Cao and spermine,
suggesting that substantially lower polyamine concentrations may be
effective agonists for endogenous CaR-expressing tissues.
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DISCUSSION |
The endogenously expressed CaR is activated at physiological levels of
Cao, allowing the parathyroid
gland and other tissues to regulate serum
Cao within a narrow concentration
range (3, 5, 19). Other agonists known to activate the CaR include polyvalent cations such as Mg2+,
Gd3+, and manganese as well as
polycationic molecules such as neomycin and polylysine (3, 4). These
other CaR agonists are not thought to act as endogenous ligands but can
be used as pharmacological agents to aid in experimentation involving
the CaR.
One set of putative endogenous ligands are the polyamines, which are
polycationic molecules produced in vivo (24). Spermine has four evenly
spaced positive charges, whereas spermidine and putrescine have three
and two positive charges, respectively. These polyamines are found
circulating in the plasma at low micromolar concentrations.
Furthermore, polyamines appear to be secreted presynaptically by some
neurons in the brain in a regulated manner (9, 25). Thus the levels of
spermine and spermidine, in particular, can reach substantially higher
concentrations in the interstitial fluid surrounding these neurons and
within synaptic clefts. Some tumors produce and secrete large amounts
of polyamines; thus the local concentrations of these polycationic
molecules may be much higher than circulating levels under these
conditions. Another CaR-expressing tissue that experiences high levels
of polyamines is the intestine, where bacteria produce large amounts of
spermine and spermidine (14, 23).
Cao and bacteria are both thought to be critical for the growth and differentiation of the intestinal mucosa. Given the polycationic nature of the polyamines and the association of high polyamine levels and expression of the CaR in
several tissues, experiments were designed to test the ability of
polyamines to act as agonists for the CaR.
Both spermine and spermidine were effective agonists for the CaR,
whereas putrescine had little or no effect at millimolar concentrations. The CaR was most sensitive to spermine, with a threshold concentration of 200 µM and an
EC50 of 500 µM in the presence
of 0.5 Cao. Spermidine was most
effective at low millimolar concentrations, regardless of the
Cao level. These data suggest that
several of the positive charges are involved in the binding of spermine
and spermidine to extracellular domains of the CaR. The steep
dose-response relationship found with spermine stimulation also implies
the presence of positive cooperativity for spermine activation of the
CaR, which may be explained by the binding of two or more spermine
molecules per CaR or multiple binding sites for each spermine molecule.
CaR agonists appear to interact with multiple cooperative binding sites
on the CaR, leading to the steep dose dependency of CaR effects on
signal transduction and hormone secretion (3, 22). Consistent with this
characteristic of CaR activation, at a more physiological
Cao of 1.5 mM, the threshold for
receptor activation by spermine was <100 µM and the
EC50 was shifted leftward to
~300 µM. Likewise, CaR sensitivity to
Cao was also enhanced in the
presence of spermine. This positive cooperativity of CaR stimulation
allows for a wider range of effective spermine concentrations and has
an impact on Ca2+ sensing at even
subthreshold concentrations of polyamine.
The sensitivity of the CaR-transfected HEK cell may not accurately
reflect the sensitivity of the endogenous CaR expressed in vivo. For
example, the parathyroid glands show exquisite sensitivity to serum
Cao, with a set point of ~1.5 mM
Cao for PTH secretion (3). Several
signal transduction events also have dose-response relationships for
Cao modulation, suggesting greater
sensitivity to Cao (3). The
CaR-transfected HEK cell has an
EC50 for
Cao-evoked Cai responses, and IP accumulation
is ~3.5 mM Cao under our
experimental conditions. This raises the possibility that the
parathyroid gland and other tissues endogenously expressing the CaR may
have a greater sensitivity to polyamines than that found for
CaR-transfected HEK cells. Indeed, PTH cells show both 50% inhibition
and maximal inhibition of PTH secretion between 100 and 200 µM
spermine in the presence of 0.5 mM
Cao. Under similar conditions, the
HEK cell displays EC50 values of
~500 µM and maximal stimulation at >1 mM spermine. Thus
it is likely that low micromolar concentrations of spermine may yield
modest changes in the activation of the CaR under physiological
conditions in tissues expressing the receptor, especially in the
presence of the normal Cao level
of ~1.5 mM.
The greatest impact of polyamines on CaR activation may occur in the
brain, where the concentrations of spermine and spermidine are likely
to achieve their highest values, particularly within the synaptic
region. The CaR is expressed in a number of regions of the rat brain,
including the hippocampus, hypothalamus, amygdala, olfactory bulb, and
cortex (7, 21). The pattern of immunoreactive staining is punctate,
suggesting that the CaR are found at nerve terminals (21); however, it
is not clear at this time whether the CaR is located pre- or
postsynaptically. Many of these brain regions are quite sensitive to
the neurotoxic effects of polyamines associated with glutamate
toxicity, ischemia, and other pathological conditions (6). In recent
experiments with rat and mouse hippocampal neurons, we have found that
Ca2+, neomycin, and spermine can
activate a nonselective cation channel having characteristics similar
to the nonselective channel in the HEK cell (29). Hippocampal neurons
from mice with targeted disruption of the CaR (11) exhibit the same
channels; however, the CaR agonists
Cao, neomycin, and spermine have
no effect, further implicating the CaR in the activation of these
nonselective cation channels (28).
The CaR and the NMDA receptor-channel complex share several common
receptor modulators, including the polyamines, neomycin, Ca2+, and
Mg2+ (17, 18). The NMDA receptor 1 (NR1) shows some modest (50%) homology to the CaR in the region of
residues 85-190 (5, 26, 31). In particular, the density and
distribution of negative charges appear to be conserved, suggesting
structural and functional homology between the CaR and NR1. There are a
number of splice variants of NR1 that insert alternative exons, with
exon 5 being of particular interest because its expression determines,
in part, the extent of glycine-independent polyamine modulation (26, 31). Exon 5 immediately follows the region (residues 85-190) that
shows homology to the CaR and encodes a 21-amino acid sequence with a
high density of positive charges. It is possible that the region of
homology between the NR1 and the CaR may be a putative polycation
binding site, with exon 5 acting as an intrinsic agonist. Recently,
studies on the NMDA receptor using site-directed mutagenesis suggest
that acidic residues at positions 339 and 342 may also be important in
glycine-independent spermine modulation (27).
In summary, the CaR is a complex receptor. Its ligands include the
physiologically important divalent cation
Ca2+ as well as polycationic
molecules such as spermine and spermidine. The CaR is expressed in
diverse tissues, including the parathyroid, the thyroid
calcitonin-secreting cells, several kidney sites, the pituitary, the
intestines, and several regions of the brain including the hippocampus.
The importance of each CaR ligand may depend on the tissue that
expresses the CaR. Several tissues may sense this polyamine through the
CaR, including the hippocampus, where spermine is concentrated in
vesicles of the presynaptic termini, can be cosecreted along with
neurotransmitters on stimulation, and could achieve a high micromolar
range within the synaptic cleft. Systemic concentrations of polyamines
are in the low micromolar range; however, production of spermine and
other polyamines can be high where cell proliferation and tissue growth
are rapid. The intestinal mucosa can also be a special site of spermine
action, since bacteria in the gut produce large amounts of polyamines and the mucosa turns over rapidly. Clearly, our understanding of the
pharmacology, molecular biology, and physiology of the CaR within the
context of polyamine stimulation will provide a more comprehensive view
of this receptor and its importance in many and varied tissues.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-41415, DK-44588, and DK-48220
(to E. Brown) and by the St. Giles Foundation.
 |
FOOTNOTES |
Address for reprint requests: S. J. Quinn, Brigham and Women's
Hospital, Endocrine-Hypertension Division, 221 Longwood Ave., Boston,
MA 02115.
Received 3 December 1996; accepted in final form 19 June 1997.
 |
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