(Received for publication, January 23, 1996)
From the
We have stably expressed cDNA for the rat brain
Ca-sensing receptor in Chinese hamster ovary cells.
Stimulation of phosphatidylinositol hydrolysis and arachidonic acid
(AA) release displayed markedly cooperative responses to Ca
with Hill coefficients of 4-5. Both phosphatidylinositol
and AA responses were not detected below a threshold of 1.5 mM Ca
. Mg
behaved as a partial
agonist with only half the maximal inositol phosphate and AA responses
displayed by Ca
and with a more shallow
concentration-response slope. The potency of Mg
in
augmenting inositol phosphate and AA responses, in the presence of 1.5
mM Ca
, implies that serum Mg
concentrations attained in clinical conditions will influence the
Ca
-sensing receptor.
The parathyroid gland monitors serum Ca levels
with great precision, thereby regulating the secretion of parathyroid
hormone. Brown and associates (1) cloned a
Ca
-sensing receptor (CaSR) (
)that responds
to physiologic extracellular Ca
levels and which is
highly concentrated in the parathyroid gland. In the brain we
identified CaSR highly localized to nerve terminals(2) , where
extracellular Ca
regulates neurotransmitter
release(3) .
The parathyroid gland responds maximally to
increases in serum Ca over a narrow range, reflecting
marked cooperativity (4) . Prior to the cloning of CaSR,
molecular tools were not available to analyze mechanisms responsible
for precise Ca
sensing. Serum Mg
levels are similar to those of Ca
(5) ,
and Mg
is detected by CaSR, though it has not been
established whether Mg
effects on CaSR occur at
physiologic concentrations(1, 4) . CaSR is linked to
activation of phospholipase C and the generation of inositol
trisphosphate, a Ca
-dependent process(4) .
The importance of precise Ca regulation by CaSR is
evident in clinical disorders arising from mutations in CaSR that cause
substantial clinical symptomatology despite only small alterations in
the set point for serum
Ca
(6, 7, 8) . To examine
mechanisms that influence CaSR responses, we have stably expressed rat
CaSR in CHO cells and evaluated in detail influences of Ca
and other divalent cations upon the generation of inositol
phosphates (IP) via phospholipase C and the formation of arachidonic
acid (AA) by the action of phospholipase A
.
The expression vector pRK5(CaSR) containing the open reading
frame encoding CaSR (2) and the pUT523 vector containing the
gene for resistance to phleomycin (Cayla, Toulouse, France) were used
(10:1) to transfect CHO-K1 cells (ATCC no., CCL61) as
described(9) . Stable transfectants were selected by serial
dilution in Ham's F-12 medium supplemented with fetal calf serum
(10%) and 100 µg/ml phleomycin and tested for CaSR expression by
Western blot analysis(2) . Expression of a 140-kDa peptide was
detected, and one clone named CHO(CaSR) was selected for
pharmacological characterization. In a control experiment, CHO-K1 cells
were transfected with pUT523 alone. A clone designated CHO(WT*) was
used as a control in pharmacological studies. Cell
Culture-CHO(CaSR) or CHO(WT*) cells were maintained at 37 °C
in a humidified atmosphere with 5% CO in basal Ham's
F-12 medium (0.5 mM Ca
, 0.5 mM Mg
) containing 10% (v/v) dialyzed fetal calf
serum and antibiotics.
CHO cells transfected with CaSR were labeled with
[H]inositol, and the formation of
[
H]IP in response to Ca
was
monitored (Fig. 1). A negligible increase occurs until 1.5
mM Ca
after which there is a precipitous
increase in [
H]IP formation with maximal levels
at 4 mM Ca
, 5-fold higher than basal values.
[
H]IP levels plateau between 4 and 10 mM Ca
. Thus, the total response to Ca
occurs between 2 and 4 mM Ca
,
reflecting prominent cooperativity with a Hill coefficient (n
) of 4. Fifty percent of the maximal response to
Ca
occurs at 2.9 mM Ca
. CHO(WT*) cells not transfected with CaSR manifest no IP response to
Ca
.
Figure 1:
Ca-induced
accumulation of [
H]IP and
[
H]AA release in CHO(WT*) or in CHO(CaSR). A, CHO cells stably expressing CaSR (CHO(CaSR)) or transfected
with the plasmid pUT523 alone (CHO(WT*)) were prelabeled overnight with
myo-[
H]inositol, washed twice with basal
Ham's F-12 (0.5 mM Ca
, 0.5 mM Mg
) supplemented with 10 mM LiCl, and
further incubated in the same medium for 15 min. Medium was removed,
and cells were incubated for 30 min in 0.5 ml of the same medium with
increasing Ca
concentrations. Mean ± S.E. of
basal [
H]IP was 392 ± 12 and 193 ±
6 cpm for CHO(CaSR) and CHO (WT*), respectively. B, cells were
prelabeled with [
H]AA overnight, washed twice
with 1 ml of basal Ham's F-12 supplemented with 0.2% BSA, and
then incubated for 30 min in 1 ml of the medium alone or with
increasing Ca
concentrations. ATP (0.1 mM)
induced [
H]AA release was evaluated in CHO(WT*)
at two Ca
concentrations (1.5 and 3 mM)
using the same experimental procedure. Mean ± S.E. of basal
[
H]AA release was 607 ± 29 and 328
± 12 cpm for CHO(CaSR) and CHO(WT*), respectively. Data shown in A and B are expressed as percent of basal
[
H]IP accumulation or [
H]AA
release, respectively, for a representative one of three to five
independent experiments performed in triplicate. Hill plots and Hill
coefficients (n
) for calcium-induced accumulation
of [
H]IP or [
H]AA release
from CHO(CaSR) cells are indicated as insets in A and B, respectively.
Mg elicits only about
50-60% as great a maximal increase in [
H]IP
as Ca
(Fig. 2A). The half-maximal
response to Mg
occurs at 4.5 mM, similar to
Ca
. The slope of the concentration-response curve for
Mg
is more shallow than for Ca
with
a maximal response requiring about 4 times the concentration of
Mg
as a minimal detectable response. Mg
is substantially more potent in stimulating
[
H]IP formation in the presence of 1.5 mM than 0.5 mM Ca
. At 0.5 mM Ca
, [
H]IP does not
increase until 4 mM Mg
, and the
concentration-response curve is shifted to the right (EC
= 7 mM). The shallow concentration-response slope
and the decreased maximal effect of Mg
are consistent
with a partial agonist effect.
Figure 2:
Magnesium-induced accumulation of
[H]IP and stimulation of
[
H]AA release from CHO(WT*) or CHO(CaSR) cells. A, after prelabeling with
myo-[
H]inositol, cells were washed twice with
basal Ham's F-12 (0.5 mM Ca
, 0.5
mM Mg
) or Ham's F-12 containing 1.5
mM Ca
, 0.5 mM Mg
supplemented with 10 mM LiCl and further incubated in
the respective medium for 15 min. Medium was removed, and cells were
incubated for 30 min in the same medium with increasing
Mg
. Mean ± S.E. of basal
[
H]IP was 107 ± 3 cpm. B, after
prelabeling with [
H]AA, cells were washed with
basal Ham's F-12 containing 1.5 mM Ca
,
0.5 mM Mg
, supplemented with 0.2% BSA, and
then incubated in this medium alone or with increasing
Mg
. Mean ± S.E. of basal
[
H]AA release was 778 ± 15 and 328
± 12 cpm for CHO(CaSR) and CHO(WT*), respectively. Data shown in A and B are expressed as the percent of basal
[
H]IP accumulation or [
H]AA
release, respectively, for a representative one of three to five
independent experiments performed in
triplicate.
In contrast to Mg,
Ba
behaves like a full agonist (Fig. 3). In
the presence of 1.5 mM Ca
, Ba
elicits a maximal IP response at 2 mM with an extremely
steep concentration-response curve that displays strong cooperativity (n
= 3). Under the same experimental
conditions, Mn
also strongly stimulates IP turnover
(EC
= 2.8 mM) with a less pronounced
effect than for Ba
. Above 5 mM concentration, Mn
precipitates, precluding a
detailed concentration-response analysis.
Figure 3:
Barium and manganese-induced accumulation
of [H]IP in CHO(CaSR) cells. After prelabeling
with myo-[
H]inositol, cells were washed twice
with Ham's F-12 containing 1.5 mM Ca
,
0.5 mM Mg
supplemented with 10 mM LiCl and further incubated in the same medium for 15 min. Medium
was removed, and cells were incubated for 30 min in the same medium
with increasing Ba
or Mn
. Basal
level (mean ± S.E.) of [
H]IP was 205
± 6 cpm. Data are expressed as the percent of
[
H]IP accumulation induced by 10 mM Ca
(1404 ± 15 cpm; mean ± S.E.)
and are a representative one of three independent experiments performed
in triplicate.
Of the various ions
tested, only Ca and Ba
behave as
full agonists with similar maximal responses (Table 1). Nickel
produces some stimulation at 3 mM but was not evaluated at
higher concentrations because it precipitates. Similarly, zinc and
cadmium could not be evaluated at concentrations greater than 0.3 and
1.0 mM, respectively, because of precipitation. Polyarginine,
which is known to activate CaSR(1) , is quite potent, tripling
[
H]IP levels at 30 nM.
We also
evaluated influences of Ca upon
[
H]AA formation from phospholipids labeled with
[
H]AA, reflecting actions of PLA
.
PLA
is activated by increases in intracellular
Ca
(12) . Activation of PLC and opening of
plasma membrane Ca
channels are the most common
triggers for PLA
activation(13) . Thus, for CaSR,
the PLA
response would likely reflect Ca
entry as well as PLC activation. [
H]AA
formation is sensitive to Ca
addition (EC
= 3.3 mM) with a 10-15-fold increase over
basal levels (Fig. 1B, Table 1). As in the PLC
response, no effect is detected in CHO(CaSR) cells until a threshold of
2 mM Ca
is attained. The slope of the
concentration-response curve indicates pronounced cooperativity (n
= 5) for [
H]AA,
which is quite similar to results with [
H]IP. No
[
H]AA response is evident in untransfected cells.
In the presence of 1.5 mM Ca,
Mg
behaves as a partial agonist for
[
H]AA as for [
H]IP with a
maximal increase of [
H]AA to about 4 times basal
levels (Fig. 2). It is not possible to compare directly maximal
responses to Mg
and Ca
, as higher
concentrations of Ca
directly increase PLA
activity, which is highly sensitive to stimulation by
Ca
. Thus, in CHO(WT*) cells, which possess endogenous
P2 purinergic receptors(14) , stimulation of
[
H]AA formation by ATP (0.1 mM) is
severalfold greater at 3 mM Ca
than at 1.5
mM Ca
(Fig. 1B). As with the
[
H]IP response, [
H]AA
release in response to Mg
is substantially greater at
1.5 than 0.5 mM Ca
(Table 2).
To
establish that [H]AA formation reflects PLA
activity, we showed that 100 µM quinacrine, a
PLA
inhibitor, reduces Ca
(3 mM)
induced [
H]AA formation by 75%. By contrast 30
µM RHC-80267, a diacylglycerol lipase
inhibitor(15) , has no effect (data not shown).
Of the other
ions examined, barium and manganese produce a lesser effect on
[H]AA than Mg
, whereas they
display similar effects on [
H]IP (Table 1).
Lithium (10 mM) has no effect on [
H]AA
formation, implying that it does not influence directly PLC or
PLA
and accordingly can be employed to inhibit inositol
phosphatases in studies monitoring [
H]IP
formation. Polylysine (30 nM) doubles
[
H]AA formation. Polyarginine (10 and 30
nM) augments [
H]AA formation similar to
its actions upon [
H]IP. None of these agents
influence [
H]IP turnover or
[
H]AA formation in CHO(WT*) cells (data not
shown).
Parathyroid responses to Ca have long been
known to be cooperative, but the site of cooperativity has not been
definitively established(16) . Our studies reveal dramatic
cooperativity that presumably occurs at the level of CaSR. CaSR is a
seven-transmembrane G protein-coupled receptor, and marked
cooperativity has not previously been demonstrated in responses to
agonists of any other receptors in this family(17) . Among
G-protein-coupled receptors, the structure of CaSR most closely
resembles the metabotropic glutamate receptor. Cloned and expressed
metabotropic glutamate responses are not cooperative (18) . The
site of CaSR cooperativity is not evident. Possibilities include the
Ca
recognition site, coupling to G proteins or
interactions among two or more CaSR.
Neither
[H]IP nor [
H]AA responses
occur until a threshold of 1.5 mM Ca
.
Conceivably, CaSR does not detect Ca
until this
threshold is attained. This would fit with a role of parathyroid CaSR
in responding primarily to increases above minimal physiologic
extracellular Ca
. Alternatively, this level of
Ca
may be necessary to activate PLC. In response to
stimulation of most receptors that act through PLC, the enzyme can be
activated to form inositol trisphosphate and elicit an initial
Ca
spike in the absence of external
Ca
. However, sustained production of substantial
levels of IP requires the entry of extracellular
Ca
(10) , which is triggered by an
uncharacterized mechanism that is thought to depend on the initial
intracellular Ca
spike. The threshold levels of
extracellular Ca
to support pronounced IP formation
in response to receptor stimulation have not been established.
We
found Mg to stimulate CaSR with a similar potency to
Ca
, though Mg
behaved as a partial
agonist. Brown and associates (1) found Mg
to
be one-third as potent as Ca
in stimulating second
messengers in frog oocytes. Reasons for these discrepancies are
unclear. Mg
behaves as a partial agonist of CaSR, in
contrast to Ca
and Ba
that are full
agonists. These results may indicate that Mg
acts at
a different site on CaSR than does Ca
.
Responses
to Mg are greater when one reaches a threshold of 1.5
mM Ca
. This suggests that physiologic and
pathophysiologic effects of Mg
will be more
pronounced in clinical situations where Ca
levels are
also elevated. Mg
serum concentrations reach those
that would activate CaSR in various clinical conditions(5) .
AA and IP formation display similar sensitivity to Ca levels. Concentrations of Ca
that directly
activate PLC and PLA
enzyme activity and regulate these
second messenger systems are similar to those that are detected by
CaSR. Accordingly, it is likely that in physiologic conditions, overall
responses will be determined by the integrative effects of
Ca
upon these enzymes as well as upon CaSR. In the
brain the substantial number of neuronal Ca
channels
of different types, together with the panoply of
Ca
-dependent second messengers, would be expected to
influence CaSR responses.