©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloned and Expressed Rat Ca-sensing Receptor
DIFFERENTIAL COOPERATIVE RESPONSES TO CALCIUM AND MAGNESIUM (*)

(Received for publication, January 23, 1996)

Martial Ruat (§) Adele M. Snowman Lynda D. Hester Solomon H. Snyder (¶)

From the Departments of Neuroscience, Pharmacology, and Molecular Sciences and Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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(2).


EXPERIMENTAL PROCEDURES

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(2) 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.

[^3H]AA Release

Cells were seeded in 24-well plates and cultured overnight in culture medium without the antibiotics and containing 0.5 µCi/ml [^3H]AA (DuPont NEN). Cells were washed twice in the presence of 0.2% BSA (fatty acid free) with 1 ml of basal Ham's F-12 or in Ham's F-12 supplemented with CaCl(2) to a final concentration of 1.5 mM to remove unincorporated [^3H]AA. Cells were further incubated at 37 °C for 30 min with the appropriate ions or drugs in 1 ml of the same buffer. [^3H]AA release was determined by liquid scintillation counting of a 0.5-ml sample of the incubation medium. We have shown in previous studies that 90% of the released radioactivity corresponds to authentic [^3H]AA(9) .

[^3H]IP Formation

Cells, in 24-well plates, were cultured overnight in Ham's F-12 medium (without the antibiotics) containing O.5 µCi/ml myo-[^3H]inositol (Amersham Corp.). Cells were washed twice with 1 ml of basal Ham's F-12 or with 1 ml of Ham's F-12 supplemented with CaCl(2) to a final concentration of 1.5 mM and containing 10 mM LiCl. Cells were further incubated in the appropriate medium for 15 min at 37 °C. Medium was removed, and cells were incubated for 30 min at 37 °C in 0.5 ml of the same medium containing the indicated drugs or ions. Reactions were stopped as described (10) and [^3H]inositol phosphates were isolated(11) . The ions (chloride form), polyarginine (M(r) = 45,000) and polylysine (M(r) = 27,000), were from Sigma. Data, expressed as means of at least triplicate determinations, varied less than 5% in any given experiment.


RESULTS AND DISCUSSION

CHO cells transfected with CaSR were labeled with [^3H]inositol, and the formation of [^3H]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 [^3H]IP formation with maximal levels at 4 mM Ca, 5-fold higher than basal values. [^3H]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(H)) 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 [^3H]IP and [^3H]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-[^3H]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 [^3H]IP was 392 ± 12 and 193 ± 6 cpm for CHO(CaSR) and CHO (WT*), respectively. B, cells were prelabeled with [^3H]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 [^3H]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 [^3H]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 [^3H]IP accumulation or [^3H]AA release, respectively, for a representative one of three to five independent experiments performed in triplicate. Hill plots and Hill coefficients (n(H)) for calcium-induced accumulation of [^3H]IP or [^3H]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 [^3H]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 [^3H]IP formation in the presence of 1.5 mM than 0.5 mM Ca. At 0.5 mM Ca, [^3H]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 [^3H]IP and stimulation of [^3H]AA release from CHO(WT*) or CHO(CaSR) cells. A, after prelabeling with myo-[^3H]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 [^3H]IP was 107 ± 3 cpm. B, after prelabeling with [^3H]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 [^3H]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 [^3H]IP accumulation or [^3H]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(H) = 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 [^3H]IP in CHO(CaSR) cells. After prelabeling with myo-[^3H]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 [^3H]IP was 205 ± 6 cpm. Data are expressed as the percent of [^3H]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 [^3H]IP levels at 30 nM.



We also evaluated influences of Ca upon [^3H]AA formation from phospholipids labeled with [^3H]AA, reflecting actions of PLA(2). PLA(2) 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(2) activation(13) . Thus, for CaSR, the PLA(2) response would likely reflect Ca entry as well as PLC activation. [^3H]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(H) = 5) for [^3H]AA, which is quite similar to results with [^3H]IP. No [^3H]AA response is evident in untransfected cells.

In the presence of 1.5 mM Ca, Mg behaves as a partial agonist for [^3H]AA as for [^3H]IP with a maximal increase of [^3H]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(2) activity, which is highly sensitive to stimulation by Ca. Thus, in CHO(WT*) cells, which possess endogenous P2 purinergic receptors(14) , stimulation of [^3H]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 [^3H]IP response, [^3H]AA release in response to Mg is substantially greater at 1.5 than 0.5 mM Ca (Table 2).



To establish that [^3H]AA formation reflects PLA(2) activity, we showed that 100 µM quinacrine, a PLA(2) inhibitor, reduces Ca (3 mM) induced [^3H]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 [^3H]AA than Mg, whereas they display similar effects on [^3H]IP (Table 1). Lithium (10 mM) has no effect on [^3H]AA formation, implying that it does not influence directly PLC or PLA(2) and accordingly can be employed to inhibit inositol phosphatases in studies monitoring [^3H]IP formation. Polylysine (30 nM) doubles [^3H]AA formation. Polyarginine (10 and 30 nM) augments [^3H]AA formation similar to its actions upon [^3H]IP. None of these agents influence [^3H]IP turnover or [^3H]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 [^3H]IP nor [^3H]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(2) 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.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant DA-00266 (to S. H. S.), Research Scientist Award DA-00074 (to S. H. S.), and a grant from the W. M. Keck Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Institut National de la Santé et de la Recherche Médicale and a grant from Elf Aquitaine, Inc.

To whom correspondence and reprint requests should be addressed. Tel.: 410-955-3024; Fax: 410-955-3623.

(^1)
The abbreviations used are: CaSR, calcium-sensing receptor; AA, arachidonic acid; BSA, bovine serum albumin; CHO(CaSR), Chinese hamster ovary cells transfected with the calcium-sensing receptor; CHO(WT*), Chinese hamster ovary cells not transfected with CaSR; IP, inositol phosphates; PI, phosphatidylinositol; PLA(2), phospholipase A(2); PLC, phospholipase C.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.