Paradoxical Block of Parathormone Secretion Is Mediated by Increased Activity of Galpha Subunits*

Ursula QuittererDagger §, Michaela HoffmannDagger , Marc Freichel, and Martin J. LohseDagger

From the Dagger  Institut für Pharmakologie und Toxikologie, Universität Würzburg, 97078 Würzburg, Germany and the  Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, 66421 Homburg, Germany

Received for publication, August 24, 2000, and in revised form, October 17, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The paradox of blunted parathormone (PTH) secretion in patients with severe hypomagnesemia has been known for more than 20 years, but the underlying mechanism is not deciphered. We determined the effect of low magnesium on in vitro PTH release and on the signals triggered by activation of the calcium-sensing receptor (CaSR). Analogous to the in vivo situation, PTH release from dispersed parathyroid cells was suppressed under low magnesium. In parallel, the two major signaling pathways responsible for CaSR-triggered block of PTH secretion, the generation of inositol phosphates, and the inhibition of cAMP were enhanced. Desensitization or pertussis toxin-mediated inhibition of CaSR-stimulated signaling suppressed the effect of low magnesium, further confirming that magnesium acts within the axis CaSR-G-protein. However, the magnesium binding site responsible for inhibition of PTH secretion is not identical with the extracellular ion binding site of the CaSR, because the magnesium deficiency-dependent signal enhancement was not altered on CaSR receptor mutants with increased or decreased affinity for calcium and magnesium. By contrast, when the magnesium affinity of the Galpha subunit was decreased, CaSR activation was no longer affected by magnesium. Thus, the paradoxical block of PTH release under magnesium deficiency seems to be mediated through a novel mechanism involving an increase in the activity of Galpha subunits of heterotrimeric G-proteins.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Parathormone (PTH)1 secretion from the parathyroid gland is suppressed by high extracellular calcium and magnesium (1). The calcium-sensing receptor (CaSR) is responsible for the calcium-dependent inhibition of PTH secretion (2). Direct binding of calcium or magnesium activates the CaSR (3). Activation of the CaSR triggers Galpha q/Galpha i-mediated signaling pathways (4). Several mutations have been identified with increased activation of this receptor (5, 6). CaSR mutants with increased affinity/potency for the agonist calcium and in part enhanced constitutive activity led to permanent inhibition of PTH secretion (7). Therefore, patients with activated CaSR mutants suffer from hypoparathyroidism. A similar phenotype of blunted PTH secretion is seen in patients with severe magnesium deficiency (8-10). This finding is unexpected since the effects of high magnesium on parathyroid hormone secretion are similar to those of calcium, and therefore, low magnesium should be expected to result in increased PTH secretion. And indeed, in contrast to patients, rats respond to severe hypomagnesemia with increased secretion of PTH (11, 12). It is known that hypomagnesemia reflects intracellular magnesium deficiency (9). Thus, the site of magnesium action has been assumed to lie intracellularly (9). However, causality between blunted PTH secretion and magnesium deficiency is not established, although the magnesium paradox has been known for more than 20 years (8). In search for the mechanism we investigated the relationship between magnesium deficiency, PTH secretion, and CaSR-mediated signaling. We present evidence that increased activity of Galpha subunits leads to enhanced signaling mediated by constitutive activation of the human CaSR. Enhanced CaSR-mediated signaling may thus constitute the link between severe hypomagnesemia and blunted PTH secretion.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfection-- Human embryonic kidney cells (HEK-293) were cultivated and transfected with plasmids encoding the wild-type and the different CaSR mutants (7, 13-15) under the control of the cytomegalovirus promotor as described (16).

Construction of Galpha i and CaSR Mutants-- For mutagenesis of Galpha i we used the Escherichia coli expression vector pQE60 containing the Galpha i1-cDNA with an internal nucleotide sequence after amino acid 121 encoding a hexahistidine tag. The codon coding for arginine 209 of Galpha i1 was exchanged to cysteine by site-directed mutagenesis (16). For mutagenesis of the human CaSR, an additional restriction site for XhoI was introduced (silent mutation of leucine 276). The mutated cDNAs were sequenced entirely to confirm the identity of the mutants.

Expression and Purification of Galpha s and Galpha i1-- The Galpha s and Galpha i1 proteins (wild-type and R209C) were expressed in E. coli BL21(DE3) and purified by Ni2+-agarose according to the manufacturer's protocol (Qiagen). Purified proteins were desalted and concentrated by centrifugation through a Centricon concentrator with an exclusion limit of 30,000 Da (Amicon). After the addition of 20% (v/v) glycerol, the purified proteins were frozen in liquid nitrogen and stored in aliquots at -80 °C at a protein concentration of 2-10 µg/µl.

Purification of Galpha o from Bovine Brain-- Galpha o from bovine brain was prepared as described (17).

Expression of Galpha q in Sf9 Cells-- Galpha q was expressed in Sf9 cells together with Gbeta 1gamma 2 using recombinant baculoviruses. Forty-eight hours after infection, cells were harvested, and membranes of baculovirus-infected cells were prepared as described (16). Membranes were directly assayed for [35S]GTPgamma S binding in comparison to control membranes expressing Gbeta 1gamma 2 alone, since Galpha q was not stable during further purification.

Preparation of HEK-293 Membranes-- Membranes of HEK-293 cells transfected with the cDNA encoding the human CaSR were prepared at 4 °C by sucrose density gradient centrifugation as described (18). The membrane pellet was treated with 5 M urea, washed, and stored at a protein concentration of 0.1-0.5 µg/µl at -80 °C .

Determination of [35S]GTPgamma S Binding to Galpha Subunits-- Basal and receptor-stimulated binding of [35S]GTPgamma S to Galpha i, Galpha s, Galpha o, and Galpha q was determined as described (18, 19). Briefly, membranes of HEK-293 cells (control or CaSR-transfected, 5-10 µg of protein/assay in 50 µl) were reconstituted with the indicated Galpha subunits (40 nM) and bovine brain Gbeta gamma (100 nM) for 30 min on ice in reaction buffer (25 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM dithiothreitol, 160 nM GDP, and MgCl2 as indicated). Similar results were obtained with MgSO4. For determination of basal guanine nucleotide binding to Galpha , membranes and Gbeta gamma were omitted. The experiment was started by the addition of [35S]GTPgamma S (60 nM; 2 × 106 cpm/50 µl). The reaction tubes were placed at 25 °C, and at different time points, samples (50 µl) were withdrawn and passed over nitrocellulose filters followed by three washes with ice-cold washing buffer (20 mM Tris, 100 mM NaCl, 25 mM MgCl2, pH 7.4), and filter-bound radioactivity was determined.

Determination of Cellular Inositol Phosphate Levels-- Inositol phosphate levels of dispersed parathyroid or of HEK-293 cells were determined as described (19) with minor modifications. Before the experiment, cells were washed with buffer (138 mM NaCl, 0.5 mM CaCl2, 5 mM KCl, 20 mM Na+-HEPES, pH 7.4) containing the indicated concentration of MgCl2 and stored in the same buffer for 15 min to equilibrate the cells with the Mg2+ concentration tested. Then 10 mM LiCl was added. After 20 min at 37 °C, cellular inositol phosphates were extracted and determined.

Determination of Cellular Levels of Cyclic AMP-- Cellular cAMP levels were determined in dispersed parathyroid cells or in HEK-293 cells 48 h after transfection. Before the experiment the cells were washed with buffer (138 mM NaCl, 0.5 mM CaCl2, 5 mM KCl, 20 mM Na+-HEPES, pH 7.4) containing the indicated concentration of MgCl2. Cells were allowed to equilibrate with the indicated Mg2+ concentration for 15 min. Then buffer with the specified Mg2+ concentration and supplemented with 0.2 mM isobutylmethylxanthine was added, and the experiment was started by the addition of forskolin (10 µM), isoproterenol (100 nM), or buffer as a control. After 20 min at 37 °C, cellular cAMP was extracted and determined by radioimmunoassay (Immunotech).

PTH Release from Dispersed Human Parathyroid Cells-- Human adenomatous or primary hyperplastic parathyroid glands removed during surgery from patients with hyperparathyroidism were immediately placed in ice-cold RPMI-medium. Dispersed parathyroid cells from parathyroid tissue were prepared by digestion with collagenase and DNase similarly as described (20, 21). For determination of PTH release, dispersed parathyroid cells (1 × 105) were equilibrated for 1 h in incubation buffer (138 mM NaCl, 5 mM KCl, 20 mM Na+-Hepes, pH 7.4) supplemented with CaCl2 and MgCl2 as indicated. After washing, PTH release was performed for 10 min at 37 °C in the specified buffer. The concentration of immunoreactive PTH in the incubation medium was measured by immunoradioactive assay determining intact human parathyroid hormone (Intact PTH, Nichols Institute Diagnostics). Inositol phosphate and cAMP levels were determined in dispersed parathyroid cells similarly as described above.

Measurement of the Intracellular Free Mg2+ Concentration, [Mg2+]i-- For determination of changes in [Mg2+]i, HEK-293 cells in incubation buffer (138 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM Na+-Hepes, pH 7.4) were loaded with 5 µM mag-fura-2/AM dissolved in dimethyl sulfoxide with 0.02% pluronic acid for 30 min at 37 °C in a humidified incubator. Cells were washed three times with warmed incubation buffer and incubated for 30 min at 37 °C to ensure complete deesterification. Cells were finally washed once with fresh incubation buffer supplemented with CaCl2 and MgCl2 as indicated. Fluorescence was recorded with a PerkinElmer fluorescence photometer (LS50B) at an emission wavelength of 520 nm and an excitation wavelength alternating between 340 and 380 nm. [Mg2+]i was determined from the ratio between 340 and 380 nm as described previously (22).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of Magnesium Deficiency on PTH Release from Human Parathyroid Cells-- In patients with severe hypomagnesemia, PTH secretion is blocked (8-10). To analyze whether this in vivo paradox is related to the release of PTH from parathyroid cells, we determined the effect of low magnesium on PTH secretion in vitro on dispersed parathyroid cells. As a control for calcium-dependent stimulus-secretion coupling of the parathyroid tissue from patients with hyperparathyroidism, stimulation of the CaSR by calcium was measured. Calcium as a CaSR agonist blocked the release of PTH with an EC50 of 1.5 ± 0.2 mM (n = 4) when the Mg2+ concentration was 1 mM (Fig. 1A). This EC50 value is in close correlation with the set-point values reported previously for human adenomas or primary hyperplasias (23, 24). Severe magnesium deficiency was simulated by decreasing the extracellular Mg2+ to 0.1 mM. When Mg2+ was decreased, PTH secretion was blocked independently of the extracellular Ca2+ concentration (Fig. 1A). These findings resemble the in vivo situation in patients; magnesium deficiency blocks PTH release, leading to concomitant hypocalcemia (8-10), and calcium replenishment cannot overcome this inhibition of PTH secretion (9). The IC50 value of magnesium for the inhibition of PTH release was 0.25 ± 0.04 mM, and inhibition of PTH release is not seen before the Mg2+ concentration falls below 0.5 mM (Fig. 1B). This finding correlates again with in vivo data demonstrating that the magnesium levels in patients that induce PTH secretion block are generally very low, with concentrations varying between 0.4 and 0.1 mM (8-10).



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Fig. 1.   Effect of magnesium on PTH release from dispersed parathyroid cells. A and B, dispersed parathyroid cells (1 × 105) were incubated for 10 min in incubation buffer with the indicated concentration of Mg2+ and Ca2+, and PTH release was determined by radioimmunoassay. Basal PTH release at 0.5 mM Ca2+ and 1 mM Mg2+ was 4.9 ± 1.5 ng/105 cells/10 min. C, inhibition of basal cAMP-levels (=100%) in dispersed parathyroid cells incubated for 10 min in buffer with 0.5 mM Ca2+ and the indicated concentration of Mg2+. D, inositol phosphate levels of dispersed parathyroid cells labeled with myo[3H]inositol for 12 h. Cells were incubated in buffer with 0.5 mM Ca2+ and the indicated concentrations of Mg2+ for 20 min at 37 °C. Maximum stimulation (100%) was determined with 5 mM CaCl2. Results are the means ± S.D. (n = 4).

Enhanced Second Messenger Generation under Magnesium Deficiency-- PTH secretion is inhibited by higher concentrations (>1 mM) of Ca2+ and Mg2+, which are agonists of the CaSR. Two different signaling pathways are activated by the CaSR, generation of inositol phosphates and inhibition of cAMP. Under magnesium deficiency, both signaling pathways were enhanced in dispersed parathyroid cells (Fig. 1, C and D). Generation of cAMP was inhibited to about 80-85% that of the maximum inhibition observed after stimulation with the CaSR agonist calcium (Fig. 1C and not shown), and basal inositol phosphates were increased to 30-35% that of the maximum stimulation by magnesium or calcium (Fig. 1D). The IC50 values of the magnesium deficiency-dependent signal enhancement were similar as for the PTH secretion, i.e. 0.19 ± 0.03 mM for the cAMP pathway and 0.2 ± 0.02 mM for the inositol phosphate production (Fig. 1, C and D). These findings demonstrate that magnesium deficiency affects similar signaling pathways as those activated by the CaSR.

Desensitization of CaSR-mediated Signaling Affects PTH Release under Magnesium Deficiency-- Is the CaSR necessary for mediating the effect of magnesium deficiency on PTH release? To address this question, we desensitized the CaSR on parathyroid cells by prolonged calcium stimulation. Desensitization of the CaSR was visible by a substantially decreased response to the agonist magnesium (6 mM) and calcium (2 mM), i.e. after desensitization, magnesium and calcium inhibited PTH release by only approx 10% (Fig. 2, lower panel) compared with a approx 60% inhibition of PTH release in nondesensitized control cells (Fig. 2, upper panel). Interestingly, the effect of magnesium deficiency on PTH release was suppressed similar to the response to the CaSR agonists (Fig. 2, lower panel). This finding strongly suggests that active cell surface-localized CaSRs are a prerequisite for the effect of magnesium deficiency on PTH release.



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Fig. 2.   Desensitization of the CaSR. The CaSR of human parathyroid cells was desensitized by incubation of the cells for 6 h in medium containing 5 mM Ca2+. PTH release from parathyroid control cells (upper panel) or from cells pretreated for 6 h with 5 mM Ca2+ (lower panel) was determined in cells incubated in buffer with 0.5 mM Ca2+ and the indicated concentration of Mg2+ or in buffer with 2 mM Ca2+ and 1 mM Mg2+. Results are the means ± S.D. (n = 4).

Effects of Low Magnesium on Inositol Phosphate Generation Mediated by Basal Activation of Recombinantly Expressed Human CaSR-- Since the previous experiments suggested that the site of magnesium action lies within the axis CaSR-G-protein-effector, we further analyzed the mechanism of the magnesium paradox in a transfected cell system. Direct effects of magnesium on the signaling of the CaSR were analyzed compared with control cells without this receptor. Basal inositol phosphate levels of HEK-293 cells expressing the CaSR increased up to 2.8-fold when the Mg2+ concentration in the buffer was decreased from 1 to 0.1 mM, whereas mock-transfected cells without CaSR expression did not show any significant increase in basal inositol phosphate levels under these conditions (Fig. 3A). The IC50 value of magnesium was 0.18 ± 0.02 mM. The CaSR was responsible for the increase in basal inositol phosphate levels under magnesium deficiency, since basal inositol phosphate levels increased with increasing CaSR expression levels (Fig. 3B). Thus, the effect of magnesium deficiency on basal activation of recombinantly expressed human CaSR paralleled the effects of magnesium deficiency on parathyroid cell signaling.



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Fig. 3.   Effect of magnesium on the human CaSR expressed in HEK-293 cells. A, HEK-293 cells (1 × 106) were transfected with 10 µg of a plasmid encoding the human CaSR. Mock-transfected cells served as a control. The increase in basal inositol phosphate levels compared with the mock control was determined in buffer containing 0.5 mM Ca2+ and the indicated concentration of Mg2+. B, HEK-293 cells were transfected with increasing amounts of a plasmid encoding the human CaSR, and basal inositol phosphate levels were determined in buffer containing 0.5 mM CaCl2 and either 0.1 mM MgCl2 (black-square) or 1 mM MgCl2 (). The total amount of DNA was held constant with plasmid pcDNA3. C, CaSR-mediated inhibition of cAMP levels. Concentration-response relationship of MgCl2 for the CaSR-mediated inhibition of forskolin-stimulated (10 µM) cAMP levels in HEK-293 cells expressing the human CaSR () or in mock-transfected control cells (open circle ). Cells were kept in buffer containing 0.5 mM CaCl2 and the indicated concentrations of MgCl2. Results are the means ± S.D. (n = 6).

CaSR-mediated Inhibition of Adenylyl Cyclase-- CaSR-mediated inhibition of adenylyl cyclase activity was determined. In HEK-293 cells, forskolin-stimulated adenylyl cyclase activity was inhibited by the CaSR. Maximal inhibition was obtained with 5 mM Ca2+ in buffer with 0.5 mM or 0.1 mM Mg2+ (not shown). Low magnesium alone resulted in partial inhibition (Fig. 3C). The CaSR-mediated inhibition of cAMP levels at 0.1 mM Mg2+ was 64 ± 8% that of the maximum inhibition by 5 mM Ca2+ (not shown). The IC50 value of magnesium was 0.25 ± 0.05 mM (Fig. 3C). In contrast, the forskolin-stimulated cAMP levels of mock-transfected control cells were barely affected by a decrease in magnesium (Fig. 3C). Together these findings demonstrate that the Galpha i- and Galpha q-mediated pathways triggered by constitutive activity of the CaSR were affected by magnesium deficiency in HEK-293 cells similarly as in dispersed parathyroid cells (cf. Fig. 1).

Effect of Magnesium on the Activation of a CaSR Mutant with Altered Ion Binding Properties-- Magnesium is an agonist of the CaSR. Therefore we asked whether the extracellular magnesium binding site(s) of the CaSR is (are) involved in mediating the signal enhancement under magnesium deficiency. Different CaSR receptor mutants were expressed (Fig. 4A). Similar expression levels of the different CaSR mutants were verified in immunoblot (Fig. 4A). Although CaSRR185Q has a nearly 10-fold decreased potency for calcium (data not shown and Ref. 15) and magnesium (Fig. 4B, panel II, and not shown), the IC50 value of magnesium in inhibiting basal receptor activation was similar between cells expressing mutated CaSRR185Q and the wild-type CaSR (Fig. 4B, panel I). This finding strongly suggests that the extracellular ion binding site of the CaSR for calcium and magnesium is not involved in mediating the signal enhancement under low magnesium.



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Fig. 4.   Effect of magnesium deficiency on CaSR mutants with altered affinity for the extracellular binding of magnesium and calcium or with altered basal activity. Immunoblot (IB) of the wild-type human CaSR (lane 2), CaSRR185Q (lane 3), CaSRF128L (lane 4), CaSRR795W (lane 5), and the rat CaSR (lane 6) detected in membranes of transfected HEK-293 cells. Mock-transfected HEK-293 cells served as a control (lane 1). B, inositol phosphate levels of HEK-293 cells expressing the indicated CaSR incubated in buffer containing 0.5 mM Ca2+ and the indicated concentrations of Mg2+. Data are expressed as % of maximum, i.e. the increase in inositol phosphate levels after stimulation of the wild-type CaSR by 5 mM CaCl2. Results are the means ± S.D. (n = 4).

Magnesium Deficiency Increases Signaling Mediated by the Basal Activity of Different CaSR Mutants-- CaSR mutants with altered basal activity were analyzed. Basal activity of CaSRF128L was increased 1.6-fold compared with the wild-type receptor (Fig. 4B, panel IV). The maximum signal enhancement observed under magnesium deficiency (0.1 mM) in CaSRF128L-expressing cells was also increased 1.5-1.8-fold compared with the wild-type receptor (Fig. 4B, panel III), whereas the IC50 value of magnesium in inhibiting basal CaSRF128L activation was similar between the wild-type CaSR and the mutated CaSRF128L (Fig. 4B, panel III). As a control, the EC50 value for the activation of CaSRF128L by magnesium was 3.5 ± 0.2 mM (Fig. 4, panel B, panel IV). Thus, signaling mediated by the basal activity of the wild-type CaSR and of CaSRF128L is enhanced under magnesium deficiency with similar IC50 values, but the absolute extent of this enhancement is different and depends on the basal activity of the respective CaSR.

To further analyze whether basal CaSR activity determines the absolute extent of the signal enhancement by low magnesium, we coexpressed wild-type CaSR together with mutant CaSRR795W, resulting in receptor heterodimers with altered functional properties (18, 25), e.g. decreased basal activity. CaSRR795W displays defective G-protein-coupling. Coexpression of CaSRR795W with wild-type CaSR decreased the affinity/potency for calcium (not shown) or magnesium about 2-fold (Fig. 4B, panel IV). In parallel, the signal generated by the basal activity of CaSR-CaSRR795W heterodimers was suppressed to 20-25% that of the signal of the wild-type receptor at 0.5 mM and at 0.1 mM Mg2+ (Fig. 4B, panel III). By contrast, the IC50 value of the magnesium decrease-dependent signal enhancement was not altered. Thus, a decrease in the basal activity of the CaSR is accompanied by a decrease in the absolute extent of the signal enhancement under magnesium deficiency but no change in the IC50 value of magnesium. Together these findings confirm that magnesium deficiency enhances signaling mediated by the basal activity of the CaSR.

Comparison of the Signals Generated by the Basal Activity of the Rat and the Human CaSR-- Is the basal activity of the CaSR related to the magnesium paradox of blunted PTH secretion in vivo? Since the paradox of blunted PTH secretion under severe magnesium deficiency has been observed in patients but not in rats (11, 12), we compared the basal activity of the rat and the human CaSR. With 0.5 mM Mg2+, the increase in basal inositol phosphate levels of rat CaSR-expressing cells was only 15-20% that of the increase of cells expressing the human CaSR (Fig. 4B, panels V and VI). Interestingly, a decrease in the extracellular magnesium also led to an enhancement of the signaling mediated by basal rat CaSR activity, with an IC50 value for magnesium similar to that of the human CaSR (Fig. 4B, panel V). However, the absolute extent of the signal enhancement of the rat CaSR at 0.1 mM Mg2+ was again only 15-20% that of the human CaSR (Fig. 4B, panel V). Equally effective levels of the human and the rat CaSR were expressed in these experiments, as determined by maximum stimulation with 10 mM Mg2+ (Fig. 4B, panel VI) and by similar concentration response relationships with EC50 values of 2.8 ± 0.3 and 5.6 ± 1 mM for calcium (not shown) and magnesium (Fig. 4B), respectively. Together these experiments demonstrate that differences in PTH secretion under magnesium deficiency reported for patients and rats correlate with differences in the basal activity of the respective CaSRs and with differences in the absolute extent of the signal enhancement under magnesium deficiency. The findings that the IC50 values of magnesium for the signal enhancement were similar between the human and the rat CaSR and between different CaSR mutants with different basal activity or with different affinity for extracellular magnesium binding suggest a common magnesium binding site different from the extracellular ion binding site of the CaSR.

CaSR-mediated Activation of G-proteins-- Since the extracellular magnesium binding site of the CaSR did not seem to be involved in the magnesium paradox, we asked whether magnesium acted on the intracellular side of the CaSR, at the CaSR- G-protein interface. To address this question, CaSR-mediated activation of G-proteins was determined by the receptor-stimulated enhancement of [35S]GTPgamma S binding to recombinant Galpha i-protein. A decrease of the Mg2+ concentration from 1 to 0.1 mM or 0.01 mM increased the rate of CaSR-mediated binding of [35S]GTPgamma S nearly 2-fold (Table I and Fig. 5A). The IC50 value for the magnesium-dependent inhibition of CaSR-stimulated GTPgamma S binding was 0.18 ± 0.04 mM (Table I). Thus, magnesium suppresses the CaSR-mediated G-protein activation and the CaSR-stimulated activation of Galpha q- and Galpha i-coupled pathways with similar concentration-response relationships. These findings also demonstrate that magnesium acts within the axis receptor-G-protein.


                              
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Table I
IC50 [Mg2+] and t1/2 of the CaSR-stimulated binding of [35S]GTPgamma S to wild-type Galpha i and to Galpha <UP><SUB><IT>i</IT></SUB><SUP><IT>R209C</IT></SUP></UP> reconstituted with membranes from CaSR-expressing HEK-293 cells as described under "Experimental Procedures."
±S.E.; n = 6.



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Fig. 5.   CaSR-stimulated [35S]GTPgamma S binding. Membranes of HEK-293 cells transfected with the CaSR-cDNA were prepared and reconstituted with wild-type Galpha i (A) or mutant Galpha <UP><SUB>i</SUB><SUP>R209C</SUP></UP> (B). CaSR-stimulated GTPgamma S binding was determined at t = 5 min in the absence of Ca2+ in buffer containing the indicated concentrations of MgCl2. The CaSR-stimulated increase in [35S]GTPgamma S binding is given, i.e. the difference between the [35S]GTPgamma S bound to CaSR-transfected cell membranes and control membranes. Results are the means ± S.E. (n = 6).

CaSR-mediated G-protein Activation of Galpha <UP><SUB>i</SUB><SUP>R209C</SUP></UP>-- Is the magnesium binding site responsible for this inhibition of basal CaSR activation localized on the CaSR or on Galpha ? Since the IC50 value of magnesium for the inhibition of basal CaSR activation was not affected by CaSR mutants with increased or decreased affinity for extracellular magnesium or calcium (cf. Fig. 4), we focused on the magnesium binding site of the Galpha subunit (26, 27). Although magnesium effects on the activation of Galpha subunits have not been detected on Galpha i/o isolated from bovine brain (28), the fact that magnesium suppresses the guanine nucleotide exchange of small GTP-binding proteins (29) suggests the possibility of a similar mechanism for Galpha subunits. And indeed, on wild-type Galpha i, magnesium inhibited basal guanine nucleotide exchange, with an IC50 of 0.18 mM (Table II). By contrast, basal guanine nucleotide exchange of a Galpha i mutant, Galpha <UP><SUB>i</SUB><SUP>R209C</SUP></UP> with impaired magnesium binding (30) was not affected by 1 mM Mg2+ (Table II). The mutant had a decreased affinity for magnesium, with an IC50 value of 2.8 ± 0.4 mM for the magnesium-dependent inhibition of basal [35S]GTPgamma S binding (Table II).


                              
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Table II
IC50 and Imax (%) determined at 1 mM Mg2+ of the magnesium-induced inhibition of basal [35S]GTPgamma S binding to different purified Galpha subunits determined as described under "Experimental Procedures."
±S.E.; n = 6.

CaSR-stimulated [35S]GTPgamma S binding was determined also for the Galpha <UP><SUB>i</SUB><SUP>R209C</SUP></UP> mutant. In contrast to the wild-type Galpha i, CaSR-stimulated [35S]GTPgamma S binding to Galpha <UP><SUB>i</SUB><SUP>R209C</SUP></UP> was not significantly suppressed by 1 mM Mg2+ compared with 0.1 mM Mg2+ (Fig. 5B). Also, the t1/2 were similar at 0.1 and 1 mM Mg2+ (4.8 ± 0.3 and 4.5 ± 0.4 min, Table I). Together these data are compatible with the concept that the magnesium binding site responsible for the enhancement of CaSR-mediated G-protein activation under low magnesium is localized on the G-protein.

Determination of the Intracellular Free Mg2+ Concentration at Different Extracellular Mg2+ Concentrations-- Since the previous experiments suggested that the magnesium binding site responsible for the enhancement of CaSR-mediated G-protein activation was localized intracellularly on the Galpha subunit, we asked whether a decrease in the extracellular free Mg2+ concentration, [Mg2+]e, was followed by a decrease in the intracellular free Mg2+ concentration, [Mg2+]i. HEK-293 cells were loaded with mag-fura-2, and [Mg2+]i was determined. The [Mg2+]i of resting HEK-293 cells incubated in buffer with 1.0 mM Mg2+ was 0.7 ± 0.06 mM (Fig. 6A). This value is in good agreement with the [Mg2+]i of resting vascular smooth muscle cells (22). By contrast, when the HEK-293 cells were incubated for 20 min in buffer with 0.1 mM Mg2+, the [Mg2+]i was reduced to 0.15 ± 0.03 mM (Fig. 6B). This finding demonstrates that a decrease in [Mg2+]e leads to a concomitant decrease in [Mg2+]i. Upon the addition of 1.0 mM extracellular Mg2+, the [Mg2+]i of magnesium-depleted HEK-293 cells increased to the initial value within 15 min (Fig. 6C). These experiments are in agreement with previous data demonstrating that alterations in [Mg2+]e are accompanied by concomitant alterations in [Mg2+]i (31). Considering that effects of magnesium on guanine nucleotide exchange were observed with a Mg2+ concentration below 1 mM (cf. Fig. 5, Tables I and II), the observed decrease in [Mg2+]i upon a decrease of [Mg2+]e is sufficient to mediate effects on guanine nucleotide exchange of G-proteins in intact cells.



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Fig. 6.   Measurement of [Mg2+]i at different extracellular Mg2+ concentrations. [Mg2+]i of HEK-293 cells loaded with mag-fura-2 was determined with cells incubated in buffer supplemented with 0.5 mM CaCl2 and 1.0 mM Mg2+ (A) or 0.1 mM Mg2+ (B and C). In panel C, the cells received 1.0 mM Mg2+ at the time point indicated by an arrow.

Inhibition of Galpha i and the Release of PTH from Parathyroid Cells-- To further analyze whether G-proteins are indeed involved in mediating the magnesium paradox of blunted PTH secretion, we determined the effect of Galpha i/o-protein inhibition on parathyroid cells (Fig. 7). Pertussis toxin treatment abolished the effect of high magnesium (6 mM) in suppressing PTH release (Fig. 7, B versus A). Pertussis toxin also abolished the effect of low magnesium (0.1 mM) on PTH release (Fig. 7B). These findings demonstrate that Galpha i/o proteins are essentially involved in mediating the magnesium paradox of PTH release.



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Fig. 7.   Effect of pertussis toxin pretreatment on PTH release from dispersed human parathyroid cells. PTH release was determined in buffer containing 0.5 mM Ca2+ and the indicated concentrations of Mg2+ in the absence (A and B) or presence (C and D) of 100 nM isoproterenol. In panels B and D, cells were pretreated with pertussis toxin (100 ng/ml) for 8 h. Results are means ± S.E. (n = 6) (*p < 0.05; **p < 0.01).

Pertussis Toxin Treatment Reveals Magnesium Effects of Galpha s-mediated Signaling-- The magnesium binding site on the Galpha subunit that seems responsible for the enhancement of CaSR-mediated signaling under magnesium deficiency is conserved between various Galpha subunits (26). In accordance with this fact, GTPgamma S binding to various Galpha subunits was inhibited by magnesium with similar concentration response relationships (Table II). However, in parathyroid cells, magnesium deficiency selectively enhanced CaSR-mediated inhibitory signaling, whereas the stimulation of PTH secretion by isoproterenol was not enhanced under magnesium deficiency (Fig. 7C). Since Galpha i/o proteins are the most abundant Galpha subunits in the cell, we asked whether inhibition of Galpha i/o proteins could reveal effects of low magnesium on Galpha s-mediated signaling. And indeed, when Galpha i/o proteins were blocked by pertussis toxin, isoproterenol-stimulated PTH release was slightly but significantly enhanced under 0.1 mM Mg2+ compared with 1 mM or 6 mM Mg2+ (Fig. 7D). Thus, in parathyroid cells the enhancement of Galpha s-mediated signaling under magnesium deficiency is overcome by the increased activity of Galpha i/o proteins.

Effect of Magnesium Deficiency on Signals Generated by Activation of Different Galpha q/i-coupled Receptors-- If the magnesium binding site responsible for the enhancement of CaSR-mediated signaling under magnesium deficiency is localized on the Galpha subunit, signals mediated by the basal activity of receptors other than the CaSR should be similarly enhanced under magnesium deficiency. To analyze whether magnesium deficiency enhances the basal activity of Galpha q/i-coupled receptors in intact cells, we determined the effect of magnesium deficiency on signals generated by the basal activity of two different Galpha q/i-coupled receptors, the angiotensin II AT1 and the bradykinin B2 receptor. Both receptors were expressed at high levels in HEK-293 cells to increase signaling mediated by basal receptor activity. Inositol phosphate levels stimulated by basal receptor activity of the AT1 and the B2 receptor were increased 2-3-fold under low magnesium (0.1 mM) compared with 1 mM Mg2+ (Table III). Since extracellular magnesium is not an agonist of the AT1 and the B2 receptor as of the CaSR, we finally asked whether signals generated by agonist-mediated receptor activation were enhanced similarly under magnesium deficiency as were the signals generated by constitutive receptor activity. And indeed, signaling of angiotensin II and bradykinin was enhanced under magnesium deficiency (0.1 mM Mg2+) compared with 1 mM Mg2+ as detected by a 2-3-fold decrease in the EC50 values of angiotensin II and bradykinin (Table III). Together these experiments demonstrate that signaling mediated by activation of different Galpha q/i-coupled receptors is enhanced under magnesium deficiency. Furthermore, these data are in agreement with the newly identified mechanism of magnesium suppressing the guanine nucleotide exchange of Galpha subunits in intact cells.


                              
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Table III
Magnesium effects on Galpha q-stimulated signaling
The basal or agonist-stimulated increase in inositol phosphate levels of HEK-293 cells expressing the angiotensin II AT1 or the bradykinin B2 receptor was determined in buffer supplemented with 1 or 0.1 mM Mg2+. Constitutive receptor activity is expressed as % of maximum agonist stimulation with 1 µM angiotensin II or bradykinin and was similar at 1 and 0.1 mM Mg2+.

±SD; n = 3.




    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of a CaSR-cDNA (3) and the identification of several CaSR mutants from patients with hypo- and hyperparathyroidism (1, 5-7) clearly established the mechanistic relationship between serum calcium and magnesium and PTH secretion. Despite this progress, the paradox of blunted PTH secretion under magnesium deficiency is still an open question. Previous work suggested that the target of magnesium action was intracellular (9). Since PTH synthesis was not affected by magnesium deficiency (32) and PTH levels rose within minutes after parenteral magnesium replacement (33), the defect was pinpointed to the level of PTH secretion.

And indeed, we found that in vitro PTH secretion from parathyroid cells was blocked under low magnesium similarly to that reported in patients. Since the effect of low magnesium was dependent on the axis CaSR-G-protein, i.e. desensitization of the CaSR or inhibition of Galpha i-proteins by pertussis toxin also suppressed the effect of magnesium deficiency on PTH secretion, we reconstituted this system of CaSR-G-protein in vitro in a cell line other than the parathyroid cell. The characteristics of the magnesium deficiency-mediated signal enhancement of basal CaSR activity were similar in parathyroid and in CaSR-expressing HEK-293 cells. The magnesium binding site was localized on the Galpha subunit; the presence of a Galpha i mutant with decreased affinity for magnesium abolished the effect of magnesium on CaSR-mediated G-protein activation, whereas on CaSR mutants with increased or decreased affinity for magnesium, the IC50 value of the magnesium deficiency-mediated signal enhancement did not change. Together these findings let us conclude that the enhancement of G-protein activation under magnesium deficiency enhances signals mediated by constitutive CaSR activity. The absolute extent of this enhancement is greater for the human CaSR than for the rat CaSR because the human receptor has a higher constitutive activity. This difference between the rat and the human CaSR leads to blunted PTH secretion under severe hypomagnesemia in patients but not in rats. Although our data suggest that the magnesium binding site responsible for this effect is located on the Galpha , we cannot rule out the possibility that the human CaSR differs from the rat CaSR by an additional yet unknown magnesium binding site acting in concert with the magnesium binding site on the Galpha subunit.

Magnesium-dependent inhibition of guanine nucleotide exchange by stabilizing guanine nucleotide binding is a common feature of small GTP-binding proteins (29). Our finding that magnesium suppresses the release of GDP on Galpha subunits parallels the role of magnesium on c-Ras mechanistically (34). In contrast to GDP-bound Ras, which has a more than 10-fold higher affinity for magnesium than Galpha (34), the affinity of magnesium for GDP-bound Galpha subunits is in the submillimolar range. Therefore pathophysiological alterations in the magnesium homeostasis will affect selectively the activity of heterotrimeric G-proteins. Guanine nucleotide exchange of Galpha i, Galpha o, Galpha s, and Galpha q was similarly affected by magnesium. Therefore the detected mechanism of magnesium stabilizing the GDP-bound Galpha may define a novel function of magnesium on heterotrimeric G-proteins.

Why does magnesium deficiency in vivo selectively enhance signaling mediated by the CaSR although low magnesium enhances guanine nucleotide exchange of many different Galpha -proteins. Several characteristics of receptor-mediated signaling may be responsible for this phenotype. In vivo, Galpha i/o proteins are the most abundant G-proteins. Therefore the enhancement of Galpha i/o-mediated signaling will be predominant. This suggestion is true for parathyroid cells, where magnesium effects on Galpha s-mediated signaling are not detectable unless Galpha i/o proteins are inhibited. The absence of magnesium effects on Galpha s-mediated signaling has also been found for Galpha s-dependent secretion processes other than PTH (35). Concerning Galpha q/11-mediated signaling, minor or absent effects of magnesium deficiency on Galpha q/11-stimulated responses in vivo may also be due to enhanced activation of Galpha i/o because many Galpha q/11-coupled receptor systems are functionally antagonized by Galpha i-mediated responses and vice versa (36-38). Therefore magnesium deficiency-dependent signal enhancement of Galpha q/11-coupled receptor systems may only become apparent in vitro when compensatory mechanisms are not effective (39) and/or under conditions of overexpression (Table III). By contrast, CaSR-mediated inhibition of PTH release is one of the few physiological systems that needs simultaneous stimulation of Galpha i and Galpha q proteins. Since there exists no functional antagonism of CaSR-mediated signaling, as demonstrated by the phenotype of activating CaSR mutations from patients with hypoparathyroidism (7), magnesium deficiency in vivo may selectively affect CaSR-governed PTH release due to an increase in Galpha i/o/q/11 activation.


    ACKNOWLEDGEMENTS

We thank Dr. Kleuss, University of Berlin, Berlin, Germany for Galpha i1- and Galpha s- cDNAs, Dr. Seuben, Novartis AG, Basel, Switzerland for the rat CaSR-cDNA, and Dr. Timmermann, University of Würzburg, Germany, for human material.


    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft and the European Union InversA program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Institut für Pharmakologie und Toxikologie, Versbacher Strasse 9, 97078 Würzburg, Germany. Tel.: 49-(0)931-201-3982; Fax: 49-(0)931-201-3539; E-mail: toph029@rzbox.uni-wuerzburg.de.

Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M007727200


    ABBREVIATIONS

The abbreviations used are: PTH, parathormone; CaSR, calcium-sensing receptor; [Mg2+]i, intracellular free Mg2+ concentration; [Mg2+]e, extracellular free Mg2+ concentration; GTPgamma S, guanosine 5'-3O-(thio)triphosphate.


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