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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 MOmega . Junction potential was compensated using the electronic circuit of the amplifier. Seal resistances of >10 GOmega 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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

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.

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.

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.

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.

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.

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.

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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(4):C1315-C1323
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