Materials and Methods

Assays for Assessing Potency and Selectivity of Compounds on Ca²⁺ Receptor.

HEK 293 cells engineered to express the human parathyroid Ca²⁺ receptor have been described in detail previously (Nemeth et al., 1998). This clonal cell line, referred to as HEK 293 4.0-7 cells, has been used in a high-throughput screening format to detect agonists and allosteric activators (calcimimetics) of the Ca²⁺ receptor (Nemeth et al., 1998). Changes in the concentration of cytoplasmic Ca²⁺([Ca²⁺]_i) provide a quantitative and functional assessment of Ca²⁺receptor activity in these cells and the results using this assay parallel those obtained using a homologous expression system of bovine parathyroid cells. On-line continuous measurements of fluorescence in fluo-3- or fura-2-loaded HEK 293 4.0-7 cells were obtained using a custom-built spectrofluorimeter (Racke and Nemeth, 1993) or a fluorescence imaging plate reader instrument (Molecular Devices, Sunnyvale, CA). Test compounds were incubated with cells for 1 min before increasing the concentration of extracellular Ca²⁺ from 1.0 to 1.75 mM. Compounds were tested individually at a concentration of 100 μg/ml (20–80 μM) and those causing more than a 40% inhibition of the control response were considered to be biologically active.

To determine the potencies (IC₅₀) of compounds with biological activity, concentration-response curves were obtained and then, as an initial assessment of selectivity, the effects of compounds on [Ca²⁺]_ievoked by other G protein-coupled receptors were examined at a concentration several times their IC₅₀. Wild-type HEK 293 cells (and HEK 293 4.0-7 cells) express receptors for thrombin, bradykinin, and ATP, which couple to the mobilization of intracellular Ca²⁺. These responses can be studied to quickly assess any nonselective action of compounds on G protein-coupled receptors. Additional assays for selectivity included HEK 293 cells engineered to express receptors most homologous in sequence and topology to the Ca²⁺ receptor. These included native or chimeric receptors for various metabotropic glutamate (mGluRs) and γ-aminobutyric acid type B receptors (GABA_BRs). Chimeric receptors were created using partial sequences of metabotropic glutamate receptors and Ca²⁺ receptors, engineered to couple to activation of phospholipase C and release of intracellular Ca²⁺ in HEK 293 cells as described in the legend to Fig. 4 and in Table 1. Compounds lacking pan-activity were then subjected to structural modifications and their potencies and selectivities monitored using these HEK 293 4.0-7 cell assays in an iterative process.

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Figure 4

NPS 2143 does not affect the activity of mGluR1. A chimeric rat mGluR1 receptor containing the human mGluR1 transmembrane domain was transiently expressed in HEK 293 cells. Recordings show the [Ca²⁺]_i responses elicited by 3,5-dihydroxyphenylglycine (DHPG) (10 μM), a selective group 1 mGluR agonist, in the absence (a) or presence (b) of 10 μM NPS 2390, a small molecule antagonist of group I mGluRs, or 3 μM NPS 2143 (c). Experiments were performed in buffer containing 1.0 mM extracellular Ca²⁺ and fluorescence was recorded using the fluorescence imaging plate reader. Each trace is from a single well of a 96-well plate and is representative of that recorded in 3 to 11 other wells treated identically. The numbers accompanying each trace are arbitrary units of fluorescence and, when measured in wells from a single plate, are directly comparable. DMSO, dimethyl sulfoxide.

Screening of compound libraries using measurements of [Ca²⁺]_i in HEK 293 4.0-7 cells resulted in the identification of a number of structurally diverse compounds. Many of these compounds, however, failed to meet subsequent criteria and were eliminated. Several of these original compounds did possess the requisite potency and selectivity; structural modifications to one of these (IC₅₀ of 11 μM) led to a number of compounds that were profiled in detail. The pharmacodynamic properties of one of these, NPS 2143 (Fig. 1), is described in this report.

Assays of Ca²⁺ Receptor Activity Using Bovine Parathyroid Cells.

The effect of NPS 2143 on Ca²⁺ receptor-dependent regulation of PTH secretion and cyclic AMP formation was assessed using primary cultures of dissociated bovine parathyroid cells. Following overnight culture, the cells were removed from the flasks by decanting and washed with buffer containing 126 mM NaCl, 4 mM KCl, 1 mM MgSO₄, 0.5 mM CaCl₂, 0.7 mM K₂HPO₄/KH₂PO₄, 20 mM Na-HEPES, pH 7.45, 1 mg/ml glucose, and 0.1% bovine serum albumin (ICN Biomedicals, Cleveland, OH). Portions (0.2 ml) of this cellular suspension were added to 12- × 75-mm polystyrene tubes with or without NPS 2143 and/or varying concentrations of CaCl₂. Incubations (in triplicate) at 37°C for 20 or 30 min were terminated by placing the tubes on ice. Cells were pelleted by centrifugation (500g for 10 min at 4°C) and 0.1 ml of supernatant was immediately assayed for PTH content. A portion of the cellular suspension was left on ice during the incubation period and then processed in parallel with the other tubes. The amount of PTH in the supernatant from the tubes maintained on ice was defined as “basal release” and was subtracted from all other samples. PTH levels were quantified using a rat PTH(1-34) immunoradiometric assay kit, which also detects bovine PTH (Immutopics, San Clemente, CA). For each experiment, results were expressed as picograms of PTH released/10⁶ cells and then normalized to PTH released in 0.5 mM Ca²⁺. Cell numbers were determined by counting nuclei in a hemocytometer after lysing the cells and staining the nuclei with cresyl violet.

For measurements of cyclic AMP formation, parathyroid cells were incubated for 15 min at 37°C in 96-well plates (120,000 cells/well) in buffer containing 0.5 or 2 mM CaCl₂ in the presence or absence of isoproterenol (1 μM) and/or NPS 2143 (300 nM) before cells were lysed. All wells additionally contained 0.5 mM isobutylmethylxanthine. Levels of total cyclic AMP (cells plus medium) were determined using the BIOTRAK cyclic AMP scintillation proximity assay kit (Amersham Pharmacia Biotech, Piscataway, NJ). Luminescence was measured on a Microbeta 1450 Tri-Lux instrument (Wallac, Gaithersburg, MD).

Plasma Levels of PTH in Rats.

Normal male Sprague-Dawley rats (250–275 g) with unrestricted access to commercial rodent chow (Teklad 8640; Harlan Teklad, Madison, WI) and tap water were used. The animals were anesthetized by intraperitoneal injection of ketamine/xylazine (90:7 mg/kg) and chronic indwelling catheters were implanted in the inferior vena cava (for compound infusion) and in the abdominal aorta (for blood sampling) accessed by the femoral vein and artery, respectively. Following catheterization, the rats were housed individually and allowed to recover for at least 3 days before study. The protocol was approved by the Institutional Animal Care and Use Committee of NPS Pharmaceuticals, Inc. (Salt Lake City, UT).

On the day of study, the rats were infused intravenously (0.1 ml/kg · min) for 120 min with NPS 2143 (0.1 μmol/kg · min) or vehicle, a 20% aqueous solution of 2-hydroxypropyl-β-cyclodextrin (Sigma/RBI, Natick, MA). Blood samples (0.5 ml) were collected before and at various times after the start of the infusion for measurements of plasma levels of PTH and Ca²⁺. To prevent excessive blood volume loss during the course of the experiment, for each blood sample the erythrocyte pellet was resuspended in an equal volume of normal rat plasma and reinjected. Plasma levels of Ca²⁺ were measured immediately after collection using a model 634 ionized calcium analyzer (Ciba Corning Diagnostics, Medford, MA). PTH levels were measured using the Immutopics rat PTH(1-34) immunoradiometric assay kit.

Statistical Analyses.

The potency of NPS 2143 for stimulating PTH secretion in parathyroid cells (EC₅₀) or inhibiting [Ca²⁺]_i responses in HEK 293 4.0-7 cells (IC₅₀) was determined by fitting a curve to the data from each series of experiments with the Levenberg-Marquardt algorithm using the KaleidaGraph program (Synergy Software, Reading, PA). The differences in PTH secretion and cyclic AMP accumulation by parathyroid cells treated with agonists and antagonists of the Ca²⁺ receptor were analyzed by analysis of variance followed by Fisher's least-significant difference procedure (StatView; SAS Institute, Cary, NC). The plasma PTH and Ca²⁺ levels in the in vivo studies were analyzed by repeated measures analysis of variance, followed by a ttest to determine the significance of differences at each time point (StatView).

Results

Potency and Selectivity of NPS 2143.

Increasing the concentration of extracellular Ca²⁺ from 1.0 to 1.75 mM caused a rapid and transient increase followed by a lower yet more prolonged increase in [Ca²⁺]_i in HEK 293 4.0-7 cells expressing the human Ca²⁺ receptor (Fig.2a). Preincubation of these cells with NPS 2143 caused a concentration-dependent inhibition of the cytoplasmic Ca²⁺ response to extracellular Ca²⁺ (Fig. 2a). When NPS 2143 (300 nM) was added to cells during the prolonged phase of the response, there was an immediate fall in [Ca²⁺]_ito baseline values (Fig. 2a). Concentration-response curves, obtained using a change in agonist concentration equivalent to the EC₈₀ (a 1.0–1.75 mM increase in the concentration of extracellular Ca²⁺) yielded an IC₅₀ for NPS 2143 of 43 ± 5 nM.

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Figure 2

NPS 2143 blocks increases in [Ca²⁺]_i elicited by increases in extracellular Ca²⁺ but not those induced by ATP or thrombin in HEK 293 cells expressing the human Ca²⁺ receptor. Fluorescence recordings were obtained from fura-2-loaded cells in a cuvette with continuous stirring. Receptor agonists and NPS 2143 were added to the cuvette as indicated by the arrows. Breaks in the fluorescence traces at the arrows are addition artifacts resulting from opening the cuvette compartment. In the traces shown here, and in those of Figs. 3 and 6, these artifacts have been intentionally eliminated. The recordings in a are superimposed traces in the absence of NPS 2143 (top trace) or after pretreatment with 30 (middle trace) or 300 nM (bottom trace) NPS 2143 before increasing the concentration of extracellular Ca²⁺ from 1.0 to 1.75 mM. The recordings in b are superimposed traces showing responses to 1 U/ml thrombin in the absence or presence of 10 μM NPS 2143 and 1.0 mM extracellular Ca²⁺. Traces in c and d show responses to ATP (100 nM) and to extracellular Ca²⁺ (1.0–1.75 mM) in the presence or absence of 300 nM NPS 2143. The numbers accompanying each trace are estimates of [Ca²⁺]_i in nanomolar concentration. Shown are representative recordings from two or three separate experiments.

The prompt fall in sustained levels of cytoplasmic Ca²⁺ caused by the addition of NPS 2143 (Fig. 2, a and c) is similar to that caused by inorganic trivalent cations, which at low micromolar concentrations, block influx of extracellular Ca²⁺ through both voltage-sensitive and -insensitive channels, including those permitting influx of extracellular Ca²⁺ in parathyroid cells (Nemeth and Scarpa, 1987). Thapsigargin was used to release Ca²⁺ from intracellular stores and to promote capacitive Ca²⁺ influx independently of receptor activation. Neither La³⁺ nor NPS 2143 affected the rapid increase in [Ca²⁺]_i evoked by thapsigargin, which results from release of Ca²⁺from intracellular stores (Fig. 3, b and c). In contrast, sustained increases in [Ca²⁺]_i elicited by thapsigargin were promptly lowered by the addition of La³⁺ but not by NPS 2143, whether added prior to or after thapsigargin (Fig. 3). NPS 2143 and La³⁺thus lower sustained increases in [Ca²⁺]_i elicited by activation of the Ca²⁺ receptor by different mechanisms.

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Figure 3

Influx of extracellular Ca²⁺ elicited by thapsigargin is blocked by La³⁺ but not by NPS 2143. Fluorescence was recorded from a cuvette containing HEK 293 4.0-7 cells loaded with fura-2. Thapsigargin, NPS 2143, and La³⁺ were each used at a final concentration of 1 μM. The numbers accompanying each trace are estimates of [Ca²⁺]_i in nanomolar concentration. The traces shown are from a single preparation of cells and, with the exception of b, are representative of the results obtained using a separate cell preparation. In b, basal [Ca²⁺]_i was slightly elevated compared with those in a or c and those in another cell preparation.

HEK 293 cells normally express purinergic, thrombin, and bradykinin receptors, all of which couple to phospholipase C and evoke the mobilization of intracellular Ca²⁺. NPS 2143 did not affect responses elicited by thrombin (Fig. 2b) or ATP (Fig. 2, c and d) even when tested at concentrations as high as 10 μM. Preincubation with ATP tended to reduce the cytoplasmic Ca²⁺ response to a subsequent challenge with extracellular Ca²⁺, a not unexpected result suggesting that both agonists draw on a common store of intracellular Ca²⁺. Again, the addition of NPS 2143 (300 nM) caused a prompt decrease in the sustained phase of [Ca²⁺]_i elicited by extracellular Ca²⁺ (Fig. 2c). Increases in [Ca²⁺]_i elicited by bradykinin were similarly unaffected by 10 μM NPS 2143 (data not shown).

More stringent tests for assessing the selectivity of NPS 2143 used those receptors that are more structurally homologous to the Ca²⁺ receptor, i.e., mGluRs and GABA_BRs. In this report we present the results with cell lines expressing group 1 mGluRs (mGluR1 and mGluR5) and a cell line coexpressing GABA_BR2 and GABA_BR1, the latter engineered to couple to phospholipase C and mobilization of intracellular Ca²⁺. Representative results are presented in Fig. 4 and summarized in Table 1. At concentrations as high as 3 μM, NPS 2143 did not block [Ca²⁺]_i responses elicited either by glutamate or the selective group 1 mGluR agonist 3,5-dihydroxyphenylglycine in cells expressing mGluR1 or mGluR5 (Fig.4; Table 1); nor did it affect responses to GABA or baclofen, a selective GABA_BR agonist, in cells coexpressing GABA_BR1 and GABA_BR2. Responses to each of these agonists, however, were blocked by known antagonists of these receptors, NPS 2390 (for mGluR1 and for mGluR5) or SCH 50911 (for GABA_BR1) (Bolser et al., 1995; van Wagenen et al., 1998). Importantly, NPS 2143 by itself did not increase [Ca²⁺]_i in cells expressing a mGluR or coexpressing GABA_Breceptors R1 and R2. Thus, NPS 2143 did not demonstrate any agonist or antagonist activity at these structurally homologous receptors.

Mechanism of Action of NPS 2143.

Concentration-response curves to increasing concentrations of extracellular Ca²⁺ were generated using data derived from studies with HEK 293 4.0-7 cells in the presence or absence of NPS 2143. The addition of NPS 2143 shifted the extracellular Ca²⁺ concentration-response relationship to the right in a concentration-dependent manner (Fig.5). The maximal response to extracellular Ca²⁺ was not affected by treatment with NPS 2143 nor did NPS 2143 decrease [Ca²⁺]_i below that caused by lowered levels of extracellular Ca²⁺. The inhibitory potency of NPS 2143 is thus dependent on the concentration of extracellular Ca²⁺ and this compound has little effect at levels of extracellular Ca²⁺outside the range of those occurring physiologically. In this respect, the effects of NPS 2143 parallel those of type II calcimimetic compounds.

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Figure 5

NPS 2143 shifts the concentration-response curve to extracellular Ca²⁺ in HEK 293 cells expressing the human parathyroid Ca²⁺ receptor. HEK 293 4.0–7 cells were incubated for 40 to 60 sec in the absence (○) or presence of 30 (▪), 100 (●), 300 (▴), or 1000 (▾) nM of NPS 2143 before increasing the concentration of extracellular Ca²⁺ to the final indicated level. Each point is the mean of duplicate determinations from a single experiment and representative of the results obtained in two other separate experiments.

Type II calcimimetic compounds act by a different mechanism of action than does extracellular Ca²⁺ and provide a means of activating the Ca²⁺ receptor without changing the concentration of extracellular Ca²⁺. The effects of NPS 2143 on responses elicited by one such calcimimetic, NPS R-467, were initially examined in HEK 293 4.0-7 cells. The addition of NPS R-467 caused a rapid increase in [Ca²⁺]_i, which was reduced in a concentration-dependent manner by pretreatment with NPS 2143 (Fig. 6b). Cumulative concentration-response curves to NPS R-467 were generated in the presence of increasing concentrations of NPS 2143 (Fig. 6, a–c). NPS 2143, when used at the lower concentrations, decreased the potency of NPS R-467 without affecting maximal responses.

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Figure 6

NPS 2143 blocks cytoplasmic Ca²⁺responses to NPS R-467 in HEK 293 4.0-7 cells. Cells were preincubated with 300 nM NPS 2143 or vehicle before cumulative additions of NPS R-467 (arrowheads) to final concentrations of 0.1, 1.1, or 4.1 μM. The numbers accompanying each trace are estimates of [Ca²⁺]_i in nanomolar concentration.

NPS 2143 Affects Functional Responses of Parathyroid Cells.

The above-mentioned results showed that NPS 2143 was a potent antagonist of the Ca²⁺ receptor with some degree of selectivity, enough, at least, to test the suitability of this compound as a tool to alter Ca²⁺ receptor activity in cells normally expressing this receptor. Bovine parathyroid cells were used as a homologous expression system, and two distinct functional readouts of Ca²⁺ receptor activity were monitored: secretion of PTH and formation of cyclic AMP.

The effect of NPS 2143 on the release of PTH in vitro was studied using three different experimental designs. In the first series of experiments, cells were incubated under normocalcemic conditions (1.25 mM) in the absence or presence of increasing concentrations of NPS 2143. NPS 2143 augmented the amount of PTH appearing in the medium during a 20-min incubation (Fig. 7a). The threshold concentration for this effect was around 10 nM; the EC₅₀ for NPS 2143 under these conditions was 41 ± 9 nM.

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Figure 7

NPS 2143 stimulates PTH secretion from bovine parathyroid cells. a, parathyroid cells were suspended in buffer containing 1.25 mM Ca²⁺ and incubated for 20 min in the absence (○) or presence (●) of increasing concentrations of NPS 2143. b, parathyroid cells were initially suspended in buffer containing 0.5 mM Ca²⁺ and then transferred to tubes containing a small volume of CaCl₂ to obtain the final indicated concentration of extracellular Ca²⁺. Incubation in the absence (○) or presence (●) of 300 nM NPS 2143 was for 20 min. Each point is the mean ± S.E. of four separate experiments each performed in triplicate.

The second series of experiments examined the effects of NPS 2143 on the regulation of PTH secretion by extracellular Ca²⁺. The IC₅₀ for extracellular Ca²⁺on PTH secretion was increased from 0.99 ± 0.02 mM in the absence to 1.29 ± 0.07 mM in the presence of 300 nM NPS 2143 (Fig. 7b). Similar to cytoplasmic Ca²⁺ responses in HEK 293 4.0-7 cells, neither the minimal nor maximal PTH secretory responses to extracellular Ca²⁺ were affected by NPS 2143.

The final experiments assessed whether NPS 2143 would affect the inhibitory effects of NPS R-467 on PTH secretion. The results are presented in Table 2 and show that NPS 2143 reversed the depressive effect of NPS R-467 on the secretion of PTH.

In addition to secretion of PTH, the Ca²⁺receptor in parathyroid cells couples through a G_i-like protein to the inhibition of adenylate cyclase and this effect is most pronounced when cyclic AMP levels have been increased by activating the β-adrenergic or D₁-dopaminergic receptor on parathyroid cells. In the present study, we used isoproterenol to activate the β-adrenergic receptor. Increases in cyclic AMP elicited by isoproterenol were inhibited by 80% when the concentration of extracellular Ca²⁺ was increased from 0.5 to 2 mM (Fig.8). NPS 2143 did not affect the levels of cyclic AMP in the absence of isoproterenol when tested at either low (0.5 mM) or high (2 mM) concentrations of extracellular Ca²⁺ (data not shown). In the presence of isoproterenol, however, NPS 2143 tended to augment cyclic AMP levels at low concentrations of extracellular Ca²⁺ and completely blocked the inhibitory effects of 2 mM extracellular Ca²⁺ on evoked increases in cyclic AMP levels (Fig. 8).

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Figure 8

NPS 2143 blocks the inhibitory effects of extracellular Ca²⁺ on isoproterenol-stimulated increases in cyclic AMP formation in bovine parathyroid cells. Dissociated parathyroid cells were incubated with or without isoproterenol in buffer containing 0.5 or 2 mM CaCl₂ in the presence or absence of 300 nM NPS 2143. Control levels of cyclic AMP (in buffer containing 0.5 mM CaCl₂ and isoproterenol) were 5.6 ± 1.6 pg/well (n = 3). a, p < 0.05 versus control. b, p < 0.05 versus 2 mM calcium in the absence of NPS 2143.

NPS 2143 Increases Plasma Levels of PTH in Rats.

The in vitro experiments described above clearly demonstrate that NPS 2143 increases the release of PTH from parathyroid cells. However, from these experiments it is not clear whether the increased amount of PTH appearing in the medium results from either a rapid stimulation of regulated exocytotic PTH secretion or increased PTH synthesis or some combination of both. To help distinguish between these mechanisms, NPS 2143 was administered intravenously to normal rats. This route of administration was chosen to avoid pharmacokinetic features of this molecule that could limit its plasma concentration to subthreshold levels. This study, performed under physiological conditions, also provides information relevant to the therapeutic feasibility of using Ca²⁺ receptor antagonists to stimulate bone formation.

The intravenous infusion of NPS 2143 resulted in a rapid increase in plasma PTH levels that were maximal within 15 to 30 min of the start of infusion (Fig. 9). Plasma PTH levels were elevated throughout the 120-min infusion, but were approaching baseline levels 60 min after the infusion was terminated. There were no significant differences in plasma PTH levels between vehicle-treated animals and those infused with NPS 2143 at later time points (4–6 h).

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Figure 9

Plasma levels of PTH and Ca²⁺ in normal rats infused i.v. with vehicle (○) or NPS 2143 (●) (0.1 μmol/kg · min) for 2 h. Values are mean ± S.E.,n = 3/group. *p < 0.05 versus vehicle-infused controls.

The rapid, 4- to 5-fold increase in plasma PTH levels caused by infusion of NPS 2143 was associated with a transient increase in plasma Ca²⁺ levels (Fig. 9). However, the two responses were clearly dissociated temporally. Plasma levels of Ca²⁺ were not elevated significantly until 90 min after the start of the infusion with NPS 2143 and then rose more slowly than those of PTH. Similarly, the return of plasma Ca²⁺ levels to baseline also appeared slower and levels were still significantly elevated 2 h after the end of infusion when plasma PTH levels were no longer elevated. The rapidity of the changes in PTH levels suggests that the appearance of PTH in culture medium in the in vitro experiments is associated with the secretion of PTH stored in the cells, and not to new hormone synthesis.

Discussion

The present study describes the salient pharmacodynamic properties of the first compound shown to possess antagonist activity at the Ca²⁺ receptor. For most G protein-coupled receptors, potent and selective antagonists have typically preceded the discovery of corresponding agonists. The converse has been the case with the Ca²⁺ receptor and a plethora of inorganic ions and organic compounds, acting either as true agonists or allosteric activators of this receptor, have been reported (Nemeth and Fox, 1999). Many of these calcimimetics lack potency and selectivity yet a ligand possessing even such meager properties, but acting as an antagonist, has not been identified. In fact, NPS 2143 is the first substance, either atomic or molecular, shown to block Ca²⁺ receptor activity. We call such blocking ligands “calcilytics” and, together with calcimimetics, they encompass the known pharmacological means of altering Ca²⁺ receptor activity.

While G protein-coupled receptors in general are hardly ideal candidates for receptor-based drug design, the Ca²⁺ receptor is considerably more challenging because Ca²⁺ ions fail to offer much structural information for molecular ligand-based drug design. High-throughput screening of small molecule libraries seemed to offer the best chance of discovering a Ca²⁺ receptor antagonist. Although the initial hit detected was not potent and clearly lacked specificity, it was not pan-active. Importantly, its structure suggested that it might have pharmaceutically acceptable properties. The compounds that emerged from the ensuing medicinal chemistry effort have potencies that span three orders of magnitude and differ markedly in selectivity. NPS 2143 was chosen as representative of the compounds in this family based on its potency and the initial results suggesting some degree of selectivity.

We initially tested for actions that might interfere with the readout of Ca²⁺ receptor activity and/or those that might generally affect the activity of G protein-coupled receptors. Because NPS 2143 did not inhibit responses to a number of different receptors, all of which couple to the mobilization of intracellular Ca²⁺ in HEK 293 cells, NPS 2143 affects neither receptor activity nor the signal transduction pathways leading from these receptors to the increase in [Ca²⁺]_i. Likewise, the failure of NPS 2143 to alter cytoplasmic Ca²⁺responses to thapsigargin suggests that this compound does not affect ATPase activity or mechanisms comprising capacitive Ca²⁺ influx. In this respect, NPS 2143 differs from La³⁺, which decreases the sustained increases in [Ca²⁺]_iinduced by thapsigargin. Thus, NPS 2143 does not block extracellular Ca²⁺ influx under these conditions, as does La³⁺. This, in turn, suggests that the ability of NPS 2143 to rapidly lower [Ca²⁺]_i (Fig. 2, a and c) does not result from inhibition of Ca²⁺ influx. If the sole action of NPS 2143 in these experiments is the inhibition of the Ca²⁺ receptor then the rapid fall in [Ca²⁺]_i might indicate that continuous activation of the Ca²⁺ receptor is required to maintain increased levels of [Ca²⁺]_i.

NPS 2143 did not affect the activity of several G protein-coupled receptors from class C that are structurally similar to Ca²⁺ receptors. NPS 2143 neither activated nor inhibited group I mGluRs, which like the parathyroid Ca²⁺ receptor, couple to phospholipase C and mobilize intracellular Ca²⁺. 1To test for actions on the GABA_B receptors, a GABA_BR1 fusion construct was used. Like the native GABA_BRs, coexpression of GABA_BR2 and the R1 fusion construct was required to obtain a response to GABA or baclofen. The requirement for coexpression implies that affecting either receptor would alter the response, and the failure of NPS 2143 to do so suggests that it affects neither the R1 nor the R2 GABA_B receptors. In the aggregate, the results show that NPS 2143 is a compound with reasonable selectivity and, when used appropriately, might be a useful compound to probe the functions of Ca²⁺ receptors in nonhuman animals.2

Despite clear evidence for potent and selective inhibitory actions of NPS 2143 on the Ca²⁺ receptor, it was not a certainty that a calcilytic compound would in fact stimulate PTH secretion. While intuitively appealing, this assumption rests solely on the finding that lowering the level of extracellular Ca²⁺ stimulates PTH secretion. This is not, however, equivalent to blocking the activity of the receptor in a normocalcemic setting. Moreover, our screening assay used measurements of [Ca²⁺]_i as a functional readout of Ca²⁺ receptor activity. In the parathyroid cell, increases in [Ca²⁺]_i are associated with an inhibition of PTH secretion. The mechanisms giving rise to this unusual relationship between [Ca²⁺]_i and hormone secretion are not understood and the possibility remains that the Ca²⁺ receptor couples to an additional or alternative intracellular signal that regulates PTH secretion. Thus, it was not a certainty that compounds detected using measures of [Ca²⁺]_i would also affect PTH secretion. It was therefore rewarding to discover that NPS 2143 stimulated PTH secretion in vitro in the absence of changes in the level of extracellular Ca²⁺ and also in normocalcemic rats.

Curiously, the effects of NPS 2143 on PTH secretion from bovine parathyroid cells mirror those of type II calcimimetic compounds. These calcimimetics are positive allosteric modulators that shift the concentration-response curve of extracellular Ca²⁺ to the left without changing the maximum or minimum responses (Nemeth et al., 1998). NPS 2143 affects the agonist concentration-response curve in a converse manner: there is a shift to the right that is unaccompanied by changes in either the maximal or minimal response. These changes in the agonist concentration-response curves suggest that NPS 2143 decreases, whereas type II calcimimetic compounds increase the sensitivity of the Ca²⁺receptor to activation by extracellular Ca²⁺. Although the rightward shift of the concentration-response curve could indicate competitive inhibition by NPS 2143, the shifts in the concentration-response curves shown in Fig. 5 would also be produced by a noncompetitive antagonist acting on a tissue with a large receptor reserve. Additional studies are underway to understand further the molecular actions of NPS 2143.

The stimulatory effects of NPS 2143 on PTH secretion in vitro and on circulating levels in vivo underscore the key role of the Ca²⁺ receptor in controlling PTH secretion. These findings are additionally relevant for therapies, which by controlling circulating levels of endogenous PTH, seek to promote new bone growth. In this latter respect, it is instructive to compare the magnitude and rate of changes in plasma levels of PTH elicited by intravenous infusion of NPS 2143 with those elicited by subcutaneous administration of PTH in rats or humans. The 4-fold increase in plasma PTH levels following infusion of NPS 2143 falls within the range of levels produced by doses of exogenous PTH that stimulate new bone formation and increase bone mineral density in osteopenic rats (Fox et al., 1997) and osteoporotic humans (Lindsay et al., 1993, 1997). The increase in circulating levels of PTH is also rapid and indicates that NPS 2143 affects the regulated, rather than the constitutive pathway of PTH secretion. This finding mirrors that obtained with calcimimetics, which inhibit only the secretory responses sensitive to extracellular Ca²⁺ (Nemeth et al., 1998). The decrease in circulating levels of PTH following the end of infusion is likewise comparable with that seen following administration of exogenous hormone. Moreover, the elicited increase in plasma PTH levels induced the appropriate physiological response, i.e., a delayed increase in the plasma levels of Ca²⁺. Thus, NPS 2143 causes a pattern of change in circulating levels of PTH that is quite similar to that seen following doses of PTH that stimulate bone growth. Calcilytic compounds might therefore prove useful as anabolic therapies for bone diseases such as osteoporosis. Indeed, we have shown that NPS 2143 when administered daily by oral gavage to estrogen-treated osteopenic rats for 5 weeks increases plasma PTH levels and bone formation sufficiently to increase bone mineral density and trabecular bone volume (Gowen et al., 2000).