Introduction

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The Ca²⁺ receptor is a cell surface G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to small changes in the concentration of extracellular Ca²⁺ (Brown et al., 1995; Nemeth, 1996). In parathyroid cells, the Ca²⁺ receptor monitors changes in the level of serum Ca²⁺ and is coupled to the regulation of PTH secretion. Activation of the parathyroid Ca²⁺ receptor by increased levels of extracellular Ca²⁺ results in the rapid formation of inositol 1,4,5-trisphosphate, the mobilization of intracellular Ca²⁺, and the inhibition of PTH secretion (Brown, 1991). This reciprocal relationship between extracellular Ca²⁺ levels and PTH secretion is largely responsible for maintaining systemic Ca²⁺ homeostasis. Because of its central role in this homeostatic mechanism, the Ca²⁺ receptor is a promising molecular target for drugs designed to alter circulating levels of PTH. At present, however, no potent and selective compounds are known to act at this novel receptor.

The Ca²⁺ receptor responds not only to extracellular Ca²⁺, but also to a variety of inorganic and organic cations, such as Mg²⁺, La³⁺, spermine, and neomycin (Nemeth and Scarpa, 1987; Brown et al., 1991a, 1991b). Although some of the organic cations, such as polylysine, activate the Ca²⁺ receptor at nanomolar concentrations, neither the inorganic nor the organic cations possess desirable pharmaceutical properties. We have synthesized a series of phenylalkylamine compounds, typified by NPS 467 and NPS 568 (Fig. 1), that mobilize intracellular Ca²⁺ and inhibit PTH secretion from bovine or human parathyroid cells in vitro (Steffey et al., 1993). These effects are similar to those obtained by increasing the concentration of extracellular Ca²⁺. To determine if these compounds act directly on the Ca²⁺ receptor, we have expressed the bovine or human parathyroid Ca²⁺ receptor in Xenopus laevis oocytes and have assessed the effects of NPS 467 and NPS 568 on Ca²⁺-activated Cl currents. The results provide evidence that these phenylalkylamine compounds act to potentiate the effects of cationic agonists of the Ca²⁺ receptor, but do so differently than all other known agonists of this receptor. The results suggest that NPS 467 and NPS 568 behave as positive allosteric modulators to increase the sensitivity of the Ca²⁺ receptor to extracellular Ca²⁺.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   The chemical structures of NPS 467 (A) and NPS 568 (B), shown as the R-enantromers.

	Materials and Methods

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Preparation of cRNA. The plasmid cDNA clones used were BoPCaR (Brown et al., 1993), hPCaR 4.0 (Garrett et al., 1995), bovine SKR (Nakanishi, 1991), and mGluR1a isolated from rat olfactory bulb cDNA (Masu et al., 1991). Plasmid DNA was linearized by NotI digestion, and used as template for transcription of sense-strand cRNA using T7 RNA polymerase. Transcription reactions were done as previously described (Garrett et al., 1995).

Oocyte isolation and cRNA injection. Adult female X. laevis toads were anesthetized in 0.1% tricaine according to an animal use protocol approved by the Institutional Animal Use and Care Committee of NPS Pharmaceuticals in accordance with federal animal welfare regulations. Pieces of ovarian lobe were surgically removed and incubated for 30-60 min in Ca²⁺-free MBS containing 1.5 mg/ml Collagenase P (Boehringer Mannheim, Indianapolis, IN). The MBS contained 88 mM NaCl, 1 mM KCl, 0.82 mM MgSO₄, 10 mM HEPES, and 2.4 mM NaCO₃, pH value 7.5. Stage V or VI oocytes were separated manually and washed with MBS containing 0.8 mM CaCl₂ before injection. The cRNAs of Ca²⁺ receptors, mGluR1a and SKR cRNAs were dissolved in water and 50 nl (12.5 ng/oocyte) of the RNA solution was injected into individual oocytes. Control oocytes were injected with water. After injection, oocytes were incubated at 16° in MBS containing 0.5 mM CaCl₂ for 2-7 days before electrophysiological recording (Goldin, 1992).

Two-electrode voltage-clamp. Voltage-recording and current-passing electrodes were filled with 3 M KCl and had resistances of 0.5-2 M. Oocytes were voltage-clamped at a holding potential of 60 mV with an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA) by using standard two-electrode voltage-clamp techniques (Stuhmer, 1992). Currents were recorded on a chart recorder. The standard control buffer was MBS containing 0.3 mM CaCl₂ and 0.8 mM MgCl₂, except where otherwise noted, and all concentrations shown are final. The 0 Ca²⁺ solutions contained no added Ca²⁺, and no chelating agents were used. Test substances were applied by superfusion at a flow rate of about 5 ml/min. All experiments were done at room temperature. The activity of NPS 568 and NPS 467 was determined by their effects on agonist-evoked increases in the amplitude of I_Cl. Activation of I_Cl was quantified by measuring the peak inward current stimulated by agonist or drug, relative to the holding current at 60 mV.

Concentration-response study of agonist-mediated increases in I_Cl. When multiple agonist concentrations were applied to the same oocyte, the maximal increase in I_Cl amplitude varied considerably among different oocytes. The same degree of variability was observed in oocytes expressing BoPCaR or hPCaR and is characteristic of the oocyte expression system. Therefore, the data for each oocyte were normalized to the maximum value obtained for each series of applications. A curve was fit to the data for each experiment with the Levenberg-Marquardt algorithm using the Kaleidograph fitting program (Synergy Software, Reading, PA). The curve for each set of data was fit to the equation I_Cl = A / [1 + (EC₅₀ / [agonist])^n_H], where A represents the dynamic range for the stimulation of I_Cl and n_H is the Hill coefficient. The fitted value of the dynamic range was then used to calculate the percent of maximum response to agonists. All results expressed as percent of maximum response were pooled and fit to the equation: % of maximal response = 100 / [1 + (EC₅₀ / [agonist])^n_H].

	Results

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In oocytes injected with cRNA encoding BoPCaR, increasing the concentration of extracellular Ca²⁺ to levels >1.5 mM activated an inward current. The reversal potential of this current was 33 ± 3 mV (n = 4). This reversal potential corresponds well with the equilibrium potential for Clin this system (Goldin, 1992) and indicates that the channels activated by extracellular Ca²⁺ in oocytes injected with cRNA encoding BoPCaR are the endogenous Ca²⁺-dependent Cl channels. These Ca²⁺ receptor-mediated increases in I_Cl were transient and concentration-dependent (Fig. 2). In contrast, water-injected oocytes did not respond to application of Ca²⁺ in quantities up to 20 mM (data not shown). Concentration-response characteristics of BoPCaR were determined by exposing oocytes to 1, 1.7, 3, 5.6, and 10 mM extracellular Ca²⁺ in a cumulative manner. Oocytes expressing hPCaR 4.0 typically responded only to extracellular Ca²⁺ concentrations >3 mM. These oocytes were therefore exposed to 1.7, 3, 5.6, 10, and 15 mM Ca²⁺ for concentration-response analysis. The results of concentration-response analysis for both BoPCaR and hPCaR are shown in Fig. 3 and indicate that the bovine receptor may be somewhat more sensitive to extracellular Ca²⁺ than the human homolog when studied in this heterologous expression system.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent increases in ICl amplitude evoked by extracellular Ca2+ in X. laevis oocytes injected 2-4 days before assay with cRNA encoding A, BoPCaR, or B, hPCaR 4.0. Tracings show ICl recorded at a holding potential of 60 mV. Oocytes were bathed in MBS containing 0.3 mM CaCl2 and exposed to higher levels of Ca2+ for the periods indicated by horizontal bars above current tracings.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Concentration-response characteristics of BoPCaR and hPCaR. Data shown are the normalized mean ICl amplitude ± standard error for 16 and 5 oocytes, respectively.

The activation of I_Cl by elevated concentrations of extracellular Ca²⁺ was potentiated in the presence of 1 µM NPS R-568 (Fig. 4). In contrast, noninjected oocytes did not respond to NPS R-467 or NPS R-568 at concentrations up to 100 µM, in either the absence or the presence of added Ca²⁺ (not shown). However, when extracellular Ca²⁺ was omitted, the stimulatory activity of NPS R-467 and NPS R-568 was abolished (Fig. 5). Oocytes expressing hPCaR 4.0 also responded to application of extracellular Ca²⁺ and these responses were also potentiated by NPS R-568. These results suggest that NPS R-568 may act by sensitizing Ca²⁺ receptors to activation by extracellular Ca²⁺. The effects of NPS R-467 and of NPS R-568 on the Ca²⁺ concentration-response relationship were determined in oocytes expressing BoPCaR in the presence of NPS R-467 or R-568 at concentrations of 1 µM, 3 µM, or 10 µM (Fig. 6A). Either compound caused a dose-dependent, leftward shift in the Ca²⁺ concentration-response curve. In oocytes expressing hPCaR 4.0, the effect of elevated extracellular Ca²⁺ was determined in the presence of 3 µM NPS R-568 and this also resulted in a leftward shift in the Ca²⁺ concentration-response curve (Fig. 6B).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   NPS R-568 potentiation of Ca2+ receptor-dependent activation of ICl in oocyte injected with A, hPCaR 4.0, or B, BoPCaR cRNA 2-4 days earlier. Horizontal bars indicate the duration of substance application. Extracellular Ca2+ concentration was increased transiently to 3 mM or 5.6 mM and then, after washout, 3 mM or 5.6 mM calcium was applied in the presence of 1 µM or 3 µM NPS R-568.

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Ca2+ receptor-mediated increase in ICl by NPS R-568 requires the presence of extracellular Ca2+. Tracing shows current recorded at a holding potential of 60 mV in an X. laevis oocyte injected with BoPCaR 4 days before assay. Dotted horizontal bar, duration of 1 mM Ca2+ application; bold solid bar, period during which Ca2+ was removed; plain solid bar, period when 1 µM NPS R-568 was present.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   A, Concentration-response curves for Ca2+ alone () and for Ca2+ plus 1 µM (), 3 µM (), and 10 µM () NPS R-568 and 10 µM NPS R-467 () in oocytes that were injected at least 2 days earlier with BoPCaR cRNA. Data shown are the mean ICl amplitude ± standard error (n = 16, 5, 5, 4, and 4 for each concentration, respectively). EC50 values in millimolar for Ca2+, Ca2+ plus 1 µM NPS R-568, Ca2+ plus 3 µM NPS R-568, and Ca2+ plus 10 µM NPS R-568 were 5.1 ± 0.1, 3.6 ± 0.2, 3.2 ± 0.1, and 1.5 ± 0.1, respectively. Hill coefficients for Ca2+, Ca2+ plus 1 µM NPS R-568, Ca2+ plus 3 µM NPS R-568, and Ca2+ plus 10 µM NPS R-568 were 4.5 ± 0.3, 3.3 ± 0.5, 2.3 ± 0.1, and 3.4 ± 0.7, respectively. B, Concentration-response curve for Ca2+ alone and for Ca2+ plus 3 µM NPS R-568 in oocytes expressing hPCaR 4.0. EC50 values in millimolar for Ca2+ and Ca2+ plus 3 µM NPS R-568 were 7.6 ± 1.7 and 3.2 ± 0.2 with Hill coefficients of 3.6 ± 0.2 and 5.4 ± 1.7, respectively.

The stereoselectivity in the activation of I_Cl by NPS 568 (which contains a single chiral carbon, Fig. 1) was examined in X. laevis oocytes expressing BoPCaR. Extracellular Ca²⁺ (3 mM) was applied alone, then in the presence of various concentrations of NPS S-568. After washout, Ca²⁺ was reapplied together with NPS R-568 and the response amplitudes were compared. Application of NPS R-568 (1 µM) greatly enhanced the response to extracellular Ca²⁺, but NPS S-568 was effective only at augmenting responses at concentrations 3 µM. Overall, the potentiation of Ca²⁺ responses by 10 µM NPS S-568 was slightly less than that evoked by 1 µM NPS R-568 (Fig. 7). When 10 µM NPS S-568 was coapplied with 3 mM Ca²⁺, responses were increased by 150 ± 42% (n = 3) over responses to 3 mM Ca²⁺ alone, whereas 1 µM NPS R-568 increased the response to 3 mM Ca²⁺ alone by 235 ± 67% (n = 3).

View larger version (10K): [in this window] [in a new window]   Fig. 7.   NPS R-568 is a more potent activator than NPS S-568 of Ca2+ receptor-mediated increases in ICl amplitude. In this representative trace the oocyte was injected with BoPCaR cRNA 3 days before assay. Horizontal bars above tracing, substances tested and duration of their application.

The parathyroid Ca²⁺ receptor can also be activated by elevated concentrations of certain other inorganic cations, such as Mg²⁺ and Gd³⁺, as well as organic polycations such as neomycin and spermine (Brown, 1991; Brown et al., 1991a). To determine whether NPS R-568 potentiated Ca²⁺ receptor activation evoked by other cation agonists, the effects of NPS R-568 were examined on Ca²⁺ receptor-mediated responses to Mg²⁺, Gd³⁺, or neomycin in oocytes injected with BoPCaR or hPCaR 4.0 cRNA. Oocytes expressing BoPCaR were responsive to application of 10 mM Mg²⁺, but a lower concentration (4 mM Mg²⁺) did not increase I_Cl, nor did the same oocytes respond when challenged with a Ca²⁺-free saline that contained 10 µM NPS R-467. However, all five oocytes tested responded when challenged with 4 mM Mg²⁺ plus 10 µM NPS R-467. (Fig. 8A). Gadolinium-evoked responses were also potentiated by NPS R-568 in oocytes expressing BoPCaR and hPCaR 4.0 (Fig. 8B). In the absence of added Ca²⁺, 30 µM neomycin evoked increases in I_Cl in all four cells tested, whereas the application of 5 µM neomycin did not elicit a response in any of the four oocytes. However, when 5 µM neomycin was applied together with 1 µM NPS R-568, all cells responded with large increases in I_Cl (Fig. 8C).

View larger version (13K): [in this window] [in a new window]   Fig. 8.   A, Effects of NPS R-568 on Mg2+-evoked increases in ICl. Tracing shows ICl amplitude at a holding potential of 60 mV in an X. laevis oocyte injected with BoPCaR cRNA 4 days earlier. Shown are changes in the concentration of Mg2+ in the absence of added Ca2+ and with the addition of NPS R-568 (bars above tracing). B, Effects of NPS R-467 on Gd3+-evoked increases in ICl in oocytes injected with BoPCaR. In oocytes expressing hPCaR 4.0, the response to Gd3+ is also potentiated by NPS R-568. C. Effects of NPS R-568 on neomycin-evoked increases in ICl in the absence of extracellular Ca2+.

The receptor specificity of NPS R-568 was examined in oocytes injected with cRNA encoding bovine SKR or rat mGluR1a. The SKR, the mGluR1a, and the Ca²⁺ receptor are coupled to inositol triphosphate-mediated Ca²⁺ mobilization and, therefore, produce qualitatively similar responses (increases in I_Cl amplitude) when these receptors are activated by their cognate ligand (Masu et al., 1991; Nakanishi, 1991; Brown et al., 1993; Garrett et al., 1995). Further, the Ca²⁺ receptor and mGluRs share limited sequence homology (Masu et al., 1991; Brown et al., 1993; Garrett et al., 1995). Oocytes expressing SKR did not respond to 10 µM NPS 568 either alone or when added in the presence of 10 mM Ca²⁺ (n = 5). In those oocytes expressing the SKR, increases in I_Cl were evoked in response to substance K concentrations ranging from 0.3 to 10 nM and those responses were unaffected by NPS R-568 (10 µM). Further, the sensitivity of mGluR1a to activation by L-glutamate was not affected by NPS R-467. Responses to 3 µM L-glutamate approximated those to 3 µM L-glutamate plus 10 µM NPS R-467. (Fig. 9).

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Elevated Ca2+ and NPS 467 or NPS 568 do not affect SKR or mGluR1a expressed in X. laevis oocytes. A, Tracing shows the application of 10 mM Ca2+ and of 10 mM Ca2+ in the presence of NPS 568 to an oocyte injected 3 days earlier with cRNA encoding the SKR. After washout of Ca2+ and NPS 568, substance K is applied. B. Tracing shows mGluR1a responses to 3 µM L-glutamate, alone and in the presence of 10 µM NPS R-467, and to 30 µM L-glutamate for comparison. The breaks in the mGluR1a tracings represent 7 minutes. Horizontal bars, duration of substance application.

	Discussion

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Expression of G protein-coupled receptors in heterologous cellular systems like X. laevis oocytes has been used to obtain evidence for direct actions of compounds on receptors and to further define their molecular pharmacology (Barnard and Bilbe, 1987). The X. laevis oocyte system is particularly useful for exploring the pharmacology of receptors that couple through phospholipase C to the mobilization of intracellular Ca²⁺ because increases in the concentration of cytoplasmic Ca²⁺ are readily assessed by measuring currents through the Ca²⁺-activated chloride channels (Ji et al., 1991; Feng et al., 1996). In the present series of experiments, this system has been used to define the mechanism of action of a novel class of compounds believed to act on the Ca²⁺ receptor.

The present study has confirmed the results obtained with extracellular Ca²⁺ using the cloned bovine parathyroid Ca²⁺ receptor and has now included the human Ca²⁺ receptor, which shows some differences in sensitivity to extracellular Ca²⁺ (Brown et al., 1993). Comparison of the extracellular Ca²⁺ concentration-response curves for BoPCaR and for hPCaR 4.0 suggests that the bovine parathyroid receptor is more sensitive to Ca²⁺ than is the human receptor, although maximal responses of each receptor were similar. These differences in sensitivity to extracellular Ca²⁺ are not understood, but may be related to the fidelity of expression or to species differences. The bovine and human Ca²⁺ receptors differ in 74 of 1078 amino acids (93% identity) and 43 of these differences are found in the cytoplasmic tail region containing amino acids 920 through 1078. The other differences are distributed throughout the remainder of the protein and many of these are conservative amino acid substitutions (Brown et al., 1993; Garrett et al., 1995). One possibility is that the clustering of differences within the carboxyl-terminal region (amino acids 920-1078) may account for the differences in sensitivity to agonists in this system. The mGluR1a, which shares significant homology with the Ca²⁺ receptor, exists in at least three alternative splice variants that differ in the length and sequence of the carboxyl-terminal tail. Pharmacological analysis of these splice variants has showed that although the rank order of potencies of agonists is identical for all three variants, agonists are consistently more potent on the mGluR1a than on the mGluR1b and the mGluR1c (Flor et al., 1996). It may be that those differences in the carboxyl-terminal tail between the human and bovine Ca²⁺ receptors, which are unlikely to have direct effects on agonist binding, do affect the overall sensitivity of the receptor to agonist stimulation.

The ability of NPS R-467 and NPS R-568 to elicit responses in oocytes injected with cRNA encoding the Ca²⁺ receptor, but not in noninjected or water-injected oocytes, provides compelling evidence that these compounds act directly on the Ca²⁺ receptor. The response in oocytes injected with the Ca²⁺ receptor cRNA does not result simply from expression of an exogenous G protein-coupled receptor because oocytes injected with substance K receptor cRNA or mGluR1a cRNA do not respond to either compound, but readily respond to their respective ligands. Although an indirect action of these compounds is possible, it would have to result from the interactions of a molecule endogenous to the oocyte that displays no similar activity on other G protein-coupled receptors. Further, the effect of these compounds is stereoselective, as it is in authentic bovine or human parathyroid cells (Steffey et al., 1993). In the aggregate then, the results offer strong evidence for an action of these compounds on the Ca²⁺ receptor.

Compounds that directly activate the Ca²⁺ receptor are called "calcimimetics." Polycations like spermine and neomycin are calcimimetics that activate the Ca²⁺ receptor in the absence of extracellular Ca²⁺, whether expressed in X. laevis oocytes or in authentic parathyroid cells (Brown et al., 1991a). Compounds like NPS 467 and NPS 568, however, fail to elicit responses in the absence of extracellular Ca²⁺. Rather, they potentiate responses to extracellular Ca²⁺ as well as to other extracellular di- or trivalent cations known to act on the Ca²⁺ receptor. In each case, NPS 467 and NPS 568 shift the concentration-response curve for extracellular Ca²⁺ to the left. The most parsimonious explanation for these effects is that these phenylalkylamine compounds behave as positive allosteric modulators to increase the sensitivity of the Ca²⁺ receptor to activation by extracellular Ca²⁺. Our recent findings that extracellular Ca²⁺ acts in the extracellular domain (Hammerland et al., 1995), whereas these compounds act in the transmembrane region of the Ca²⁺ receptor (Hammerland et al., 1996), are consistent with this mechanism of action. However, it remains uncertain if the phenylalkylamine compounds bind to the receptor in the absence of extracellular Ca²⁺ or if the binding of Ca²⁺ unmasks a cryptic binding site for these compounds. In either case, it is clear that their action is dependent on extracellular Ca²⁺ and that they potentiate responses to physiological and other Ca²⁺ receptor ligands.

Calcimimetic compounds therefore include compounds that mimic or potentiate the actions of extracellular Ca²⁺ by acting directly on the Ca²⁺ receptor. We can thus describe calcimimetics as being either Type I or Type II. Type I calcimimetics act in the absence of extracellular Ca²⁺ and the Type II calcimimetics act only in the presence of Type I calcimimetics. These structurally novel Type II calcimimetics are the first compounds that selectively target the Ca²⁺ receptor. As such, they may be suitable as drugs or drug leads to treat various bone and mineral disorders, such as hyperparathyroidism, where it is desirable to lower plasma levels of PTH (Silverberg et al., 1997).