text:
Drug Metabolism and Disposition
Journal of Pharmacology and Experimental Therapeutics
Molecular Pharmacology
Pharmacological Reviews
Pharmacology Research & Perspectives
ASPET
Visit molpharm on Facebook
Follow molpharm on Twitter
Follow molpharm on LinkedIn
Research ArticleArticle
Allosteric Activation of the Ca2+ Receptor Expressed in Xenopus laevis Oocytes by NPS 467 or NPS 568
Lance G. Hammerland, James E. Garrett, Benjamin C. P. Hung, Cynthia Levinthal and Edward F. Nemeth
Molecular Pharmacology June 1998, 53 (6) 1083-1088;
Lance G. Hammerland
James E. Garrett
Benjamin C. P. Hung
Cynthia Levinthal
Edward F. Nemeth
The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the concentration of extracellular Ca2+. In this study, two novel phenylalkylamine compounds, NPS 467 and NPS 568, were examined for effects on Xenopus laevis oocytes expressing the bovine or human parathyroid Ca2+ receptors. Increases in chloride current (ICl) were elicited in oocytes expressing the bovine Ca2+ receptor when the extracellular Ca2+concentration was raised above 1.5 mm, whereas Ca2+ concentrations > 3 mm were generally necessary to elicit responses in oocytes expressing the human Ca2+ receptor. NPS 467 and NPS 568 potentiated the activation of ICl by extracellular Ca2+ in oocytes expressing either Ca2+ receptor homolog, and this resulted in a leftward shift of the Ca2+concentration-response curve. Neither compound was active in the absence of extracellular Ca2+. Certain inorganic and organic cations known to activate the Ca2+ receptor were substituted for elevated levels of extracellular Ca2+ to increase ICl and the effects of these agonists were also potentiated by NPS 568 or NPS 467. The effects of NPS 568 were stereoselective and the R-enantiomer was about 10-fold more potent than the corresponding S-enantiomer. Neither NPS 467 nor 568 affected ICl in water-injected oocytes or in oocytes expressing the substance K receptor or the metabotropic glutamate receptor 1a. These results provide compelling evidence that NPS 467 and NPS 568 act directly upon the parathyroid Ca2+receptor to increase its sensitivity to activation by extracellular Ca2+. This activity suggests that these compounds are positive allosteric modulators of the Ca2+ receptor. As such, these compounds define a new class of pharmacological agents with potent and selective actions on the Ca2+ receptor.
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 laevisoocytes 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+.
The chemical structures of NPS 467 (A) and NPS 568 (B), shown as the R-enantromers.
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 (Garrettet 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 mmNaCO3, pH value 7.5. Stage V or VI oocytes were separated manually and washed with MBS containing 0.8 mmCaCl2 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 MΩ. 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 mmCaCl2 and 0.8 mmMgCl2, 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 ICl. Activation of ICl 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 ICl.
When multiple agonist concentrations were applied to the same oocyte, the maximal increase in ICl 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 ICl = A / [1 + ( EC50/ [ agonist])nH] , where A represents the dynamic range for the stimulation of ICl and nH 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])nH].
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 Cl−in 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 ICl 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 mmextracellular 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.
Concentration-dependent increases in ICl amplitude evoked by extracellular Ca2+ inX. laevis oocytes injected 2–4 days before assay with cRNA encoding A, BoPCaR, or B, hPCaR 4.0. Tracings show IClrecorded 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 byhorizontal bars above current tracings.
Concentration-response characteristics of BoPCaR and hPCaR. Data shown are the normalized mean IClamplitude ± standard error for 16 and 5 oocytes, respectively.
The activation of ICl 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).
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.
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.
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 ICl by NPS 568 (which contains a single chiral carbon, Fig. 1) was examined inX. 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 mmCa2+, responses were increased by 150 ± 42% (n = 3) over responses to 3 mmCa2+ alone, whereas 1 μm NPS R-568 increased the response to 3 mm Ca2+alone by 235 ± 67% (n = 3).
NPS R-568 is a more potent activator than NPS S-568 of Ca2+ receptor-mediated increases in IClamplitude. 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 mmMg2+, but a lower concentration (4 mmMg2+) did not increase ICl, 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 ICl 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 μmneomycin was applied together with 1 μm NPS R-568, all cells responded with large increases in ICl (Fig.8C).
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 ICl 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 ICl 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 μml-glutamate approximated those to 3 μml-glutamate plus 10 μm NPS R-467. (Fig.9).
Elevated Ca2+ and NPS 467 or NPS 568 do not affect SKR or mGluR1a expressed in X. laevisoocytes. A, Tracing shows the application of 10 mmCa2+ 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 μml-glutamate, alone and in the presence of 10 μm NPS R-467, and to 30 μml-glutamate for comparison. The breaks in the mGluR1a tracings represent 7 minutes. Horizontal bars, duration of substance application.
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). TheX. 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 (Jiet 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 (Floret 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 (Hammerlandet 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).
We thank Dr. Shigetada Nakanishi for providing the SKR clone, Karen Krapcho and Rachel Simin for providing the mGluR1a clone and Sharon Bennett for assistance in preparing the manuscript.
Send reprint requests to: Lance Hammerland, Ph.D., NPS Pharmaceuticals, Inc., 420 Chipeta Way, Salt Lake City, UT 84108. E-mail: lhammerland{at}npsp.com
parathyroid hormone
BoPCaR
bovine parathyroid Ca2+ receptor
hPCaR
human parathyroid Ca2+ receptor
substance K receptor
mGluR
metabotropic glutamate receptor
modified Barth's solution
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
chloride current
Received November 6, 1997.
Accepted February 13, 1998.
The American Society for Pharmacology and Experimental Therapeutics
Turner AJ,
Bachelard HS
Barnard EA,
Bilbe G
(1987) Functional expression in the Xenopus oocyte of mRNAs for receptors and ion channels. in Neurochemistry: A Practical Approach, eds Turner AJ, Bachelard HS (ICL Press, Oxford, Washington, DC), pp 243–270.
Brown EM
(1991) Extracellular Ca2+ sensing, regulation of parathyroid cell function and role of Ca2+ and other ions as extracellular (first) messengers. Physiol Rev 71:371–411.
Brown EM,
Butters R,
Katz C,
Kifor O
(1991a) Neomycin mimics the effects of high extracellular calcium concentrations on parathyroid function in dispersed bovine parathyroid cells. Endocrinology 128:3047–3054.
Gamba G,
Riccardi D,
Lombardi M,
Kifor O,
Sun A,
Hediger J.,
Lytton MA,
Hebert SC
(1993) Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature (Lond) 366:575–580.
(1991b) Polyarginine, polylysine, and protamine mimic the effects of high extracellular calcium concentrations on dispersed bovine parathyroid cells. J Bone Miner Res 6:1217–1225.
Pollak M,
Seidman CE,
Seidman JG,
Chou Y-HW,
(1995) Calcium ion-sensing cell-surface receptors. N Eng J Med 333:234–240.
Feng G,
Hannan F,
Reale V,
Hon YY,
Kousky CT,
Evans PD,
Hall LM
(1996) Cloning and functional characterization of a novel dopamine receptor from Drosophila melanogaster. J Neurosci 16:3925–3933.
Flor PJ,
Gomeza J,
Tones MA,
Kuhn R,
Pin J-P,
Knopfel T
(1996) The C-terminal domain of the mGluR1 metabotropic glutamate receptor affects sensitivity to agonists. J Neurochem 67:58–63.
Garrett JE,
Capuano IV,
Hammerland LG,
Hung BCP,
Hebert SC,
Fuller F,
Nemeth EF
(1995) Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. J Biol Chem 270:12919–12925.
Goldin AL
(1992) Maintenance of Xenopus laevis and oocyte injection. Methods Enzymol 207:266–279.
Krapcho KJ,
Simin R,
Alasti N,
Nemeth EF,
Fuller FH
(1996) NPS R-467 activation of chimeric calcium-metabotropic glutamate receptors and a calcium receptor deletion mutant indicates a site of action within the transmembrane of the calcium receptor. J Bone Miner Res 11:P272.
(1995) Cation binding determinants of the calcium receptor revealed by functional analysis of chimeric receptors and a deletion mutant. J Bone Miner Res 10:70.
Ji H,
Sandberg K,
Catt KJ
(1991) Novel angiotensin II antagonists distinguish amphibian from mammalian angiotensin II receptors expressed in Xenopus laevis oocytes. Mol Pharmacol 39:120–123.
Masu M,
Tanabe Y,
Tsuchida K,
Shigemoto R,
Nakanishi S
(1991) Sequence and expression of a metabotropic glutamate receptor. Nature (Lond) 349:760–765.
(1991) Mammalian tachykinin receptors. Annu Rev Neurosci 14:123–136.
Bilezikian JP,
Raisz LG,
Rodan GA
(1996) Calcium receptors as novel drug targets. in Principles of Bone Biology, eds Bilezikian JP, Raisz LG, Rodan GA (Academic Press, San Diego), pp 1019–1035.
Scarpa A
(1987) Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor. J Biol Chem 262:5188–5196.
Silverberg SJ,
Bone HG,
Marriott TB,
Locker FG,
Thys-Jacobs S,
Dziem G,
Kaatz S,
Sanguinetti EL,
Bilezikian JP
(1997) Short-term inhibition of parathyroid hormone secretion by a calcium-receptor agonist in patients with primary hyperparathyroidism. N Engl J Med 337:1506–1510.
Steffey ME,
Fox J,
VanWagenen BC,
Delmar EG,
Balandrin MF,
(1993) Calcimimetics: structurally and mechanistically novel compounds that inhibit parathyroid hormone secretion from parathyroid cells. J Bone Miner Res 8:S175.
Stuhmer W
(1992) Electrophysiological recording from Xenopus oocytes. Methods Enzymol 207:319–339.
Thank you for sharing this Molecular Pharmacology article.
You are going to email the following Allosteric Activation of the Ca2+ Receptor Expressed in Xenopus laevis Oocytes by NPS 467 or NPS 568
Message Subject (Your Name) has forwarded a page to you from Molecular Pharmacology
Message Body (Your Name) thought you would be interested in this article in Molecular Pharmacology.
Molecular Pharmacology June 1, 1998, 53 (6) 1083-1088;
Inhibition of GABAAR by Sulfated Steroids
Extracellular Control of Signaling Bias at the β2AR
Fluorizoline Induces p21 via PHB2-GGCT Interaction Blockade
Show more Article
Fast Forward by date
Fast Forward by section
About Molecular Pharmacology
Customized Alerts
ASPET's Other Journals
Copyright © 2022 by the American Society for Pharmacology and Experimental Therapeutics


text:
RE-RELEASE Julia Fischer Mendelssohn
Julia Fischer, Daniel Müller-Schott, Jonathan Gilad
Felix Mendelssohn-Bartholdy
ConcertoNet
In a rather lively rhythm, the performers seize the First Trio (1839) with a dramatism accentuated in the vertical passages, admirably contrasted with the milder moments.
Piano Trio No. 1 in D minor, Op. 49
1) Molto allegro agitato
2) Andante con moto tranquillo
3) Scherzo – Leggiero e vivace
4) Finale – Allegro assai appassionato
Piano Trio No. 2 in C minor, Op. 66
5) Allegro energico e con fuoco
6) Andante espressivo
7) Scherzo – Molto allegro quasi presto
8) Finale – Allegro appassionato
There are few composers who simultaneously exemplify both restraint and innovation as well as Felix Mendelssohn-Bartholdy. Heavily inspired by both the Classical and Romantic traditions, Mendelssohn saw his life's purpose as being to "continue working on the legacy left to me by the great masters… The work should also carry on from where it left off in accordance with one's creative powers, and not be solely an unimaginative repetition of what has been."
Mendelssohn's two piano trios provide some of the finest examples of the balance he was able to achieve between deference to the great masters, in this case Ludwig van Beethoven, and a more Romantic sensibility for chromaticism, harmony and orchestration. A noted pianist in his own right, Mendelssohn performed the piano part on the premieres of both trios, along with Ferdinand David on violin and Carl Wittmann on cello. In this release these roles are filled by violinist Julia Fischer, cellist Daniel Müller-Schott and pianist Jonathan Gilad.
The contrast between the two piano works is striking. The Piano Trio in D minor was written in 1839, during the artistic and personal zenith of Mendelssohn's career. After years of remarkable success all over Europe, his appointment as director of the Leipzig Gewandhaus concerts in 1835 ensured that he could finally began to settle down in Germany, secure in his financial prospects. Two years later, Mendelssohn married and appeared to relish his new life as a husband and father of five. The first piano trio clearly reflects this elated mood, with lively dance movements and joyful, lyrical themes that this release manages to capture perfectly.
In contrast the Piano Trio in C minor, written only five years later, reflects a much more tumultuous period in Mendelssohn's life. His mother had died in 1843 and his health was beginning to turn for the worse, culminating in a fatal stroke on November 4th, 1847. Musically, this contributed to a period of dark timbres, tense tempos and increased chromaticism. Even during this gloomy time the tone is more understated than melodramatic, tending towards a more subtle form of subversion which is even more difficult to pull off, and all the more enjoyable when performed as convincingly as it is by Fischer, Müller-Schott and Gilad.
The digital files can be downloaded from the original release with catalogue number PTC 5186 085
Khachaturian / Prokofiev / Glazunov - Russian Violin Concertos
Brahms - Violin Concerto in D & Double Concerto in A minor
Tchaikovsky - Violin Concerto in D / Sérénade mélancolique / Valse-Scherzo / Souvenir d'un lieu cher

redpajama_set_name: RedPajamaCommonCrawl


Includes: Padded jumpsuit with boot tops and plastic face mask with elasticated strap. Please click on the arrows to view size guide.
Includes: Padded jumpsuit with over the head mask. Please click on the arrows to view size guide.
Includes: Padded jumpsuit with Infinity gauntlet & hard plastic face mask. Please click on the arrows to view size guide.
Includes: 3D muscle chest jumpsuit with boot tops & overhead material mask with mesh eyes. Please click on the arrows to view size guide.
Includes: Padded printed muscle chest jumpsuit with gold glow effect & plastic face mask. Please click on the arrows to view size guide.
Includes: Jumpsuit with quilted jacket and knee pads, face mask & boot covers. Please click on the arrows to view size guide.
Includes: Spiderman hoodie, & sweatpants with attached boot tops & snood with goggles. This is a US product. Please click on the arrows to view size guide.
Includes: Padded jumpsuit with over the head latex character mask with iconic tongue. Please click on the arrows to view the size guide.


text:
Q: Object Orientated Rust (The rust book chapter 17 blog) I want to implement the last bullet point on the options of going further for creating the blog from The Rust Programming Language:

Allow users to add text content only when a post is in the Draft state. Hint: have the state object responsible for what might change about the content but not responsible for modifying the Post.

I want to implement the add_text method that would have a default implementation that does nothing in the State trait but would push the string into post.content if the state was Draft:
pub struct Post {
    state: Option<Box<dyn State>>,
    content: String,
}

impl Post {
    pub fn new() -> Post {
        Post {
            state: Some(Box::new(Draft {})),
            content: String::new(),
        }
    }

    pub fn add_text(&mut self, string: &str) {
        self.state.as_ref().unwrap().add_text(&mut self, string)
    }

    pub fn content(&self) -> &str {
        self.state.as_ref().unwrap().content(&self)
    }
}

trait State {
    fn content<'a>(&self, post: &'a Post) -> &'a str {
        ""
    }
    fn add_text<'a>(&self, post: &'a mut Post, string: &str) {
        "";
    }
}

impl State for Draft {
    fn add_text<'a>(&self, post: &'a mut Post, string: &str) {
        &mut post.content.push_str(string);
    }
}

My issue is around this line of Post::add_text
self.state.as_ref().unwrap().add_text(&mut self, string)

I get a cannot borrow as mutable try removing &mut.
However:
self.state.as_ref().unwrap().add_text(self, string)

Gives me 'cannot borrow *self as mutable because it is also borrowed as immutable'.
Can someone help me with this? I am not sure whether I am going about this in the right way.

A: The problem is that state is a member of Post. If you borrow state then you cannot mutably borrow self (that is the Post) at the same time, and that is right. Consider what would happen if your Draft::add_text() function were to assign to the received post.state:
impl State for Draft {
    fn add_text<'a>(&self, post: &'a mut Post, string: &str) {
        post.state = None; //killed self!
    }
}

That would make self disappear while this function is running! That would be terribly unsafe.
This usage pattern is not allowed in Rust, but you have several workarounds available. The nicest, IMO, would be to separate the data to be mutated into another type:
pub struct PostData {
    content: String,
}
pub struct Post {
    state: Option<Box<dyn State>>,
    data: PostData,
}
trait State {
    fn add_text<'a>(&self, post: &'a mut PostData, string: &str) {
        "";
    }
    //...
}
impl Post {
    pub fn add_text(&mut self, string: &str) {
        self.state.as_ref().unwrap().add_text(&mut self.data, string)
    }
}

This works because you are borrowing separated pieces of self, that is self.state on one hand and self.data on the other.
Another easy workaround would be to remove the status from the Post and re-add it later:
impl Post {
    pub fn add_text(&mut self, string: &str) {
        let state = self.state.take();
        state.as_ref().unwrap().add_text(&mut self, string);
        assert!(self.state.is_none());
        self.state = Some(state);
    }
}

Personally, I find this solution a bit hacky.

A: There is a solution provided here
But this solution has a few deprecation warnings. Here's my version which fixes the warnings
For posterity's sake, here is the code in the provided playground link:
pub struct Post {
    state: Option<Box<dyn State>>,
    content: String,
}

impl Post {
    pub fn new() -> Post {
        Post {
            state: Some(Box::new(Draft {})),
            content: String::new(),
        }
    }

    pub fn add_text(&mut self, text: &str) {
        self.content = self.state.as_ref().unwrap().add_text(&self.content, text);
    }

    pub fn content(&self) -> &str {
        self.state.as_ref().unwrap().content(&self)
    }

    pub fn request_review(&mut self) {
        if let Some(s) = self.state.take() {
            self.state = Some(s.request_review())
        }
    }

    pub fn approve(&mut self) {
        if let Some(s) = self.state.take() {
            self.state = Some(s.approve())
        }
    }
}

trait State {
    fn request_review(self: Box<Self>) -> Box<dyn State>;
    fn approve(self: Box<Self>) -> Box<dyn State>;

    fn content<'a>(&self, _post: &'a Post) -> &'a str {
        ""
    }

    fn add_text(&self, original_text: &str, _text_to_add: &str) -> String {
        original_text.to_string()
    }
}

struct Draft {}

impl State for Draft {
    fn request_review(self: Box<Self>) -> Box<dyn State> {
        Box::new(PendingReview {})
    }

    fn approve(self: Box<Self>) -> Box<dyn State> {
        self
    }

    fn add_text(&self, original_text: &str, text_to_add: &str) -> String {
        format!("{}{}", original_text, text_to_add)
    }
}

struct PendingReview {}

impl State for PendingReview {
    fn request_review(self: Box<Self>) -> Box<dyn State> {
        self
    }

    fn approve(self: Box<Self>) -> Box<dyn State> {
        Box::new(Published {})
    }
}

struct Published {}

impl State for Published {
    fn request_review(self: Box<Self>) -> Box<dyn State> {
        self
    }

    fn approve(self: Box<Self>) -> Box<dyn State> {
        self
    }

    fn content<'a>(&self, post: &'a Post) -> &'a str {
        &post.content
    }
}

fn main() {
    let mut post = Post::new();

    post.add_text("I ate a salad for lunch today");
    post.add_text("\nAnd a steak!");
    assert_eq!("", post.content());

    post.request_review();
    post.add_text("\nAnd dessert!");
    assert_eq!("", post.content());

    post.approve();
    post.add_text("\nAnd coffee!");
    assert_eq!("I ate a salad for lunch today\nAnd a steak!", post.content());
}

A: Here's my take.
Since it was hinted that the state will be responsible of what might change and not for modifying the Post, add add_text (or a much more appropriate function name) to the State trait. Make it accept a string slice.
trait State {
    // Some codes here
    fn add_text<'a>(&self, _text: &'a str) -> &'a str {
        ""
    }
}

Implement the add_text method for the Draft struct:
impl State for Draft {
    // Some codes here
    fn add_text<'a>(&self, text: &'a str) -> &'a str {
        text
    }
}

Re-write the add_text of Post struct in such a way that it will call add_text of the state and return the "change" that should be added to the current content.
impl Post {
    // Some codes here
    pub fn add_text(&mut self, text: &str) {
        if let Some(s) = self.state.as_ref() {
            let text = s.add_text(text);
            self.content.push_str(text)
        }
    }
    // Some codes here
}

Only Draft state will return the string slice we pass to it. We'll use the add_text of the state to determine what to append to the current content.
fn main() {
    let mut post = Post::new();

    post.add_text("I ate a salad for lunch today");
    post.add_text("\nThen slept");
    assert_eq!("", post.content());

    post.request_review();
    post.add_text("\nThen woke up");
    assert_eq!("", post.content());

    post.approve();
    post.add_text("\nThen got back to work");
    assert_eq!("I ate a salad for lunch today\nThen slept", post.content());
}

redpajama_set_name: RedPajamaStackExchange


As a supervisor or manager, you will probably be responsible for evaluating the performance of your employees. Most companies conduct annual Performance Appraisals and many companies also offer a probationary review, to ensure the new employee understands what he/she is doing well, or may need to focus upon to improve.
Also, a performance evaluation may be accompanied by a merit increase, so it is important that the review is accurate and objective. Equally important is how you conduct the performance appraisal meeting. The review is designed to increase the employee's effectiveness and efficiency, and also to recognize his/her accomplishments and contributions to the company.
The following tips will help to make the review process a win-win situation for both parties.
Focus on the areas of positive performance by first describing what the employee is doing right. It's important to point out employee successes as a thank you for hard work and dedication. Introducing the report with the positives keeps the employee from putting their guard up and persuades them to keep reading.
Provide performance feedback that increases employee engagement, by asking questions about the areas in which you believe the employee should improve, and soliciting the employee's input on how he/she could enhance their performance.
Use coaching techniques to deliver negative feedback in a helpful manner, by providing constructive feedback. Don't be critical; use specific examples of behaviors that need to change or could improve and give examples of how the behavior should change.
Develop a rapport with the employee to allow them to provide you with their opinions about the company, their job, and their performance.
Encourage the employee to manage their own performance by developing goals & objectives with timelines to improve.
Help the employee to prepare in advance for their annual review by suggesting that they come to the discussion with a list of their strengths and weaknesses.
Enable the employee to leave the discussion with a plan that will help him/her grow and improve.
A professional performance review process helps define the responsibilities of the job and is important to retaining good employees and keeping them happy.


The ACT Mop Set provides infection control and is designed for environmental and ergonomic friendliness. The mops made of ultra microfiber materials are the Dry Mop, the green one, and the Damp Mop, the blue one. Static electricity on the Dry Mop catches hair and dust. The Damp Mop uses water and capillary action to catch dirt and stains and removes 99% of the bacteria. The handle is ergonomically designed so it does not stress your waist. The frame has Velcro, which facilitates the attachment and detachment of the mop without your needing to bend at the waist or touch the dirty mop surface.


text:
Denis William Hanrahan (1 November 1933 – 1 February 1987) was the 6th Roman Catholic Bishop of Christchurch, New Zealand. He was appointed Coadjutor Bishop of Christchurch on 25 March 1984 by Pope John Paul II, succeeded to the see of Christchurch  on 4 July 1985 on the retirement of his predecessor Bishop Ashby and died in office on 1 February 1987.

Early life
Hanrahan was born in Greymouth, New Zealand and was educated there by the Sisters of Mercy and the Marist Brothers. He trained for the priesthood at Holy Cross College, Mosgiel.

Priesthood
Hanrahan was ordained a priest by Bishop Joyce on 21 July 1957 in Greymouth. In 1970 he went to Manila to study for a year at the East Asian Pastoral Institute. On returning to Christchurch in 1971, he lived with the Brothers of St John of God, at what was then Marylands, at Halswell. Hanrahan was appointed Diocesan Director for Religious Education in 1971. He remained in this position until becoming Parish Priest at Bishopdale in 1979. He was also chairman of the Christchurch Priests' Senate from 1979 until 1983.

Episcopacy
Hanrahan was appointed Coadjutor Bishop of Christchurch 25 March 1984 and was consecrated on 6 June 1984 by Bishop Ashby, Archbishop Magnoni (the Pro-Nuncio) and Cardinal Williams. When Bishop Ashby retired on 4 July 1985, Hanrahan succeeded him as Bishop of Christchurch and he was formally enthroned in the Cathedral of the Blessed Sacrament on 5 August 1985. On 24 November 1986, he hosted Pope John Paul II at his Cathedral during the Papal visit to New Zealand at an ecumenical service during which the Pope dedicated a chapel for Christian Unity. In 1987 Hanrahan took over the role of Episcopal Vicar for New Zealand Catholic Education, a role previously held by Bishop Kavanagh who had died in 1985.

Death
Hanrahan died completely unexpectedly after a game of tennis at the age of 53 in the evening of Sunday, 1 February 1987. The chief celebrant of his concelebrated Requiem Mass was Cardinal Williams. Hanrahans predecessor as Bishop of Christchurch, Bishop Ashby, was also one of the celebrants. Bishop Hanrahan is buried at Bromley Cemetery.

Notes

Holy Cross College, New Zealand alumni
20th-century Roman Catholic bishops in New Zealand
People from Greymouth
Roman Catholic bishops of Christchurch
1933 births
1987 deaths
Burials at Bromley Cemetery

redpajama_set_name: RedPajamaWikipedia


The first contemporary African artist to have his work purchased by the Museum of Modern Art.
Skunder Boghossian was born in Addis Ababa in 1937 and died in Washington DC. in 2003.
He studied art informally at Tafari Mekonnen secondary school. In 1954 - 55 Skunder won the second prize at an art exhibition held as part of HIM, Haile Selalssies's jubilee Anniversary Celebration. As a result he was awarded an imperial scholarship to study in London. He attended St. Martin's School, The Central School, and The Slade School of fine Art in London. In 1957 he moved to Paris and spent nine years studying and teaching. He studied at the Ecole Nationale Superieure de Beaux-are and the Académie de la Grande Chaumière. Skunder's compositions from the Paris years, with their kaleidoscopic layering of form, meaning, color and design, reflects his interest in pan-African themes as well as his Ethiopian heritage.
Skunder returned to Addis Ababa in 1966 and joined the faculty at the Addis Ababa school of fine Arts until 1969. In 1970 he emigrated to the United States, teaching at Howard University from 1972 until 2001. Although he taught in Ethiopia for only three years Skunder's impact was lasting.
Early in his career Skunder's work entered the collection of the Muse`e d`Art modern de la ville de Paris and the museum of modern Art in New York. Boghossian was the first contemporary African artist to have his work purchased by the Museum of Modern Art; his paintings are also in the collections of the National Museum of African Art in Washington, DC. His paintings talk about supernatural forces, mystical transformations, filled with imagery of sprit and nightmarish metamorphoses, political opposition, violence, and destruction as in the 1972 painting DMZ (for demilitarized zone) in the collection of Howard University museum.
the first women Included In the Art collection of the National Museum of Ethiopia.
Desta Hagos was born in Adwa in the Tigray region of northern Ethiopia 1952. She moved to Addis Ababa at age nine to attend boarding school and later attended the prestigious Empress Menen High school, a school of girls. When she received her diploma from the Addis Ababa Fine Arts School in 1969, Desta's graduation exhibition was the first solo show by a woman artist in Ethiopia. She then received her BFA from California Lutheran University in 1973. She was the first woman artist to be represented in the collection of the National Museum of Ethiopia.
The Stage, Desta Hagos, 83.8 cm x 108.9cm Oil and collage on board, collection of Desta Hagos.