Allosteric Activation of the Ca Receptor Expressed in Xenopus laevis Oocytes
نویسندگان
چکیده
The Ca 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 Ca. 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 Ca receptors. Increases in chloride current (ICl) were elicited in oocytes expressing the bovine Ca receptor when the extracellular Ca concentration was raised above 1.5 mM, whereas Ca concentrations . 3 mM were generally necessary to elicit responses in oocytes expressing the human Ca receptor. NPS 467 and NPS 568 potentiated the activation of ICl by extracellular Ca in oocytes expressing either Ca receptor homolog, and this resulted in a leftward shift of the Ca concentrationresponse curve. Neither compound was active in the absence of extracellular Ca. Certain inorganic and organic cations known to activate the Ca receptor were substituted for elevated levels of extracellular Ca 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 Ca receptor to increase its sensitivity to activation by extracellular Ca. This activity suggests that these compounds are positive allosteric modulators of the Ca receptor. As such, these compounds define a new class of pharmacological agents with potent and selective actions on the Ca receptor. The Ca 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 Ca (Brown et al., 1995; Nemeth, 1996). In parathyroid cells, the Ca receptor monitors changes in the level of serum Ca and is coupled to the regulation of PTH secretion. Activation of the parathyroid Ca receptor by increased levels of extracellular Ca results in the rapid formation of inositol 1,4,5-trisphosphate, the mobilization of intracellular Ca, and the inhibition of PTH secretion (Brown, 1991). This reciprocal relationship between extracellular Ca levels and PTH secretion is largely responsible for maintaining systemic Ca homeostasis. Because of its central role in this homeostatic mechanism, the Ca 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 Ca receptor responds not only to extracellular Ca, but also to a variety of inorganic and organic cations, such as Mg, La, spermine, and neomycin (Nemeth and Scarpa, 1987; Brown et al., 1991a, 1991b). Although some of the organic cations, such as polylysine, activate the Ca 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 Ca 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 Ca. To determine if these compounds act directly on the Ca receptor, we have expressed the bovine or human parathyroid Ca receptor in Xenopus laevis oocytes and have assessed the effects of NPS 467 and NPS 568 on Ca-activated Cl currents. The results provide evidence that these phenylalkylamine compounds act to potentiate the effects of cationic agonists of the Ca 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 Ca receptor to extracellular Ca. ABBREVIATIONS: PTH, parathyroid hormone; BoPCaR, bovine parathyroid Ca receptor; hPCaR, human parathyroid Ca receptor; SKR, substance K receptor; mGluR, metabotropic glutamate receptor; MBS, modified Barth’s solution; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; ICl, chloride current 0026-895X/98/061083-06$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:1083–1088 (1998). 1083 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from Materials and Methods 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 Ca-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 Ca 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 currentpassing electrodes were filled with 3 M KCl and had resistances of 0.5–2 MV. Oocytes were voltage-clamped at a holding potential of 260 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 Ca solutions contained no added Ca, 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
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تاریخ انتشار 1998