Effects of Positive Allosteric Modulators on Single-Cell Oscillatory Ca Signaling Initiated by the Type 5 Metabotropic Glutamate Receptor

Agonist stimulation of the type 5 metabotropic glutamate (mGlu5) receptor initiates robust oscillatory changes in cytosolic Ca concentration ([Ca ]i) in single cells by rapid, repeated cycles of phosphorylation/dephosphorylation of the mGlu5 receptor, involving protein kinase C and as-yet-unspecified protein phosphatase activities. An emergent property of this type of Ca oscillation-generating mechanism (termed “dynamic uncoupling”) is that once a threshold concentration has been reached to initiate the Ca oscillation, its frequency is largely insensitive to further increases in orthosteric agonist concentration. Here, we report the effects of positive allosteric modulators (PAMs) on the patterns of single-cell Ca signaling in recombinant and native mGlu5 receptor-expressing systems. In a Chinese hamster ovary cell-line (CHO-lac-mGlu5a), none of the mGlu5 receptor PAMs studied [3,3 -difluorobenzaldazine (DFB), N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl]phenyl}-2-hydroxy-benzamide (CPPHA), 3-cyano-N-(1, 3-diphenyl-1H-prazol-5-yl)benzamide (CDPPB), S-(4-fluorophenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl1-yl}-methanone (ADX47273)], stimulated a Ca response when applied alone, but each PAM concentration-dependently increased the frequency, without affecting the amplitude, of Ca oscillations induced by glutamate or quisqualate. Therefore, PAMs can cause graded increases (and negative allosteric modulator-graded decreases) in the Ca oscillation frequency stimulated by orthosteric agonist. Initial data in rat cerebrocortical astrocytes demonstrated that similar effects of PAMs could be observed in a native cell background, although at high orthosteric agonist concentrations, PAM addition could much more often be seen to drive rapid Ca oscillations into peakplateau responses. These data demonstrate that allosteric modulators can “tune” the Ca oscillation frequency initiated by mGlu5 receptor activation, and this might allow pharmacological modification of the downstream processes (e.g., transcriptional regulation) that is unachievable through orthosteric ligand interactions. Glutamate, the major excitatory neurotransmitter in the central nervous system, acts on ionotropic glutamate receptors to elicit fast excitatory responses and on metabotropic glutamate (mGlu) receptors to modulate and fine tune synaptic transmission (Conn and Pin, 1997). The eight subtypes of the mammalian mGlu receptor can be divided into three subgroups, based on sequence homologies, agonist and antagonist binding profiles, and preferred coupling to signal transduction pathways. The group I mGlu receptors, mGlu1 and mGlu5, both preferentially couple via Gq/11 proteins to stimulate phospholipase C activity but are differentially localized and probably fulfill distinct physiological functions (Hermans and Challiss, 2001; Mannaioni et al., 2001; Ferraguti and Shigemoto, 2006; Kumar et al., 2008). Activation of these receptors also elicits highly distinct Ca responses at a single cell level, the mGlu1 receptor primarily eliciting a peak-plateau type of Ca response, whereas mGlu5 receptor activation leads to oscillatory changes in intracellular Ca concentration ([Ca ]i) in both recombinant and native (e.g., astrocyte) cell backgrounds (Kawabata et al., 1996; NakaThis work was supported by the Biotechnology and Biological Sciences Research Council by a CASE PhD studentship (to S.J.B.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.059170. ABBREVIATIONS: mGlu, metabotropic glutamate; NAM, negative allosteric modulator; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; PAM, positive allosteric modulator; DFB, 3,3 -difluorobenzaldazine; CPPHA, N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide; CDPPB, 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide; ADX47273, S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl-1-yl}-methanone; 5MPEP, 5-methyl-2-(phenylethynyl)pyridine; M-5MPEP, 2-(2-(3-methoxyphenyl)ethynyl)-5-methylpyridine; CHO, Chinese hamster ovary; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; EBSS, Earle’s balanced salt solution; DIV, day(s) in vitro; ANOVA, analysis of variance; FLIPR, fluorometric imaging plate reader; NR, nonresponder; SP, single peak; OS, oscillatory; PP, peak-and-plateau. 0026-895X/09/7606-1302–1313$20.00 MOLECULAR PHARMACOLOGY Vol. 76, No. 6 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 59170/3533028 Mol Pharmacol 76:1302–1313, 2009 Printed in U.S.A. 1302 at A PE T Jornals on A ril 9, 2016 m oharm .aspeurnals.org D ow nladed from hara et al., 1997; Nash et al., 2001, 2002; Atkinson et al., 2006). The robust oscillatory pattern of Ca signaling initiated by the mGlu5 receptor has been proposed to be a result of a “dynamic uncoupling” mechanism involving rapid cycles of receptor phosphorylation and dephosphorylation (Kawabata et al., 1996), Ser-839 being implicated most recently as the site of reversible covalent modification (Kim et al., 2005). After agonist stimulation, the mGlu5 receptor is rapidly phosphorylated by protein kinase C, disabling productive receptor-G protein coupling (Kawabata et al., 1996; Uchino et al., 2004; Kim et al., 2005). A protein phosphatase, perhaps tightly associated with the receptor, efficiently dephosphorylates Ser-839, allowing reactivation of the receptor, and through the rapid enabling and disabling of receptor activity, a Ca oscillation is generated (Nash et al., 2001, 2002; Atkinson et al., 2006). This mechanism is probably similar to that reported for Ca oscillations initiated by Ca -sensing receptor activation (Young et al., 2002), but it is clearly different from the Ca -induced Ca release mechanism proposed to explain the majority of Ca oscillatory behaviors elicited by (submaximal) agonist stimulation of a variety of G protein-coupled receptors (Berridge et al., 2000). An intriguing property of mGlu5 receptor-stimulated Ca oscillations is that once a concentration of agonist [e.g., glutamate, quisqualate, (S)-3,5-dihydroxyphenylglycine] has been reached to initiate a response, both the frequency and amplitude of the Ca oscillation is essentially insensitive to further increases in agonist concentration (i.e., an “all-ornothing” response) (Nash et al., 2002). In contrast, altering the expression level of the mGlu5 receptor has marked effects on the frequency of the Ca oscillation stimulated by agonist, and Ca oscillation frequency can be reduced by addition of submaximally effective concentrations of the negative allosteric modulator (NAM) MPEP (1–100 nM) (Nash et al., 2002). Positive allosteric modulators (PAMs) of the mGlu5 receptor are thought to be of potential clinical use in a variety of neurological and psychiatric disorders, including schizophrenia (Gasparini et al., 2002; Kew, 2004). The mGlu5 receptor is known to potentiate the function of N-methyl-D-aspartate receptors in various brain regions, and so PAMs, which bind to an allosteric site on the mGlu5 receptor and increase the response of the receptor to glutamate, may be able to counteract the N-methyl-D-aspartate receptor hypofunction proposed to be associated with this condition (Lindsley et al., 2006). Several PAMs acting at the mGlu5 receptor have been identified, including DFB (O’Brien et al., 2003), CPPHA (O’Brien et al., 2004), CDPPB (Kinney et al., 2005), and ADX47273 (Liu et al., 2008). Like MPEP, these PAMs bind to seven-transmembrane domain of the mGlu5 receptor to modulate the effects of orthosteric agonists (Pagano et al., 2000; Malherbe et al., 2003; Mühlemann et al., 2006). In addition, an MPEP analog, 5MPEP, antagonizes the actions of both NAMs and PAMs at the mGlu5 receptor and thus acts as a neutral allosteric site ligand (Rodriguez et al., 2005). Considering the mechanism used by the mGlu5 receptor to generate Ca oscillations and the unusual emergent pharmacology, we have systematically investigated the effects of PAMs on orthosteric mGlu5 receptor agonist-stimulated responses at a single cell level. These new data add an important new dimension to previous investigations of the pharmacological properties of PAMs at the mGlu5 receptor, which to date have relied on signaling readouts that quantify cell population responses. Materials and Methods Compounds. L-Quisqualic acid, L-glutamic acid, 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and 3,3 -difluorobenzaldazine (DFB) were obtained from Tocris Cookson Ltd. (Bristol, UK). N-{4-Chloro2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2hydroxybenzamide (CPPHA), 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB), S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl-1-yl}-methanone (ADX47273), and 5-methyl2-(phenylethynyl)pyridine (5MPEP) were synthesized in-house by GlaxoSmithKline (Harlow, UK). 2-(2-(3-methoxyphenyl)ethynyl)-5methylpyridine (M-5MPEP) was a kind gift from Dr. P. J. Conn (Vanderbilt Program in Drug Discovery, Nashville, TN). Cell Culture. Chinese hamster ovary (CHO) cells expressing the human mGlu5a receptor under the control of a lac-repressor system (Hermans et al., 1998; Nash et al., 2002) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing GlutaMAX-1 with sodium pyruvate, 4.5 g/l glucose, 10% fetal bovine serum (FBS), 44 g/ml proline, 2.5 g/ml amphotericin B, 10 units/ml penicillin, 100 g/ml streptomycin, and 300 g/ml G418. Once confluent, flasks of CHO-lac-mGlu5a cells were washed twice with phosphate-buffered saline (without Ca /Mg ) and harvested with 0.25% (w/v) trypsin and 0.02% (w/v) EDTA. Cells were maintained at 37°C in a humidified 5% CO2:air atmosphere. For experiments, cells were seeded on to multiwell plates in medium with dialyzed FBS (substituted for FBS) and devoid of G418. mGlu5 receptor expression was induced by incubating CHO-lac-mGlu5a cells with 100 M isopropyl-D-thiogalactoside/10 mM sodium butyrate for 24 h before experimentation. Rat Cerebrocortical Astrocyte Preparation. Wistar rats (1–2 days of age) were decapitated, and the cortices were removed. During the dissection, cortices were placed into ice-cold Earle’s balanced salt solution (EBSS; Invitrogen, Carlsbad, CA), supplemented with 3.2 mM MgSO4, 0.3% (w/v) BSA (fraction V), and 16.7 mM glucose. Tissue was cut up into small pieces and incubated at 37°C for 15 min in 10 ml of EBSS solution containing 0.025% (w/v) (bovine pancreatic) trypsin with gentle agitation. After 15 min, 10-ml modified EBSS solution [containing 50 M MgSO4, DNase I (type IV, 150 Kunitz units), and 0.02% (w/v) trypsin inhibitor] was added, and the suspension left to settle for 5 min. The supernatant was subsequently decanted, and 2.5 ml EBSS solution containing 320 M MgSO4, DNase I (800 Kunitz units), and 0.12% (w/v) trypsin inhibitor was added. Tissue was slowly triturated using a glass firepolished Pasteur pipette and 2.5 ml of EBSS solution [supplemented with 0.4% (w/v) BSA and 250 M MgSO4] added. The cell suspension was centrifuged (1000 rpm; 8 min), and pellet resuspended in DMEM containing GlutaMAX-1 with sodium pyruvate, 4.5 mg/l glucose, 15% heat-inactivated FBS, 2.5 g/ml Fungizone, and 0.1 g/ml gentamicin. Cells were plated into poly-D-lysine-coated cell culture flasks and incubated at 37°C in a 5% CO2, humidified air atmosphere for 7 days, with medium being replaced after 4 days. At 7 DIV, medium was replaced again and flasks were transferred to a shaking incubator overnight (37°C; 320 rpm). On the following day (8 DIV), cells were washed twice with phosphate-buffered saline (without Ca /Mg ) and harvested with 0.25% (w/v) trypsin and 0.02% (w/v) EDTA. Cells were subsequently seeded onto precoated poly-D-lysine tissue-culture plates for experiments. After 24 h (DIV 9), medium was replaced with DMEM containing GlutaMAX-1 with sodium pyruvate, 4.5 mg/l glucose, 2.5 g/ml Fungizone, and 0.1 g/ml gentamicin and G-5 supplement. Cells were used for experiments at DIV 11 to 13. Single-Cell Intracellular Ca Concentration Assay. CHOlac-mGlu5a cells were seeded onto 22-mm borosilicate coverslips and grown to approximately 80% confluence. Cells were loaded with Fura-2 acetoxymethyl ester (2 M) in Krebs-Henseleit buffer (composition: 118 mM NaCl, 4.7 mM KCl, 4 mM NaHCO3, 1.3 mM CaCl2, Allosteric Modulation of mGlu5 Receptor Signaling 1303 at A PE T Jornals on A ril 9, 2016 m oharm .aspeurnals.org D ow nladed from 1.2 mM MgSO4, 1.2 mM KH2PO4, 11.7 mM glucose, and 8.5 mM HEPES, pH 7.4) containing 1 mg/ml bovine serum albumin for 60–90 min at room temperature. Coverslips of astrocyte (DIV 11–13) were incubated with Fura-2 acetoxymethyl ester (2 M) similarly to the CHO-lac cells, except that the loading period was 40 min at room temperature. Coverslips were then transferred to the stage of an inverted epifluorescence microscope (Diaphot; Nikon, Tokyo, Japan) with an oil immersion objective (40 ) and a SpectraMASTER II module (PerkinElmer Life and Analytical Sciences, Waltham, MA). Cells were excited at wavelengths of 340 and 380 nm using a SpectraMASTER II monochromator, and emission was recorded at wavelengths above 520 nm. The ratio of fluorescence intensities at these wavelengths is given as an index of [Ca ]i. All experiments were performed at 37°C; drug additions were made via a perfusion line. Cell Population [Ca ]i Assay. CHO-lac-mGlu5a cells were seeded onto 96-well black-walled cell culture plates (Costar; Corning Life Sciences, Lowell, MA) and induced on the following day with isopropyl -D-thiogalactoside (100 M) and sodium butyrate (10 mM) for 24 h before experimentation. Cells were loaded in Tyrode’s solution containing the Ca sensitive fluorescent dye, calcium-3 (Calcium 3 assay kit; Molecular Devices, Sunnyvale, CA), 1.5 mM CaCl2, and 2.5 mM probenecid for 1 h. Allosteric modulators were preincubated for 30 min before the addition of the agonist on the FLIPR. Changes in fluorescence intensity are recorded as an index of [Ca ]i. Data Analysis. Concentration-response relationships were analyzed by nonlinear regression using Prism 5.0 software (GraphPad Software, San Diego, CA). For statistical tests, where only two datasets were being compared an unpaired Student’s t test (two-tailed) was used, where P 0.05 was deemed statistically significant. Where more than two datasets were compared, oneor two-way analysis of variance (ANOVA) tests were used with P 0.05 being accepted as significantly different. ANOVA tests were followed by the Bonferroni’s post hoc test. All statistical analyses were performed using Prism 5.0 software.