![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
NEUROPHARMACOLOGY
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba, Japan
Received April 20, 2007; accepted July 2, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
15 (IC50 = 20 and 26 nM). The maximal response in agonist concentration-response curves was reduced in the presence of MMPIP, and its antagonism is reversible. MMPIP did not displace [3H](2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495) bound to mGluR7. These results suggested that these isoxazolopyridone derivatives are allosteric antagonists. In CHO cells expressing rat mGluR7, MDIP and MMPIP inhibited L-AP4-induced inhibition of forskolin-stimulated cAMP accumulation (IC50 = 99 and 220 nM). In CHO cells coexpressing human mGluR7 with G15, MDIP and MMPIP also inhibited the L-AP4-induced cAMP response. The maximal degree of inhibition by MMPIP was higher than that by MDIP in a cAMP assay. MMPIP was able to antagonize an allosteric agonist, the N,N'-dibenzhydryl-ethane-1,2-diamine dihydrochloride (AMN082)-induced inhibition of cAMP accumulation. In the absence of these agonists, MMPIP caused a further increase in forskolin-stimulated cAMP levels in CHO cells expressing mGluR7, whereas a competitive antagonist, LY341495, did not. This result indicates that MMPIP has an inverse agonistic activity. The intrinsic activity of MMPIP was pertussis toxin-sensitive and mGluR7-dependent. MMPIP at concentrations of at least 1 µM had no significant effect on mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, and mGluR8. MMPIP is the first allosteric mGluR7-selective antagonist that could potentially be useful as a pharmacological tool for elucidating the roles of mGluR7 on central nervous system functions.
).
mGluR7 is widely expressed in the central nervous system (CNS) (Okamoto et al., 1994; Saugstad et al., 1994; Kinoshita et al., 1998; Kosinski et al., 1999). Interestingly, mGluR7 is primary localized on presynaptic terminals where it is thought to regulate neurotransmitter release, and it is highly concentrated at specific neuronal terminals (Shigemoto et al., 1996). Based on its specific presynaptic localization and much lower L-glutamate affinity compared with other mGluR subtypes, mGluR7 seems to function as a low-pass filter, inhibiting synapses from firing above a certain frequency (Shigemoto et al., 1996). mGluR7 knockout mice showed reduced levels of anxiety, increased susceptibility to convulsants, and impaired working memory (Masugi et al., 1999; Sansig et al., 2001, Bough et al., 2004; Hölscher et al., 2004, 2005; Callaerts-Vegh et al., 2006; Mitsukawa et al., 2006). However, the interpretation of phenotype analyses using genetically manipulated mGluR7 knockout mice might be limited by gene compensation, developmental effects, and variance among strains. Therefore, the pharmacological manipulation of mGluR7 by agonists and antagonists is useful to explore the physiological and pathophysiological roles of mGluR7. Recently, AMN082 was identified as the first mGluR7-selective allosteric agonist, and activation of mGluR7 with AMN082 was shown to modulate plasma stress hormone concentrations (Mitsukawa et al., 2005). Therefore, mGluR7 antagonists may be useful for treating conditions involving chronic stress, such as depression and anxiety disorders (Conn and Niswender, 2006). However, no mGluR7-selective antagonist has been discovered to date.
Extensive efforts to identify subtype-selective mGluR ligands by competitive binding assays have been unsuccessful, probably due to the fact that the amino acid sequences of L-glutamate binding sites are highly conserved among mGluR subtypes (Kunishima et al., 2000). In contrast, high-throughput functional assays for detecting Ca2+ mobilization have led to the identification of subtype-selective ligands in Gq-coupled mGluR1 and mGluR5 (Varney et al., 1999; Suzuki et al., 2007). However, it is difficult to directly apply this type of high-throughput functional assay to the identification of subtype-selective ligands for Gi-coupled group II or group III mGluR subtypes. Promiscuous G proteins such as G15 and G16 are known to allow Gi-coupled receptors to couple to phospholipase C, resulting in Ca2+ mobilization in response to agonist stimulation (Offermanns and Simon, 1995). In addition, mGluR7 is known to efficiently couple with G15, but not with G16 (Parmentier et al., 1998). To identify mGluR7-selective ligands, we generated CHO cells stably coexpressing mGluR7 with G15, and we screened chemical libraries using the cells and fluorometric imaging plate reader (FLIPR). Because mGluR7 seems to be coupled to Gi rather than to G15 in neurons (Wright and Schoepp, 1996), their activities were further confirmed by Gi-coupled cAMP response. Evaluation of mGluR7 ligands on mGluR7-mediated cAMP response could be a physiologically relevant assay, because mGluR7 is known to regulate L-glutamate release through modulation of cAMP levels in cerebrocortical nerve terminals (Millán et al., 2002).
Using the screening strategy mentioned above, we have identified isozazolopyridone derivatives as mGluR7 antagonists. Based on descriptions in the patent application (Nakamura et al., 2002), several isozazolopyridone derivatives have been synthesized, and preliminary pharmacological characterization of the compounds has been reported (Niswender et al., 2006). In the present study, we describe the comprehensive in vitro pharmacological characterization of two isozazolopyridone derivatives. 5-Methyl-3,6-diphenylisoxazolo[4,5-c]pyridin-4(5H)-one (MDIP) was identified as a novel mGluR7 antagonist by random high-throughput functional screening, whereas 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP) was obtained by subsequent chemical modification of MDIP. These isoxazolopyridone derivatives were pharmacologically characterized using recombinant rat and human mGluR7-expressing cells.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-cyclopropyl-4-phosphonophenylglycine (CPPG), LY341495, and [3H]LY341495 (36.5 Ci/mmol) were purchased from Tocris Cookson Inc. (Bristol, UK). L-Glutamate, 5-hydroxytryptamine (5-HT), 3-isobutyl-1-methylxanthine (IBMX), pertussis toxin (PTX), and human calcitonin (hCT; thyrocalcitonin) were purchased from Sigma-Aldrich (St. Louis, MO). AMN082 (Fig. 1C) was purchased from Ascent Scientific (North Somerset, UK). FTIDC was synthesized in-house (Suzuki et al., 2007). L-Proline and forskolin were purchased from Wako Pure Chemicals (Osaka, Japan). Dialyzed fetal bovine serum, culture media, and other reagents used for cell culture were purchased from Invitrogen (Carlsbad, CA). All other reagents used were of molecular or analytical grade, where appropriate.
|
Methods
Stable Cell Lines. Rat mGluR7 cDNA was kindly donated by Dr. S. Nakanishi (Osaka Bioscience Institute, Osaka, Japan). CHO-NFAT-bla cells from Aurora Biosciences (San Diego, CA) were transfected with rat mGluR7 cDNA cloned into pIRESneo (Clontech, Palo Alto, CA), and they were selected in medium [Dulbecco's modified Eagle's medium with 10% dialyzed fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, and 1% proline supplemented with 500 µg/ml Geneticin (G-418; Invitrogen)]. The stable cell lines were isolated, and they were selected by their abilities to inhibit forskolin-stimulated cAMP accumulation following L-AP4 addition. CHO-NFAT-bla cells expressing rat mGluR7 (CHO-rat mGluR7) were transfected with G15 cDNA (Aurora Biosciences) cloned into pIREShyg (Clontech), and they were selected in medium supplemented with 500 µg/ml hygromycin B (Invitrogen) and 500 µg/ml G-418. The stable cell lines were isolated, and they were selected by their abilities to elicit Ca2+ mobilization following L-AP4 addition (CHO-rat mGluR7/G15). CHO cells stably coexpressing human mGluR7 with G15 (CHO-human mGluR7/G15) and expressing human mGluR5 were obtained as described previously by O'Brien et al. (2004). CHO-dhfr– cells stably expressing human mGluR1a were obtained as described previously by Ohashi et al. (2002). CHO-dhfr– cells stably expressing rat mGluR3 and rat mGluR4 were kindly donated by Dr. S. Nakanishi. CHO-dhfr– cells stably coexpressing human mGluR2 with G16 and CHO-K1 cells stably expressing human mGluR8 were obtained as described previously by Suzuki et al. (2007).
Intracellular Ca2+ Mobilization. Intracellular Ca2+ mobilization was measured according to the method described by Suzuki et al. (2007). In brief, CHO cells coexpressing rat mGluR7 with G15 were seeded at 5 x 104 cells/well in a 96-well black-well/clear-bottomed plate (PerkinElmer Life and Analytical Sciences, Boston, MA), and they were cultured overnight. The cells were then incubated with 4 µM Fluo-3 in assay buffer (Hanks' balanced salt solution containing 20 mM HEPES and 2.5 mM probenecid) containing 1% dialyzed fetal bovine serum for 1 h at 37°C with 5% CO2 in a humidified atmosphere. The extracellular dye was removed, and Ca2+ flux was measured using a FLIPR (Molecular Devices, Sunnyvale, CA). The cells were preincubated with a test compound for 5 min to evaluate its agonistic activity. After preincubation, antagonistic activity was evaluated for 3 min after addition of an agonist. The final concentration of L-AP4 was 500 µM in the antagonist assay for rat mGluR7. In the antagonist assay for human mGluR1a, human mGluR5, and human mGluR2, the final concentration of L-glutamate was 10 µM. CHO cells expressing human mGluR5 were seeded at 7.5 x 104 cells/well, and they were loaded with 4 µM Fluo-4. To access the reversibility of the antagonism by MMPIP, Fluo-3-loaded CHO cells coexpressing rat mGluR7 with G15 were preincubated for 5 min in the absence or presence of MMPIP, and then they were washed three times with 250 µl of assay buffer before agonist stimulation. An agonist (0.5 mM L-AP4) was added 5 min after washout in the FLIPR.
[3H]LY341495 Binding. CHO cells expressing rat mGluR7 were seeded at 3 x 105 cells/well in a 24-well plate, and they were cultured overnight. The culture medium was then removed, and the cells were incubated in 250 µl of culture medium containing 100 nM [3H]LY341495 in the presence or absence of test compounds at 37°C with 5% CO2 in a humidified atmosphere. After 1 h, the cells were washed three times with ice-cold phosphate-buffered saline and solubilized in 2 M NaOH. Radioactivity was measured using Tri-carb2500 (PerkinElmer Life and Analytical Sciences) after addition of Ultima Gold XR (PerkinElmer Life and Analytical Sciences). Nonspecific binding was defined as binding in the presence of 100 µM LY341495.
Intracellular cAMP Measurements. Intracellular cAMP was measured by a modification of the method of Tanabe et al. (1992). CHO cells expressing rat mGluR7, rat mGluR3, rat mGluR4, and human mGluR8 were seeded at 5 x 104 cells/well in a 96-well clear-bottomed plate, and they were cultured overnight. The culture medium was then replaced with Locke's buffer (154 mM NaCl, 5.6 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 3.6 mM NaHCO3, 5.6 mM glucose, and 10 mM HEPES, pH 7.4) containing 1 mM IBMX, and the cells were incubated for 20 min at 37°C with 5% CO2 in a humidified atmosphere. The cells were then incubated with Locke's buffer containing 1 mM IBMX and 10 µM forskolin in the presence or absence of test compounds for an additional 20 min. The amount of intracellular cAMP was determined using the cAMP enzyme immunoassay Biotrak (enzyme immunoassay) system (GE Healthcare, Piscataway, NJ). CHO cells stably coexpressing human mGluR7 with G15 were seeded at 4 x 104 cells/well in a 96-well clear-bottomed plate, and they were cultured overnight. The final concentrations of forskolin and IBMX were 3 µM and 0.5 mM, respectively. Intracellular cAMP levels were determined using an AlphaScreen cAMP Assay kit (PerkinElmer Life and Analytical Sciences) according to the manufacturer's instructions. In the antagonist assay for rat and human mGluR7, the final concentrations of L-AP4 were 0.5 and 1 mM, respectively, whereas in the antagonist assay for mGluR3, mGluR4, and mGluR8, the final concentrations of L-glutamate were 100, 70, and 10 µM, respectively. To evaluate the effects of compounds on cAMP response in PTX-treated CHO cells expressing rat mGluR7, the cells were incubated with 100 ng/ml PTX for 24 h before the cAMP assay.
Knockdown of mGluR7 Using siRNA. Four siRNA 21-mers matching the human mGluR7 sequence (referred to as mGluR7 siRNA) were purchased in a pooled form (siGENOME SMARTpool, M-005622-00) from Dharmacon RNA Technologies (Lafayette, CO). An siRNA 21-mer matching the luciferase GL2 sequence was used as a negative control siRNA (referred to as control siRNA) (Elbashir et al., 2002). CHO cells coexpressing human mGluR7 with G15 were seeded at 2.5 x 105 cells in a 25-cm2 flask, and they were cultured overnight in the culture medium described above, but without antibiotics. siRNA was transfected into cells at a final concentration of 25 nM using DharmaFECT 4 (Dharmacon RNA Technologies) according to the manufacturer's instructions. After 2 days, the cells were seeded at 4 x 104 cells in a 96-well white-well/clear-bottomed plate (PerkinElmer Life and Analytical Sciences), and they were cultured overnight. Intracellular cAMP levels were measured as described above.
Total RNA was extracted from the cells 3 days after transfection using an RNeasy Mini kit (QIAGEN GmbH, Hilden, Germany). TaqMan Gene Expression Assays (Assay ID, Hs00356067_m1) and TaqMan Rodent GAPDH Control Reagents (Applied Biosystems, Foster City, CA) were used to quantify mRNA expression of mGluR7 and GAPDH, respectively. Reverse transcription of 0.5 µg of total RNA was performed in a total volume of 25 µl using random hexamers and a TaqMan Reverse Transcription Reagent kit (Applied Biosystems) according to the manufacturer's protocol. The resultant cDNA sample (25 µl) was diluted with 75 µl of nuclease-free water. Quantitative real-time PCR was performed in a total volume of 25 µl containing 12.5 µl of TaqMan Universal PCR Master Mix, 1.25 µl of TaqMan probe/primers mixture, and 3 µl of the diluted cDNA templates using the ABI Prism 7700 cycler (Applied Biosystems). The cycling profile was 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C, as recommended by the manufacturer. Relative quantification of the real-time PCR results was performed using the comparative Ct method with GAPDH as the internal control, according to the manufacturer's protocol (User Bulletin 2; ABI Prism 7700 Sequence Detection system; Applied Biosystems).
Data Analyses and Statistics
Data analyses were performed using Prism, version 4.03, from GraphPad Software Inc. (San Diego, CA). Concentration-response curves for Ca2+ mobilization and cAMP accumulation were fitted using nonlinear regression analysis. To determine the potency of a noncompetitive antagonist, the KB value was calculated from the equation KB = [B]/(slope – 1), where [B] is the concentration of antagonist (B), and slope is that of a double-reciprocal plot of equieffective concentrations of agonist (A) in the absence (1/[A]) and presence (1/[A']) of antagonist (Kenakin, 1997). Competition binding experiments were analyzed using nonlinear regression analysis. Student's t test was used to analyze data obtained from studies with PTX and siRNA. A probability level of <0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
15 (CHO-rat mGluR7/G15), the mGluR7 agonists L-AP4 and L-CCG-I were able to increase intracellular Ca2+ concentrations, with EC50 values of 140 ± 18 µM (n = 5) and 70 ± 6.4 µM(n = 4), respectively. The maximum response of L-CCG-I was 53 ± 8.7% (n = 4, a percentage of the response to 3 mM L-AP4) (Fig. 2A). Relative fluorescence units in CHO-rat mGluR7/G15 were 16,000 ± 1200 (n = 5) and 8500 ± 1400 (n = 4) in the presence of 3 mM L-AP4 and 3mM L-CCG-I, respectively. MDIP was identified from an in-house chemical library by random screening with CHO-rat mGluR7/G15, and MMPIP was obtained by successive chemical modification of MDIP. In CHO-rat mGluR7/G15, MDIP and MMPIP inhibited 0.5 mM L-AP4-induced Ca2+ mobilization, with IC50 values of 20 ± 2.4 and 26 ± 3.4 nM (n = 8), respectively (Fig. 2B). These compounds did not show any agonist activity in CHO-rat mGluR7/G15. To analyze the mode of action of these isoxazolopyridone derivatives, the effect of MMPIP on agonist concentration-response curves of intracellular Ca2+ mobilization was evaluated in CHO-rat mGluR7/G15. Agonist concentration-response curves for L-AP4 and L-CCG-I-induced increases in intracellular Ca2+ concentrations were obtained in the presence or absence of MMPIP. The maximal responses of L-AP4 and L-CCG-I were reduced in the presence of MMPIP (Fig. 3, A and B), whereas an orthosteric antagonist, CPPG, caused a parallel rightward shift in the L-AP4 concentration-response curves with no effect on maximum response (Fig. 3C). The antagonism of MMPIP on L-AP4- and L-CCG-I-induced Ca2+ responses was analyzed by a model of noncompetitive antagonism (Kenakin, 1997). To estimate the KB values of MMPIP, we used a double-reciprocal plot of equally effective concentrations of L-AP4 or L-CCG-I (A) in the absence (1/[A]) and presence (1/[A']) of MMPIP (Supplemental Fig. 1, A and B). Equally effective concentrations were calculated from curves in the absence or presence of 100 nM MMPIP shown in Fig. 3A and in the absence or presence of 30 nM MMPIP shown in Fig. 3B, respectively. The KB values of MMPIP calculated from the double-reciprocal plots of L-AP4 and L-CCG-I were 24 ± 3.4 and 30 ± 2.7 nM, respectively. Schild plot analysis of the antagonism produced by CPPG yielded a pA2 of 4.7 ± 0.053 and a slope factor of 0.91 ± 0.055 (Supplemental Fig. 1C). The reversibility of the antagonism by MMPIP was determined by comparing Ca2+ responses to 0.5 mM L-AP4 in CHO-rat mGluR7/G15, with and without washout procedure. MMPIP (0.01–1 µM) dose-dependently inhibited L-AP4-induced Ca2+ response. The agonist-induced responses were recovered within 5 min after washout of MMPIP (Fig. 3D). [3H]LY341495 binding assays, carried out to evaluate whether MMPIP binds to the L-glutamate binding site of mGluR7, showed that it did not displace [3H]LY341495 bound to CHO-rat mGluR7 (Fig. 3E). In contrast to MMPIP, both LY341495 and CPPG displaced [3H]LY341495 bound to CHO-rat mGluR7.
|
|
Effects of Isoxazolopyridone Derivatives on Agonist-Induced Inhibition of Forskolin-Stimulated cAMP Accumulation in CHO Cells Expressing mGluR7. The effects of isoxazolopyridone derivatives on cAMP accumulation were evaluated using CHO-rat mGluR7 and CHO-human mGluR7/G15. L-AP4 inhibited forskolin-stimulated cAMP accumulation with EC50 values of 86 ± 12 µM(n = 12) and 170 ± 76 µM(n = 4) in CHO-rat mGluR7 and CHO-human mGluR7/G15, respectively (Fig. 4, A and B). In CHO-rat mGluR7, MDIP and MMPIP dose-dependently antagonized L-AP4-induced inhibition of cAMP accumulation with IC50 values of 99 ± 25 nM (n = 6) and 220 ± 23 nM (n = 5), respectively (Fig. 4C). MDIP and MMPIP also antagonized L-AP4-induced inhibition of cAMP accumulation with IC50 values of 140 ± 18 nM (n = 6) and 610 ± 130 nM (n = 5), respectively, in CHO-human mGluR7/G15 (Fig. 4D). The orthosteric mGluR antagonist LY341495 antagonized L-AP4-induced inhibition of cAMP accumulation in CHO-rat mGluR7 and CHO-human mGluR7/G15, with IC50 values of 1600 ± 360 nM (n = 3) and 2300 ± 1300 nM (n = 3), respectively. Because the maximal degree of inhibition by MMPIP was higher than that by MDIP, as shown in Fig. 4, C and D, MMPIP was selected for further pharmacological characterization.
|
An allosteric mGluR7 agonist, AMN082 (Fig. 1C), inhibited forskolin-stimulated cAMP accumulation, with an EC50 value of 95 ± 36 nM (n = 4) in CHO-human mGluR7/G15 (Fig. 5A), whereas AMN082 up to 10 µM did not induce intracellular Ca2+ mobilization (Supplemental Fig. 2). MMPIP dose-dependently antagonized AMN082-induced inhibition of cAMP accumulation in CHO-human mGluR7/G15 (Fig. 5B). In contrast, even at 1 mM, an orthosteric mGluR antagonist, CPPG, did not antagonize AMN082-induced inhibition of cAMP accumulation in CHO-human mGluR7/G15, whereas L-AP4-induced inhibition of cAMP accumulation was antagonized by CPPG.
|
15 (Fig. 6B), respectively. In contrast, LY341495 did not increase forskolin-induced cAMP accumulation in either cell type (Fig. 6, A and B). In CHO-rat mGluR7 treated with 100 ng/ml PTX, L-AP4 did not inhibit forskolin-stimulated cAMP accumulation, and MMPIP did not increase forskolin-stimulated cAMP accumulation in the absence of agonist. Differences in the activities of these compounds acting on control- and PTX-treated cells were statistically significant (Fig. 6C).
|
Effect of mGluR7 Knockdown on Intrinsic Activity of MMPIP. To confirm the intrinsic activity of MMPIP on mGluR7, its activity was evaluated in CHO-human mGluR7/G15 transfected with mGluR7 siRNA. mGluR7 siRNA and negative control siRNA were transfected into CHO-human mGluR7/G15. Three days after transfection, mGluR7 mRNA levels were measured using quantitative real-time reverse transcription-PCR (Fig. 7A). In CHO-human mGluR7/G15 transfected with mGluR7 siRNA (mGluR7 knockdown cells), mGluR7 mRNA levels were decreased to 34 ± 6.7% (n = 4; percentage of nontransfected cells), whereas mGluR7 mRNA levels in CHO-human mGluR7/G15 transfected with negative control siRNA (control mGluR7 cells) were unaffected (102 ± 21%; n = 4; percentage of nontransfected cells). The difference in mGluR7 mRNA levels between mGluR7 knockdown cells and the control cells was statistically significant (P < 0.05). In mGluR7 control cells, 1 µM AMN082 inhibited forskolin-stimulated cAMP accumulation to 37 ± 9.2% (n = 4) (percentage of forskolin-stimulated cAMP accumulation). In contrast, cAMP levels in the presence of 1 µM AMN083 were 78 ± 12% (n = 4) in mGluR7 knockdown cells (Fig. 7B). MMPIP increased forskolin-stimulated cAMP accumulation in the mGluR7 control cells, whereas the compound had little effect on mGluR7 knockdown cells (Fig. 7C). The differences in the activities of AMN082 and MMPIP between mGluR7 control cells and knockdown cells were statistically significant (P < 0.05). To confirm the specificity of knockdown by mGluR7 siRNA, the effects of 5-HT and hCT on cAMP accumulation were evaluated in mGluR7 control cells and knockdown cells endogenously expressing Gi-coupled 5-HT receptors and Gs-coupled calcitonin receptors (Fig. 7B). 5-HT comparably inhibited forskolin-stimulated cAMP accumulation in both mGluR7 knockdown cells and mGluR7 control cells, whereas hCT increased cAMP accumulation with comparable efficacy in mGluR7 knockdown cells as well as the mGluR7 control cells.
|
Selectivity of MMPIP toward Other mGluR Subtypes. L-Glutamate induced intracellular Ca2+ mobilization in CHO cells expressing mGluR1 or mGluR5, and in CHO cells coexpressing mGluR2 with G16 (mGluR2/G16). FTIDC (an mGluR1 antagonist; 0.1 µM), 0.1 µM MPEP (an mGluR5 antagonist), and 1 µM LY341495 (a group II/III mGluR antagonist) inhibited L-glutamate-induced increases in Ca2+ concentrations in CHO cells expressing mGluR1, mGluR5, and mGluR2/G16, respectively. In contrast, MMPIP did not show either antagonistic or agonistic activity toward these mGluR subtypes (Fig. 8). In CHO cells expressing mGluR3, mGluR4, or mGluR8, L-glutamate inhibited forskolin-stimulated cAMP accumulation, whereas MMPIP did not. MMPIP also did not antagonize L-glutamate-induced inhibition of cAMP accumulation in CHO cells expressing mGluR3, mGluR4, or mGluR8, whereas LY341495 acted as an antagonist (Fig. 9). The selectivity of MMPIP was also tested against 168 target molecules, including enzymes, neurotransmitter receptors, transporters, and ion channels; these included ionotropic glutamate receptors (N-methyl-D-aspartate, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate) and other neurotransmitter receptors (dopamine, serotonin, acetylcholine, GABA, adrenalin, and histamine) (MDS Pharma, Bothell, WA). The IC50 values of MMPIP were higher than 10 µM against all these targets, except for 51% inhibition at 10 µM toward monoamine oxidase-A (data not shown).
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
15), and coexpression was shown to be coupled to intracellular Ca2+ mobilization. Using CHO-rat mGluR7/G15, the isoxazolopyridone derivatives MDIP and MMPIP were identified as mGluR7 antagonists. These isoxazolopyridone derivatives fully inhibited L-AP4-induced Ca2+ mobilization in CHO-rat mGluR7/G15. MMPIP caused the reduced maximal Ca2+ response to agonist in CHO-rat mGluR7/G15, whereas an orthosteric antagonist, CPPG, did not reduce the maximum response. Antagonism of MMPIP was analyzed using a model of noncompetitive antagonism, indicating that MMPIP has a potent mGluR7 antagonist (KB values = 24–30 nM). An insurmountable antagonism in the measurement of transient Ca2+ responses could be caused not only by an allosteric antagonist but also by a slowly dissociating or irreversible orthosteric antagonist (Kenakin, 1997; Christopoulos et al., 1999). The agonist-induced Ca2+ mobilization in CHO-rat mGluR7/G15 was fully recovered within 5 min after washout of MMPIP, suggesting that MMPIP is a reversible antagonist with fast dissociation kinetics. [3H]LY341495 is an orthosteric radioligand that binds to the L-glutamate binding site of mGluR7 (Wright et al., 2000); CPPG displaced [3H]LY341495 bound to rat mGluR7 expressed in CHO, whereas MMPIP did not. These results suggested that the antagonism by MMPIP could be exerted via an allosteric mechanism. Chimeric and point-mutated receptors of mGluR7 will be useful for further characterization of an allosteric inhibitory mechanism by MMPIP.
AMN082 was recently identified as an mGluR7-selective agonist that activates mGluR7 via an allosteric site in the transmembrane domain (Mitsukawa et al., 2005). In the present study, MMPIP antagonized AMN082-induced inhibition of cAMP accumulation. In contrast, an orthosteric mGluR antagonist, CPPG, did not inhibit the effect of AMN082, consistent with previous observations (Mitsukawa et al., 2005). These results support that MMPIP is an allosteric mGluR7 antagonist, and they suggest that the binding regions of MMPIP might be shared with those of AMN082. However, further studies, such as a binding assay with radiolabeled MMPIP or AMN082, are necessary before this interpretation can be validated.
mGluR7 is expressed in many regions of the CNS, whereas G15 expression is normally limited to certain cells derived from the hematopoietic lineage (Offermanns and Simon, 1995). Thus, mGluR7 is not likely to couple with G15 in the CNS under physiological conditions. In heterologous expression systems, mGluR7 is negatively coupled with adenylate cyclase via Gi protein, resulting in inhibition of forskolin-stimulated cAMP accumulation (Okamoto et al., 1994; Saugstad et al., 1994; Wu et al., 1998). In addition, a group III mGluR agonist, L-AP4, inhibits forskolin-stimulated cAMP accumulation in neuronal cells. The inhibitory effect is biphasic, with the low-affinity component probably mediated by mGluR7 and the high-affinity component possibly mediated by mGluR4 (Wright and Schoepp, 1996). In the present study, MMPIP antagonized L-AP4-induced inhibition of forskolin-stimulated cAMP accumulation in CHO cells expressing mGluR7. L-AP4 is known to inhibit forskolin-stimulated cAMP levels and resultant L-glutamate release in cerebrocortical nerve terminals, presumably via mGluR7 (Millán et al., 2002). Therefore, the inhibitory activity of MMPIP shown in CHO cells expressing mGluR7 could be biologically relevant, because the compound exhibits inhibitory activities not only in G15-coupled Ca2+ mobilization but also in Gi-coupled cAMP response.
In the absence of an agonist, MMPIP caused a further increase in forskolin-stimulated cAMP levels in CHO cells expressing mGluR7. This is in contrast to the effect of an agonist such as L-AP4, suggesting that MMPIP exhibits inverse agonist activity and thus it might inhibit the agonist-independent constitutive activity of mGluR7. An alternative interpretation could be that MMPIP inhibits activation of mGluR7 caused by either residual L-glutamate in the assay medium or endogenous L-glutamate released from cells. However, an orthosteric mGluR antagonist, LY341495, did not increase forskolin-stimulated cAMP in CHO cells expressing mGluR7 in the absence of agonist. These results support the conclusion that the activity of MMPIP in the absence of agonist comes from inhibition of the constitutive activity of mGluR7, and not from inhibition of mGluR7 activation due to L-glutamate contamination from the medium or cells. Treatment of CHO cells expressing mGluR7 with PTX diminished the intrinsic activity of MMPIP in the absence of an agonist. This result suggests that the intrinsic activity of MMPIP is mediated via PTX-sensitive Gi/o proteins and that the compound does not directly activate endogenous adenylate cyclase in the cells. To date, the constitutive activity of mGluR7 has not been directly demonstrated under physiological conditions in the CNS. However, in the absence of an agonist, mGluR7 constitutively inhibited voltage-sensitive Ca2+ channels via G
in cerebellar granule neurons transfected with mGluR7 cDNA (Bertaso et al., 2006). This mGluR7-dependent constitutive inhibition of voltage-sensitive Ca2+ channels might be mediated via constitutive activity of mGluR7, and it suggests that a constitutively active mGluR7 might have physiological roles in the cerebellum. Therefore, MMPIP could be useful for revealing the functions of the agonist-independent activity of mGluR7.
RNA interference using siRNA was used to confirm whether the intrinsic activity of MMPIP was dependent on mGluR7 expression. mGluR7 knockdown cells exhibited decreased agonistic activity of AMN082, suggesting that mGluR7 knockdown cells have lost mGluR7 receptor function. In addition, the intrinsic activity of MMPIP was significantly diminished in the same mGluR7 knockdown cells. These results indicate that the intrinsic activity of MMPIP is mediated via mGluR7. To confirm the specificity of mGluR7 knockdown, the effects of 5-HT and hCT on cAMP levels were compared between mGluR7 knockdown cells and mGluR7 control cells. CHO cells endogenously express the 5-HT1B receptor, and 5-HT inhibits forskolin-stimulated cAMP accumulation via Gi protein in CHO cells (Giles et al., 1996). In the present study, forskolin-stimulated cAMP accumulation was comparably inhibited by 5-HT in both mGluR7 knockdown cells and in mGluR7 control cells. hCT increased cAMP accumulation via activation of endogenous calcitonin receptors in CHO cells (George et al., 1997). In the present study, hCT increased cAMP accumulation in mGluR7 knockdown cells as well as in mGluR7 control cells. These results indicate that the diminished intrinsic activity of MMPIP toward mGluR7 knockdown cells is due to specific mGluR7 knockdown and not to nonspecific effects of mGluR7 siRNA.
The results presented here show that both MDIP and MMPIP fully inhibit agonist-induced Ca2+ mobilization via the Gq pathway. However, the maximum degree of inhibition by MDIP was less than that of MMPIP in eliciting a cAMP response from Gi protein in both human and rat mGluR7. Although the exact reason for this difference is not presently clear, it might be related to the property of allosteric antagonists that allow orthosteric agonist binding to the receptor. Allosteric antagonists might block specific signaling pathways while permitting other intracellular signaling, an idea recently proposed as "permissive antagonism" (Kenakin, 2005). The difference in maximum degree of inhibition toward mGluR7-mediated Ca2+ mobilization and cAMP response might be explained by permissive antagonism, suggesting that isoxazolopyridone derivatives could exhibit signal-pathway-dependent antagonistic activity. In addition, the antagonist potencies (IC50 values) of MMPIP and MDIP on cAMP response were less potent than those on Ca2+ mobilization. The permissive antagonism could also explain the difference between the two readouts. Alternatively, the discrepancy could be interpreted by a three-state receptor model (Leff et al., 1997). In the model, the receptor may be inactive (R) or can adopt two active confirmations (R* and R**) that preferentially interact with different G proteins (G1 and G2, respectively). MMPIP and MDIP might have different affinities for two confirmations of mGluR7 coupling to G15 and Gi/o. It may therefore be worth evaluating the activities of isoxazolopyridone derivatives toward other functional responses (Saugstad et al., 1996, Perroy et al., 2000; Millán et al., 2002, 2003) to further understand their signal pathway-dependent activities.
Counter assays using CHO cells expressing other mGluR subtypes showed that MMPIP is selective for mGluR7. MMPIP did not exhibit agonistic or antagonistic activity toward Gq-coupled mGluR1 and mGluR5. Furthermore, MMPIP did not inhibit agonist-induced Ca2+ mobilization in CHO cells coexpressing mGluR2 with promiscuous G protein. These results further suggest that the inhibitory activity of MMPIP in CHO-rat mGluR7/G15 does not arise from nonselective inhibition of the signaling pathway via Gq proteins, including promiscuous G protein. Furthermore, MMPIP at concentrations of at least 1 µM exhibited no significant effect on cAMP response mediated via mGluR3, mGluR4, or mGluR8.
The limitation of the present study was that all the results were obtained using recombinant systems; thus, the mode of action of these compounds might be different in native tissues where expression levels of mGluR7 could be lower than those in the recombinant systems. It will be necessary to confirm their actions in native mGluR7 in future studies.
In conclusion, MMPIP is a potent mGluR7 antagonist against agonist-induced Gq-coupled Ca2+ mobilization and Gi-coupled cAMP pathway. The inhibitory mode is noncompetitive and allosteric. In the absence of agonist, MMPIP showed PTX-sensitive and mGluR7-dependent intrinsic activities, suggesting inverse agonist activity. MMPIP showed no significant effect on other mGluR subtypes or on other molecules tested. This is the first detailed description of allosteric mGluR7-selective antagonists. It is expected that MMPIP will be a useful pharmacological tool for elucidating the role of mGluR7 on CNS functions.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: mGluR, metabotropic glutamate receptor; CNS, central nervous system; AMN082, N,N'-dibenzhydryl-ethane-1,2-diamine dihydrochloride; hCT, human calcitonin; CHO, Chinese hamster ovary; CHO-rat mGluR7, Chinese hamster ovary cells expressing rat mGluR7; CHO-rat mGluR7/G15, Chinese hamster ovary cells coexpressing rat mGluR7 with G15; CHO-human mGluR7/G15, Chinese hamster ovary cells coexpressing human mGluR7 with G15; FLIPR, fluorometric imaging plate reader; MDIP, 5-methyl-3,6-diphenylisoxazolo[4,5-c]pyridin-4(5H)-one; MMPIP, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one; L-AP4, L-(+)-2-amino-4-phosphonobutyric acid; L-CCG-I, (2S, 1'S,2'S)-2-(carboxycyclopropyl)glycine; MPEP, 2-methyl-6-(phenylethynyl)pyridine; CPPG, (R,S)--cyclopropyl-4-phosphonophenylglycine; LY341495 (LY), (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; 5-HT, 5-hydroxytryptamine; IBMX, 3-isobutyl-1-methylxanthine; PTX, pertussis toxin; FTIDC, 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide; NFAT, nuclear factor of activated T cells; dhfr, dihydrofolate reductase; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Gentaroh Suzuki, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan. E-mail: gentaroh_suzuki{at}merck.com
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bertaso F, Lill Y, Airas JM, Espeut J, Blahos J, Bockaert J, Fagni L, Betz H, and El-Far O (2006) MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels. J Neurochem 99: 288–298.[CrossRef][Medline]
Bough KJ, Mott DD, and Dingledine RJ (2004) Medial perforant path inhibition mediated by mGluR7 is reduced after status epilepticus. J Neurophysiol 92: 1549–1557.
Callaerts-Vegh Z, Beckers T, Ball SM, Baeyens F, Callaerts PF, Cryan JF, Molnar E, and D'Hooge R (2006) Concomitant deficits in working memory and fear extinction are functionally dissociated from reduced anxiety in metabotropic glutamate receptor 7-deficient mice. J Neurosci 26: 6573–6582.
Conn PJ and Niswender CM (2006) mGluR7's lucky number. Proc Natl Acad Sci U S A 103: 251–252.
Conn PJ and Pin JP (1997) Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol 37: 205–237.[CrossRef][Medline]
Christopoulos A, Parsons AM, Lew MJ, and El-Fakahany EE (1999) The assessment of antagonist potency under conditions of transient response kinetics. Eur J Pharmacol 382: 217–227.[CrossRef][Medline]
Elbashir SM, Harborth J, Weber K, and Tuschl T (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26: 199–213.[CrossRef][Medline]
George SE, Bungay PJ, and Naylor LH (1997) Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene. J Neurochem 69: 1278–1285.[Medline]
Giles H, Lansdell SJ, Bolofo ML, Wilson HL, and Martin GR (1996) Characterization of a 5-HT1B receptor on CHO cells: functional responses in the absence of radioligand binding. Br J Pharmacol 117: 1119–1126.[Medline]
Hölscher C, Schmid S, Pilz PK, Sansig G, van der Putten H, and Plappert CF (2004) Lack of the metabotropic glutamate receptor subtype 7 selectively impairs short-term working memory but not long-term memory. Behav Brain Res 154: 473–481.[CrossRef][Medline]
Hölscher C, Schmid S, Pilz PK, Sansig G, van der Putten H, and Plappert CF (2005) Lack of the metabotropic glutamate receptor subtype 7 selectively modulates theta rhythm and working memory. Learn Mem 12: 450–455.
Kenakin T (1997) Allotopic, noncompetitive, and irreversible antagonism, in Pharmacological Analysis of Drug-Receptor Interaction, 3rd ed (Kenakin T ed) pp 374–395, Lippincott-Raven Publishers, Philadelphia, PA.
Kenakin T (2005) New concepts in drug discovery: collateral efficacy and permissive antagonism. Nat Rev Drug Discov 4: 919–927.[CrossRef][Medline]
Kinoshita A, Shigemoto R, Ohishi H, van der Putten H, and Mizuno N (1998) Immunohistochemical localization of metabotropic glutamate receptors, mGluR7a and mGluR7b, in the central nervous system of the adult rat and mouse: a light and electron microscopic study. J Comp Neurol 393: 332–352.[CrossRef][Medline]
Kosinski CM, Risso Bradley S, Conn PJ, Levey AI, Landwehrmeyer GB, Penney JB Jr, Young AB, and Standaert DG (1999) Localization of metabotropic glutamate receptor 7 mRNA and mGluR7a protein in the rat basal ganglia. J Comp Neurol 415: 266–284.[CrossRef][Medline]
Kunishima N, Shimada Y, Tsuji Y, Sato T, Yamamoto M, Kumasaka T, Nakanishi S, Jingami H, and Morikawa K (2000) Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407: 971–977.[CrossRef][Medline]
Leff P, Scaramellini C, Law C, and McKechnie K (1997) A three-state receptor model of agonist action. Trends Pharmacol Sci 18: 355–362.[Medline]
Masugi M, Yokoi M, Shigemoto R, Muguruma K, Watanabe Y, Sansig G, van der Putten H, and Nakanishi S (1999) Metabotropic glutamate receptor subtype 7 ablation causes deficit in fear response and conditioned taste aversion. J Neurosci 19: 955–963.
Millán C, Lujan R, Shigemoto R, and Sanchez-Prieto J (2002) The inhibition of glutamate release by metabotropic glutamate receptor 7 affects both [Ca2+]c and cAMP: evidence for a strong reduction of Ca2+ entry in single nerve terminals. J Biol Chem 277: 14092–14101.
Millán C, Castro E, Torres M, Shigemoto R, and Sanchez-Prieto J (2003) Coexpression of metabotropic glutamate receptor 7 and N-type Ca2+ channels in single cerebrocortical nerve terminals of adult rats. J Biol Chem 278: 23955–23962.
Mitsukawa K, Yamamoto R, Ofner S, Nozulak J, Pescott O, Lukic S, Stoehr N, Mombereau C, Kuhn R, McAllister KH, et al. (2005) A selective metabotropic glutamate receptor 7 agonist: activation of receptor signaling via an allosteric site modulates stress parameters in vivo. Proc Natl Acad Sci U S A 102: 18712–18717.
Mitsukawa K, Mombereau C, Lotscher E, Uzunov DP, van der Putten H, Flor PJ, and Cryan JF (2006) Metabotropic glutamate receptor subtype 7 ablation causes dysregulation of the HPA axis and increases hippocampal BDNF protein levels: implications for stress-related psychiatric disorders. Neuropsychopharmacology 31: 1112–1122.[Medline]
Nakamura M, Kurihara H, Ohkubo M, and Tsukamoto N (2002), inventors; Banyu Pharmaceutical Co., Ltd., assignee. Isoxazolopyridone derivatives and their use. World patent WO 02/102807. 2002 Dec 27.
Niswender CM, Myers KA, Williams R, Ayala JE, Luo Q, Saleh S, Jones CK, Weaver CD, Orton D, and Conn PJ (2006) Permissive antagonism induced by novel allosteric antagonists of metabotropic glutamate receptor 7. Neuropsychopharmacology 31: S127–S128.
O'Brien JA, Lemaire W, Wittmann M, Jacobson MA, Ha SN, Wisnoski DD, Lindsley CW, Schaffhauser HJ, Rowe B, Sur C, et al. (2004) A novel selective allosteric modulator potentiates the activity of native metabotropic glutamate receptor subtype 5 in rat forebrain. J Pharmacol Exp Ther 309: 568–577.
Offermanns S and Simon MI (1995) G15 and G16 couple a wide variety of receptors to phospholipase C. J Biol Chem 270: 15175–15180.
Ohashi H, Maruyama T, Higashi-Matsumoto H, Nomoto T, Nishimura S, and Takeuchi Y (2002) A novel binding assay for metabotropic glutamate receptors using [3H] L-quisqualic acid and recombinant receptors. Z Naturforsch [C] 57: 348–355.[Medline]
Okamoto N, Hori S, Akazawa C, Hayashi Y, Shigemoto R, Mizuno N, and Nakanishi S (1994) Molecular characterization of a new metabotropic glutamate receptor mGluR7 coupled to inhibitory cyclic AMP signal transduction. J Biol Chem 269: 1231–1236.
Parmentier ML, Joly C, Restituito S, Bockaert J, Grau Y, and Pin JP (1998) The G protein-coupling profile of metabotropic glutamate receptors, as determined with exogenous G proteins, is independent of their ligand recognition domain. Mol Pharmacol 53: 778–786.
Perroy J, Prezeau L, De Waard M, Shigemoto R, Bockaert J, and Fagni L (2000) Selective blockade of P/Q-type calcium channels by the metabotropic glutamate receptor type 7 involves a phospholipase C pathway in neurons. J Neurosci 20: 7896–7904.
Sansig G, Bushell TJ, Clarke VR, Rozov A, Burnashev N, Portet C, Gasparini F, Schmutz M, Klebs K, Shigemoto R, et al. (2001) Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7. J Neurosci 21: 8734–8745.
Saugstad JA, Kinzie JM, Mulvihill ER, Segerson TP, and Westbrook GL (1994) Cloning and expression of a new member of the L-2-amino-4-phosphonobutyric acid-sensitive class of metabotropic glutamate receptors. Mol Pharmacol 45: 367–372.[Abstract]
Saugstad JA, Segerson TP, and Westbrook GL (1996) Metabotropic glutamate receptors activate G-protein-coupled inwardly rectifying potassium channels in Xenopus oocytes. J Neurosci 16: 5979–5985.
Shigemoto R, Kulik A, Roberts JD, Ohishi H, Nusser Z, Kaneko T, and Somogyi P (1996) Target-cell-specific concentration of a metabotropic glutamate receptor in the presynaptic active zone. Nature 381: 523–525.[CrossRef][Medline]
Suzuki G, Kimura T, Satow A, Kaneko N, Fukuda J, Hikichi H, Sakai N, Maehara S, Kawagoe-Takaki H, Hata M, et al. (2007) Pharmacological characterization of a new, orally active and potent allosteric mGluR1 antagonist, 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide (FTIDC). J Pharmacol Exp Ther 321: 1144–1153.
Tanabe Y, Masu M, Ishii T, Shigemoto R, and Nakanishi S (1992) A family of metabotropic glutamate receptors. Neuron 8: 169–179.[CrossRef][Medline]
Varney MA, Cosford ND, Jachec C, Rao SP, Sacaan A, Lin FF, Bleicher L, Santori EM, Flor PJ, Allgeier H, et al. (1999) SIB-1757 and SIB-1893: selective, noncompetitive antagonists of metabotropic glutamate receptor type 5. J Pharmacol Exp Ther 290: 170–181.
Wright RA, Arnold MB, Wheeler WJ, Ornstein PL, and Schoepp DD (2000) Binding of [3H](2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl) glycine ([3H]LY341495) to cell membranes expressing recombinant human group III metabotropic glutamate receptor subtypes. Naunyn Schmiedebergs Arch Pharmacol 362: 546–554.[CrossRef][Medline]
Wright RA and Schoepp DD (1996) Differentiation of group 2 and group 3 metabotropic glutamate receptor cAMP responses in the rat hippocampus. Eur J Pharmacol 297: 275–282.[CrossRef][Medline]
Wu S, Wright RA, Rockey PK, Burgett SG, Arnold JS, Rosteck PR Jr, Johnson BG, Schoepp DD, and Belagaje RM (1998) Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells. Brain Res Mol Brain Res 53: 88–97.[Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
