Abstract
Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of excitatory amino acids, via mechanisms of presynaptic inhibition, such as regulation of neurotransmitter release. Here, we describe (R,S)-4-phosphonophenylglycine (PPG) as a novel, potent, and selective agonist for group III mGluRs. In recombinant cell lines expressing the human receptors hmGluR4a, hmGluR6, hmGluR7b, or hmGluR8a, EC50 values for (R,S)-PPG of 5.2 ± 0.7 μM, 4.7 ± 0.9 μM, 185 ± 42 μM, and 0.2 ± 0.1 μM, respectively, were measured. The compound showed EC50 and IC50 values of ≥200 μM at group I and II hmGluRs and was inactive at cloned humanN-methyl-d-aspartate, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, and kainate receptors (>300 μM). On the other hand, it showed micromolar affinity for a Ca2+/Cl−-dependentl-glutamate binding site in rat brain, similar to other phosphono-substituted amino acids likel-2-amino-4-phosphonobutyrate. In cultured cortical neurons, (R,S)-PPG provided protection against a toxic pulse ofN-methyl-d-aspartate (EC50 = 12 μM), which was reversed by the group III mGluR antagonist (R,S)-α-methylserine-O-phosphate but not by the group II antagonist (2S)-α-ethylglutamate. Moreover, (R,S)-PPG protected againstN-methyl-d-aspartate- and quinolinic acid-induced striatal lesions in rats and was anticonvulsive in the maximal electroshock model in mice. In contrast to the group III mGluR agonists l-2-amino-4-phosphonobutyrate andl-serine-O-phosphate, (R,S)-PPG showed no proconvulsive effects (2200 nmol i.c.v.). These data provide novel in vivo evidence for group III mGluRs as attractive targets for neuroprotective and anticonvulsive therapy. Also, (R,S)-PPG represents an attractive tool to analyze the roles of group III mGluRs in nervous system physiology and pathology.
The neurotoxicity of excitatory amino acids such as l-glutamate and some of its analogs, e.g., kainate andN-methyl-d-aspartic acid (NMDA), is well established in the central nervous system (Lipton and Rosenberg, 1994). Analogs of l-glutamate, which have been investigated in neural systems, share with their parent compound the α-amino acid moiety and a distal, negatively ionizable group (Watkins et al., 1990). These structural features are considered essential forl-glutamate to interact with each member of its large family of ionotropic and metabotropic neurotransmitter receptors (Hollmann and Heinemann, 1994).
In an effort to discover new agents interfering with the glutamatergic system, a large panel of phosphono-substituted α-amino acid derivatives has been generated.d-2-amino-5-phosphonopentanoic acid (d-AP5),d-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 40116; Fig. 1),d-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid (d-CPPene), and 2-amino-3-(2′-chloro-5-phosphonomethyl-biphenyl-3-yl)-propionic acid) (SDZ 220-581), for instance, are potent, selective, and competitive antagonists for NMDA receptors, which constitute one pharmacological group within the class of ionotropic glutamate receptors (iGluRs). Those and many related compounds served as tools to elucidate the role of NMDA receptors in brain disorders, such as neurodegenerative processes following ischemia and epileptic seizures (Sauer et al., 1992; Urwyler et al., 1996a). On the other hand, many phosphono-substituted α-amino acids, likel-2-amino-4-phosphonobutyrate(l-AP4),l-serine-O-phosphate(l-SOP), and 4-phosphono-phenylglycine [(R,S)-PPG; Bigge et al., 1989], were found to be inactive as NMDA receptor ligands.
l-AP4 and l-SOP, however, are potent and selective agonists at a group of metabotropic glutamate receptors (mGluRs). Eight mGluR subtypes are currently known, which are numbered according to the order of their molecular discovery, and are subdivided into three distinct groups (Tanabe et al., 1992; Conn and Pin, 1997). Group I mGluRs (mGluR1 and mGluR5) are positively coupled to the phosphoinositide/Ca2+ cascade. Group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7, and mGluR8) receptors are both negatively coupled to adenylate cyclase in heterologous expression assays. The three groups can be discriminated pharmacologically with the use of selective agonists. 3,5-Dihydroxyphenylglycine selectively activates group I mGluRs, whereas 2R,4R-aminopyrrolidine-2,4-dicarboxylate and LY-354740 are examples for group II selective agonists (Conn and Pin, 1997; Monn et al., 1997). l-AP4,l-SOP, and close analogs are the only selective agonists known for group III mGluRs, with low micromolar potency (EC50 values, 0.1–7 μM) at mGluR4, mGluR6, and mGluR8; mGluR7 can only be activated at concentrations higher than 100 μM (Okamoto et al., 1994; Johansen et al., 1995; Conn and Pin, 1997;Flor et al., 1997). Group III (and group II) mGluRs are thought to mediate presynaptic depression of glutamatergic synaptic potentials in several brain areas, most likely via inhibition of voltage-gated calcium entry and regulation of glutamate release (Trombley and Westbrook, 1992; Conn and Pin, 1997). Moreover, selective activation of group III mGluRs results in neuroprotection in vitro; agonists likel-AP4 and l-SOP promote survival of rat cerebellar granule cells and protect cultured cortical and cerebellar neurons against toxic insults, such as prolonged β-amyloid peptide exposure, transient iGluR activation, or mechanical damage (Graham and Burgoyne, 1994; Copani et al., 1995; Bruno et al., 1996; Faden et al., 1997). In contrast to the findings with NMDA receptor antagonists, in vivo neuroprotection with group III mGluR agonists has not yet been reported to our knowledge.
Both, proconvulsive and anticonvulsive effects of group III mGluR agonists have been observed, depending not only on the animal model used but also on timing and dosage of the drug treatment (e.g., Graham and Burgoyne, 1994; Abdul-Ghani et al., 1997; Ghauri et al., 1996; Tang et al., 1997).
Here, we report in vitro and in vivo neuroprotective actions of (R,S)-PPG, a compound with structural similarity to competitive NMDA receptor antagonists and group III mGluR agonists (Fig. 1). (R,S)-PPG was also tested for anticonvulsive properties in the maximal electroshock-induced convulsion model in mice, in comparison withl-AP4 and l-SOP. In an effort to characterize activity and selectivity of (R,S)-PPG at the molecular level, the compound was tested for interaction with all eight mGluR subtypes, with a representative selection of recombinant human iGluRs and with a Ca2+/Cl−-dependentl-glutamate binding site of rat brain. Group III mGluRs as attractive drug targets for the treatment of neurological disorders, such as epilepsy and Huntington’s disease, will be discussed.
Experimental Procedures
Chemical Synthesis of (R,S)-PPG.
(R,S)-PPG was synthetized in four steps starting from 4-hydroxybenzaldehyde using a different synthetic pathway than the one described (Bigge et al., 1989). The starting material was first esterified with trifluoromethanesulfonicanhydride. The trifluoromethanesulfonate ester was then converted to the corresponding phosphonate ester using a palladium-catalyzed coupling. Conversion of the aldehyde to the amino nitrile and subsequent hydrolysis performed in concentrated HCl gave (R,S)-PPG with an overall yield of about 30%. Further details of the synthesis will be published elsewhere.
Cloning of hmGluR8a cDNA.
The sequence encoding the human metabotropic glutamate receptor subtype 8a (hmGluR8a) was constructed from clones obtained by library screening in combination with polymerase chain reaction (PCR).
Library Screening.
Five × 105plaques each of two human cDNA libraries from whole adult brain (in λgt10; Clontech, Palo Alto, CA) and adult hippocampus (in λZAPII; Stratagene, Heidelberg, Germany) were screened with 5′ and 3′ probes from the rat mGluR4 sequence (Tanabe et al., 1992) as described previously (Laurie et al., 1997). After a second round of screening, individual cDNA inserts were rescued into Bluescript SK(−) phagemids (Stratagene) by in vitro (λgt10) or in vivo (λZAPII) excision. cDNA inserts were characterized by restriction enzyme mapping and DNA sequencing (ABI systems, Langen, Germany). Two nonoverlapping clones were identified as homologous to portions of the mouse mGluR8 sequence (Duvoisin et al., 1995): HMGBr7 (homologous to bases 576–799 of mouse mGluR8) and HMGHi7 (homologous to bases 2059–2830 of mouse mGluR8).
PCR.
The 5′ end of the hmGluR8 coding sequence was amplified from human retinal cDNA. Thermocycling conditions were: 94°C for 1 min, 45°C for 1 min, 72°C for 1 min, 10 cycles, then 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, 38 cycles, using “Expand-High Fidelity” polymerase (Boehringer Mannheim, Mannheim, Germany). PCR oligos were 5′-GTCGCTGACTGCAATACCACCTGCGGAGAAAATG-3′ [sense oligo from mouse mGluR8 sequence (Duvoisin et al., 1995); translation initiation codon underlined] and 5′-CAACTATCTGAGCCAATCCAG-3′. The resultant 900-bp amplicon, “PCR5p8,” was subcloned into the PCR cloning vector pCRII (Invitrogen, San Diego, CA).
The missing sequence between clones HMGBr7 and HMGHi7 was obtained by PCR from human retinal cDNA, and the resultant 1570-bp amplicon was also subcloned into the vector pCRII. Thermocycling conditions were 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, 38 cycles, using Expand-High Fidelity polymerase (Boehringer Mannheim). PCR oligos were 5′-GACTCCTACCAAGCCCAAGCCATG-3′ and 5′-CGCTGCTCTCCATAGTCAATGATG-3′. This intervening sequence, “PCRint8”, was digested into three fragments with BamHI and the largest (1214-bp) fragment, “3′PCRint8” ligated to HMGHi7 by a common BamHI site, forming 3′PCRint8-HMGHi7. A parallel digest of PCRint8 withMunI released the 420-bp fragment “5′PCRint8”.
The full-length hmGluR8a sequence was constructed by ligating 3′PCRint8-HMGHi7 to 5′PCRint8 via a common MunI site, ligating the product to PCR5p8 via an NcoI site and finally ligating the whole sequence into the expression vector pCIneo (Clontech) using XbaI and SalI sites. The coding region of the assembled hmGluR8a clone (hmGluR8a.pCIneo) was sequenced on both strands. In comparison with the very recently published hmGluR8a sequence (Wu et al., 1998), we find only one amino acid difference: Asp-768 to Ile, which is encoded by the library-derived clone HMGHi7.
Stable Expression of hmGluR8a in HEK293 Cells.
The construct hmGluR8a.pCIneo was linearized by digestion with Asp-700. One microgram of the linearized DNA was used to transfect 107HEK293 cells. Selection for stable integration was made by addition of 0.8 mg/ml G418 (Life Technologies, Basel, Switzerland) to the medium (minimal essential medium with 2 mM l-glutamine supplemented with 10% dialyzed fetal calf serum; Life Technologies), and 30 G418-resistant clonal cell lines were isolated as described previously (Laurie et al., 1995). Further selection was performed by measuring the glutamate (0.1 mM)-induced depression of forskolin (10 μM)-elevated cAMP accumulation in cells grown in collagen-coated wells. Responses ranged from no depression to about 85% depression. Using this approach, two cell lines, HEK-hmGluR8a-2 and HEK-hmGluR8a-20, were identified as giving consistently good responses for up to at least 20 passages of subculturing.
Stable Mammalian Cell Lines for Cloned mGluR1 to mGluR7 and Ionotropic Glutamate Receptors.
Generation, culture, and pharmacological characterization of stable cell lines for hmGluR1b, -2, -4a, -5a, -6, -7b, rat mGluR3, hNMDAR1A/2A, hNMDAR1A/2B, hGluR3i, and hGluR6 have been described recently (Knöpfel et al., 1995; Laurie et al., 1995, 1997; Daggett et al., 1996; Varney et al., 1996, 1998;Flor et al., 1997 and references therein; Lin et al., 1997).
In Vitro Pharmacological Assays for Cloned Glutamate Receptors.
Measurements of cyclic AMP accumulation (Flor et al., 1997), inositol monophosphate formation (Knöpfel et al., 1995), and cytoplasmic calcium elevation (Flor et al., 1996) were performed as described previously.
l-[3H]Glutamate-Binding Assay for mGluR3.
HEK293 cells stably transfected with the cDNA encoding rat mGluR3 were cultured and harvested as described previously (Laurie et al., 1995). Membranes from these cells were washed by five cycles of centrifugation (10 min at 50,000g, 4°C) and resuspension in assay buffer before being frozen and stored at −80°C until their use in the binding experiments. After thawing, membranes were washed five times by centrifugation and resuspension in ice-cold assay buffer as above. Thel-[3H]glutamate-binding assay was performed in 0.6 ml of 50 mM Tris-HCl buffer (pH 7.5 at 0°C) containing an aliquot of the membrane suspension (about 50 μg of protein), 5 nMl-[3H]glutamate (NEN, Regensdorf, Switzerland), 2.5 mM CaCl2, and the test compounds at the appropriate concentrations. Nonspecific binding was determined by including 0.5 mM unlabeledl-glutamate. The samples were incubated at 0°C for 4 h before bound and free radioligand were separated by centrifugation at 4°C for 20 min at approximately 10,000g. The supernatant was decanted, and the pellets were quickly and superficially rinsed with ice-cold assay buffer and then added to scintillation fluid containing tissue solubilizer (Solvable; NEN). After solubilization at 50°C overnight, the radioactivity was measured by liquid scintillation counting. Competition curves were analyzed, and IC50 values were determined by nonlinear curve fitting using the program GraphPad Prism (GraphPad Software, Inc., San Diego, CA).
Ca2+/Cl−-Dependentl-[3H]Glutamate Binding to Rat Brain Membranes.
This assay was essentially performed as described previously (Urwyler et al., 1996b). In brief, the assay mixture (in a final volume of 1.1 ml) contained 50 mM Tris-HCl (pH 7.4), 2.5 mM CaCl2, extensively washed rat hippocampal membranes (freshly prepared on the day of the experiment) corresponding to approximately 3 mg of original tissue (wet weight), 5 nMl-[3H]glutamate, and the test compounds at the desired concentrations. Nonspecific binding was defined with 0.2 mM dl-AP7. The samples were incubated for 25 min at 37°C before bound and free radioligand were separated by centrifugation at 12,000g for 4 min. The pellets were quickly and superficially rinsed with 100 μl of ice-cold incubation buffer and then added to scintillation fluid containing tissue solubilizer (Solvable; NEN). After solubilization at 50°C overnight, the radioactivity was measured by liquid scintillation counting.
Preparation of Cultured Cortical Cells and Examination of NMDA Toxicity.
Mixed cultures of cortical cells were prepared from fetal mice (14–16 days of gestation), as described (Bruno et al., 1996), and used 13 to 14 days after plating. Cultures were exposed to 100 μM NMDA for 10 min at room temperature in a HEPES-buffered salt solution. After extensive washing, cultures were incubated for 18 to 24 h at 37°C in minimal essential medium-Eagle’s buffer (Life Technologies) supplemented with 25 mM NaHCO3 and 21 mM glucose. Neuronal toxicity was examined by phase-contrast microscopy and quantitated after staining with trypan blue. Stained neurones were counted from three random fields per well. Lactate dehydrogenase release into the medium was also measured as described previously (Bruno et al., 1996).
Examination of Neuronal Toxicity after Intrastriatal Infusion with NMDA and Quinolinic Acid.
Male Sprague-Dawley rats (250–300 g) were anesthetized with pentobarbital (50 mg/kg, i.p.) and infused with NMDA (100 nmol/0.5 μl/2 min) or NMDA + group III mGluR agonists (balanced to neutral pH) in the left caudate nucleus, at +2.0 mm AP, 2.6 mm L, and 5 mm V, according to the Pellegrino and Cushman atlas. The injection was repeated at a second site (1 mm posterior to the first site) to increase the extent of striatal toxicity. Seven days later, animals were sacrificed, and neuronal damage was assessed by either histological analysis or measurements of striatal glutamate decarboxylase (GAD) activity. For histological analysis, the brains were rapidly frozen in isopentane at −40°C and then stored at 0°C. Twenty-micrometer cryostat sections were Nissl-stained and examined in light microscopy. For measurements of GAD activity, the corpus striatum was dissected bilaterally and homogenized in 5 mM imidazol buffer containing 0.2% Triton X-100 and 10 mM dithiothreitol. An aliquot of the homogenate was incubated in 400 μl of 10 mM phosphate buffer (pH 7.0) containing 10 mM 2-mercaptoethanol, 0.02 mM pyridoxalphosphate, and 1 μCi of l-[3H]glutamate (Amersham Intl., Buckinghamshire, UK; specific activity 46 Ci/mmol) for 1 h at 37°C; the reaction was stopped with 15 μl of ice-cold 11.8 N HClO4. After centrifugation in a microfuge at maximal speed, 10 μl of the supernatant were diluted with 0.01 N HCl and derivatized with O-phtalaldehyde and mercaptoethanol for 1 min at room temperature before injection into HPLC. The HPLC apparatus consisted of a programmable solvent module 126 (Beckman Instruments, Inc., Fullerton, CA), an analytical C18 reversed-phase column kept at 30°C (Ultrasphere ODS 5 μm spherical, 80 Å pore, 2 mm × 15 cm; Beckman Instruments Inc.), and an RF-551 spectrofluorimetric detector (Shimadzu Corp., Tokyo, Japan). Excitation and emission were set at 360 and 450 nm, respectively. The mobile phase consisted of 50 mM sodium phosphate, 10% methanol, pH 7.2 (A), and 50 mM sodium phosphate, 70% methanol, pH 7.2 (B). After 8 min of isocratic conditions with 98% (A) and 2% (B), (B) was increased up to 40% within 30 min and then to 98% within 1 min and then maintained at 98% for 11 min before returning to the initial conditions. The radioactivity coeluting with γ-aminobutyric acid (GABA) was collected and counted by scintillation spectrometry. Protein concentrations in the original samples were determined by using a commercially available kit (Bio-Rad, Richmond, CA).
Intrastriatal injection of quinolinic acid and evaluation of excitotoxic neurodegeneration by magnetic resonance imaging was performed as previously described (Sauer et al., 1992).
Maximal Electroshock Test (MES).
Experiments were conducted on 19- to 25-g male mice [Tif:MAGf (SpF)] at 21–22°C. Generalized tonic-clonic convulsions of the hind extremities were induced by passing alternating electrical currents of 50 Hz and 18 mA through corneal electrodes (for reference, see Kupferberg and Schmutz, 1997).l-AP4, l-SOP, and (R,S)-PPG were dissolved in 0.9% saline, the pH corrected to 7.0 and administered i.c.v., i.p., or i.v. with pretreatment times of 15 or 30 min. Five to 10 animals per dose were used; ED50 values were calculated on the basis of at least five doses, and each experiment was done at least twice. The number of animals protected from tonic hind limb extension seizure and the duration of hind limb tonus were determined in each dose group.
Statistics.
Significant differences were estimated using the two-tailed Dunnett’s t test by comparing test-drug groups with the control group. Values of 2P < 0.05 were considered as statistically significant.
Materials.
Molecular biology reagents and enzymes were purchased from Amersham, Bio-Rad, Boehringer Mannheim, Invitrogen, New England Biolabs, and Stratagene. Tissue culture reagents were from Life Technologies and Sigma. Forskolin, 3-isobutyl-1-methylxanthine, andl-SOP were obtained from Sigma. All other reference agonists and antagonists were purchased from Tocris (Anawa Trading SA, Zürich, Switzerland). CGP 40116 was synthesized within Novartis Pharma AG by Dr. Roland Heckendorn. All other chemicals were of reagent grade and were obtained from Fluka (Buchs, Switzerland), Merck (Darmstadt, Germany), Serva (Heidelberg, Germany), or Sigma.
Results
Profiling of (R,S)-PPG against All Eight Cloned mGluR Subtypes.
As shown in Fig.2, (R,S)-PPG most potently inhibited forskolin-stimulated cAMP accumulation in recombinant cells stably expressing hmGluR8a, producing 70 to 80% inhibition at a maximally effective concentration of 100 μM and an EC50 value of 0.2 ± 0.1 μM (mean ± S.E.M.). Concentration-response curves for agonist activity at the other known group III mGluR subtypes were measured, and EC50 values of 5.2 ± 0.7 μM, 4.7 ± 0.9 μM, and 185 ± 42 μM (means ± S.E.M.) were found for hmGluR4a, hmGluR6, and hmGluR7b, respectively (Fig. 2; Table1). In contrast, (R,S)-PPG showed no significant inhibition of forskolin-stimulated cAMP accumulation in untransfected CHO-K1 and HEK293 cells when tested at 300 μM and 100 μM, respectively (not shown). Different extents of maximal inhibition of cAMP formation by (R,S)-PPG were observed in the four cell lines expressing recombinant group III mGluRs, ranging from 45 to 80% inhibition. The Hill coefficients for the interaction of (R,S)-PPG with the four group III mGluRs were between 1.5 and 2.5 (Table 1).
To address specificity of (R,S)-PPG, the compound was tested for agonist and antagonist activity at all known group I, II, and III mGluR subtypes. In recombinant cells expressing hmGluR1b or hmGluR5a, quisqualate produced 5- to 15-fold stimulation of phosphoinositide hydrolysis at concentrations of 20 μM and 0.3 μM, which are approximate EC80 concentrations for these two clonal lines. (R,S)-PPG, tested at 500 μM, neither stimulated phosphoinositide hydrolysis on its own nor reversed the quisqualate effects in either hmGluR1b- or hmGluR5a-expressing cells (Fig. 3A). Moreover, at concentrations of 10 μM and 100 μM, (R,S)-PPG was also found inactive at hmGluR1b and hmGluR5a (data not shown).
In Chinese hamster ovary (CHO) cells stably expressing hmGluR2, 1-aminocyclopentane-1S,3R-dicarboxylic acid [(1S,3R)-ACPD], at 30 μM, depressed forskolin-stimulated cAMP accumulation by approximately 65%, which represents about 80% of the maximal response (Fig. 3B). Five hundred micromolar (R,S)-PPG showed no significant depression of cAMP when applied alone but showed 52% reversal of cAMP depression when coapplied with 30 μM (1S,3R)-ACPD (Fig. 3B), indicating antagonist activity at hmGluR2. At 300 μM, the antagonist activity of (R,S)-PPG was reduced to 45%; at 100 μM, no significant antagonist activity of (R,S)-PPG at hmGluR2 was observed (2P > .05, Dunnett’st test, n = 8). At recombinant rat mGluR3 expressed in human embryonic kidney (HEK) cells, (R,S)-PPG as well as l-AP4 had only very weak affinity in anl-[3H]glutamate binding assay. (R,S)-PPG, l-AP4, and l-glutamate showed around 50% inhibition ofl-[3H]glutamate binding at 200 μM, 300 μM, and 200 nM, respectively (data not shown).
As shown in Fig. 3, B and C, and Table 1, inhibition of forskolin-stimulated cAMP formation in recombinant cells expressing hmGluR4a, hmGluR6, hmGluR7b, or hmGluR8a by (R,S)-PPG was at least as efficacious as withl-AP4, applied at EC80. Coapplication of l-AP4 and (R,S)-PPG (300 or 1000 μM) did not reverse thel-AP4 response.
Characterization of (R,S)-PPG at Ionotropic Glutamate Receptors.
(R,S)-PPG was originally designed and characterized for NMDA-receptor binding (Bigge et al., 1989). However, the compound was found to be inactive up to 100 μM in the [3H]-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphoric acid receptor binding assay on rat cortical brain membranes, indicating that (R,S)-PPG does not bind to thel-glutamate site of native rat NMDA receptors. Here, we extend the characterization by employing stable cell lines transfected with the hNMDAR1A/2B subunit combination (Varney et al., 1996) to address functional agonist and antagonist activity of (R,S)-PPG at cloned human NMDA receptors. In this cell line, l-glutamate (applied at 1 μM) induced a rise in intracellular calcium concentration. (R,S)-PPG neither induced a calcium rise when applied at 300 μM nor antagonized thel-glutamate-induced calcium signal (Fig.4). In contrast, the competitive NMDA-receptor antagonist CGP 40116 concentration-dependently prevented the l-glutamate-evoked rise in intracellular calcium (Fig. 4). Similar lack of activity of 300 μM (R,S)-PPG was observed in calcium assays for cloned hNMDAR1A/2A (data not shown, Table 1).
Moreover, stable cell lines expressing human AMPA(GluR3) or kainate(GluR6) receptors were utilized in the same assay (Daggett et al., 1996; Varney et al., 1998). Application of l-glutamate resulted in robust increases of cytoplasmic calcium, whereas (R,S)-PPG (300 μM) did neither evoke calcium signals on its own nor antagonize l-glutamate in either of the two cell lines (data not shown; Table 1).
Ca2+/Cl−-Dependentl-[3H]Glutamate Binding to Rat Brain Membranes.
l-AP4, l-SOP, and (R,S)-PPG displacedl-[3H]glutamate binding at rat hippocampal membranes, measured in the presence of CaCl2, with IC50 values of 0.5 μM, 3.1 μM, and 1.9 μM, respectively (Table2). Thus, l-AP4 was the most potent among the three compounds, having a 4-fold higher affinity than (R,S)-PPG, which was about equipotent with l-SOP.
Neuroprotective Activity of (R,S)-PPG in Neuronal Culture.
In primary cultures of cortical neurons cocultured with glia, (R,S)-PPG was highly neuroprotective when applied during the NMDA-induced excitotoxic pulse (10 min). The action of (R,S)-PPG was concentration-dependent, with an apparent EC50value of 12 μM (Fig. 5A). Maximally effective concentrations of (R,S)-PPG rescued slightly more than 50% of the neuronal population from excitotoxic degeneration (Fig. 5). (R,S)-α-Methylserine-O-phosphate (MSOP), a preferential antagonist of group III mGluRs (Thomas et al., 1996), prevented the neuroprotective activity of (R,S)-PPG. In contrast, the selective group II mGluR antagonist (2S)-α-ethylglutamic acid (EGlu) (Jane et al., 1996; Thomas et al., 1996) did not affect the action of (R,S)-PPG (Fig. 5B). Neither (R,S)-PPG nor MSOP or EGlu had any effect on neuronal viability when applied to the cultures in the absence of NMDA (not shown).
Protection by (R,S)-PPG against NMDA- and Quinolinic Acid-Induced Striatal Lesions.
Intrastriatal infusion of various ionotropic glutamate receptor agonists results in neurochemical and neuropathological changes resembling Huntington’s disease (DiFiglia, 1990). First, we have used infusion of 100 nmol of NMDA which induces an extended area of necrosis characterized by neuronal loss, reactive gliosis, edema, and neuronal pyknosis (Fig.6A). Neuronal damage was visible across the extension of the caudate nucleus, up to 3 mm posterior to the injection site. Coinfusion of 250 nmol of (R,S)-PPG with NMDA resulted in efficacious neuroprotection against excitotoxic neuronal damage; particularly in the more lateral parts of the caudate nucleus, drastically reduced neuronal loss and pyknosis as well as less edema formation were seen (Fig. 6B). To quantitate the protective effect of (R,S)-PPG, we have measured striatal GAD activity as a biochemical marker of viable GABAergic neurones (Fig.7), which has been widely used as post-mortem assessment of lesion size (e.g., Urwyler et al., 1996a). NMDA infusion led to a 45 to 50% decrease in GAD activity as compared with the respective contralateral side. No reduction in GAD activity was observed in animals coinfused with NMDA plus (R,S)-PPG. The protective activity of (R,S)-PPG against NMDA toxicity was mimicked byl-AP4 (50 or 250 nmol, Fig. 7).
Furthermore, we tested (R,S)-PPG for protection against intrastriatal infusion of quinolinic acid (200 nmol), which produced larger lesions than NMDA. Lesion size was quantitated by magnetic resonance imaging as described (Sauer et al., 1992). Compared with the control group receiving 200 nmol of quinolinic acid (11 animals treated), coinfusion of (R,S)-PPG (250 nmol) showed significant protection (2P < .005, Dunnett’s t test) with a reduction of the lesion size by 58.4% ± 13% (mean ± S.E.M., n = 13 animals).
Protection by (R,S)-PPG against MES-Induced Convulsions in Mice.
MES is commonly used as a basic in vivo test for anticonvulsive compounds (Kupferberg and Schmutz, 1997and references therein). In this test, with i.c.v. injections ofl-AP4 and l-SOP, a pretreatment time of 15 min, and doses between 60 and 220 nmol, no anticonvulsive effects were seen. When doses were increased to approximately 2000 nmol, l-AP4 and l-SOP induced clonic/clonic-tonic seizures at 5 to 10 min after drug administration in 40 to 60% of the treated animals (Table 3). In contrast, (R,S)-PPG up to 2200 nmol did not show any proconvulsant effect. Moreover, (R,S)-PPG when applied at 173 nmol (i.c.v.) produced 100% protection against MES with an ED50 value of 78 nmol (Table 3). At doses above 2000 nmol (i.c.v.), all three compounds were lethal in 20 to 60% of the animals. (R,S)-PPG given i.p. or i.v. was inactive against MES-induced convulsions up to 100 mg/kg and 10 mg/kg, respectively.
Discussion
In Vitro Pharmacology.
Recent cDNA cloning and recombinant expression of the heterogeneous family of G protein-coupled (metabotropic) glutamate receptors has created a large gap between the molecular knowledge of mGluR subtypes and the understanding of their role in brain function and dysfunction. Thus, the discovery of subtype-selective compounds and their testing in experimental models for nervous system physiology and pathology has become increasingly important.
In the present study, using 12 different cell lines stably expressing cloned ionotropic and metabotropic glutamate receptor subtypes, we have characterized the pharmacological profile of (R,S)-PPG. This compound behaved as a potent agonist at hmGluR8a (EC50 = 200 nM), hmGluR6 (EC50 = 4.7 μM), and hmGluR4a (EC50 = 5.2 μM), with no appreciable activity (up to 200 μM) at hmGluR1, hmGluR2, rat mGluR3, hmGluR5, and hNMDAR1A/2B, hNMDAR1A/2A, AMPAR(hGluR3), and kainateR(hGluR6).
(R,S)-PPG therefore exhibited approximately 1000-fold selectivity for hmGluR8 and ≥40-fold selectivity for hmGluR4a and hmGluR6 versus group I/II mGluRs and all tested iGluRs. (R,S)-PPG activated hmGluR7b only at high micromolar concentrations (EC50 = 185 μM), which is only slightly more potent than the compound’s weak antagonist activity at hmGluR2 and its activity against glutamate binding at rat mGluR3 (Table 1). In addition to its potent activity at group III mGluRs, (R,S)-PPG, as well asl-AP4 and l-SOP, displayed micromolar affinity for a Ca2+/Cl−-dependentl-[3H]glutamate binding site in rat brain (Fagg et al., 1982; Urwyler et al., 1996b). The functional role of this binding site was proposed to be that of a glutamate transporter, but alternative explanations are also possible (see Urwyler et al., 1996b and references therein for detailed discussion). The affinity of (R,S)-PPG at this site was found to be 4-fold lower than the affinity ofl-AP4. Whether this difference is reflected at the level of endogenous glutamate accumulation, and thus could contribute to explain the discrepancy we have found betweenl-AP4 and (R,S)-PPG in the MES model in mice (see below), is currently unclear, especially because the explanation cannot be extended to l-SOP.
In summary, (R,S)-PPG represents a novel group III mGluR agonist, with in vitro pharmacological properties indistinguishable from the current agonistsl-SOP, l-AP4, and cyclopropyl-AP4 (Johansen et al., 1995; Okamoto et al., 1994; Conn and Pin, 1997; Flor et al., 1997;). Interestingly, however, (R,S)-PPG differs structurally from all of these compounds but shares close similarity with classical competitive NMDA receptor antagonists like CGP 40116, as the distance between the amino acid moiety and the phosphonate group is comparable. Because this distance is substantially smaller in l-AP4 andl-SOP, the potency and selectivity of (R,S)-PPG for group III mGluRs is quite surprising and suggests a particular mode of binding of (R,S)-PPG, involving the phenyl spacer between the α-amino acid and phosphonic acid moieties.
α-Methylphosphonophenyl glycine, a mixed antagonist of group II and III mGluRs (Bedingfield et al., 1996), is structurally the most closely related compound and differs from (R,S)-PPG only by a methyl group in the α-position. Thus, replacement of that group with the hydrogen of (R,S)-PPG has changed the antagonistic properties into selective agonist activity. Similar properties have been shown for linear l-glutamate analogs and cyclopropylglycine derivatives (Jane et al., 1996; Thomas et al., 1996), where the introduction of a methyl group at the α-position changed agonists into antagonists.
Neuroprotection and Anticonvulsive Actions Mediated by (R,S)-PPG.
Neuroprotective effects of l-AP4 and l-SOP observed in several in vitro paradigms (see Introduction) prompted us to examine (R,S)-PPG, as a structurally different but pharmacologically indistinguishable compound, in the model of NMDA-induced degeneration of mouse cortical neurons cocultured with glia (Bruno et al., 1996). Here, we found (R,S)-PPG highly neuroprotective, and its action was antagonized by MSOP, which is a group III mGluR antagonist (Thomas et al., 1996), but not by the group II mGluR antagonist EGlu. These results therefore strengthen the suggestion that activation of group III mGluRs is neuroprotective in vitro (e.g., Copani et al., 1995;Bruno et al., 1996; Faden et al., 1997).
To investigate neuroprotective effects of (R,S)-PPG also in vivo, we analyzed striatal degeneration following local infusion of NMDA and quinolinic acid into the rat caudate nucleus. The use of such excitotoxic injury models, to produce neuronal depletion, reactive gliosis, and alterations of neurotransmitter levels, has been highly valuable for examining pathological patterns reminiscent of Huntington’s disease (HD). Even if the primary cause of HD is unrelated, excitotoxic injury mediated by iGluR activation may play a role in progressive neuronal depletion (DiFiglia, 1990).
We found (R,S)-PPG protective against NMDA- and quinolinic acid-induced striatal lesions; to our knowledge, this provides the first in vivo evidence that activation of group III mGluRs is neuroprotective in animal models.
Inhibition of glutamate release by presynaptic mGluR4, -7 and/or -8 (Shigemoto et al., 1997) may represent a common mechanism of neuroprotection in vitro and in vivo. Accordingly, an enhanced release of endogenous glutamate has been shown to facilitate the progression of NMDA toxicity in cortical cultures (Monyer et al., 1992). Additionally, in vivo striatal toxicity induced by kainate or NMDA receptor agonists such as quinolinic acid and NMDA critically involves the presence of cortical glutamatergic fibers afferent to the caudate nucleus of striatum (Colwell et al., 1996 and references therein). Although little recurrent excitation exists in the striatum, the endogenously releasedl-glutamate may have a permissive role on NMDA and quinolinic acid toxicity, perhaps by activating postsynaptic group I mGluRs or other facilitatory receptors.
Upon depletion of the caudate neuronal population during the progression of HD, the resulting “excess” of corticostriatal glutamatergic input may cause further neuronal loss (DiFiglia, 1990), and inhibition of this input via group III mGluRs may provide protection. In addition to the regulation of presynaptic glutamate release, an inhibition of NMDA receptors by postsynaptic group III mGluRs via a protein phosphorylation cascade (Martin et al., 1997) may also be involved in their neuroprotective effects. Thus, activation of group III mGluRs could open several novel strategies to interfere with the progressive course of neurodegenerative disorders.
The potency at cloned mGluRs relative to the protective activity of (R,S)-PPG in primary culture (EC50 = 12 μM) suggests that the neuroprotective action of (R,S)-PPG preferentially involves mGluR4, mGluR6, and/or mGluR8, which are all expressed by cultured cortical neurons (Faden et al., 1997). For in vivo neuroprotection, however, mGluR6 is probably irrelevant because of its restricted expression in the retinal bipolar cells layer. A more detailed examination of the relative contribution of individual group III mGluR subtypes to in vitro and in vivo neuroprotection awaits the discovery of more selective agonists and the utilization of group III mGluR subtype-deficient mice.
Modulation of epileptic seizures by group III mGluRs has been frequently reported (e.g., Ghauri et al., 1996; Abdul-Ghani et al., 1997; Tang et al., 1997). Thus, we were tempted to test (R,S)-PPG, in comparison withl-AP4 and l-SOP, in the MES model in mice. MES is a basic screening test for anticonvulsive drugs, it is indicative of drug activity primarily against generalized tonic-clonic and, secondarily, also partial seizures, and it led to the discovery of several clinically approved antiepileptics, e.g., carbamazepine, oxcarbazepine, and phenytoin (Kupferberg and Schmutz, 1997 and references therein). Surprisingly, our results for (R,S)-PPG obtained with this model revealed important differences as compared with the prototypic group III mGluR agonists l-AP4 and l-SOP. In agreement with previous results (Ghauri et al., 1996; Tang et al., 1997), both l-AP4 and l-SOP were proconvulsive at high doses (around 2000 nmol) and did not protect against MES-induced seizures at any of the given doses (60–2400 nmol). In contrast, (R,S)-PPG exhibited substantial anticonvulsive activity with an ED50 value of 78 nmol, full protection at 173 nmol, and did not exhibit any proconvulsive effect up to 2200 nmol. These discrepancies could possibly be explained by physicochemical properties and/or divergent in vivo metabolism of the structurally quite different group III mGluR agonists l-AP4, l-SOP, and (R,S)-PPG. Although we cannot exclude that anticonvulsive properties of (R,S)-PPG are mediated by a mechanism distinct from group III mGluRs, other reports also support group III mGluRs as mediators of anticonvulsive and antiepileptogenic effects. Abdul-Ghani et al. (1997) reported protective effects of l-AP4 on development of electrical kindling and also in fully kindled rats. Furthermore, Tang et al. (1997) reported for l-SOP an immediate, transient (<10 min) proconvulsive effect followed by a prolonged (>1 day) anticonvulsive effect against sound-induced seizures with an anticonvulsant ED50 value of 36 nmol. Moreover, recent experimental evidence from mGluR7-deficient mice that exhibit spontaneous epileptic seizures upon certain olfactory stimuli indicates that at least one group III mGluR is critically involved in maintaining the delicate balance between neuronal inhibition and excitation (H. van der Putten, personal communication).
In conclusion, (R,S)-PPG represents a novel pharmacological tool to analyze the role of group III mGluRs in nervous system physiology and pathology, and our in vivo data support a neuroprotective and anticonvulsive role for group III mGluRs and encourage the search for systemically active group III mGluR agonists as promising drugs for the treatment of neurological disorders, such as Huntington’s disease and epilepsies.
Acknowledgments
We thank H. Allgeier for critically reading the manuscript and helpful discussion, R. Heckendorn for providing CGP 40116, H. van der Putten for sharing unpublished data, and P. Schoeffter for initial cAMP measurements with the HEK-hmGluR8a cell lines.
Footnotes
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Send reprint requests to: Dr. Peter J. Flor, K-125.6.08, Nervous System Research, Novartis Pharma AG, CH-4002 Basel, Switzerland. E-mail:peter-josef.flor{at}pharma.novartis.com
- Abbreviations:
- (1S,3R)-ACPD
- 1-aminocyclopentane-1S,3R-dicarboxylic acid
- CHO
- Chinese hamster ovary
- EGlu
- (2S)-α-ethylglutamic acid
- HEK
- human embryonic kidney
- iGluR
- ionotropic glutamate receptor
- l-AP4
- l-2-amino-4-phosphonobutyrate
- mGluR
- metabotropic glutamate receptor
- MSOP
- (R,S)-α-methylserine-O-phosphate
- PCR
- polymerase chain reaction
- PPG
- 4-phosphonophenylglycine
- MES
- maximal electroshock test
- GAD
- glutamate decarboxylase
- NMDA
- N-methyl-d-aspartic acid
- GABA
- γ-aminobutyric acid
- Received October 19, 1998.
- Accepted January 25, 1999.
- The American Society for Pharmacology and Experimental Therapeutics