Introduction

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The neurotoxicity of excitatory amino acids such as L-glutamate and some of its analogs, e.g., kainate and N-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 -amino acid moiety and a distal, negatively ionizable group (Watkins et al., 1990). These structural features are considered essential for L-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 -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 -amino acids, like L-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.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Chemical structures of phosphono-amino acid derivatives. (R,S)-PPG, D-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 40116), and two reference agonists for group III mGluRs, L-AP4 and L-SOP, are depicted. The carbon atom backbone of each structure is numbered in italics. All four compounds contain the amino acid function and a distal phosphonic acid. CGP 40116, a classical competitive NMDA receptor antagonist, and (R,S)-PPG have a considerably larger distance between the two acidic groups than the glutamic acid analogs L-AP4 and L-SOP.

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 like L-AP4 and L-SOP promote survival of rat cerebellar granule cells and protect cultured cortical and cerebellar neurons against toxic insults, such as prolonged -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 with L-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-dependent L-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

Top Abstract Introduction Experimental Procedures Results Discussion References

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 × 10⁵ plaques each of two human cDNA libraries from whole adult brain (in gt10; Clontech, Palo Alto, CA) and adult hippocampus (in ZAPII; 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 (gt10) or in vivo (ZAPII) 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).

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 10⁷ HEK293 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. The L-[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 nM L-[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 unlabeled L-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-Dependent L-[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 nM L-[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 -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).

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, and L-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

Top Abstract Introduction Experimental Procedures Results Discussion References

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; Table 1). 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).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Agonist activity of (R,S)-PPG at group III hmGluRs. Concentration-response curves for inhibition of forskolin (10 µM)-stimulated cAMP accumulation by (R,S)-PPG at CHO cells expressing hmGluR4a, hmGluR6, and hmGluR7b as well as HEK cells expressing hmGluR8a. Each data point represents mean value ± S.E.M. from at least three independent experiments (n  6). Sigmoidal curves were fit using the GraphPad Prism program (GraphPad Software, Inc., San Diego, CA).

View this table: [in this window] [in a new window]   TABLE 1 Activity of (R,S)-PPG on metabotropic and ionotropic glutamate receptors expressed in recombinant mammalian cells Concentration-response curve fitting and statistical analysis were done using the GraphPad Prism program (GraphPad Software, Inc.). EC50 values are given ± S.E.M. (n  3); nH, Hill coefficient.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Activity of (R,S)-PPG at group I, II, and III hmGluRs. A, inositol monophosphate formation of CHO cells expressing hmGluR1b and Ltk cells expressing hmGluR5a. Exposure to buffer was taken as control and set to 1.0; for submaximal and maximal stimulation of hmGluR1b, 20 µM quisqualate and 1000 µM quisqualate were used, respectively; 0.3 µM quisqualate gave submaximal stimulation of hmGluR5a, and 10 µM quisqualate showed the maximal effect. To test for agonist activity at group I mGluRs, (R,S)-PPG was applied at 500 µM to CHO-hmGluR1b and Ltk-hmGluR5a cells. Antagonist activity of 500 µM (R,S)-PPG at hmGluR1b and hmGluR5a was determined by coapplication of the submaximal concentrations of quisqualate (around EC80). Mean values ± S.E.M. from at least two independent experiments are shown (n  4). B, inhibition of forskolin-stimulated cAMP accumulation in CHO cells expressing either hmGluR2 or hmGluR4a. Forskolin (10 µM) stimulated cAMP formation about 40-fold (taken as control). All values are given as fraction of control. The effect of forskolin is inhibited by 30 µM (1S,3R)-ACPD in hmGluR2-expressing cells and by 1 µM L-AP4 in hmGluR4a-expressing cells. These submaximal agonist concentrations represent approximately EC80. To test for agonist activity, (R,S)-PPG was applied to forskolin-stimulated CHO-hmGluR2 and CHO-hmGluR4a cells. Antagonist activity of (R,S)-PPG at hmGluR2 and hmGluR4a was determined by coapplication of submaximal concentrations of (1S,3R)-ACPD and L-AP4, respectively. Columns represent mean values ± S.E.M. of at least two independent experiments (n  5). Asterisks indicate statistically significant antagonism or agonist activity of (R,S)-PPG (2P < .01; Dunnett's t test). C, inhibition of forskolin (10 µM)-stimulated cAMP accumulation in CHO cells expressing hmGluR7b and HEK cells expressing hmGluR8a. Forskolin-stimulated cAMP formation was taken as control. To obtain submaximal depression of the forskolin-stimulated cAMP accumulation, 500 µM L-AP4 and 1 µM L-AP4 were used for hmGluR7b and hmGluR8a, respectively (this represents about EC80). (R,S)-PPG was tested for agonist activity as well as antagonist activity of submaximal L-AP4. Asterisks indicate statistically significant agonist activity of (R,S)-PPG (2P < .01; Dunnett's t test, n  4).

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 the L-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 the L-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).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Lack of activity at NMDA-type ionotropic glutamate receptors by (R,S)-PPG. Elevation of cytoplasmic calcium ion concentrations, [Ca2+]i, in Ltk cells expressing cloned human NMDA receptors (subunit combination 1A/2B). A, increase in the fluorescent response ratio F340/F380 corresponds to an elevation in [Ca2+]i. Three single calcium measurements are shown: application of 1 µM L-glutamate (L-Glu) alone, coapplication of L-Glu (1 µM) and (R,S)-PPG (300 µM), and 300 µM (R,S)-PPG alone. The duration of the applications is indicated with a column. B, each column represents calcium signals (mean values ± S.E.M.) of at least six measurements of two independent experiments. The response of 1 µM L-Glu was taken as control and set to 100%. Asterisks indicate statistically significant antagonist activity (2P < .01; Dunnett's t test).

Ca²⁺/Cl-Dependent L-[³H]Glutamate Binding to Rat Brain Membranes. L-AP4, L-SOP, and (R,S)-PPG displaced L-[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 (Table 2). 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.

View this table: [in this window] [in a new window]   TABLE 2 Activity of group III mGluR agonists at a Ca2+/CI-dependent L-glutamate binding site of rat brain The affinities of L-AP4, L-SOP, and (R,S)-PPG as displacers of L-[3H]glutamate binding to rat hippocampal membranes were measured in the presence of calcium chloride as described (Urwyler et al., 1997b). The results shown are means ± S.E.M. from three independent experiments, each performed in triplicate; nH, Hill coefficient.

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 EC₅₀ value 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)--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)--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).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Protection of cultured cortical neurons against NMDA-induced excitotoxic degeneration by (R,S)-PPG. A, concentration-protection relationship of (R,S)-PPG against NMDA (100 µM)-induced degeneration in mixed cortical cultures assessed by extracellular lactate dehydrogenase activity (measured as mOD/min). The value measured with 100 µM NMDA was taken as 100%. To determine EC50 and Hill coefficient (nH), a sigmoidal curve was fit using the GraphPad Prism program (GraphPad Software, Inc.). B, neuronal degeneration in mixed cortical cultures was induced by 100 µM NMDA (resulting in approximately 85% of maximal NMDA toxicity) and assessed by extracellular lactate dehydrogenase activity (measured as mOD/min, set to 100%). Statistically significant neuroprotection by (R,S)-PPG is indicated by asterisks (2P < .01; Dunnett's t test, n  4, at least two independent experiments). (R,S)-PPG neuroprotection can be antagonized by MSOP but not by EGlu.

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 by L-AP4 (50 or 250 nmol, Fig. 7).

View larger version (126K): [in this window] [in a new window]   Fig. 6.   (R,S)-PPG protects against NMDA-induced striatal lesions in vivo. Nissl staining of 20-µm cryostat sections from rat caudate nucleus lateral to the site of NMDA (100 nmol) injection (A); note the large area of extensive necrotic damage at the left side of the micrograph (white arrowheads) and widespread neuronal pyknosis; black arrowheads point at two pyknotic neurones. When (R,S)-PPG (250 nmol) is coinjected with NMDA (B), necrotic damage and neuronal pyknosis is drastically reduced; open arrowheads indicate two healthy neurons. Also, note the higher neuronal density in B compared with A.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Quantitation of in vivo neuroprotection by (R,S)-PPG. Degeneration of GABAergic neurones in rat corpus striatum was induced by NMDA (100 nmol) injection and assessed by measuring the GAD activity. GAD activity of rat corpus striatum contralateral to the site of NMDA injection was taken as control and set to 100%. Each column represents mean values ± S.E.M. Statistically significant neuroprotection by (R,S)-PPG and L-AP4 is indicated by asterisks (2P < .01, Dunnett's t test; the number of tested animals in each group is indicated within the columns; for each group, at least two independent experiments were performed).

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, 1997 and references therein). In this test, with i.c.v. injections of L-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

Top Abstract Introduction Experimental Procedures Results Discussion References

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.

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).