Felbamate Increases [H]Glycine Binding in Rat Brain and Sections of Human Postmortem Brain

The anticonvulsant compound felbamate (2-phenyl-1,3-propanediol dicarbamate; FBM) appears to inhibit the function of the N-methyl-D-aspartate (NMDA) receptor complex through an interaction with the strychnine-insensitive glycine recognition site. Since we have demonstrated previously that FBM inhibits the binding of [H]5,7-dichlorokynurenic acid (DCKA), a competitive antagonist at the glycine site, we assessed the ability of FBM to modulate the binding of an agonist, [H]glycine, to rat forebrain membranes and human brain sections. In contrast to its ability to inhibit [H]5,7-DCKA binding, FBM increased [H]glycine binding (20 nM; EC50 5 485 mM; Emax 5 211% of control; nH 5 1.8). FBM, but not carbamazepine, phenytoin, valproic acid or phenobarbital, also increased [H]glycine binding (50 nM; EC50 5 142 mM; Emax 5 157% of control; nH 5 1.6) in human cortex sections. Autoradiographic analysis of human brain slices demonstrated that FBM produced the largest increases in [H]glycine binding in the cortex, hippocampus and the parahippocampal gyrus. Because various ions can influence the binding of glycine-site ligands, we assessed their effects on FBM-modulation of [H]glycine binding. FBM-enhanced [H]glycine binding was attenuated by Zn and not inhibited by Mg in human brain. These results suggest that FBM increases [H]glycine binding in a manner sensitive to ions which modulate the NMDA receptor. These data support the hypothesis that FBM produces anticonvulsant and neuroprotective effects by inhibiting NMDA receptor function, likely through an allosteric modulation of the glycine site. The dicarbamate compound FBM (2-phenyl-1,3-propanediol dicarbamate) has been shown to have anticonvulsant activity in several animal seizure models including maximal electroshock-, pentylenetetrazoland picrotoxininduced seizures in rodents (Swinyard et al., 1986; Coffin et al., 1994), and focal seizures induced by aluminum hydroxide injection into preand postcentral gyri in rhesus monkeys (Perhach et al., 1986). Moreover, FBM is efficacious in the treatment of human seizures including partial complex seizures and Lennox-Gastaut syndrome (Bourgeois et al., 1993; Burdette et al., 1992; Faught et al., 1993; Leppik et al., 1991; Sachdeo et al., 1992; The Felbamate Study Group in LennoxGastaut Syndrome, 1993; Theodore et al., 1991). Although the primary mechanism of action has not been firmly established, the neuropharmacological profile of this compound appears to be distinct from more classic anticonvulsant medications (McCabe et al., 1993; Porter, 1989; Rho et al., 1994; Sofia et al., 1991; White et al., 1992). FBM appears to act as a functional antagonist of the NMDA receptor-ionophore complex, a multi-subunit heterooligomer (Kutsuwada et al., 1992) with multiple, allosterically coupled recognition sites for glutamate, glycine, polyamines, ions and use-dependent channel blockers (for review see McBain and Mayer, 1994). Furthermore, converging lines of evidence have shown that FBM may produce this effect through an interaction with the strychnine-insensitive glycine recognition site of the NMDA receptor. First, FBM reduces the increase in intracellular [Ca]i stimulated by NMDA and glycine (Taylor et al., 1995; White et al., 1995). Second, the anticonvulsant effects of FBM in mice are reversed by glycine (Coffin et al., 1994; De Sarro et al., 1994) and D-serine, a glycine site agonist (De Sarro et al., 1994; White et al., 1995). Third, FBM acts as a neuroprotectant (Wasterlain et al., 1992; Wallis et al., 1992) in a glycine reversible fashion (Wallis and Panizzon, 1993). Fourth, FBM is capable of inhibiting the binding of DCKA, a high affinity glycine site antagonist, in membranes from both rat (McCabe Received for publication August 8, 1997. ABBREVIATIONS: 5,7-DCKA, 5,7-dichlorokynurenic acid; CARB, carbamazepine; dentate gyr, dentate gyrus; FBM, felbamate; NMDA, N-methyD-aspartate; para. gyr., parahippocampal gyrus; PHEN, phenobarbital; PHNY, phenytoin; pyr, stratum pyramidale; rad, stratum radiatum; VAL, valproic acid. 0022-3565/98/2862-0991$00.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 286, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 286:991–999, 1998 991 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from et al., 1993) and human brain (Wamsley et al., 1994). Because occupancy of both the glycine and glutamate sites are necessary for NMDA-channel opening, and agonist activation of these sites causes neuronal depolarization (Monaghan et al., 1989), inhibition of glycine action at strychnine-insensitive glycine receptors would be expected to result in a decreased frequency of channel opening and a reduction of neuronal activation (Wong and Kemp, 1991). These observations strongly support the hypothesis that the anticonvulsant activity of FBM involves an action at the strychnine-insensitive glycine recognition site of the NMDA receptor. Because our previous studies (McCabe et al., 1993) have demonstrated that FBM inhibits the binding of the competitive antagonist [H]5,7-DCKA, we assessed the ability of FBM to modulate the binding of an agonist, [H]glycine, to rat forebrain membranes and human brain sections. In addition, since various ions are capable of influencing the binding of glycine-site ligands, we assessed the effects of several ions on the modulation of [H]glycine binding by FBM. We now report that, in contrast to its ability to inhibit [H]5,7DCKA binding, pharmacologically relevant concentrations of FBM increase [H]glycine binding to both rat membrane homogenates and human cortical sections, and furthermore that this increase in binding is sensitive to ions which modulate the NMDA receptor. Materials and Methods Rat tissue preparation. Membranes were prepared from forebrains of male Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 150 to 200 g. Animals were housed under a 12-hr light/dark cycle (lights on 0700) with access to food and water ad libitum. Animals were anesthetized with CO2 and killed by decapitation. Tissues were assayed according to previously described methods (McCabe et al., 1993). Brains were immediately removed and forebrains rapidly dissected, weighed and placed in 10 volumes (original wet weight:volume) of 5 mM HEPES/4.5 mM Tris buffer (HTS; pH 7.8 at room temperature) containing 0.32 M sucrose. Tissue preparation was performed at 4°C. Tissues were homogenized by 8 to 10 passes of a motor-driven Teflon pestle in a glass tube, diluted to 50 volumes in HTS-sucrose, and centrifuged at 1,000 3 g for 10 min. The pellet (P1) was discarded and supernatant centrifuged at 20,000 3 g for 20 min. The pellet (P2) was resuspended in HTS using a Brinkmann Polytron (setting 6, 5 sec) and centrifuged at 8000 3 g for 20 min. Subsequently, the supernatant and outer buffy coat (remaining pellet core discarded) was centrifuged at 20,000 3 g for 20 min. The resultant pellet (P2/P3) was resuspended in HTS containing 1 mM EDTA and centrifuged at 20,000 3 g for 20 min. The P2/P3 pellet was resuspended in HTS and the “washing” procedure repeated two more times. The P2/P3 pellet was then resuspended in 5 volumes of HTS, frozen on dry-ice and stored at -80°C for at least 72 hr before binding assay. [H]Glycine binding to rat brain membrane homogenates. Radioligand binding assays using [H]glycine were performed as described (Baron et al., 1991) with minor modifications. On the day of the assay, the tissue was washed twice by resuspension (50 mM HEPES-KOH buffer, pH 7.4 at 4°C) and centrifugation at 20,000 3 g for 20 min. Assays were performed at 4°C using quadruplicate samples in a total volume/tube of 0.5 ml consisting of: 250 ml membrane suspension (200 mg protein/assay tube) in 50 mM HEPESKOH buffer (pH 7.4 at 4°C), 50 ml [H]glycine (20 nM; 48.4 Ci/mmol; Du Pont/NEN, Boston, MA) solution, 50 ml drug or buffer and 150 ml buffer. FBM (10 mM stock) was dissolved 75% dimethylsulfoxide, and serial dilutions in buffer were prepared for assay. Pilot experiments revealed that this concentration of dimethylsulfoxide did not interfere with the assay (data not shown). Glycine or D-serine (100 mM) were used to define nonspecific binding. The ability of FBM to modulate [H]glycine (20 nM) binding was also studied in rat lung tissue (prepared as described for rat forebrain) or heat-inactivated rat forebrain tissue (heated to 70°C for 60 min). Binding reactions were terminated after 60 min by centrifugation (20,000 3 g for 20 min at 4°C). The supernatants were aspirated and pellets rinsed with 2 3 1-ml aliquots of ice-cold buffer. The pellets were solubilized in Solvable (25 ml; Packard Instrument Co., Meriden, CT). Scintillation cocktail (4 ml; F989, Du Pont/NEN) was added, and radioactivity measured in a Beckman LS 5801 liquid scintillation counter. Where appropriate, radioligand binding data were analyzed via iterative curve fitting software (GraphPad Prism Version 1.03, San Diego, CA). [H]Glycine binding to slices of human brain. Dissected blocks of human brain were obtained from the brain bank at the University of Auckland. These tissues were kept frozen at 270°C. For binding studies, blocks of cerebral cortex (middle temporal gyrus) from two female individuals (ages 62 and 66 yr with postmortem times of 14 and 15.5 hr) were thawed, pooled and lightly homogenized. These cortical tissues were placed in small cylindrical centrifuge tubes and refrozen. The frozen cylinders of tissue were cut into sections (18 mm in thickness) in a cryostat and thaw-mounted onto microscope slides. Binding of [H]glycine to these sections was accomplished as previously described (McDonald et al., 1990). Sections were preincubated for 30 min (2 3 15 min) in ice-cold 50 mM Tris-citrate buffer (pH 7.4). The slides were then transferred to coplin jars containing the same buffer plus 50 nM [H]glycine (45.1 Ci/mmol, Du Pont NEN) for a 35-min incubation period followed by a dip in fresh buffer (without radioactivity), three rinses (1 sec each) and a final dip in distilled water. The sections were wiped from the slides with microfiber glass filter discs and the radioactivity was determined by liquid scintillation counting as described above. The effect of various concentrations of FBM (10-10 M) on the binding of [H]glycine under these conditions was determined. Nonspecific binding was detected by incubating sections in the presence of 100 mM unlabeled glycine. The ability of several other anticonvulsant drugs (300 mM; carbamazepine, phenobarbital, phenytoin and valproic acid) to modify the binding of [H]glycine was assessed and compared to that of 300 mM FBM. The ability of various ions to modify the modulation of [H]glycine binding by FBM (300 or 600 mM) was examined by including 10 mM concentrations of various salts [MgSO4, MgCl2, CaCl2, CaPO4, NaCl, KOH, Ca(NO3)2, ZnCl2 and Zn(C2H3O2)2] in the binding assay. Several experiments were also performed to examine the potential contribution of glycine uptake sites to the binding of [H]glycine. Slices were preloaded with glycine agonists by preincubation in the presence of 100 mM glycine, D, L-serine or D-serine. In addition, incubation of sections (with [H]glycine and 600 mM FBM) with and without 100 mM taurine was performed. Autoradiographic localization of [H]glycine binding was performed in sections of human brain from five male individuals (no history of neurological disease) ages 38 to 66 yr with postmortem time to freezing varying from 7.5 to 15.5 hr. These tissues we cut in a cryostat, thaw-mounted onto microscope slides and incubated under the same conditions described above. The incubations were performed in the presence of [H]glycine alone or the addition of 600 mM FBM or 100 mM carbamazepine, phenytoin, valproic acid or phenobarbital. Nonspecific binding was detected by incubating sections in the presence of 100 mM unlabeled glycine. The labeled sections were rapidly dried, desiccated overnight, and placed against sheets of tritium sensitive film (Hyperfilm, Amersham Corp., Arlington Heights, IL). After a 2mo exposure period, the films were developed and analyzed on a MCID imaging system (Imaging Research, Inc.; St. Catharines, Ontario, Canada). Optical densities from each region were compared with that of standards (Microscales, Amersham) for determination of the femtomoles of ligand bound per milligram of tissue. 992 McCabe et al. Vol. 286 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from