The Anticonvulsant Gabapentin (Neurontin) Does Not Act through -Aminobutyric Acid-B Receptors

The actions of the anticonvulsant gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin] have been somewhat enigmatic until recently, when it was claimed to be a -aminobutyric acid-B (GABAB) receptor agonist acting exclusively at a heterodimeric complex containing the GABAB(1a) splice variant (Mol Pharmacol 2001;59:144–152). In this study, we have investigated the effects of gabapentin on recombinant GABAB(1a) and GABAB(1b) receptors coexpressed with GABAB(2) in five different functional recombinant assays, its ability to inhibit [H]GABA binding in a GABAB receptor-selective binding assay using rat synaptic membranes, and its ability to inhibit transient lower esophageal sphincter relaxations in Labrador retriever dogs. Up to a concentration of 1 mM, gabapentin displayed no agonistic effects on either the GABAB(1a,2) or the GABAB(1b,2) heterodimer, when these were expressed in Xenopus laevis oocytes or mammalian cells and assayed by means of electrophysiology, calcium mobilization, inositol phosphate, and fluorometry assays. Gabapentin did not displace [H]GABA from GABAB receptor sites in rat synaptic membranes. Finally, in contrast to the classic GABAB receptor agonist baclofen, gabapentin was unable to inhibit transient lower esophageal sphincter relaxations in dogs. Because of high levels of GABAB(1a) in the canine nodose ganglion, this finding indirectly supports the inactivity of gabapentin on the GABAB(1a,2) heterodimer demonstrated in various in vitro assays. In light of these results, we find it highly questionable that gabapentin is a GABAB receptor agonist. Hence, the anticonvulsive effects of the compound have to arise from GABAB receptor-independent mechanisms. This also implies that the first GABAB receptor splice variantselective ligand remains to be discovered. -Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system, where it exerts its effect through the ionotropic GABAA receptors and the metabotropic GABAB receptors. The GABAB receptors belong to the family C of the G-protein-coupled receptor superfamily (Möhler and Fritschy, 1999; Marshall et al., 2000). Two receptors, GABAB(1) and GABAB(2), have recently been cloned, and several splice variants of both receptors have been identified (Kaupmann et al., 1997, 1998; Jones et al., 1998; White et al., 1998; Pfaff et al., 1999; Billinton et al., 2001). GABAB(1) and GABAB(2) form heterodimers (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998). Gene knockout studies have shown that GABAB(1) is a necessary part for virtually all central GABAB receptors (Prosser et al., 2001; Schuler et al., 2001). The majority of the GABAB heterodimer complexes are either of a GABAB(1a,2) or a GABAB(1b,2) composition, and the two GABAB(1) splice variants differ in their expression pattern and their preand postsynaptic localization (Kaupmann et al., 1997; Benke et al., 1999; Poorkhalkali et al., 2000; Prosser et al., 2001; Schuler et al., 2001). Agonist binding to the GABAB(1,2) heterodimer has been demonstrated to take place in the amino-terminal domain of the GABAB(1) subunit (Galvez et al., 1999, 2000; Malitschek et al., 1999). The major part of this region shares a weak amino acid sequence similarity with a family of bacterial periplasmic binding proteins, as is the case for other family C A.A.J. and H.B.-O. were supported by the Danish Medical Research Council. T.L. was supported by Fundação de Amparo á Pesquisa do Estado de São Paulo (FAPESP). 1 Present address: Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland. ABBREVIATIONS: GABA, aminobutyric acid; VFT, Venus flytrap; TLESR, transient lower esophageal sphincter relaxation; AEBSF, 3,4-(2aminoethyl)benzenesulfonylfluoride; [Ca ]i, intracellular calcium concentration; FLIPR, fluorescence imaging plate reader; HBSS, Hanks’ buffered saline solution; CHO, Chinese hamster ovary; HEK, human embryonic kidney; IP, inositol phosphate; TC, Tris-calcium; HPLC, high-performance liquid chromatography; CGP54626, [S-(R*,R*)]-[3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl)phosphinic acid; Kir, inwardly rectifying potassium channel. 0026-895X/02/6106-1377–1384$7.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 6 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1419/986979 Mol Pharmacol 61:1377–1384, 2002 Printed in U.S.A. 1377 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from receptors such as the metabotropic glutamate receptors and the calcium-sensing receptor (O’Hara et al., 1993; Kaupmann et al., 1997; Bräuner-Osborne et al., 1999b). This “Venus flytrap” (VFT) region is believed to consist of two globular lobes, and the endogenous agonist binds to residues in the cleft between these lobes (Galvez et al., 1999, 2000; Kunishima et al., 2000). The VFT region is conserved in its entirety in all GABAB(1) splice variants. Thus, the only molecular difference between splice variants GABAB(1a) and GABAB(1b) is the presence of two “Sushi domains” upstream of the VFT region in GABAB(1a) that are not found in GABAB(1b) (Kaupmann et al., 1997). The human GABAB(1c) is characterized by the absence of the second Sushi domain compared with GABAB(1a) (Billinton et al., 2001). Gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin] is a frequently administered anticonvulsant that has been shown to prevent partial seizures and generalized tonicclonic seizures in epileptics (Taylor et al., 1998). Furthermore, the compound has displayed promising results in animal models of various forms of pain, amyotrophic sclerosis, bipolar disorder, and anxiety (Taylor et al., 1998). Although it was developed as an analog of GABA, gabapentin was originally claimed not to interact directly with either GABAA or GABAB receptors or with the high-affinity Na -dependent GABA transporters (Taylor, 1995; Taylor et al., 1998). Thus, the site of action of gabapentin has been somewhat of an enigma, although the compound displays high-affinity binding to the 2 subunit of a calcium channel (Gee et al., 1996; Marais et al., 2001). In contrast to the previous beliefs, Ng et al. (2001) recently claimed that gabapentin is, in fact, a GABAB receptor agonist. Furthermore, the authors postulated that gabapentin acts exclusively on the GABAB(1a,2) heterodimer complex and has no effect on the GABAB(1b,2) and GABAB(1c,2) heterodimers (Ng et al., 2001). Therefore, gabapentin is the first GABAB receptor splice variant-selective compound to be identified in a published study. The GABAB(1a,2) activity of gabapentin was supported by a follow-up study in which the compound was reported to inhibit the high K -evoked activation of voltage-dependent calcium channels in a murine mIL-tsA58 cell line that endogenously expresses the GABAB(1a,2) heterodimer (Bertrand et al., 2001). The proposed splice variant selectivity of gabapentin is quite remarkable, considering that the “Sushi domains” differentiating the GABAB(1) splice variants are located outside of the VFT region responsible for the binding of the endogenous agonist. Hence, to elucidate these observations further, we have characterized gabapentin pharmacologically at GABAB(1a,2) and GABAB(1b,2) heterodimers in five different functional recombinant assays, in a [H]GABA binding assay to rat synaptic membranes, and in a model measuring transient lower esophageal sphincter relaxations (TLESRs) in dogs. Experimental Procedures Materials. Culture media, serum, antibiotics, and buffers for cell culture were obtained from Invitrogen (Paisley, Scotland, UK). 3.4(2-Aminoethyl)benzenesulfonylfluoride (AEBSF) was obtained from Calbiochem (La Jolla, CA). The gabapentin used in the study was obtained from Sigma-Aldrich (St. Louis, MO) or from Goedecke/ Parke-Davis (Freiburg, Germany) (hereafter termed “Goedecke”), or it was extracted from Neurontin 100 capsules (Pfizer, New York, NY). myo-[2-H]Inositol and 4-amino-N-[2,3-H]butyric acid ([H]GABA) were obtained from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK and Uppsala, Sweden). Isoguvacine was purchased from Sigma/RBI (Natick, MA), and all other chemicals were obtained from Sigma-Aldrich. The tsA cells were a generous gift from Dr. Penelope S.V. Jones (University of California, San Diego, CA). Electrophysiology on Oocytes. Oocyte preparation and injection were done essentially as described previously (Mosbacher et al., 1998; Lingenhoehl et al., 1999). In brief, ovarian lobes containing oocytes were surgically removed from anesthetized (1.2 g/l 3-aminobenzoic acid ethyl ester) female Xenopus laevis frogs. Oocytes were separated and defolliculated by treatment with collagenase type II (Sigma-Aldrich) and with a subsequent incubation in 4 mM EGTA, pH 8.5. They were injected 3 h later with 10 to 50 ng of mRNA coding for either of the two rat splice variants GABAB(1a) or GABAB(1b) (Kaupmann et al., 1997), together with GABAB(2) and rat inwardly rectifying potassium channels (Kir3.1, Kir3.2, and Kir3.4), and incubated at 18°C for 3 to 8 days. Two-electrode voltage-clamp recordings were made with a Geneclamp 500 amplifier (Axon Instruments, Union City, CA) using electrodes filled with 3 M KCl. Oocytes were continuously perfused with normal frog Ringer (115 mM NaCl, 10 mM HEPES, 2.5 mM KCl, and 1.8 mM CaCl2, pH 7.2) or highpotassium Ringer (90 mM KCl, 27.5 mM NaCl, 10 mM HEPES, and 1.8 mM CaCl2, pH 7.2). The compounds were dissolved in water at 100 mM and perfused in high-potassium Ringer at the given concentrations. No corrections for changes in the osmolarity were performed. Currents were recorded using LabVIEW-based software (KooL; New Vision Engineering, Winterthur, Switzerland) and analyzed using Prism 3.0 software (GraphPad, San Diego, CA). Baseline current drifts were corrected using linear interpolations. For concentration-response curves, the induced inward current was measured 2 s before the application of the next higher concentration. Data from different oocytes were pooled. Values are mean S.E.M. Measurement of [Ca ]i by Fluorescence Imaging Plate Reader. For measurement of changes in intracellular calcium concentrations, human embryonic kidney (HEK) 293 cells were transiently transfected with rat or human GABAB(1a,2) or GABAB(1b,2). All transfections included G qzic to couple the receptors to phospholipase C (Franek et al., 1999) and were made as described in detail previously (Pagano et al., 2001). Transfected HEK 293 cells were plated into poly(D-lysine)-coated 96-well plates (BD Biosciences, San Jose, CA). At 24 to 72 h after transfection, cells were loaded for 45 min with 2 M fluo-4 acetoxymethyl ester (Molecular Probes, Eugene, OR) in Hanks’ buffered saline solution (HBSS) (Invitrogen, Basel, Switzerland) containing 50 M probenecid (Sigma-Aldrich, Buchs, Switzerland). Plates were washed twice in HBSS and transferred to a FLIPR (Molecular Devices, Crawley, UK). Fluorescence was measured at room temperature for 3 min after the addition of agonists. Relative fluorescence changes over baseline ( F/F) were determined. Concentration-response curves were recorded with three to eight wells per concentration and experiment, and the data were pooled and fitted using Igor Pro (Wavemetrics, Lake Oswego, OR) with a logistic equation using weighted nonlinear regression. The abilities of GABA and gabapentin to evoke changes in the intracellular calcium concentrations were also measured in Chinese hamster ovary (CHO-K1) cells stably expressing GABAB(1a) and GABAB(2) and in CHO-K1 cells stably expressing a GABAB(1a)-G q-i5 fusion protein and GABAB(2). Fusion proteins of G-protein–coupled receptors and G-proteins have been demonstrated in numerous studies to exhibit pharmacological profiles similar to those of the wildtype receptors (Seifert et al., 1999). The cells were assayed in a FLIPR (Molecular Devices, Menlo Park, CA), using procedures similar to those described above. Calcium Measurements by Fluorometer. HEK 293 cells (1.5 10) were electroporated (250 V, 300 F; Gene Pulser; Bio-Rad, Hercules, CA) with 5 g of rat GABAB(1a) or GABAB(1b) cDNA with 1378 Jensen et al. at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from GABAB(2) and G qzic (Franek et al., 1999) cDNAs in a total volume of 150 l of buffer (50 mM K2HPO4, 20 mM CH3COOK, and 20 mM KOH, pH 7.4). Transfected cells were resuspended in culture medium and plated on poly(D-lysine)-coated glass coverslips. Twentyfour hours after transfection, the cells were incubated at room temperature for 1 h in a HEPES buffer, pH 7.6 (Invitrogen), containing the calcium indicator fura-2 acetoxymethyl ester (10 g/ml), 0.5% Pluronic F-127 (Molecular Probes), and 1% (v/v) dimethyl sulfoxide. Glass coverslips carrying dye-loaded cells were mounted into a perfusing cuvette (2 ml/min) in a fluorescence spectrophotometer (F4500; Hitachi, Yokohama, Japan). Changes in [Ca ]i were recorded as the fluorescence ratio at 380 nm versus that at 360 nm. The viability of transfected cells was tested by application of 10 M ATP. Inositol Phosphate Assay. The tsA cells [a transformed HEK 293 cell line (Chahine et al., 1994)] were maintained at 37°C in a humidified 5% CO2 incubator in Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, and 10% calf serum. tsA cells (3 10) were plated in a 6-cm tissue culture plate and transfected the following day with 0.25 g of GABAB(1a)-pCDNA3.1 or GABAB(1b)-pCDNA3.1, 1.2 g of GABAB(2)pCDNA3.1, and 0.25 g of G q-z5-pCDNA3.1, using Polyfect as DNA carrier according to the protocol of the manufacturer (QIAGEN GmbH, Hilden, Germany). The day after transfection, the cells were transferred to 24 wells in a 96-well cluster plate in growth medium containing 2 Ci/ml myo-[2-H]inositol. After 16 to 20 h, the cells were washed in HBSS and incubated for 20 min in HBSS supplemented with 0.9 mM CaCl2 and 1.05 mM MgCl2. The cells were then incubated for another 20 min in phosphate-buffered saline supplemented with 0.9 mM CaCl2, 1.05 mM MgCl2, and 10 mM LiCl. Finally, the cells were incubated for 40 min in phosphate-buffered saline supplemented with 0.9 mM CaCl2, 1.05 mM MgCl2, 10 mM LiCl, and various concentrations of GABA and gabapentin (from Sigma-Aldrich or Goedecke). The reactions were stopped by exchanging the buffer with 200 l of ice-cold 20 M formic acid, and separation of total [H]inositol phosphates was carried out by ion-exchange chromatography as described previously (Nanevicz et al., 1996; Bräuner-Osborne et al., 1999a). [H]GABA Filtration Binding Assay. Rat synaptic membranes were prepared using the method described by Zukin et al. (1974), with some modifications. Whole brains from Sprague-Dawley male rats (about 300 g) were homogenized in 10 volumes of ice-cold buffer containing 0.32 M sucrose, 10 mM Tris, 0.1 mM AEBSF, and 20 g/ml bacitracin, pH 7.4. The homogenate was centrifuged at 1,000g for 10 min, and the supernatant was then centrifuged at 20,000g for 20 min. The pellet was resuspended (by vortex) in 6 volumes of ice-cold distilled water containing 0.1 mM AEBSF and 20 g/ml bacitracin (pH set to 7.4), and centrifuged at 8,000g for 20 min. The supernatant and the upper layer of the pellet were centrifuged at 33,000g for 20 min. The pellet was resuspended in 6 volumes of 50 mM Tris, pH 7.4, containing 1 mM AEBSF and 20 g/ml bacitracin and centrifuged at 33,000g for 20 min. The last centrifugation step was repeated one more time, and finally the pellet was snap-frozen in methanol/dry ice and stored overnight at 70°C. The frozen pellet was thawed and washed six times in 6 volumes of 50 mM Tris, pH 7.4, by centrifugation at 8,000g for 10 min at 18°C. The resulting pellet was resuspended in TC buffer (50 mM Tris/2.5 mM CaCl2, pH 7.4), snap-frozen in methanol/dry ice, and stored at 70°C. Membranes to be used in the radioligand binding assay were further treated (washed) as follows. The membranes were thawed in lukewarm water followed by resuspension in TC buffer and homogenization using a Polytron PT 3000 from Kinematika AG (Basel, Switzerland) five times for 5 s each. The membranes were washed three times in TC buffer by centrifugation at 8,000g for 10 min, resuspended in TC buffer, and homogenized 10 times in a Teflon/glass homogenizer. The membranes were suspended in aliquots, snapfrozen in methanol/dry ice, and stored at 70°C. Protein concentration was determined according to the method of Bradford (1976) using the Bio-Rad protein assay kit with bovine -globulin as a standard. The [H]GABA competition assay, modified from the method of Olpe et al. (1990), was performed in 96-well plates in 200 l of TC-isoguvacine buffer (TC buffer supplemented with 40 M isoguvacine to saturate the GABAA receptor sites) containing 20 nM [H]GABA (3.48 TBq/mmol), 80 l of test compound (at indicated concentrations, diluted in water), and 80 g of synaptic membrane (diluted in TC-isoguvacine buffer) and incubated for 10 min at room temperature before addition). The mixture was then incubated on a microplate shaker (Denley Instruments, Billinghurst-West Sussex, UK) for 20 min at room temperature, followed by rapid filtration through a glass fiber filter (Printed Filtermat B filters; PerkinElmer Wallac, Gaithersburg, MD) that had been presoaked in 0.3% polyethyleneimine, followed by a wash with TC buffer using a Tomtec cell harvester (Tomtec, Orange, CT). The filters were dried in a microwave at maximal effect for 1.5 min followed by incubation at 55°C for 45 min. MeltiLex B/HS scintillation sheets from PerkinElmer Wallac (Turku, Finland) were melted onto the filter, and radioactivity was determined in a Microbeta scintillation counter (PerkinElmer Wallac). Measurements of TLESRs in Dogs. TLESRs were measured in adult Labrador retriever dogs using Dentsleeve manometry as described previously (Lehmann et al., 1999). Gabapentin (20 mg/kg) was administered directly into the stomach through the manometric assembly. Thirty minutes after administration, TLESRs were stimulated by liquid nutrient infusion and air insufflation and quantitated during a 45-min period. The effect of the compound was compared with the average of the five preceding control experiments for each individual dog. Gabapentin used for these experiments was obtained from Neurontin capsules (Pfizer), and a suspension was made in 0.9% NaCl immediately before the experiment. Chemical Analyses of Gabapentin. Melting points of the gabapentin samples obtained from Sigma-Aldrich and Goedecke were determined in capillary tubes. H NMR spectra of both samples were recorded on a 300-MHz Varian Gemini-2000 BB spectrometer (Varian, Palo Alto, CA) in CD3OD using the solvent residual peak as internal standard. Elemental analyses of the same gabapentin samples were performed at the Department of Physical Chemistry, University of Vienna, Austria, and were within 0.4% of the theoretical values for zwitterionic gabapentin. HPLC analyses of the gabapentin sample from Sigma-Aldrich were performed on a Knauer Vertex Spherisorb ODS2 column (5 m, 4.0 120 mm) using a TSP HPLC system (Bie & Berntsen A/S, Copenhagen, Denmark) consisting of a P2000 pump, an AC 3000 autoinjector, and an SM 5000 PDA detector. The column was eluted at 1.0 ml/min with aqueous trifluoroacetic acid, pH 2.0.