Noncompetitive Metabotropic Glutamate5 Receptor Antagonist (E)-2-Methyl-6-styryl-pyridine (SIB1893) Depresses Glutamate Release through Inhibition of Voltage-Dependent Ca Entry in Rat Cerebrocortical Nerve Terminals (Synaptosomes)

The effect of (E)-2-methyl-6-styryl-pyridine (SIB1893), a selective metabotropic glutamate (subtype 5) receptor (mGlu5R) antagonist, on glutamate release from isolated nerve terminals (synaptosomes) was examined. SIB1893 caused a potent inhibition of the Ca -dependent release of glutamate evoked by 4-aminopyridine (4AP). That the implied mGlu5R-mediated modulation was contingent on diacylglycerol stimulation of protein kinase C (PKC) was indicated by PKC activator phorbol dibutyrate and PKC inhibitor Ro 32-0432 (bisindolylmaleimide XI), respectively, superceding or suppressing the inhibitory effect of SIB1893. The inhibitory action of SIB1893 was not due to it decreasing synaptosomal excitability or directly interfering with the release process at some point subsequent to Ca influx, because SIB1893 did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca ionophore ionomycin-induced glutamate release. Rather, examination of the effect of SIB1893 on cytosolic [Ca ] revealed that the diminution of glutamate release could be attributed to a reduction in voltage-dependent Ca influx. Consistent with this, the SIB1893-mediated inhibition of glutamate release was completely prevented in synaptosomes pretreated with a combination of the Nand P/Q-type Ca channel blockers, -conotoxin GVIA, and -agatoxin IVA. Together, these results suggest that noncompetitive antagonism of mGlu5Rs using SIB1893 effects a decrease in PKC activation, which subsequently attenuates the Ca entry through voltage-dependent Nand P/Q-type Ca channels to cause a decrease in evoked glutamate release. These actions of SIB1893 and related agents may contribute to their neuroprotective effects in excitotoxic injury. Metabotropic glutamate receptors (mGluRs) are a family of seven-transmembrane region, G protein-coupled receptors (GPCRs) having a variety of modulatory functions in neuronal excitability, neurotransmitter release, and synaptic plasticity in the central nervous system (CNS) (Pin and Duvoisin, 1995). Based on pharmacological profiles and signal transduction mechanisms, eight different mGluR subtypes have been catagorized into three groups: group I receptors (mGlu1 5R) being positively coupled to phospholipase C (PLC), with groups II (mGlu2 3R) and III (mGlu4/6/7 8R,) receptor both evincing negative coupling to adenylate cyclase (Conn and Pin, 1997). Group I mGluR activation leads to facilitation of excitatory synaptic neurotransmission in the CNS (McBain et al., 1994), an effect thought to be mediated by modulation of the probability of glutamate release through presynaptic mGluR activity (Conn and Pin, 1997). This is consistent with the presynaptic location of mGlu1 5R immunoreactivity in several brain regions such as cerebral cortex and hippocampus (Shigemoto et al., 1993; Romano et al., 1995), as well as functional studies showing an enhancement of glutamate release with group I mGluR activation (Herrero et al., 1992; Reid et al., 1999; Thomas et al., 2000; Fazal et al., 2003). The mechanism underlying this facilitation likely involves protein kinase C (PKC) activation by diacylglycerol (DAG) produced by receptor stimulated-PLC T.S.S. is supported by funding from the Wellcome Trust and Biotechnology and Biological Sciences Research Council. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.064881. ABBREVIATIONS: mGluR, metabotropic glutamate receptor; GPCR, G protein-coupled receptor; CNS, central nervous system; PLC, phospholipase C; PKC, protein kinase C; DAG, diacylglycerol; SIB1893, (E)-2-methyl-6-styryl-pyridine; 4AP, 4-aminopyridine; VDCC, voltage-dependent Ca channel; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; DiSC5(3), 3,3 -dipropylthiadicarbocyanine iodide; HBM, HEPES buffer medium; BSA, bovine serum albumin; [Ca ]c, cytosolic free Ca concentration; PDBu, phorbol dibutyrate; Ro 32-0432, bisindolylmaleimide XI; -CgTX, -conotoxin GVIA; -AgTX, -agatoxin IVA. 0022-3565/04/3093-951–958$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 309, No. 3 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 64881/1149323 JPET 309:951–958, 2004 Printed in U.S.A. 951 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from activity; however, the precise molecular targets of PKC in the nerve terminal underlying this regulation remain to be elucidated. Occurring inappropriately, group I mGluR autoreceptor-mediated facilitation at excitatory synapses may contribute to a number of pathological states, including ischemic brain damage, epilepsy, and neurodegenerative disorders. Consistent with this, a number of studies have been shown that group I mGluR activation can exacerbate neuronal injury both in vitro and in vivo (Agrawal et al., 1998; Bruno et al., 1995; Mukhin et al., 1997) and thus may be implicit in the pathogenesis of neuronal cell death produced by excessive glutamate release (Choi, 1992; Lipton and Rosenberg, 1994). This potential for group I mGluR-mediated excitoxicity therefore invokes the utility of antagonists of these receptors as neuroprotective agents. Indeed, antagonists of mGlu5Rs have recently been proposed to protect cultured cortical neurons against excitotoxic neuronal death (Bruno et al., 2000) and exert anticonvulsant activity in an in vivo model (Chapman et al., 2000). Whether the inhibition of glutamate release from nerve terminals underlies this neuroprotective action of mGlu5R blockade specifically is subject of debate given that the relative contributions of the two group I mGluRs (mGlu1R and mGlu5R) to the facilitation of glutamate remain contentious (Sistiaga et al., 1998; Reid et al., 1999; Fazal et al., 2003). Another connected issue arises from studies with isolated cerebrocortical nerve terminals (synaptosomes) reporting remarkably high PKC activity even in unstimulated conditions and in the effective absence of ligand (Coffey et al., 1994). This latter observation begs the question as to whether the high basal PKC activity reflects a basal, “constitutive” activity of mGluRs reported in the absence of ligand (Gasparini et al., 2002). The advent of newer subtype selective receptor antagonists for mGluRs, particularly those possessing noncompetitive activity at mGlu5Rs, have therefore led us to examine these issues. The isolated nerve terminal preparation is a well established model for studying the presynaptic regulation of neurotransmitter release by drugs in the absence of any complication of interpretation produced by concomitant postsynaptic effects. Using an established method for looking at endogenous glutamate release (Nicholls and Sihra, 1986), in the present study we characterized the effect SIB1893, a selective and noncompetitive mGlu5R antagonist, on the 4-aminopyridine (4AP)-evoked release of glutamate from cerebrocortical synaptosomes. We found that SIB1893 potently inhibits glutamate release in the absence of any endogenous or exogenous ligand. This effect seems to be through a reduction of voltage-dependent Ca channel (VDCC) activity and subsequent decrease of Ca influx into nerve terminals, rather than any upstream effect on nerve terminal excitability (Herrero et al., 1992; Herrero et al., 1994). We conclude the presence of a SIB1893-sensitive mGlu5R activity in nerve terminals, which through a mechanism involving PKC, modulates VDCCs coupled to glutamate exocytosis. Materials and Methods Materials. (E)-2-Methyl-6-styryl-pyridine (SIB1893) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) were obtained from Tocris Cookson Inc. (Bristol, UK). Fura-2-acetoxymethyl ester and DiSC3(5) were obtained from Molecular Probes (Eugene, OR). Percoll was obtained from Pharmacia (Peapack, NJ). Glutamate dehydrogenase and all other reagents were obtained from Sigma Chemical (Poole, Dorset, UK) or Merck (Poole, Dorset, UK). Synaptosomal Preparation. Synaptosomes were purified as described previously (Sihra, 1997). Briefly, the cerebral cortex from 2-month-old male Sprague-Dawley rats was isolated and homogenized in a medium containing 320 mM sucrose, pH 7.4. The homogenate was centrifuged at 3000g for 2 min (at 4°C), and the supernatant was centrifuged again at 14,500g for 12 min (at 4°C). The pellet was gently resuspended in 8 ml of 320 mM sucrose, pH 7.4. Two milliliters of this synaptosomal suspension was placed into 3-ml Percoll discontinuous gradients containing 320 mM sucrose, 1 mM EDTA, 0.25 mM DL-dithiothreitol, and 3, 10, and 23% Percoll, pH 7.4. The gradients were centrifuged at 32,500g for 7 min (at 4°C). Synaptosomes sedimenting between the 10 and the 23% Percoll bands were collected and diluted in a final volume of 30 ml of HEPES buffer medium (HBM) consisting of 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM MgCl2 6H2O, 1.2 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES (pH 7.4) before centrifugation at 27,000g for 10 min (at 4°C). The pellets thus formed were resuspended in 3 ml of HBM, and the protein content was determined by the Bradford assay. Aliquots of 0.5 mg of synaptosomal suspension were diluted in 10 ml of HBM and spun at 3000g for 10 min (at 4°C). The supernatants were discarded, and the synaptosomal pellets were stored on ice and used within 4 to 6 h. Glutamate Release Assay. Glutamate release was assayed by on-line fluorimetry as described previously (Nicholls and Sihra, 1986). Synaptosomal pellets were resuspended in HBM containing 16 M bovine serum albumin (BSA) and incubated in a stirred and thermostated cuvette maintained at 37°C in a PerkinElmer LS-50B spectrofluorimeter. NADP (2 mM), glutamate dehydrogenase (50 units/ml), and CaCl2 (1 mM) were added after 3 min. After 10 min of incubation, 4AP (3 mM) or ionomycin (5 M) was added to stimulate glutamate release. Glutamate release was monitored by measuring the increase of fluorescence (excitation and emission wavelengths of 340 and 460 nm, respectively) due to NADPH being produced by the oxidative deamination of released glutamate by glutamate dehydrogenase. Data were accumulated at 2-s intervals. A standard of exogenous glutamate (5 nmol) was added at the end of each experiment, and the fluorescence response was used to calculate released glutamate, expressed as nanomoles of glutamate per milligram of synaptosomal protein (nanomoles per milligram). Values quoted in the text represent levels of glutamate cumulatively release after 4-min depolarization, i.e., nanomoles per milligram per 4 min, unless indicated otherwise. Cumulative data were analyzed using Lotus 1-2-3 and MicroCal Origin. Statistical analysis was performed by two-tailed