Abstract
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 Ca2+-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 Ca2+ influx, because SIB1893 did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca2+ ionophore ionomycin-induced glutamate release. Rather, examination of the effect of SIB1893 on cytosolic [Ca2+] revealed that the diminution of glutamate release could be attributed to a reduction in voltage-dependent Ca2+ influx. Consistent with this, the SIB1893-mediated inhibition of glutamate release was completely prevented in synaptosomes pretreated with a combination of the N- and P/Q-type Ca2+ channel blockers, ω-conotoxin GVIA, and ω-agatoxin IVA. Together, these results suggest that noncompetitive antagonism of mGlu5Rs using SIB1893 effects a decrease in PKC activation, which subsequently attenuates the Ca2+ entry through voltage-dependent N- and P/Q-type Ca2+ 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.
Footnotes
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T.S.S. is supported by funding from the Wellcome Trust and Biotechnology and Biological Sciences Research Council.
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DOI: 10.1124/jpet.103.064881.
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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 Ca2+ channel; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; DiSC5(3), 3,3′-dipropylthiadicarbocyanine iodide; HBM, HEPES buffer medium; BSA, bovine serum albumin; [Ca2+]c, cytosolic free Ca2+ concentration; PDBu, phorbol dibutyrate; Ro 32-0432, bisindolylmaleimide XI; ω-CgTX, ω-conotoxin GVIA; ω-AgTX, ω-agatoxin IVA.
- Received December 23, 2003.
- Accepted February 20, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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