Satoshi Hayashibe

Diversity of Calcium Signaling by Metabotropic Glutamate Receptors

During prolonged application of glutamate (20 min), patterns of increase in intracellular Ca2+concentration ([Ca2+] i ) were studied in HEK-293 cells expressing metabotropic glutamate receptor, mGluR1α or mGluR5a. Stimulation of mGluR1α induced an increase in [Ca2+] i that consisted of an initial transient peak with a subsequent steady plateau or an oscillatory increase in [Ca2+] i . The transient phase was largely attributed to Ca2+mobilization from the intracellular Ca2+ stores, but the sustained phase was solely due to Ca2+ influx through the mGluR1α receptor-operated Ca2+ channel. Prolonged stimulation of mGluR5a continuously induced [Ca2+] i oscillations through mobilization of Ca2+ from the intracellular Ca2+ stores. Studies on mutant receptors of mGluR1α and mGluR5a revealed that the coupling mechanism in the sustained phase of Ca2+ response is determined by oscillatory/non-oscillatory patterns of the initial Ca2+response but not by the receptor identity. In mGluR1α-expressing cells, activation of protein kinase C selectively desensitized the pathway for intracellular Ca2+ mobilization, but the mGluR1α-operated Ca2+ channel remained active. In mGluR5a-expressing cells, phosphorylation of mGluR5a by protein kinase C, which accounts for the mechanism of mGluR5a-controlled [Ca2+] i oscillations, might prevent desensitization and result in constant oscillatory mobilization of Ca2+ from intracellular Ca2+ stores. Our results provide a novel concept in which oscillatory/non-oscillatory mobilizations of Ca2+ induce different coupling mechanisms during prolonged stimulation of mGluRs.

Neuroprotective effects of the selective type 1 metabotropic glutamate receptor antagonist YM-202074 in rat stroke models

We describe in vitro properties and in vivo neuroprotective effects of a newly synthesized, high-affinity, selective allosteric metabotropic glutamate receptor type 1 (mGluR(1)) antagonist, N-cyclohexyl-6-{[(2-methoxyethyl)(methyl)amino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-202074). YM-202074 bound an allosteric site of rat mGluR(1) with a K(i) value of 4.8+/-0.37 nM. YM-202074 also inhibited the mGluR(1)-mediated inositol phosphates production in rat cerebellar granule cells with an IC(50) value of 8.6+/-0.9 nM, while showing selectivity over mGluR(2-7). When YM-202074 was infused intravenously at an initial dose of 20 mg/kg/h for 0.5 h followed by a dose of 5 mg/kg/h for 7.5 h, the free concentration of YM-202074 in the brain rapidly (<12 min) reached approximately 0.3 microM, reaching a steady-state phase within 1.5 h. We first treated rats such that they developed transient middle cerebral artery (MCA) occlusion. Results clearly demonstrate a dose-dependent improvement of neurological deficit and reduction of the infarct volume in both the hemisphere and cortex when YM-202074 was infused intravenously immediately after occlusion at a dose of 10 or 20 mg/kg/h for 0.5 h followed by a dose of 2.5 or 5 mg/kg/h for 23.5 h, respectively. Significant neuroprotection was maintained even when the administration of drugs was delayed by up to 2 h following the onset of ischemia. Furthermore, the improvement of neurological deficit and the reduction of infarct volume were sustained for 1 week following the onset of ischemia. These results suggest that YM-202074 exhibits great potential as a novel neuroprotective agent for the treatment of stroke.