Neuropharmacology and AnalgesiaMetabotropic glutamate mGlu5 receptor-mediated serine phosphorylation of NMDA receptor subunit NR1 in hippocampal CA1 region after transient global ischemia in rats
Introduction
Ionotropic glutamate receptors of both the N-methyl-d-aspartate (NMDA) and the non-NMDA type play pivotal roles in excitatory neuronal transmission for neuronal development, as well as for learning and memory. These receptors are also involved in excitotoxic cell death in several types of neurodegenerative diseases. Particularly, the excessive Ca2+ influx via the NMDA receptor has been implicated in the pathogenesis of cerebral ischemia. The NMDA receptors are heteromeric proteins that consist of complexes of NR1 and NR2 or in some cases NR3 subunits, and their functions are regulated by phosphorylation. For example, protein kinase C (PKC) phosphorylates 2 residues (S890 and S896) in the intracellular C-terminal domain of the NR1 subunit and regulates NMDA receptor-mediated currents (Chen and Huang, 1992). It is likely that phosphorylation of NR1 induced by PKC also regulates surface expression of the receptor (Lan et al., 2001, Scott et al., 2003, Scott et al., 2001). Therefore, changes in phosphorylation of this subunit may be involved in the pathogenesis of cerebral ischemia, as phosphorylation of the NR1 subunit is altered in cerebral ischemia (Cheung et al., 2001, Cheung et al., 2003).
In addition to ionotropic glutamate receptors, metabotropic glutamate (mGlu) receptors also play a key role in the regulation of several physiological responses in the central nervous system. mGlu receptors are divided into 3 groups on the basis of their sequence homology, intracellular signal transduction, and pharmacological properties (Nakanishi, 1992, Pin and Duvoisin, 1995, Schoepp and Conn, 1993). Group I mGlu (mGlu1 and mGlu5) receptors preferentially couple with Gq-protein to activate phospholipase C (PLC) and localize to the perisynapse at the postsynaptic membrane of glutamatergic synapses (Lujan et al., 1996). Excessive release of glutamate induced by brain ischemia may stimulate group I mGlu receptors localized at the perisynapse and induce pathophysiological alterations in intracellular signaling. As PKC regulates the ion channel activities of the NMDA receptor by phosphorylation of NR1 subunits, group I mGlu receptors can be involved in regulating NMDA receptor function. Indeed, NMDA current in hippocampal and subthalamic nucleus cells is enhanced by the activation of group I mGlu5 receptor (Awad et al., 2000, Benquet et al., 2002, Mannaioni et al., 2001).
Although the stimulation of group I mGlu receptors in the ischemic brain may contribute to the enhancement of phosphorylation of the NMDA receptor and its activity, which may lead to cell damage, mGlu5 receptor-mediated phosphorylation of the NR1 subunit after brain ischemia remains to be determined. Cells in the hippocampal CA1 region, which is vulnerable to ischemic insults, preferentially express mGlu5 receptors (Ferraguti et al., 1998, Lujan et al., 1996, Lujan et al., 1997). The NMDA receptor of the postsynaptic density (PSD) has been shown to be largely insoluble in 1% Triton X-100 (Blahos and Wenthold, 1996). In addition, the deoxycholate (DOC)-insoluble fraction is enriched in proteins derived from the postsynaptic density (Matus and Taff-Jones, 1978, Takagi et al., 2000). These findings suggest that synaptic NMDA receptors could be fractionated into the DOC-insoluble fraction. The purpose of the present study was to determine if there was mGlu receptor-mediated pathophysiological alteration of the NMDA receptor NR1 subunit after cerebral ischemia. Thus, we also examined the effect of the mGlu5 receptor antagonist on phosphorylation of the NR1 subunit in the DOC-insoluble membrane fraction. The results show that the administration of the mGlu5 receptor antagonist attenuated transient global ischemia-induced phosphorylation at S890 of the NR1 subunit of the NMDA receptor, which attenuation was accompanied by a decrease in the amount of PKCγ in the deoxycholate-insoluble membrane fraction.
Section snippets
Procedure for animal surgery
Male Wistar rats, weighing between 200 and 250 g (Charles River Japan Inc., Atsugi, Japan), were housed in a cage and had free access to food and water according to the Guidelines of Experimental Animal Care issued by the Prime Minister's Office of Japan. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to use alternatives to in vivo techniques, if available. The experimental protocol was approved by the Committee of Animal Care and Use of Tokyo
Time course of changes in serine phosphorylation of NR1 subunit after transient global ischemia
First, we examined the effect of the mGlu5 receptor antagonist MPEP on cell death in the hippocampal CA1 region after 72 h of reperfusion (Fig. 1). The injection of 30 or 300 nmol MPEP prevented significantly the cell loss in the CA1 region (P ≤ 0.05). The average number of cells in the naïve control, saline-treated, and 3, 10, 30, and 300 nmol MPEP-treated ischemic groups was 497.0 ± 14.5, 19.9 ± 5.6, 34.5 ± 4.7, 118.7 ± 81.8, 406.7 ± 14.1, and 339.3 ± 17.7, respectively (n = 6 for each group). Based on these
Discussion
In the present study, we demonstrated that transient global ischemia enhanced serine phosphorylation of the NR1 subunit at 1 h after the start of the reperfusion and that this increase was maintained for at least 24 h. It has been suggested that NMDA receptor function is regulated by PKC-mediated phosphorylation, which leads to increases in surface expression of the receptor and the opening rate of receptor channels (Lan et al., 2001, Lu et al., 1999). In contrast, the activation of PKC in
Acknowledgements
This work was supported by a grant from the program Grants-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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