A Study for the Expression of the N-Methyl-D-Aspartate (NMDA) Subunit 2A(NR2A) and 2B(NR2B) of Rat Hippocampal Slices in the Hypoxic State

ABSTRACT

PURPOSE: Excessive amounts of glutamate are released into the extracelluar space during hypoxia/ischemia and contribute to neuronal injury through overactivation of the NMDA receptors. It will be expected that the changes of the NMDA receptors to decrease the injury are developed as a kind of defense mechanism. The NMDA receptors are composed of NR1 and NR2, and there are four NR2 subunits; NR2A, NR2B, NR2C, and NR2D. The NR1 is essential for the function of the NMDA receptors and multiple NR2 subunits potentiate and differentiate the function of the NMDA receptors. The NR2 subunits alone show no ability to respond to several agonists, however when coexpressed with the NR1, the NR2 subunits markedly potentiated the NR1 activity and produced functional variability of the NMDA receptors. Compared with the NR2C and the NR2D, the NR2A and the NR2B show prominent expression in the hippocampus. In an in vitro model of rat hippocampal slices after hypoxia, we investigated the changes of the NR2A and the NR2B amounts through immunoblot with anti-NR2A and anti-NR2B antisera. METHODS: Hippocampi from adult rats(Sprague Dawley) were isolated and sliced into a 500 m section in a glucose containing artificial CSF medium on ice. In order to induce a hypoxia, sample slices were treated with 95% N2/5% CO2 for 10 min, 20 min, 30 min, and 60 min, respectively and control slices were treated with 95% O2/5% CO2 to supply sufficient oxygen as the same way. And then the control and the sample slices were homogenated and 40 g of each homogenates were electrophoresed in a 6% SDS-gel, transfered to nitrocellulose, and immunostained with anti-NR2A and anti-NR2B antisera. RESULTS: 1) Anti-NR2A antisera were produced by recombinant DNA technology. 2) The NR2A and the NR2B were enriched in the order of PSD, synaptosome and brain homogenate. 3) There was no difference of the NR2A and the NR2B expression in both control and experimental group. CONCLUSIONS: At least up to one hour after hypoxic damage, it is likely that there is no prominent changes of the NR2A and the NR2B amounts. Considering that the changes could occur locally or microscopically in this experimental protocol, the relative amounts of the NR2A and the NR2B in the hippocampal homogenates are too small to be detected by immunoblot analyses. And we can not exclude the possibility of no changes in one hour after hypoxia, if these changes evolve with a extremely slow progression.