RNA EDITING OF GLUTAMATE-RECEPTOR SUBUNITS GLUR2, GLUR5, AND GLUR6 INTRANSIENT CEREBRAL-ISCHEMIA IN THE RAT

The mechanisms of ischemic cell damage are still not fully understood. It has been shown that ha-amino-3-hydroxy-5-methyl-isoxazole-4-propio nate (AMPA)/kainate receptor antagonists, such as nitro-7-sulphamoyl-b enzo-(f)-quinoxaline-2,3-dione (NBQX), an neuroprotective in models of transient forebrain ischemia, even when applied during recovery, indi cating that nonNMDA receptors may play a pivotal role in ischemic cell damage. In the present series of experiments, we studied whether tran sient cerebral ischemia causes changes in the extent of mRNA editing o f AMPA/kainate receptor subunits, a reaction critical for the control of calcium flux through nonNMDA receptor ion channels. Transient cereb ral ischemia was produced in rats using the four-vessel occlusion (4-V O) model. After 30 min of ischemia, brains were recirculated for 4, 8, or 24 h. Total RNA was extracted from the cortex, striatum, and hippo campus in order to analyze the extent of mRNA editing of the glutamate receptor subunits GluR2, GluR5, and GluR6. RNA was converted by rever se transcription into cDNA, which was used as a template for subunit-s pecific polymerase chain reaction (PCR) to amplify a product across th e edited base A (A edited to I in the second transmembrane-spanning re gions of GluR2, GluR5, and GluR6). PCR products were analyzed with the restriction enzyme Bbv 1, which recognizes the cDNA sequence GCAGC or iginating from unedited but not that originating from edited GluR2, Gl uR5, or GluR6 mRNA (GCGGC, the base I is read as G). Restriction diges ts were electrophoresed, and the bands visualized with ethidium bromid e and then photographed. The extent of mRNA editing of the different s ubunits was quantified using image analysis and appropriate standards. In all control brains studied, GluR2 mRNA was completely edited and r emained so after reversible cerebral ischemia. The extent of GluR5 mRN A editing was significantly upregulated in the striatum (from 39 +/- 6 % in controls to 57 +/- 9 and 56 +/- 7 after 4 and 8 h of recovery, re spectively, p < 0.05 versus control) but not in the cortex and hippoca mpus. The extent of GluR6 mRNA editing was significantly reduced after 24 h of recovery: in the cortex, from 92 +/- 1 to 78 +/- 6% (p < 0.01 ) in the striatum, from 91 +/- 2 to 79 +/- 1% (p < 0.001); and in the hippocampus, from 90 +/- 3 to 80 +/- 2% (p < 0.05). A significant redu ction was already apparent in the striatum after 4 h of recovery (p < 0.05). Results indicate that mRNA editing is regulated differently in each of the glutamate receptor subunits GluR2, GluR5, and GluR6 after transient cerebral ischemia. The ischemia-induced upregulation of GluR 5 mRNA editing observed in the striatum may be indicative of a higher sensitivity to transient ischemia of neurons that exhibit a large frac tion of unedited GluR5 mRNA. This assumption is corroborated by the ob servation (Mackler and Eberwine, 1993) that GluR5 mRNA is completely u nedited in neurons of the hippocampal CA1-subfield, a region most vuln erable to transient cerebral ischemia. Whether the decrease in GluR6 m RNA editing observed in all brain structures after ischemia results fr om a disturbance of the editing reaction or from glial proliferation w ill have to be established in further experiments. Studying ischemia-i nduced changes in mRNA editing of glutamate receptor subunits GluR5 an d GluR6 may help to elucidate the molecular mechanisms of ischemic cel l damage.