Abstracts

RATIONALE: Extracellular signal-regulated kinases, such as ERK1 (p44) and ERK2 (p42), are abundant in the CNS and are activated under various physiological and pathological conditions (e.g., brain ischemia and seizures). Application of the potassium channel blocker 4-aminopyridine (4AP, 50 [mu]M) to rat hippocampal slices enhances synaptic transmission and makes epileptiform discharges appear. Hence, it represents an [italic]in vitro[/italic] model commonly used for investigating the pathophysiogenesis of seizures. Here, we have studied the activation state of Mitogen-Activated Protein Kinase (MAPK) ERK1/2 in 4AP-treated rat hippocampal slices.
METHODS: Hippocampal slices were processed for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot assays. Protein loading (10 [mu]g) was monitored by staining filters with [dsquote]Ponceau[dsquote]. Immunoreactivity was detected using the chemiluminescence system (ECL-Amersham) and autoradiography. Quantitation was carried out by densitometric analysis of the films (Fluor-S, Biorad). In all experiments, membranes were first processed to visualize the phosphorylated forms of proteins then dehybridized (Restore Western blot stripping buffer, Pierce, USA) and successively reprobed with the antibody directed against total proteins for normalization.
RESULTS: We have found that hippocampal slices superfused with 4AP exhibit a marked activation (two fold [italic]vs[/italic]. control) of MAP/ERK1/2 phosphorylation that peaks 90 min after treatment and remains above the basal level for at least 4 hrs. These effects are not accompanied by any change in the activation state of other members of the MAP kinase superfamily c-Jun N-terminal kinases (JNK, also named stress-activated protein kinase, SAPK). In addition, the total kinase content remains constant during 4AP treatment suggesting no change in the expression level of ERK proteins. We have also found that the 4AP-induced ERKs phosphorylation is age-dependent. Specifically, we observed: (i) an increase in ERK protein expression with development (10 day-old [italic]vs[/italic]. 21 day-old animals); and (ii) a larger increment of the phosphorylation status in slices obtained from 10 day-old rats as compared with those prepared from 20 day-old animals.
CONCLUSIONS: Our data indicate that activation of Mitogen-Activated Protein Kinase ERKs may play a role in the enhancement of synaptic transmission induced by 4AP and thus in the implementation of epileptiform synchronization.
[Supported by: Canadian Institutes of Health Research and Savoy Foundation.]