Abstracts

Rationale: Epilepsy is a condition that affects approximately 2% of the population worldwide; therefore, there is a need not only to develop newer anti-epileptic drugs (AEDs) but also to understand their mechanism of action so as to maximize therapeutic value and develop more potent AEDs. Levetiracetam (LEV) is a novel AED used to treat both partial onset and myoclonic seizures. Despite increasing clinical use of LEV, its mechanism of actions remains largely uncharacterized. While several studies have demonstrated specific binding sites for LEV in the brain and shown that it acts in a manner distinct from other AEDs, the exact mechanism of action that prevents seizure expression remains to be determined. The ubiquitous intracellular second messenger, calcium (Ca2+) has been shown to play a critical role in the induction and maintenance of acquired epilepsy. This study focused on the effects of LEV on intracellular calcium levels ([Ca2+]i) in hippocampal neuronal cultures (HNC).Methods: Inositol-3,4,5-phosphate (IP3) and ryanodine receptor (RyR)-mediated Ca2+ release was stimulated using caffeine (10 mM) or bradykinin (1 μM). Calcium transients from vehicle and LEV pre-treated hippocampal neurons were evaluated following pharmacological stimulation of fluorescent Ca2+ indicator Fura-2AM loaded cells. Ratio images were acquired by using alternating excitation wavelengths (340/380 nm). Results: Upon stimulation with caffeine, which triggers RyR-mediated endoplasmic reticulum calcium induced calcium release (CICR), vehicle-treated control cells exhibited a marked peak in the 340/380 ratio, that corresponded to an increase in [Ca2+]i. Whereas, neurons pre-treated for 20 min with LEV (33μM) exhibited a significant reduction in caffeine induced peak height when compared to vehicle-treated control neurons. In a separate set of experiments, bradykinin was used to stimulate IP3-mediated endoplasmic reticulum CICR. Vehicle-treated control cells demonstrated a sharp peak in 340/380 ratio indicative of IP3 stimulated [Ca2+]i release. In contrast, LEV (100μM) pre-treated cells manifested a significant decrease in peak height following bradykinin stimulation. These experiments were repeated with a pre-incubation in ryanodine (50μM) to block RyR CICR prior to stimulation with bradykinin and LEV was still able to significantly decrease the amplitude of the IP3 calcium transients. Conclusions: These studies demonstrate that LEV is able to decrease CICR mediated by both IP3R and RyR. CICR mechanisms have been implicated in epileptogenesis and the maintenance of epilepsy; thus, the ability of LEV to reduce bradykinin and caffeine-induced calcium transients in hippocampal neurons may have significant implications in elucidating the mechanism by which LEV exerts some of its clinical actions. (Supported by: UCB Pharma and NIH grants R01NS051505-02, R01NS052529-01A2, U01NS058213-01)