The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca ]i Increase Induced by ATP and Bradykinin in PC12 Cells

The present study explores the hypothesis that the new antiepileptic drug levetiracetam (LEV) could interfere with the inositol 1,4,5-trisphosphate (IP3)-dependent release of intracellular Ca initiated by Gq-coupled receptor activation, a process that plays a role in triggering and maintaining seizures. We assessed the effect of LEV on the amplitude of [Ca ]i response to bradykinin (BK) and ATP in single Fura-2/acetoxymethyl ester-loaded PC12 rat pheochromocytoma cells, which express very high levels of LEV binding sites. LEV dose-dependently reduced the [Ca ]i increase, elicited either by 1 M BK or by 100 M ATP (IC50, 0.39 0.01 M for BK and 0.20 0.01 M for ATP; Hill coefficients, 1.33 0.04 for BK and 1.38 0.06 for ATP). Interestingly, although the discharge of ryanodine stores by a process of calcium-induced calcium release also took place as part of the [Ca ]i response to BK, LEV inhibitory effect was mainly exerted on the IP3-dependent stores. In fact, the drug was still effective after the pharmacological blockade of ryanodine receptors. Furthermore, LEV did not affect Ca stored in the intracellular deposits since it did not reduce the amplitude of [Ca ]i response either to thapsigargin or to ionomycin. In conclusion, LEV inhibits Ca release from the IP3sensitive stores without reducing Ca storage into these deposits. Because of the relevant implications of IP3-dependent Ca release in neuron excitability and epileptogenesis, this novel effect of LEV could provide a useful insight into the mechanisms underlying its antiepileptic properties. Levetiracetam (LEV), the S-enantiomer of -ethyl-2-oxo-1pyrrolidine acetamide, is a new antiepileptic drug (AED) with a broad-spectrum antiepileptic potential, a good tolerability, and a low propensity for pharmacokinetic drug interactions (Dooley and Plosker, 2000). Because of these pharmacological properties, LEV is becoming more and more popular among clinical neurologists. Noticeably, since its approval in 1999 for the add-on therapy of drug-resistant partial seizures in adults, almost 500,000 patients have been treated with this drug (http://www.keppra.com). Despite its widespread use in the treatment of epilepsy, the mechanisms underlying the antiseizure effect of LEV still remain to be fully elucidated (Margineanu and Klitgaard, 2002). However, it is well established that the drug binds in a reversible and stereoselective way to specific binding sites found in the brain and in neuronal cell lines, including rat pheochromocytoma PC12 cells (Fuks et al., 2003), and recently identified as the synaptic vesicle protein SV2A (Lynch et al., 2004). The functional consequences of this binding remain, however, currently unclear. LEV, in fact, does not seem to interfere with the AED classical targets in any significant way. It does not affect the activity of voltage-dependent Na channels or T-type voltage-gated Ca channels (Zona et al., 2001) while exerting a moderate inhibition of high voltage-activated Ca channels (Lukyanetz et al., 2002). Furthermore, LEV merely decreases GABAA receptor sensitivity to Zn and -carbolines (Rigo et al., 2002), without displaying any conventional GABA-enhancing activity (Margineanu and Klitgaard, 2003). This work was partially supported by a grant from UCB Pharma (BraineL’Alleud, Belgium). This work was also supported by Grant PRIN 2002 from the Ministero Italiano per l’ Università e la Ricerca Scientifica (to L.A.); by Grant PNR 2001-2003-FIRB art. 8 D. M. 199 (to L.A.); by POP and Legge 41/94 from Regione Campania, annualità 98 (to L.A.); by Programma Speciale art. 12bis comma 6, D. Lgs. 229/99 from Ministero della Salute and Regione Campania (to L.A.); by Progetto di Ricerche Finalizzato, Convenzione Ministero della Salute/Regione Campania, no. 1, 1 dicembre 2003 (to L.A.); and by POR 2006, asse 3, misura 3.16, from Centro Regionale di Competenza di Genomica Funzionale, Genomics for Applied Research (to L.A.). V.L. is a Ph.D. student supported by a fellowship grant from the Centro Regionale di Competenza di Genomica Funzionale, Genomics for Applied Research. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.079327. ABBREVIATIONS: LEV, levetiracetam; AED, antiepileptic drug; IP3, inositol 1,4,5-trisphosphate; BK, bradykinin; AM, acetoxymethyl ester; TG, thapsigargin; AUC, area under the curve; ANOVA, analysis of variance; Rya, ryanodine; Iono, ionomycin; CICR, calcium-induced calcium release; RyR, ryanodine receptor. 0022-3565/05/3132-720–730$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 313, No. 2 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 79327/1198041 JPET 313:720–730, 2005 Printed in U.S.A. 720 at A PE T Jornals on A ril 7, 2017 jpet.asjournals.org D ow nladed from