The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca²⁺]_iIncrease Induced by ATP and Bradykinin in PC12 Cells

Abstract

The present study explores the hypothesis that the new antiepileptic drug levetiracetam (LEV) could interfere with the inositol 1,4,5-trisphosphate (IP 3 )-dependent release of intracellular Ca 2ϩ initiated by G q -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 2ϩ ] 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 2ϩ ] i increase, elicited either by 1 M BK or by 100 M ATP (IC 50 , 0.39 Ϯ 0.01 M for BK and 0.20 Ϯ 0.01 M for ATP; Hill coefficients, 1.33 Ϯ 0.04 for BK and 1.38 Ϯ 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 2ϩ ] i response to BK, LEV inhibitory effect was mainly exerted on the IP 3 -dependent stores. In fact, the drug was still effective after the pharmacological blockade of ryanodine receptors. Furthermore, LEV did not affect Ca 2ϩ stored in the intracellular deposits since it did not reduce the amplitude of [Ca 2ϩ ] i response either to thapsigargin or to ionomycin. In conclusion, LEV inhibits Ca 2ϩ release from the IP 3 -sensitive stores without reducing Ca 2ϩ storage into these deposits. Because of the relevant implications of IP 3 -dependent Ca 2ϩ release in neuron excitability and epileptogenesis, this novel effect of LEV could provide a useful insight into the mechanisms underlying its antiepileptic properties.

Inactivation of the Constitutively Active Ghrelin Receptor Attenuates Limbic Seizure Activity in Rodents

“…A way to investigate this anticonvulsant mechanism of ghrelin using an in vivo setting is by testing a ligand that provides a similar GHSR activation to ghrelin, while showing a decreased tendency to desensitize the receptor. In this study, we successfully discovered that the shorter the C-terminal of ghrelin is, the less capable it is able to desensitize GHSR, while maintaining a similar pEC 50 to ghrelin. Thus, we confirmed our hypothesis by testing the ghrelin (1-5) amide in vivo, where we revealed for the first time that it is desensitization and not activation of the GHSR that is necessary to attenuate limbic seizures in vivo.…”

“…Concentrations of A778193 were added in quadruplicate to the cells and incubated for 90 minutes at 37°C in air/CO 2 (1-7), (1-9), (1-11), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), and (23)(24)(25)(26)(27)(28) were tested next to the GHSR agonist capromorelin. A sigmoidal dose response curve was fitted using GraphPad Prism software, GraphPad Software, (La Jolla, USA), yielding a pEC 50 and curve maximum. For each pretreatment condition, the maximum was expressed as a percentage of ionomycin after subtraction of the blank.…”

“…Desensitization by the first applied GHSR ligand was described as its effect on the maximum of the concentration response curve obtained after the second stimulation with ghrelin. Results were expressed as the percentage of control response (no pre-stimulation), and the negative logarithm of the pre-incubation concentration reducing the maximum response to ghrelin to 50 % of its control value was calculated (pDC 50 ). The protocol used to investigate the effect of different pretreatment times with ghrelin was the same, with the difference being that the preincubation time for desensitization varied between 3 minutes to 60 minutes.…”

“…Via in vivo microdialysis, we tested ghrelin(1-5) amide by administering it locally in the hippocampus and as based on pEC 50 and pDC 50 values (Table 1), only the 500 μM concentration showed significant anticonvulsant effects (unpaired Student's t test, p00.0253) (Fig. 6f).…”

“…Thus, we confirmed our hypothesis by testing the ghrelin (1-5) amide in vivo, where we revealed for the first time that it is desensitization and not activation of the GHSR that is necessary to attenuate limbic seizures in vivo. 50 and pDC 50 of Ghrelin, Ghrelin Fragments (1-5) Amide, (1-7), (1-9), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), (23)(24)(25)(26)(27)(28) Ghrelin and capromorelin similarly activated and desensitized the growth hormone secretagogue receptors (GHSRs). The truncated forms of ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and (1-5) amide had a similar activity (pEC 50…”