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CELLULAR AND MOLECULAR

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

Division of Pharmacology, Department of Neuroscience, School of Medicine, Federico II University of Naples, Naples, Italy

Received October 17, 2004; accepted January 7, 2005.

	Abstract

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The present study explores the hypothesis that the new anti-epileptic drug levetiracetam (LEV) could interfere with the inositol 1,4,5-trisphosphate (IP₃)-dependent release of intracellular Ca²⁺ 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 [Ca²⁺]_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 [Ca²⁺]_i increase, elicited either by 1 µM BK or by 100 µM ATP (IC₅₀, 0.39 ± 0.01 µM for BK and 0.20 ± 0.01 µM 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 [Ca²⁺]_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 Ca²⁺ stored in the intracellular deposits since it did not reduce the amplitude of [Ca²⁺]_i response either to thapsigargin or to ionomycin. In conclusion, LEV inhibits Ca²⁺ release from the IP₃-sensitive stores without reducing Ca²⁺ storage into these deposits. Because of the relevant implications of IP₃-dependent Ca²⁺ release in neuron excitability and epileptogenesis, this novel effect of LEV could provide a useful insight into the mechanisms underlying its antiepileptic properties.

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 SV_2A (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 Ca²⁺ channels (Zona et al., 2001) while exerting a moderate inhibition of high voltage-activated Ca²⁺ channels (Lukyanetz et al., 2002). Furthermore, LEV merely decreases GABA_A receptor sensitivity to Zn²⁺ and -carbolines (Rigo et al., 2002), without displaying any conventional GABA-enhancing activity (Margineanu and Klitgaard, 2003).

These findings suggest that the mechanisms of action underlying LEV's antiseizure effect are unrelated to those of classical AEDs. Therefore, given the considerable interest accruing around the identification of new pharmacological targets exploitable for the treatment of seizures unresponsive to conventional AEDs, a better understanding of the pharmacodynamic properties of LEV has become of significant importance for clinical neurology.

Actually, a number of molecular factors involved in epileptogenesis but currently orphan of specific drug approaches have already been identified. This is the case of the signaling cascade triggered by G_q-coupled receptor activation involving the IP₃-dependent Ca²⁺ release from intracellular Ca²⁺ stores. Indeed, the intracellular injection of IP₃ triggers epileptiform discharges in hippocampal neurons (Jin et al., 2000), and IP₃-dependent release of Ca²⁺ ions from the intracellular stores is regarded as a major factor responsible for the consistent elevation of [Ca²⁺]_i observed in epileptic neurons (Pal et al., 2001). Furthermore, since Honchar et al. (1983) first reported that the systemic administration of cholinergic agents induces seizures in lithium-treated rats, it has been firmly established that G_q-coupled M₁ muscarinic receptor activation is responsible for the convulsant effects of centrally acting muscarinic drugs (Bymaster et al., 2003). Similarly, glutamate proconvulsant activity can be in part ascribed to the activation of G_q-coupled group I metabotropic receptors because their specific agonist (R,S)-3,5-dihydroxyphenylglycine induces limbic seizures attenuated by dantrolene, an inhibitor of calcium release from intracellular stores (Tizzano et al., 1995).

Ca²⁺ ion release from the IP₃-sensitive intracellular stores is also triggered by neuropeptides involved in seizure generation. This is the case of bradykinin (BK), which, as suggested by the proconvulsant activity of its agonists (Bregola et al., 1999) and by the changes in its receptor density occurring in epilepsy (Arganaraz et al., 2004), could be involved in epileptogenesis.

The therapeutic potential of blocking intracellular Ca²⁺ release in epilepsy is exemplified by the suppression of bicuculline-induced epileptic bursting occurring after the depletion of intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin (TG) (Wulfert and Margineanu, 1998), and by the marked reduction of pilocarpine-induced ictal discharges induced by dantrolene and TG (Hadar et al., 2002).

The present study explores the possibility that LEV could inhibit Ca²⁺ ion release from the IP₃-sensitive stores. To address this hypothesis, we studied, by means of single-cell Fura-2 video imaging, the effect of LEV on the [Ca²⁺]_i response to BK and ATP, two neurotransmitters that induce an IP₃-dependent increase in [Ca²⁺]_i (Fatatis et al., 1994) and participate in epileptogenesis. The study was performed in PC12 cells, a neuron-like cell line with a high expression of LEV-binding sites (Fuks et al., 2003).

	Materials and Methods

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Cell Culture. Undifferentiated PC12 rat pheochromocytoma cells (courtesy of Dr. L. Colucci d' Amato, National Research Council (CNR) Institute of Endocrinology and Experimental Oncology, Naples, Italy) were grown in plastic dishes in RPMI medium supplemented with 10% horse serum, 5% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. NIH-3T3 cells (courtesy of Dr. C. Miele, Department of Biology and Cellular and Molecular Pathology, Federico II University of Naples, Naples, Italy) were cultured in plastic dishes in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Cells were cultured in a humidified 5% CO₂ atmosphere; the culture medium was changed every 2 days. For microfluorometric studies, the cells were plated on glass coverslips (Fisher Scientific Co., Pittsburgh, PA) coated with poly-L-lysine (30 µg/ml) (Sigma-Aldrich, St. Louis, MO) and were used at least 12 h after seeding. All PC12 cell experiments were performed at a cell culture passage between 10 and 25.

[Ca²⁺]_i Measurements. [Ca²⁺]_i was measured by single-cell computer-assisted videoimaging (Cataldi et al., 1996). Briefly, PC12 or NIH-3T3 cells, grown on glass coverslips, were loaded with 5 µM Fura-2/AM for 1 h at room temperature in Krebs-Ringer saline solution containing the following: 5.5 mM KCl, 160 mM NaCl, 1.2 mM MgCl₂, 1.5 mM CaCl₂, 10 mM glucose, and 10 mM Hepes-NaOH, pH 7.4. At the end of the Fura-2/AM loading period, the coverslips were placed into a perfusion chamber (Medical System, Co., Greenvale, NY) mounted onto the stage of an inverted Nikon Diaphot fluorescence microscope (Nikon, Melville, NY). A 100-W xenon lamp (Osram, Berlin, Germany) with a computer-operated filter wheel, bearing two different interference filters (340 and 380 nm), illuminated the microscopic field with UV light every 3 s, alternating the wavelengths at an interval of 500 ms. The light emitted by Fura-2-loaded cells was passed through a 400-nm dichroic mirror filtered at 510 nm and finally collected with an intensified camera (Photonic Science, Robertsbridge, UK). Images were digitized and analyzed with a Magiscan image processor (Applied Imaging Ltd., Dukesway, UK) driven by the AUTOLAB software (RBR Altair, Florence, Italy). Ratiometric values were automatically converted by the software into [Ca²⁺]_i using a preloaded calibration curve obtained in preliminary experiments (Cataldi et al., 1996). No significant overlap with Fura-2 absorption or emission was observed when the spectra of LEV were obtained using a PerkinElmer LS 50B (PerkinElmer Life and Analytical Sciences, Boston, MA) spectrofluorometer.

To test the effect of LEV on [Ca²⁺]_i increase elicited by the different drugs used in this study, the cells were incubated for 5 min with increasing concentrations of LEV or vehicle dissolved in a 1.5 mM Ca²⁺-containing Krebs' solution. Then, 5 ml of a 1.5 mM EGTA-containing Ca²⁺-free Krebs' solution with LEV (or vehicle) and the appropriate drug were quickly injected into the recording chamber. The time required for injection ranged around 5 s.

Data Calculation. [Ca²⁺]_i responses to the different pharmacological challenges used in the study were measured both as change in percentage increase above baseline [Ca²⁺]_i and as area under the curve (AUC).

For % calculation, we used the maximal value of [Ca²⁺]_i attained after stimulation and, as baseline [Ca²⁺]_i, the mean of [Ca²⁺]_i recorded during the 200 s preceding the challenge.

AUC was calculated by integrating [Ca²⁺]_i after the pharmacological challenge and was expressed as [Ca²⁺]_i (nanomolar) per second. Given the different duration of the [Ca²⁺]_i response to the different challenges, different times of integration (reported in the figure legends) were used to assess the responsiveness to different drugs. AUCs were calculated using the area below the curve macro of the Sigma plot 8.0 software, which performs integrations using the trapezoidal rule. Both % and AUCs were normalized to the mean of the respective controls and expressed as percent values.

Dose-effect curves were obtained by fitting the data to the four parameter logistic equation y = min + (max – min)/(1 + (x/IC₅₀)ⁿ), where y is the normalized value of % increase in [Ca²⁺]_i or [Ca²⁺]_i AUC, x is the LEV concentration in micromoles per liter, and n is the Hill coefficient. Data fitting was performed using the fitting routines of the Sigma Plot 8.0 software (SPSS Inc., Chicago, IL).

[³H]Bradykinin Binding and Displacement Assays. [³H]BK binding to BK plasma membrane receptors in PC12 cells and the potential interference of LEV with this binding reaction were studied using the experimental approach reported by Nardone et al. (1994). Briefly, PC12 cells were plated onto 12-well plastic dishes at the density of 1 x 10⁶ cells/well. Twenty four hours after seeding, the culture medium was removed and replaced with 2 ml of an ice-cold assay buffer containing the following: 137.5 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM glucose, 10 mM Hepes, 1 mM iodoacetic acid, 0.1% bovine serum albumin, 5 mM bacitracin, and 0.01 mM captopril. The binding reaction was started replacing this solution with 500 µl of the same buffer supplemented with different concentrations of [³H]BK (PerkinElmer Life and Analytical Sciences; specific activity 90 Ci/mM) with or without cold BK or LEV as detailed in the legend to Fig. 3. The binding reaction was left to proceed for 90 min on ice, then the medium was removed, and the cells were washed twice with 2 ml of the assay buffer and finally lysed with 1% sodium dodecyl sulfate. Cell lysates were collected and counted with a scintillation counter (LS 5000 CE; Beckman Coulter, Fullerton, CA). Nonspecific binding was determined in the presence of 20 µM cold BK. All data points were obtained in duplicate. Binding curves were fitted to the equation B = x · B_max/(K_d + x) + x · N, where N is the nonspecific binding and x is the concentration of free ligand, using the one-site saturation plus nonspecific simple ligand binding routine of the Sigma Plot 8.0 software (SPSS Inc.).

View larger version (15K): [in this window] [in a new window]   Fig. 3. LEV effect on [3H]BK binding to PC12 cells. A, [3H]BK binding to PC12 cells. The panel shows the mean ± S.E.M. values of total and nonspecific radioactivity bound to PC12 cell after a 90-min incubation on ice with different concentrations of [3H]BK. All the points reported have been obtained in duplicate, and the curve shown relates to one of two different experimental sessions performed with similar results. As detailed under Materials and Methods, curve fitting of total radioactivity was performed using the equation B = x · Bmax/(Kd + x) + x · N, where N is the nonspecific binding and x the concentration of free ligand whereas linear fitting was used in the case of nonspecific binding. B, effect of LEV on [3H]BK binding on PC12 cells. The bar plot reports the mean ± S.E.M. of total radioactivity bound on PC12 cells after a 90-min incubation on ice with 6 nM [3H]BK in the presence or in the absence of 1 µM LEV expressed as percentage of control values. LEV was added to the assay buffer simultaneously with [3H]BK. The data shown have been obtained in duplicate and relate to one experimental session of three performed with similar results.

Statistical Analysis of the Data. All data are reported as mean ± S.E.M. When comparing two data sets, the Student's t test for unpaired data was used. To compare multiple groups, we assessed the normal distribution of the data with the Bartlett test. Statistical comparisons were performed, for normally distributed data, with ANOVA followed by the Tukey-Kramer post hoc test and, for non-normally distributed data, with the Kruskal-Wallis nonparametric ANOVA followed by the Dunn's multiple comparison test. The threshold for statistical significance was set at P < 0.01. Statistical comparisons were carried out using the Graph-PAD 2.04 software suite (GraphPad Software Inc., San Diego, CA).

Drugs and Chemicals. LEV was kindly provided by UCB Pharma (Braine-l'Alleud, Belgium). It was dissolved in water as a 100 mM stock solution and kept frozen in 50-µl aliquots at –20°C until use. Culture media, horse and fetal calf sera, and antibiotics were purchased from Invitrogen (Carlsbad, CA). Fura-2/AM, iodoacetic acid, and bacitracin were obtained from Calbiochem (San Diego, CA). BK, ATP, Ryanodine (Rya), TG, Ionomycin (Iono), and all other chemicals were from Sigma-Aldrich.

	Results

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LEV Does Not Modify Basal [Ca²⁺]_i in Undifferentiated PC12 Cells. To assess whether LEV acutely affects basal [Ca²⁺]_i, we continuously monitored [Ca²⁺]_i in single Fura-2-loaded PC12 cells before and after the addition of different concentrations of this drug to the extracellular solution. In particular, after 300 s of basal [Ca²⁺]_i monitoring, LEV (0.1–10 µM) was added to the recording chamber, and [Ca²⁺]_i was determined for 300 more seconds. [Ca²⁺]_i was fairly stable all over the observation period both before and after the addition of LEV, and at all the concentrations tested, the drug failed to induce any change in [Ca²⁺]_i (control versus LEV mean basal [Ca²⁺]_i, 114.9 ± 4.1 versus 117.3 ± 3.6 nM in the 0.1 µM LEV group, n = 79; 115.1 ± 1.7 versus 116.9 ± 1.6 nM in the 0.3 µM LEV group, n = 79; 114.7 ± 3.5 versus 115.6 ± 2.6 nM in the 1 µM LEV group, n = 83; 113.5 ± 4.5 versus 113.9 ± 2.6 nM in the 10 µM LEV group, n = 72) (Fig. 1, A–D).

View larger version (24K): [in this window] [in a new window]   Fig. 1. LEV effect on basal [Ca2+]i in PC12 cells. The graph reports the time course of mean basal [Ca2+]i recorded in PC12 cells before and after the addition of (A) 0.1, (B) 0.3, (C) 1, or (D) 10 µM LEV to the extracellular solution. Basal [Ca2+]i was continuously monitored for 300 s in cells bathed with normal Krebs' solution; then, LEV was added at the concentration shown in the figure. Each point represents the mean ± S.E.M. of [Ca2+]i values detected at each time period in each cell recorded in one of the three different experimental sessions performed for each of the LEV concentration tested. The number of the cells tested in the experimental sessions represented in the figure cells was: 26, 28, 29, and 26 in 0.1, 0.3, 1, and 10 µM LEV groups.

LEV Decreases the [Ca²⁺]_i Response to BK and ATP in PC12 Cells but Not in NIH-3T3 Fibroblasts. To determine whether LEV affects the [Ca²⁺]_i response to IP₃-linked agonists, we examined the effect of this drug on the [Ca²⁺]_i response evoked in PC12 cells by BK and ATP, two neurotransmitters whose IP₃-coupled receptors are expressed in this cell line (Nardone et al., 1994; Moskvina et al., 2003). ATP and BK were delivered to PC12 cells in a nominally Ca²⁺-free Krebs' solution obtained by omitting Ca²⁺ ions and by adding 1.5 mM EGTA. This approach prevented the confounding effect of the influx of extracellular Ca²⁺ ions. Such phenomenon could be due to two different mechanisms: the first could entail the activation of the so-called plasma membrane refilling channels, which open when the intracellular calcium stores are depleted; the second, in the case of ATP, could involve the opening of the ionotropic P_2X receptors, which are also expressed in PC12 cells. A potential drawback of exposing cells to a Ca²⁺-free medium is the slow emptying of intracellular Ca²⁺ stores determined by the lack of Ca²⁺ ions in the extracellular solution and by the presence of strong Ca²⁺ ion chelators. To prevent this undesired effect, the cells were kept in a Ca²⁺-containing Krebs' solution until they were challenged with BK or ATP in the Ca²⁺-free Krebs' solution.

In control cells, 1 µM BK induced a rapid increase in [Ca²⁺]_i that reached a peak of 231 ± 11 nM (n = 149) in approximately 10 s and returned to baseline in approximately 100 s (Fig. 2A).

View larger version (27K): [in this window] [in a new window]   Fig. 2. LEV effect on [Ca2+]i increase elicited by BK in PC12 cells. A, time course of mean [Ca2+]i before and after 1 µM BK challenge in control and LEV-treated cells. Each plotted data point represents the mean ± S.E.M. of the [Ca2+]i recorded at each time in all the cells of the control (n = 29) and LEV (n = 39) groups during a single experimental session, representative of a group of four. Only the [Ca2+]i recorded during the last 50 s of the 5-min basal [Ca2+]i monitoring preceding the BK challenge are shown in the panel. B, concentration dependence curve of LEV-induced reduction in [Ca2+]i response to BK expressed as % increase above baseline [Ca2+]i. The curve reports, as a function of LEV concentration, LEV-induced reduction in [Ca2+]i increase elicited by 1 µM BK. The data were calculated as % increase above baseline [Ca2+]i. The values, obtained for each LEV concentration, were expressed as percentage of mean % in control cells (n = 149). Each data point is the mean ± S.E.M. of the data obtained in all the cells treated with each LEV concentration tested in four different experimental sessions. The number of the tested cells was: 149, 108, 108, 152, and 78 in control, 0.1, 0.3, 1, and 10 µM LEV groups. C, concentration dependence curve of LEV-induced reduction in [Ca2+]i response to BK expressed as [Ca2+]i AUC. The curve reports, as a function of LEV concentration, LEV-induced reduction in [Ca2+]i response to 1 µM BK expressed as AUC. AUC was calculated by integrating [Ca2+]i over a 100-s time period, after the BK challenge, and expressed as [Ca2+]i (nanomolar) per second. For each LEV concentration, AUCs were expressed as percentage of mean AUCs in control cells (n = 151). The number of tested cells was: 151, 106, 108, 149, and 76 in control, 0.1, 0.3, 1, and 10 µM LEV groups. Each data point is the mean ± S.E.M. of the data obtained in all the cells treated with each LEV concentration tested in four different experimental sessions. Concentration dependence curves were fitted as reported under Materials and Methods.

When the cells were exposed to increasing concentrations of LEV (ranging from 0.1–10 µM), a concentration-dependent inhibition of this response was observed (Fig. 2, B and C). LEV-induced decrease in [Ca²⁺]_i response was fitted with the four-parameter logistic Hill equation as described under Materials and Methods. When the inhibition was quantified as percentage decrease in the %, an IC₅₀ of 0.39 ± 0.01 µM and a Hill coefficient of 1.33 ± 0.04 were estimated (Fig. 2B). This Hill coefficient value suggests that LEV interacts with a single binding site. Similar results were obtained when we considered the drug-induced percentage decrease in [Ca²⁺]_i AUC. The IC₅₀ was 0.46 ± 0.01 µM, and the Hill coefficient was 1.14 ± 0.01 (Fig. 2C).

To determine whether this LEV-induced decrease in [Ca²⁺]_i response to BK was determined by an interference with the binding of this peptide to its plasma membrane receptors, we performed displacement studies aiming to assess the ability of LEV to compete with [³H]BK for its binding sites on PC12 cells. As already reported by Nardone et al. (1994), [³H]BK displayed a specific binding to PC12 cells, and the [³H]BK binding curve was adequately fitted under the assumption of a single homogeneous population of plasma membrane receptors with a K_d of 5.1 nM (Fig. 3A). When LEV was added to the assay buffer in a concentrations of 1 µM that is effective in submaximally reducing the BK-induced [Ca²⁺]_i response, no change in the binding of [³H]BK (6 nM) to PC12 cells was observed (Fig. 3B).

These results suggested that LEV effect on BK-induced intracellular Ca²⁺ release was not the mere consequence of a drug-induced displacement of this G_q-coupled ligand from its receptors. To further substantiate the idea that the ability of LEV to interfere with [Ca²⁺]_i homeostasis was a more general phenomenon not restricted to BK receptor pharmacology, we explored the effect of this drug on [Ca²⁺]_i responsiveness to another G_q-coupled agonist with a completely different chemical structure, the nucleotide ATP. To this aim, we used an experimental approach similar to that used in the case of BK. In control PC12 cells, ATP (100 µM) markedly increased the [Ca²⁺]_i that peaked at 241.7 ± 3.7 nM (n = 300) approximately 10 s after the challenge (Fig. 4A). Interestingly, although this [Ca²⁺]_i peak response was not different from that evoked by BK, the duration of [Ca²⁺]_i increase elicited by ATP was significantly shorter. In fact, [Ca²⁺]_i levels returned to baseline in approximately 50 s. When PC12 cells were exposed to LEV (0.1–10 µM) 5 min before and throughout the entire ATP stimulation, a concentration-dependent inhibition of ATP-evoked [Ca²⁺]_i response occurred (Fig. 4). The IC₅₀ for LEV-induced decrease in the [Ca²⁺]_i % was 0.24 ± 0.01 µM with a Hill coefficient of 0.9 ± 0.03 (Fig. 4B). LEV also reduced the AUC of [Ca²⁺]_i response to ATP with an IC₅₀ of 0.2 ± 0.01 µM and a Hill coefficient of 1.38 ± 0.06 (Fig. 4C).

View larger version (25K): [in this window] [in a new window]   Fig. 4. LEV effect on [Ca2+]i increase elicited by ATP in PC-12 cells. A, time course of mean [Ca2+]i before and after 100 µM ATP challenge in control and LEV-treated cells. Each data point plotted on the curves is the mean ± S.E.M. of the [Ca2+]i recorded at each time in all the cells of the control (n = 53) and LEV (n = 43) groups during a single experimental session, representative of a group of four. Only the [Ca2+]i recorded during last 50 s of the 5-min basal [Ca2+]i monitoring preceding the ATP challenge are shown in the panel. B, concentration dependence curve of LEV-induced reduction in [Ca2+]i response to ATP expressed as % increase above baseline [Ca2+]i. The curve reports, as a function of LEV concentration, LEV-induced reduction in [Ca2+]i increase elicited by 100 µM ATP. [Ca2+]i response to ATP was calculated as % increase above baseline [Ca2+]i and, for each LEV concentration, was expressed as percentage of mean % increase calculated in control cells (n = 460). Each plotted point is the mean ± S.E.M. of the data obtained in all the cells treated with each LEV concentration tested in four different experimental sessions. The number of the tested cells was 460, 154, 217, 420, and 239 in control, 0.1, 0.3, 1, and 10 µM LEV groups. Data fitting was performed as reported under Materials and Methods. C, concentration dependence curve of LEV-induced reduction in [Ca2+]i response to ATP expressed as [Ca2+]i AUC. The curve reports, as a function of LEV concentration, LEV-induced reduction in [Ca2+]i response to 100 µM ATP expressed as AUC. AUC calculation was performed integrating [Ca2+]i over a time interval of only 20 s because the very brief duration of ATP-induced [Ca2+]i spike. The number of the tested cells was 402, 99, 154, 362, and 240 in control, 0.1, 0.3, 1, and 10 µM LEV groups.

To confirm the specificity of LEV effect on IP₃-dependent Ca²⁺ release, we studied the effect of 1 µM LEV on [Ca²⁺]_i response elicited by 1 µM BK in NIH-3T3 cells since fibroblasts do express B₂ receptors (Faussner et al., 1991) but lack LEV binding sites (Fuks et al., 2003). In these cells, LEV did not induce any change either in basal [Ca²⁺]_i (131.1 ± 9.9 versus 124.5 ± 5.7 nM, respectively, in control and LEV-treated cells) or in the [Ca²⁺]_i response elicited by BK (1 µM) (Fig. 5). This result suggested that LEV affected [Ca²⁺]_i homeostasis only in those cells expressing LEV binding sites.

View larger version (26K): [in this window] [in a new window]   Fig. 5. LEV effect on [Ca2+]i increase elicited by BK in NIH-3T3 fibroblasts. A, time course of mean [Ca2+]i before and after 1 µM BK challenge in control cells and in cells treated with LEV. Each data point is the mean ± S.E.M. of the [Ca2+]i recorded at each time in all the cells of the control (n = 11) and LEV (n = 9) groups during a single experimental session, representative of a group of four. Only the [Ca2+]i recorded during last 50 s of the 5-min basal [Ca2+]i monitoring preceding the challenge with BK are shown in the panel. B, LEV-induced reduction in [Ca2+]i response to BK expressed as % increase above baseline [Ca2+]i. The bar plot reports the mean ± S.E.M. of LEV-induced reduction in [Ca2+]i increase elicited by 1 µM BK expressed as % recorded in control cells (n = 43) and cells exposed to 1 µM LEV (n = 38). Data are reported as percentage of mean % in control cells (n = 43). C, LEV-induced reduction in [Ca2+]i response to 1 µM BK expressed as [Ca2+]i AUC. The bar plot reports the mean ± S.E.M. of LEV-induced reduction in [Ca2+]i increase elicited by 1 µM BK expressed as UC. Data are reported as percentage of mean UC in control cells (n = 43). AUC calculation was performed using an integration time of 200 s because of the long duration of [Ca2+]i response to BK in these cells.

LEV Inhibitory Effect on [Ca²⁺]_i Response to BK Is Not Dependent on the Drug-Induced Inhibition of Ryanodine Stores. Another class of intracellular Ca²⁺ deposits, known as Rya stores because they express specific binding sites for the plant alkaloid ryanodine (Verkhratsky and Shmigol, 1996), are expected to take part in the [Ca²⁺]_i response triggered by the activation of G_q-coupled receptors because they are activated in the context of a calcium-induced calcium release (CICR) process (Reber et al., 1993). Therefore, to determine the extent of their involvement in our experimental system, we compared the amplitude of the [Ca²⁺]_i response to 1 µM BK in PC12 cells whose ryanodine receptors (RyRs) had been blocked by an overnight exposure to 100 µM Rya with that of control cells treated only with vehicle. As expected, the treatment with Rya significantly decreased cell responsiveness to BK. The % increase in [Ca²⁺]_i elicited by this peptide dropped, indeed, from 121.5 ± 5.8% in control cells (n = 90) to 62.1 ± 3.9% in cells exposed overnight to 100 µM Rya (n = 84) (P < 0.01; unpaired Student's t test). Noticeably, Rya not only reduced the amplitude of BK-elicited [Ca²⁺]_i response but also significantly shortened its duration (Fig. 6A).

View larger version (30K): [in this window] [in a new window]   Fig. 6. LEV does not affect Ca2+ release from the Rya stores in PC12 cells. A, LEV effect on [Ca2+]i increase elicited by BK in PC12 cells pretreated with 100 µM Rya. The left side of the panel reports the time course of mean [Ca2+]i before and after 1 µM BK challenge in control cells, in cells exposed overnight to 100 µM Rya to fully inactivate RyR, and in cells exposed overnight to 100 µM Rya and treated with LEV for 5 min before BK challenge and throughout the entire BK stimulation. Each data point is the mean ± S.E.M. of the [Ca2+]i recorded at each time in controls (n = 30), in cells pretreated with Rya but not exposed to LEV (n = 23), and in cells receiving both Rya and LEV (n = 26) during a single experimental session, representative of a group of three. Only the [Ca2+]i recorded during last 50 s of the 5-min basal [Ca2+]i monitoring preceding the BK challenge are shown in the panel. The top right side of the panel shows the [Ca2+]i response to BK expressed as % increase above baseline [Ca2+]i normalized to the mean of controls. The bar plot reports the mean ± S.E.M. of [Ca2+]i increase elicited by 1 µM BK expressed as % increase above baseline [Ca2+]i in control cells (n = 90), in cells receiving Rya but not LEV (n = 84), and in cells exposed to both drugs (n = 100). Data are reported as percentage of mean % in the control cells. The bottom left side of the panel reports the mean ± S.E.M. of [Ca2+]i increase elicited by 1 µM BK expressed as UC and normalized to the mean of control cells. AUC calculation was performed using an integration time of 30 s because of significant shortening of BK-elicited [Ca2+]i response in Rya-treated cells. **, P < 0.01 versus controls; , P < 0.01 versus Rya; ANOVA followed by the Tukey-Kramer post hoc test. B, effect of LEV on [Ca2+]i response elicited by 50 mM caffeine in PC12 cells. The left side of the panel reports the time course of mean [Ca2+]i recorded before and after the addition of 50 mM caffeine in control cells and in cells exposed to 1 µM LEV. Caffeine was dissolved in Krebs' solution with or without 1 µM LEV. To prevent the occurrence of hyperosmolarity in caffeine-containing solutions, the sodium chloride concentration was appropriately reduced. Each data point on the curves is the mean ± S.E.M. of the [Ca2+]i recorded at each time period in controls (n = 22) and in cells receiving 1 µM LEV (n = 26) during a single experimental session, representative of a group of three. The bar plots, on the left side of the panel, report the mean [Ca2+]i response to 50 mM caffeine in all the cells of the control (n = 106) and LEV (n = 106) groups. The top of the panel expresses % increase above baseline [Ca2+]i, whereas the bottom expresses the UC. AUC and % data are expressed as percentage of the respective mean values obtained in the control group. For AUC calculation, an integration time of 30 s was used because of the short duration of caffeine-elicited [Ca2+]i response.

Since LEV was reported to inhibit Ca²⁺ release from the intracellular Rya stores in neurons (Angehagen et al., 2003), we tested whether its effect on the intracellular Ca²⁺ release induced by IP₃-generating agonists was due to an inhibition of the CICR that accompanies IP₃-sensitive deposit discharges. To this aim, we studied LEV (1 µM) effect on the [Ca²⁺]_i response elicited by 1 µM BK after blocking RyR with an overnight exposure to Rya (100 µM) reasoning that, if LEV acted exclusively at the level of the Rya stores then, it should have been ineffective after the pharmacological blockade of RyR. Contrary to this hypothesis, LEV was effective in reducing the [Ca²⁺]_i response to BK in Rya-treated cells [% 41.4 ± 3.4 versus 62.1 ± 3.9% (P < 0.01); AUC, 30.5 ± 3.3 versus 46.7 ± 3.5 nM/s (P < 0.01) in cells treated with LEV (n = 100) versus controls (n = 84)] (Fig. 6A). These data suggest that, under our experimental conditions, LEV at low micromolar concentrations affects the IP₃-dependent stores but not the Rya-sensitive deposits. To verify the inefficacy of low LEV concentrations on Rya stores in PC12 cells, we directly examined the effect of 1 µM LEV on a kind of [Ca²⁺]_i response that is mediated exclusively by these deposits, using the Rya store depletor caffeine (50 mM). As expected, LEV was unable to modify the [Ca²⁺]_i response to 50 mM caffeine [%, 121.5 ± 3.9 versus 131.2 ± 3.2%, N.S.; AUC, 58.5 ± 1.6 versus 54 ± 2.2 nM/s, N.S., respectively, in control (n = 84) and in 1 µM LEV-treated (n = 100) cells] (Fig. 6B).

LEV Increases [Ca²⁺]_i Response to the Two Ca²⁺ Store-Depleting Agents TG and Iono. To assess whether LEV reduces the [Ca²⁺]_i response to BK and ATP by decreasing the amount of Ca²⁺ stored in the intracellular deposits, we studied the effect of LEV on [Ca²⁺]_i response to the store-depleting agents TG and Iono. In control PC12 cells, when TG (1 µM) and Iono (1 µM) were delivered in a Ca²⁺-free Krebs' solution, the intracellular Ca²⁺ stores were markedly depleted, as revealed by the consistent increase in [Ca²⁺]_i that peaked at 185 ± 2.7 nM (n = 94) in response to TG and at 228.9 ± 1.9 nM (n = 171) in response to Iono (Fig. 7, A and B). As expected, the duration of [Ca²⁺]_i response was significantly longer in response to TG ranging around 200 s than to Iono ranging around 30 s.

View larger version (32K): [in this window] [in a new window]   Fig. 7. LEV effect on the [Ca2+]i release elicited by TG and Iono. A, LEV effect on [Ca2+]i increase elicited by TG in PC12 cells. The left side of the panel reports the time course of mean [Ca2+]i before and after 1 µM TG challenge in control cells and in cells treated with LEV. Each data point is the mean ± S.E.M. of the [Ca2+]i recorded at each time in all the cells of the control (n = 47) and LEV (n = 36) groups during a single experimental session, representative of a group of two. Only the [Ca2+]i recorded during last 50 s of the 5-min basal [Ca2+]i monitoring preceding the TG challenge are shown in the panel. The right side of the panel reports the mean ± S.E.M. of the [Ca2+]i response to TG recorded in control cells (n = 94) and in the cells treated with LEV (n = 72) during the two experimental sessions expressed as % increase above baseline [Ca2+]i on the top and as AUC on the bottom. AUC and % data are expressed as percentage of the respective mean values obtained in the control group. Because of the very long duration of [Ca2+]i response to TG, an integration time of 200 s was used to calculate AUCs. B, LEV effect on [Ca2+]i increase elicited by Iono in PC12 cells. The left side of the panel reports the time course of mean [Ca2+]i before and after 1 µM Iono challenge in control cells and in cells treated with LEV. Each data point on the curves is the mean ± S.E.M. of the [Ca2+]i recorded at each time in all the cells of the control (n = 42) and LEV (n = 45) groups during a single experimental session, representative of a group of four. Only the [Ca2+]i recorded during last 50 s of the 5-min basal [Ca2+]i monitoring preceding the Iono challenge are shown in the panel. The right side of the panel reports the mean ± S.E.M. of the [Ca2+]i response to Iono recorded in control cells (n = 171) and in LEV-treated cells (n = 190) during the four experimental sessions. The [Ca2+]i response is expressed as % increase above baseline [Ca2+]i in the top of the panel and as AUC in the bottom. AUC and % data are expressed as percentage of the respective mean values obtained in the control group. For AUC calculation, an integration time of 30 s was used because of the short duration of Iono-elicited [Ca2+]i response. **, P < 0.01 versus controls; unpaired Student's t test.

In cells treated with 1 µM LEV for 5 min, before the TG or the Iono challenge and exposed to LEV throughout the entire stimulation, the [Ca²⁺]_i response to these store-depleting agents was significantly enhanced. Indeed, in the case of TG, the % increases were 46.38 ± 1.2 versus 36.56 ± 1.3% in cells exposed to LEV (n = 71) and in control cells (n = 93), respectively (P < 0.01; unpaired Student's t test), whereas in the same groups, the AUCs were 25.7 ± 0.9 versus 36.56 ± 1.3 nM/s (P < 0.01; unpaired Student's t test) (Fig. 7A). When the cells were challenged with 1 µM Iono with or without 1 µM LEV, the % increases in [Ca²⁺]_i above baseline were 65.95 ± 1.3% versus 55.8 ± 1.2% in cells exposed to LEV (n = 191) and in control cells (n = 171) (P < 0.01; unpaired Student's t test), whereas in the same groups, the AUCs were 62.9 ± 1.7 versus 58.8 ± 1.2 nM/s (P < 0.01; unpaired Student's t test) (Fig. 7B).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The major finding of the present study is that LEV reduces the IP₃-dependent [Ca²⁺]_i increase elicited by the activation of G_q-coupled neuropeptide and neurotransmitter receptors. This conclusion is supported by the evidence that in PC12 pheochromocytoma cells, LEV reduced, in a concentration-dependent manner, the [Ca²⁺]_i increase elicited by BK and ATP, two neurotransmitters that, in these cells, trigger IP₃ generation via the G_q-coupled B₂ (Nardone et al., 1994) and P_2Y (Moskvina et al., 2003) receptors. Actually, several arguments sustain the hypothesis that, under our experimental conditions, these responses depended on IP₃-triggered Ca²⁺ store depletion rather than on Ca²⁺ influx from the extracellular space or Ca²⁺ release from the Rya stores. Specifically, BK and ATP challenges were purposely delivered in a Ca²⁺-free solution containing EGTA (1.5 mM) so as to render extracellular Ca²⁺ unavailable for influx. By using this approach, we excluded that Ca²⁺ could enter into the cells either through the ionotropic P_2X ATP receptors, which are also expressed in PC12 cells (Moskvina et al., 2003), or through the so-called refilling channels, which are expected to open in response to the store depletion induced by BK or ATP.

Because Rya channels open during the CICR process whenever [Ca²⁺]_i significantly increases (Verkhratsky and Shmigol, 1996), it was crucial to establish whether in our system these intracellular Ca²⁺ deposits played any role in determining the intracellular Ca²⁺ response to IP₃-generating agonists. Accordingly, to obtain a pharmacological ablation of the intracellular Rya stores, we used the plant alkaloid Rya, for it irreversibly blocks RyRs whenever used at high micromolar concentrations (Verkhratsky and Shmigol, 1996). Since RyR blockade induced a consistent decrease in [Ca²⁺]_i response to BK, we evinced that CICR played a relevant role in the intracellular Ca²⁺ increase elicited by BK as already reported by Reber et al. (1993). Because of the important contribution of Rya stores in the [Ca²⁺]_i response to BK in PC12 cells, it was essential to determine whether LEV was acting directly on the IP₃-dependent Ca²⁺ release or whether it was, instead, reducing CICR. In this regard, we should consider that, in hippocampal neurons, LEV significantly reduces Ca²⁺ release from the Rya stores in response to caffeine (Angehagen et al., 2003). However, under our experimental conditions, LEV did not act at the Rya store level. In fact, its inhibitory effect persisted in PC12 cells whose RyR had been blocked with high micromolar concentrations of Rya. Furthermore, low micromolar LEV concentrations proved to be ineffective in reducing the [Ca²⁺]_i response to caffeine, whereas at high micromolar concentrations, the drug determined a small but significant decrease in this response (data not shown). This is in accordance with the data reported by Angehagen et al. (2003) who found that in hippocampal neurons, LEV did interfere with Ca²⁺ release from the Rya stores only at concentrations higher than 10 µM being ineffective in the low micromolar range.

LEV is not the first antiepileptic drug to be proposed as an inhibitor of IP₃-dependent intracellular Ca²⁺ release. In fact, Imazawa et al. (1989) demonstrated that phenytoin, phenobarbital, and carbamazepine displayed a modest ability to block IP₃-induced calcium release from microsomal fractions in vitro. However, the inhibitory effect resulting from these drugs is smaller than that exerted by LEV. Most likely, the contribution of this effect on the antiseizure efficacy of these drugs is only marginal. Indeed, these drugs, contrary to LEV, potently affect voltage-dependent channels and GABA_A receptors, and this accounts for their antiepileptic properties.

The mechanism of action through which LEV reduces IP₃-dependent intracellular Ca²⁺ release is still unclear. A direct competitive blockade of G_q-coupled receptors by LEV seems an unlikely explanation of our data for at least two different reasons. First, LEV was effective in reducing the intracellular Ca²⁺ response elicited by two structurally unrelated Gq-agonists, the peptide BK and the cyclic nucleotide ATP; second, it was not able to displace [³H]BK from its binding sites on PC12 cells. This points to an action on Ca²⁺ discharge from IP₃ stores exerted somewhere downstream the receptors. Several studies suggest that IP₃-dependent Ca²⁺ stores are composed of different compartments selectively mobilized by specific agonists (Den Hertog et al., 1992). In particular, BK and ATP mobilize two different and functionally independent IP₃-sensitive intracellular Ca²⁺ compartments (Suh et al., 1995). Because both these compartments were affected by LEV, we may suggest that the drug's effect was not compartment-specific, but it was affecting a more fundamental step of the IP₃ cascade. For example, LEV could be acting by decreasing Ca²⁺ ion accumulation in the intracellular deposits. The study results excluded this possibility. In fact, when we determined the amount of Ca²⁺ stored in the intracellular deposits by measuring the [Ca²⁺]_i response to TG and Iono, two compounds that deplete intracellular Ca²⁺ stores by acting directly on them, we found that LEV treatment did not decrease the amount of Ca²⁺ available for release from the intracellular deposits, but, on the contrary, it slightly increased it. This finding could be explained accordingly to the existing evidence on the constitutive activity of G protein-coupled plasma membrane receptors (Milligan, 2003). It can be assumed, in fact, that spontaneously active plasma membrane G_q-coupled receptors continuously release small amounts of Ca²⁺ ions from the IP₃-dependent stores in PC12 cells and that LEV could increase the amount of Ca²⁺ retained in the intracellular deposits by inhibiting this Ca²⁺ leak. Clearly, the increased availability of Ca²⁺ for release will not be evident if the cells are challenged with a G_q-coupled receptor agonist, as LEV would inhibit this response. Conversely, it will be manifest if the cells are treated with compounds directly acting on the stores, such as TG or Iono. Future studies will be necessary to establish whether LEV action on Ca²⁺ release from IP₃-sensitive stores is exerted at the level of G_q, phospholipase C, or IP₃ receptors.

LEV inhibition of IP₃-dependent intracellular Ca²⁺ release can be observed at drug concentrations in the low micromolar range that are easily reached in plasma of patients treated with LEV as mean LEV C_max at equilibrium ranges between 30 and 300 µM (Dooley and Plosker, 2000). Hence, the effect of LEV on intracellular Ca²⁺ homeostasis reported in the present study could be important in explaining the therapeutic efficacy of this drug. Indeed, the activation of several G_q-coupled receptors affects neuron excitability at central synapses and probably plays a role in epileptogenesis. For instance, presynaptic metabotropic G_q-coupled receptors depress GABA release in the hippocampus CA1 field (Gereau and Conn, 1995) and in cultured hippocampal neurons (Fitzsimonds and Dichter, 1996). Similarly, presynaptic M₁ G_q-coupled receptors negatively regulate GABA release in cerebral cortex slices (Hashimoto et al., 1994). Consistent evidence also reports that metabotropic group I glutamate receptors increase cell excitability by a postsynaptic action in different brain areas, including the cortex (Libri et al., 1997) and the hippocampus (Davies et al., 1995). Analogously, muscarinic M₁ and M₃ receptors have a definite role in promoting cell excitability by a postsynaptic mechanism in the neocortex (Cox et al., 1994). These effects on cell excitability can account for the well known proconvulsant ability of group I metabotropic glutamate receptor (Tizzano and Schoepp, 1995) and M₁ muscarinic receptor (Cruickshank et al., 1994) agonists. Accordingly, G_q-coupled receptors are very likely involved in epileptogenesis so that the antiseizure effect of LEV could be ascribed, in part, to its ability to interfere with the transduction mechanism triggered by these receptors.

Interestingly, LEV is highly effective in preventing the kindling response in rats (Löscher et al., 1998), a process involving the activation of G_q-coupled receptors, such as group I metabotropic glutamate receptors (Greenwood et al., 2000) and M₁ and M₃ muscarinic receptors (Mingo et al., 1998). Furthermore, it is worth noticing that BK is also involved in this model of epilepsy (Bregola et al., 1999).

The recent identification of LEV binding sites with the presynaptic protein SV_2A (Lynch et al., 2004) raises the important question of establishing whether LEV might affect [Ca²⁺]_i homeostasis by binding to this protein. An argument supporting this idea comes from the observation that LEV was ineffective in NIH-3T3 fibroblasts, a cell type that lacks SV_2A. Although a functional connection between LEV binding to SV_2A and [Ca²⁺]_i homeostasis has not been established, the results reported by Janz et al. (1999) suggest a role for SV₂ proteins in [Ca²⁺]_i homeostasis as the Ca²⁺ chelator EGTA-AM restores the train stimulation-induced synaptic depression which is lost in hippocampal neurons from SV_2A/SV_2B double knockout mice.

In conclusion, the present study provides evidence for a new pharmacological effect of the antiepileptic drug LEV: the inhibition of Ca²⁺ release from the IP₃-sensitive stores. Because of the relevant implications of IP₃-dependent Ca²⁺ release in neuron excitability and epileptogenesis, this effect could provide promising insights into the antiepileptic properties of LEV. More important, LEV could represent a prototypical example of a new pharmacological approach to treat epilepsy by counteracting Ca²⁺ release from the intracellular stores.

	Acknowledgements

 
We thank L. Colucci d'Amato (CNR Institute of Endocrinology and Experimental Oncology, Naples, Italy) and C. Miele (Department of Biology and Cellular and Molecular Pathology, Federico II University of Naples, Naples, Italy) for the generous gift of PC12 cells and NIH-3T3 fibroblasts; Herik Klitgaard and Doru Margineanu (UCB Pharma, Braine-L'Alleud, Belgium) for the critical reading of the manuscript and for enlightening discussions on the data; Viviana Marzaioli, Luigi Ombrato, and Anna Guacci and technician Vincenzo Grillo for the technical support in performing the experiments; and Paola Merolla and Marcella Donato for editorial help.

	Footnotes

 
This work was partially supported by a grant from UCB Pharma (Braine-L'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.

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.

Address correspondence to: Dr. Lucio Annunziato, Division of Pharmacology, Department of Neuroscience, Federico II University of Naples, Via Pansini no. 5, 80131 Naples, Italy. E-mail: lannunzi{at}unina.it

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