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NEUROPHARMACOLOGY

Modulation of Ca²⁺Channel Currents by a Novel Antidementia DrugN-(4-Acetyl-1-piperazinyl)-p-fluorobenzamide Monohydrate (FK960) in Rat Hippocampal Neurons

Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Kashima, Osaka, Japan (F.W., N.M., S.M.); and Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan (S.K.)

Received July 28, 2003; accepted October 8, 2003.

	Abstract

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N-(4-Acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate (FK960), a novel antidementia drug, has been demonstrated to ameliorate memory deficits in various experimental models of dementia. This drug selectively increases somatostatin release from hippocampal slices and augments long-term potentiation (LTP) in the CA3 area of the hippocampus. In the present study, the effects of FK960 on voltage-activated Ca²⁺channels were investigated in acutely isolated rat hippocampal neurons, using whole-cell patch-clamp technique to clarify the cellular mode of action of FK960. Application of somatostatin significantly reduced Ca²⁺currents via G protein-coupled signaling pathways. This inhibitory effect was significantly abolished by FK960 when applied in combination. In contrast, FK960 showed only modest inhibition on the reduction in Ca²⁺currents produced by baclofen, an agonist of GABA_Breceptor. Intracellular application of the protein kinase inhibitor H-7 did not alter somatostatin-induced inhibition and had no significant effect on blockade by FK960. In addition, application of FK960 alone produced modest but apparent increases in Ca²⁺currents without significant changes in the activation kinetics of the channels. The dose-response relationship on calcium current enhancement was bell-shaped with a maximum effect at 0.1 µM FK960, the same concentration as that for increasing on somatostatin release and CA3-LTP. These results show that FK960 reverses G protein-dependent inhibition of Ca²⁺currents by somatostatin in hippocampal neurons. Enhancement of Ca²⁺currents by FK960 may be due to its modulatory actions on Ca²⁺channels, rather than removal of G protein-inhibited tonic currents. Together, these mechanisms may be involved in the selective effects of FK960 on somatostatin release, excitatory transmission, and synaptic plasticity in the hippocampus.

The important regulatory mechanisms of neuronal Ca²⁺ channels in multiple cellular functions such as transmitter release, synaptic excitability and transmission, hippocampal LTP, and synapse formation have been extensively studied. The activation of voltage-activated Ca²⁺ channels is regulated by neurotransmitters and a variety of intracellular second messenger pathways (Anwyl, 1991). Many neurotransmitters, including somatostatin, are known to inhibit Ca²⁺ channels through G protein-coupled receptors and membrane-delimited pathways that may lead to autoreceptor-mediated inhibition of exocytotic release from presynaptic terminals (Dolphin 1995; Hille et al., 1995). Somatostatin selectively reduces N-type Ca²⁺ channel currents in hippocampal and other central neurons possibly via the activation of pertussis-toxin (PTX)-sensitive Gi/Go proteins (Ikeda and Schofield, 1989; Ishibashi and Akaike, 1995; Viana and Hille, 1996). Moreover, the inhibitory effect of somatostatin on Ca²⁺ channels results in the presynaptic inhibition of hippocampal synapse transmission (Boehm and Betz 1997).

To further understand the molecular basis for the ability of FK960 to enhance somatostatin release, in the present study, we investigated the effect of FK960 on voltage-dependent Ca²⁺ channels and its interaction with somatostatin using whole-cell patch-clamp recording in acutely isolated rat hippocampal neurons. Here, we report that FK960 reverses the somatostatin-induced inhibition of Ca²⁺ currents and that this effect seems to be mediated via a G protein-dependent mechanism. We also further demonstrated that FK960 has facilitatory actions on basal Ca²⁺ channel currents in hippocampal neurons.

	Materials and Methods

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Cell Preparation. Hippocampal neurons were acutely dissociated as described by Kay and Wong (1986) with slight modifications. Briefly, transverse hippocampal slices were prepared from 5- to 14-day-old male Wistar rats (Charles River Japan Inc., Yokohama, Japan). Slices were incubated at 30°C for 60 to 90 min in an oxygenated (100% O₂) solution containing 0.6 to 0.8 mg/ml trypsin (type I; Sigma-Aldrich, St. Louis, MO), 120 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 20 mM PIPES, and 25 mM glucose (pH 7.0). After rinsing with trypsin-free solution, slices were incubated at room temperature in an O₂ atmosphere. Before use, cells were triturated mechanically into individual cells with a fire-polished Pasteur pipette and were transferred to the recording chamber in Dulbecco's modified Eagle's medium. Visible pyramidal neurons with clear cellular membranes were chosen for whole-cell recording.

Current Recording and Analysis. Whole-cell recordings were performed using the conventional patch-clamp techniques (Hamill et al., 1981) with an Axopatch 200A amplifier (Axon Instruments, Union City, CA). Patch pipettes were fire-polished and had resistance of 2 to 4 M when filled with the internal pipette solution containing 100 mM CsCl, 5 mM MgCl₂, 10 mM EGTA, 40 mM HEPES, 4 mM ATP-Tris, and 0.2 mM GTP-Tris (pH 7.3). The normal external solution for recording Ca²⁺ channel currents consisted of 135 mM tetraethylammonium chloride, 10 mM BaCl₂, and 10 mM HEPES (pH 7.3). The recording chamber was continually perfused with the external solution with or without test drugs through gravity-fed flow pipes at a constant flow rate. Unless indicated otherwise, whole-cell inward currents were elicited every 10 s by depolarization to 0 mV from a holding potential of -80 mV. Currents were four-pole Bessel-filtered and digitized at 10 kHz with DigiData 1200 Interface. Data were acquired and leak subtracted using the P/4 protocol under the control of the pCLAMP (6.0) software (Axon Instruments) using a personal computer. All experiments were carried out at room temperature (21–23°C). All data are presented as mean ± S.E.M. (n = number of cells in parentheses in the figures). Statistical analysis of data was performed using Student's t test or Dunnett's multiple comparison test. A p value less than 0.05 was considered significant.

Pharmacological Materials. FK960 was synthesized by Fujisawa Pharmaceutical Co. Ltd. (Osaka, Japan). A 10 mM stock solution of FK960 was prepared daily with distilled water and diluted in the external solution to the desired final concentrations just before use. Somatostatin, baclofen, GTP S, pertussis toxin (PTX), and H-7 were obtained from Sigma-Aldrich. Somatostatin was dissolved in distilled water and frozen stored until use. Both GTP S and H-7 were dissolved in the pipette solution immediately before use. GTP in the standard pipette solution was omitted when GTP S was included.

	Results

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FK960 Disrupts Somatostatin-Induced Inhibition of Ca²⁺ Currents. Ba²⁺ was used as the charge carrier for the recording of Ca²⁺ currents in hippocampal neurons. Under whole-cell patch-clamp configuration, high-voltage-activated inward Ba²⁺ currents were completely blocked by external application of Cd²⁺, indicating that they pass through Ca²⁺ channels. Previous results have demonstrated that somatostatin selectively inhibits N-type Ca²⁺ channels in hippocampal neurons because these currents are sensitive to -conotoxin GVIA (Ishibashi and Akaike, 1995). Figure 1 illustrates the effect of FK960 on somatostatin-induced inhibition of Ca²⁺ currents in isolated rat hippocampal neurons. External application of somatostatin (0.1 µM) rapidly reduced the peak current amplitude and slowed the activation kinetics of Ca²⁺ currents (Fig. 1A). The inhibition by somatostatin was reversible after removal of somatostatin and was reproduced by repeated application of somatostatin with slight desensitization (Fig. 1B). In cells perfused with both somatostatin (0.1 µM) and FK960 (0.1 µM) concomitantly, however, the inhibition of Ca²⁺ currents by somatostatin was not observed. FK960 completely blocked the somatostatin-induced inhibition and eliminated the kinetic slowing. In contrast to the mean inhibition of 23.03 ± 1.80% (n = 12) produced by somatostatin in control conditions, there was only 2.17 ± 3.91% (n = 8) reduction in Ca²⁺ currents in the presence of FK960 (Fig. 1C).

View larger version (15K): [in this window] [in a new window]   Fig. 1. Effect of FK960 on somatostatin-induced inhibition of Ca2+ currents in hippocampal neurons. Currents were evoked every 10 s by a depolarization to 0 mV from a holding potential of -80 mV. A, superimposed current traces were obtained in control external solution and 0.1 µM somatostatin without or with 0.1 µM FK960. B, time course of the peak current in application of somatostatin (0.1 µM) in the presence or absence of 0.1 µM FK960. C, pooled results of peak current inhibited by somatostatin in the absence or presence of FK960. ***, p < 0.001; statistically significant compared to somatostatin alone group (by Student's t test). SST; somatostatin.

Considering that Ca²⁺ channels are regulated by a variety of neurotransmitters, we further investigated the effect of FK960 on inhibition of Ca²⁺ channels mediated by GABA_B receptor. As shown in Fig. 2, application of a GABA_B receptor agonist baclofen (25 µM) reduced the peak currents with an average 16.33 ± 2.88% (n = 7) inhibition in control condition and 11.38 ± 2.63% (n = 7) inhibition in the presence of 0.1 µM FK960. Although FK960 tended to reduce baclofen-induced inhibition, this effect was partial and was not statistically significant compared with baclofen alone-treated group, in contrast to the combination with somatostatin. These results suggest that FK960 selectively disrupts inhibition of Ca²⁺ currents produced by somatostatin.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Effect of FK960 on baclofen-induced inhibition of Ca2+ currents in hippocampal neurons. A, superimposed current traces were obtained in control external solution and 25 µM baclofen without or with 0.1 µM FK960. B, pooled results show the peak current inhibition produced by baclofen and concomitant application with FK960. No statistical significant change was observed between two groups (by Student's t test).

G Protein-Mediated Inhibition by Somatostatin. It is well demonstrated that voltage-dependent N-type Ca²⁺ channel activity is regulated by a G protein-coupled membrane-delimited pathway (Hille et al., 1995; Dolphin 1995) and the G protein-mediated inhibition of Ca²⁺ currents can be relieved by a large degree of depolarization (Bean, 1989; Ikeda, 1991). To test the voltage-dependent facilitation of current inhibition, currents were observed after a stronger depolarizing step to +80 mV. Under control conditions, the depolarizing prepulse did not produce characteristic facilitation of the Ca²⁺ currents. This implied the lack of tonic inhibition of Ca²⁺ currents by G proteins in these cells that has been shown in several neuronal systems, even in the absence of neurotransmitters (Ikeda, 1991; Kasai, 1991). As shown in Fig. 3A, somatostatin-induced inhibition of Ca²⁺ currents was mostly but not completely relieved, and the altered current kinetics was eliminated after the depolarizing prepulse, suggesting that voltage-dependent components reliving in somatostatin action. There was still a small portion of currents remaining after the prepulse in these cells. This may be due to the voltage protocol used here that resulted in incomplete recovery from somatostatin inhibition. In contrast, the application of FK960 abolished somatostatin inhibition before and after the prepulse was applied. Furthermore, FK960 restored the relief of the resting currents after the prepulse in these cells. These results suggest that FK960 modulates voltage-dependent inhibition of Ca²⁺ channels by somatostatin.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Involvement of G proteins in the action of somatostatin. A, effect of prepulse facilitation on somatostatin-induced inhibition. Currents were evoked by the voltage protocol as shown above. Superimposed current traces were obtained in control external solution and 0.1 µM somatostatin without (top traces) or simultaneously with 0.1 µM FK960 (bottom traces). SST; somatostatin. B effects of intracellular GTPS and PTX treatment. Peak currents were reduced by the intracellular application of 100 µM GTPS or by pretreatment of PTX. Both of them blocked significantly the somatostatin-induced inhibition. Pooled results show the peak current inhibition by somatostatin in the absence or presence of FK960. **, p < 0.01; statistically significant compared to somatostatin alone group (by Student's t test).

Line of evidence has shown that many neurotransmitters, including somatostatin, inhibit voltage-activated Ca²⁺ channels through direct activation of inhibitory G proteins (Shapiro and Hille, 1993; Hille et al., 1995; Zhang et al., 1996; Herlitze et al., 1996). In the present study, we used GTPS, a nonhydrolyzable analog of GTP, to characterize the G protein-dependent inhibition by somatostatin. When 100 µM GTPS was intracellularlly present in the pipette solution, the basal currents were reduced before somatostatin application (data not shown). As shown in Fig. 3B, the mean percentage of inhibition by somatostatin was 22.49 ± 1.96% (n = 9) in the control condition but was largely reduced to 4.51 ± 4.20% (n = 4) when GTPS was added in the pipette solution (p < 0.01). For PTX pretreatment, currents were obtained after incubation in culture medium containing high concentration of PTX (25 µg/ml) at 35°C for more than 1 h (Beech et al., 1992). Application of somatostatin had an inhibition of 2.18 ± 5.6% (n = 3), compared with 22.73 ± 2.45% (n = 7) inhibition obtained without PTX treatment (p < 0.01). Together, both of GTPS and PTX pretreatment significantly eliminated somatostatin-induced inhibition of Ca²⁺ currents, suggesting that PTX-sensitive G proteins are involved in somatostatin-induced inhibition of Ca²⁺ channel currents.

Involvement of Protein Kinase Activation. Several protein kinases are implicated in neurotransmitter receptor-mediated modulation of Ca²⁺ channels by phosphorylation of transmitter receptors themselves, the associated G proteins, and functional domains of Ca²⁺ channel subunits (Ahlijanian et al., 1991; Swartz, 1993; Stea et al., 1995; Zamponi et al., 1997; Hamid et al., 1999; Cooper et al., 2000). To test whether protein kinase activation is involved in the effects of FK960 and somatostatin, we used a broad protein kinase inhibitor H-7 by intracellular application. Figure 4 illustrates the effects on Ca²⁺ currents by somatostatin and FK960 with 50 µM H-7 in the pipette solution. In the intracellular presence of H-7, somatostatin reduced the peak current amplitudes with mean inhibition of 23.29 ± 4.33% (n = 8), similar to control conditions. FK960 again significantly abolished the somatostatin-induced inhibition of Ca²⁺ currents in the presence of H-7 treatment; however, the magnitude of the reduction by FK960 (mean somatostatin inhibition of 6.98 ± 2.73%; n = 8) was slightly smaller compared with that in control cells (Fig. 1C). Basal Ca²⁺ currents were unchanged by intracellular application of H-7 (data not shown). These results show that blocking of protein kinase activity by H-7 in our cell preparation has no significant effects on somatostatin receptor-mediated inhibition of Ca²⁺ channels.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Effect of protein kinase inhibitor H-7 on somatostatin-induced inhibition of Ca2+ currents and on the blockade by FK960. A, superimposed inward currents were recorded before and after application of 0.1 µM somatostatin in the intracellular presence of 50 µM H-7 (top trace). Time courses of peak current recorded under application of 0.1 µM somatostatin (bottom trace). B, pooled results of peak current inhibited by somatostatin in the absence or presence of FK960. **, p < 0.01, statistically significant compared with somatostatin-alone group (by Student's t test). SST; somatostatin.

FK960 Enhances the Basal Ca²⁺ Currents in Hippocampal Neurons. In addition to its modulating actions on somatostatin-induced inhibition of Ca²⁺ currents, we found that external application of FK960 reversibly enhances Ca²⁺ currents under the basal condition in some hippocampal neurons. Figure 5 illustrates the increase in the amplitude of peak currents after application of FK960. Application of FK960 did not alter the kinetics of the channels but increased the peak currents at most test potentials without measurable voltage dependence (Fig. 5B). Enhancement of Ca²⁺ currents by FK960 displayed a bell-shaped concentration dependence that is similar to that seen in our previous pharmacological studies (Matsuoka and Satoh, 1998; Inoue et al., 2001). As illustrated in Fig. 5C, application of 0.01 to 1 µM FK960 significantly enhanced the basal currents, and the maximal effect was obtained at a concentration of 0.1 µM FK960. Differences between 0.1 µM and other doses of FK960 were not statistically significant.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Enhancement of the basal Ca2+ currents by FK960. A, time course of enhancement of peak currents by 0.1 µM FK960. Inset shows superimposed currents recorded before, during, and after FK960 application. B, peak current-voltage relationship in the control condition and application of FK960 in the same cell. C, concentration-effect relationship for the enhancement of Ca2+ currents produced by FK960. Pooled results show the percentage changes of peak currents in the absence (set as 100%) and presence of FK960. **, p < 0.01; ***, p < 0.001, statistically significant compared to control group (by analysis of variance followed by Dunnett's comparison test).

	Discussion

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Regulation of neuronal voltage-activated Ca²⁺ channels by neurotransmitters and intracellular signaling pathways is an important step in the control of neurotransmitter release, synaptic transmission, and neuronal plasticity. In the present study, we have determined the effect of a novel anti-dementia drug FK960 on voltage-activated Ca²⁺ channels in isolated rat hippocampal neurons. Our results demonstrate for the first time that FK960 modulates the G protein-mediated inhibitory effect of somatostatin on Ca²⁺ channels and, furthermore, enhances the basal Ca²⁺ currents in hippocampal neurons.

It has been suggested that somatostatin receptors inhibit N-type Ca²⁺ channels via PTX-sensitive G proteins through a direct membrane-delimited model (Shapiro and Hille, 1993; Hille et al., 1995; Zhang et al., 1996). Somatostatin-induced inhibition of Ca²⁺ currents in these isolated hippocampal neurons is mediated by activation of G proteins because PTX as well as GTP S eliminated this inhibition. In addition, prepulse facilitation relieved most of the current reduction by somatostatin, a characteristic form of inhibition occurring via a direct interaction of the channel and G protein (Hille et al., 1995). Importantly, FK960 disrupted G protein-dependent inhibition of Ca²⁺ currents by somatostatin. Inhibitory effect of FK960 was more robust than that induced by depolarizing prepulse, which resulted in the recovery from somatostatin inhibition of Ca²⁺ currents. Somatostatin-induced inhibition of Ca²⁺ channels has been suggested to be one of the mechanisms underlying presynaptic inhibitory control of transmitter release (Boehm and Betz, 1997). Therefore, the ability of FK960 to modulate the inhibitory effect of somatostatin on Ca²⁺ channels may contribute to its ability to selectively enhance somatostatin release and somatostatinergic transmission in hippocampal slices (Inoue et al., 2001).

In the present study, we found that basal Ca²⁺ channel currents in hippocampal neurons were enhanced by FK960. Compared with its obvious effect on somatostatin-induced depression of Ca²⁺ currents, the enhancement of basal Ca²⁺ currents produced by FK960 was more modest, and was not observed in all cells. Although several types of voltage-activated Ca²⁺ channels have been identified in the hippocampal neurons, it has been suggested that N-type channels predominantly contribute to the transmitter-stimulated synaptic transmission (Wheeler et al., 1994). Further study is necessary to determine which type of Ca²⁺ channel current is enhanced by FK960. The FK960-induced increase in basal Ca²⁺ channel currents is unlikely to be a consequence of removal of G protein-mediated tonic inhibition, as observed in some neurons (Dolphin, 1995), because the rebound current facilitation by a large depolarization pulse was not observed in these cells. The facilitatory effect of FK960 on Ca²⁺ current demonstrated a bell-shaped concentration-response relationship comparable with our previous studies on somatostatin release and CA3-LTP enhancement, indicating the phenomenon might share common cellular mechanisms.

Voltage-activated Ca²⁺ channel activities are regulated by a variety of neurotransmitter and intracellular signaling pathways (Hille et al., 1995; Dolphin, 1995). Therefore, the modulation of Ca²⁺ channels by FK960 could be mediated at the level of neurotransmitter receptor, G proteins, Ca²⁺ channels, or other intracellular signaling pathways. Our previous studies have shown that FK960 does not bind to somatostatin receptors or a number of other neurotransmitter receptors (unpublished observations), although the possibility of an unknown protein component associated to its action could not be ruled out. Our present results demonstrated that FK960 disrupted G protein-dependent inhibition by somatostatin and enhanced the basal Ca²⁺ current, further suggesting that the modulation of FK960 is not in the level of somatostatin receptors activation and seems to be involved in the direct interaction between G protein and Ca²⁺ channels. Recently, an occluded inhibition of Ca²⁺ channels by activation of two types of transmitter receptor by opioid agonists and somatostatin has been investigated (Polo-Parada and Pilar, 1999).

Protein kinases are important for regulation of neuronal Ca²⁺ channel activity and have been shown to directly phosphorylate the channel subunit/G protein complex (Swartz, 1993; Hamid et al., 1999; Cooper et al., 2000). In central neurons, activation of PKC has been found to augment Ca²⁺ currents (Swartz et al., 1993; Stea et al., 1995; Hamid et al., 1999) and disrupt G protein-dependent inhibition of Ca²⁺ channels (Swartz, 1993; Zamponi et al., 1997; Barrett and Rittenhouse, 2000). Furthermore, PKC activators block the inhibition of Ca²⁺ currents induced by somatostatin in rat hippocampal neurons (Ishibashi and Akaike, 1995). In the present study, intracellular application of protein kinase inhibitor H-7 had no effect on somatostatin-induced inhibition of Ca²⁺ currents. In addition, H-7 slightly reduced FK960's modulation of somatostatin inhibition, but the effect was not significant. Considering that H-7 is known to be relatively more effective on cAMP (or cGMP)-dependent kinase activation than on PKC activation, however, we could not rule out the possibility of PKC-mediated regulation involved in the mechanism of action of FK960. Further studies with highly selective PKC modulators are required to clarify the contribution of intracellular phosphorylation pathways to the mechanism of FK960 action.

It is conceivable that modulation by FK960 of somatostatin-mediated inhibition of Ca²⁺ channels may be responsible for its enhancement of somatostatin release from hippocampal slices. Many studies have demonstrated the existence of negative feedback regulation via presynaptic G protein-coupled receptors (autoreceptors) as well as voltage-activated Ca²⁺ channels, leading to inhibition of transmitter release (Dolphin, 1995; Hille et al., 1995; Takahashi et al., 1996). Indeed, somatostatin release from nerve terminals is Ca²⁺-dependent and is modulated through activation of an autoreceptor located on presynaptic terminals (Iversen et al., 1978; Fontana et al., 1996; Helboe et al., 1998). Furthermore, somatostatin selectively inhibits the N-type Ca²⁺ channel among diverse subtypes of channels in isolated hippocampal neurons (Ishibashi and Akaike, 1995). It has also been reported that somatostatin inhibits excitatory neurotransmission via presynaptic receptors, by inhibition of downstream of Ca²⁺ entry at rat hippocampal synapses (Boehm and Betz, 1997). It will be important to determine whether FK960 acts exclusively on somatostatin-mediated inhibition, or whether it can disrupt the inhibition produced by other transmitters, which involve G protein-coupled receptors. In the present study, interestingly, FK960 showed only modest inhibition on the reduction in Ca²⁺ currents produced by an activation of GABA_B receptor, suggesting that FK960's action is not general for G protein-coupled receptors and could be selective for somatostatin receptor over other class of G protein-coupled receptors. Somatostatin-containing neurons are abundant in the hippocampus, and, although they often colocalize or functionally interact with other neurotransmitters such as GABA or ACh, they might regulate Ca²⁺ channels through different G protein pathways (Shapiro and Hille, 1993; Hille et al., 1995). In our studies on neurotransmitter release in rat hippocampal slices, we found that FK960 had no significant effect on ACh, GABA, noradrenaline, and serotonin release (Inoue et al., 2001). We therefore propose that FK960 could exert selective facilitatory actions on somatostatin release from hippocampal nerve terminals, as a consequence of blockade of the interplay between the somatostatin autoreceptor, inhibitory G protein, and (possibly N-type) Ca²⁺ channels.

In conclusion, we have demonstrated for the first time that the novel antidementia drug FK960 reverses the inhibitory effect of somatostatin on Ca²⁺ channels and enhances the basal activity of Ca²⁺ currents in rat hippocampal neurons. These cellular mechanisms may explain the unique mode of action of FK960 and may further provide new insights on the molecular basis for the understanding the control of neuropeptide release from presynaptic terminals.

	Footnotes

 
DOI: 10.1124/jpet.103.057687.

ABBREVIATIONS:ACh, acetylcholine; LTP, long-term potentiation; PTX, pertussis-toxin; GTPS, guanosine 5'-O-(3-thio)triphosphate; H-7, 1-(5-isoquinoline sulfonyl)-2-methylpiperazine; PKC, protein kinase C.

Address correspondence to: Dr. Nobuya Matsuoka, Department of Neuroscience, Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan. E-mail: nobuya_matsuoka{at}po.fujisawa.co.jp

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